Patent Publication Number: US-2006019269-A1

Title: Susceptibility gene for myocardial infarction, stroke, and PAOD, methods of treatment

Description:
This application is a continuation-in-part of International Application No. PCT/US05/03312, filed Jan. 31, 2005 which claims priority benefit of U.S. Application No. 60/642,909, filed Jan. 10, 2005, and is also a continuation-in part of U.S. patent application Ser. No. 10/830,477, filed Apr. 22, 2004, which is a continuation-in-part of U.S. patent application Ser. No. 10/769,744, filed Jan. 30, 2004, which is a continuation-in-part of International Application No. PCT/US03/32556, filed on Oct. 16, 2003, which claims the benefit of U.S. Provisional Application No. 60/419,433, filed on Oct. 17, 2002 and U.S. Provisional Application No. 60/449,331, filed on Feb. 21, 2003. This application is also a continuation-in-part of International Application No. PCT/US04/030582, filed Sep. 17, 2004, which claims priority benefit of U.S. Provisional Application No. 60/503,587, filed Sep. 17, 2003. Each of the priority applications is specifically incorporated herein by reference in their entirety, without prejudice or disclaimer. 
    
    
     BACKGROUND OF THE INVENTION  
      Myocardial infarction (MI) and Acute Coronary Syndrome (ACS), e.g., unstable angina, non-ST-elevation myocardial infarction (NSTEMI) or ST-elevation myocardial infarction (STEMI), are the leading causes of hospital admissions in industrialized countries. Cardiovascular disease continues to be the principle cause of death in the United States, Europe and Japan. The costs of the disease are high both in terms of morbidity and mortality, as well as in terms of the financial burden on health care systems.  
      Myocardial infarction generally occurs when there is an abrupt decrease in coronary blood flow following a thrombotic occlusion of a coronary artery previously damaged by atherosclerosis. In most cases, infarction occurs when an atherosclerotic plaque fissures, ruptures or ulcerates and when conditions favor thrombogenesis. In rare cases, infarction may be due to coronary artery occlusion caused by coronary emboli, congenital abnormalities, coronary spasm, and a wide variety of systemic, particularly inflammatory diseases. Medical risk factors for MI include cigarette smoking, diabetes, hypertension and serum total cholesterol levels &gt;200 mg/dL, elevated serum LDL cholesterol, and low serum HDL cholesterol. Event rates in individuals without a prior history of cardiovascular disease are about 1%. In individuals who have had a first MI or ACS, the risk of a repeat MI within the next year is 10-14%, despite maximal medical management including angioplasty and stent placement.  
      Atherosclerosis can affect vascular beds in many large and medium arteries. Myocardial infarction and unstable angina (acute coronary syndrome (ACS)) stem from coronary artery atherosclerosis, while ischemic stroke most frequently is a consequence of carotid or cerebral artery atherosclerosis. Limb ischemia caused by peripheral arterial occlusive disease (PAOD) may occur as a consequence of iliac, femoral and popliteal artery atherosclerosis. The atherosclerotic diseases remain common despite the wide-spread use of medications that inhibit thrombosis (aspirin) or treat medical risk factors such as elevated cholesterol levels in blood (statins), diabetes, or hypertension (diuretics and anti-hypertensives).  
      Atherosclerotic disease is initiated by the accumulation of lipids within the artery wall, and in particular, the accumulation of low-density lipoprotein (LDL) cholesterol. The trapped LDL becomes oxidized and internalized by macrophages. This causes the formation of atherosclerotic lesions containing accumulations of cholesterol-engorged macrophages, referred to as “foam cells”. As disease progresses, smooth muscle cells proliferate and grow into the artery wall forming a “fibrous cap” of extracellular matrix enclosing a lipid-rich, necrotic core. Present in the arterial walls of most people throughout their lifetimes, fibrous atherosclerotic plaques are relatively stable. Such fibrous lesions cause extensive remodeling of the arterial wall, outwardly displacing the external, elastic membrane, without reduction in luminal diameter or serious impact on delivery of oxygen to the heart. Accordingly, patients can develop large, fibrous atherosclerotic lesions without luminal narrowing until late in the disease process. However, the coronary arterial lumen can become gradually narrowed over time and in some cases compromise blood flow to the heart, especially under high demand states such as exercise. This can result in reversible ischemia causing chest pain relieved by rest called stable angina.  
      In contrast to the relative stability of fibrous atherosclerotic lesions, the culprit lesions associated with myocardial infarction and unstable angina (each of which are part of the acute coronary syndrome) are characterized by a thin fibrous cap, a large lipid core, and infiltration of inflammatory cells such as T-lymphocytes and monocyte/macrophages. Non-invasive imaging techniques have shown that most MI&#39;s occur at sites with low- or intermediate-grade stenoses, indicating that coronary artery occlusion is due most frequently to rupture of culprit lesions with consequent formation of a thrombus or blood clot and not solely due to luminal narrowing by stenosis. Plaque rupture may be due to erosion or uneven thinning of the fibrous cap, usually at the margins of the lesion where macrophages enter, accumulate, and become activated by a local inflammatory process. Thinning of the fibrous cap may result from degradation of the extracellular matrix by proteases released from activated macrophages. These changes producing plaque instability and risk of MI may be augmented by production of tissue-factor procoagulant and other factors increasing the likelihood of thrombosis.  
      In acute coronary syndrome, the culprit lesion showing rupture or erosion with local thrombosis typically is treated by angioplasty or by balloon dilation and placement of a stent to maintain luminal patency. Patients experiencing ACS are at high risk for a second coronary event due to the multi-vessel nature of coronary artery disease with event rates approaching 10-14% within 12 months after the first incident.  
      The emerging view of MI is as an inflammatory disease of the arterial vessel wall on preexisting chronic atherosclerotic lesions, sometimes triggering rupture of culprit lesions and leading to local thrombosis and subsequent myocardial infarction. The process that triggers and sustains arterial wall inflammation leading to plaque instability is unknown, however, it results in the release into the circulation of tumor necrosis factor alpha and interleukin-6. These and other cytokines or biological mediators released from the damaged vessel wall stimulate an inflammatory response in the liver causing elevation in several non-specific general inflammatory markers including C-reactive protein. Although not specific to atherosclerosis, elevated C-reactive protein (CRP) and serum amyloid A appear to predict risk for MI, perhaps as surrogates for vessel wall inflammation.  
      Although classical risk factors such as smoking, hyperlipidemia, hypertension, and diabetes are associated with many cases of coronary heart disease (CHD) and MI, many patients do not have involvement of these risk factors. In fact, many patients who exhibit one or more of these risk factors do not develop MI. Family history has long been recognized as one of the major risk factors. Although some of the familial clustering of MI reflects the genetic contribution to the other conventional risk factors, a large number of studies have suggested that there are significant genetic susceptibility factors, beyond those of the known risk factors (Friedlander Y, et al.,  Br. Heart J.  1985; 53:382-7, Shea S. et al.,  J. Am. Coll. Cardiol.  1984; 4:793-801, and Hopkins P. N., et al.,  Am. J. Cardiol.  1988; 62:703-7). Major genetic susceptibility factors have only been identified for the rare Mendelian forms of hyperlipidemia such as a familial hypercholesterolemia.  
      Genetic risk is conferred by subtle differences in genes among individuals in a population. Genes differ between individuals most frequently due to single nucleotide polymorphisms (SNP), although other variations are also important. SNP are located on average every 1000 base pairs in the human genome. Accordingly, a typical human gene containing 250,000 base pairs may contain 250 different SNP. Only a minor number of SNP are located in exons and alter the amino acid sequence of the protein encoded by the gene. Most SNP have no effect on gene function, while others may alter transcription, splicing, translation, or stability of the mRNA encoded by the gene. Additional genetic polymorphism in the human genome is caused by insertion, deletion, translocation, or inversion of either short or long stretches of DNA. Genetic polymorphisms conferring disease risk may therefore directly alter the amino acid sequence of proteins, may increase the amount of protein produced from the gene, or may decrease the amount of protein produced by the gene.  
      As genetic polymorphisms conferring risk of disease are uncovered, genetic testing for such risk factors is becoming important for clinical medicine. Examples are apolipoprotein E testing to identify genetic carriers of the apoE4 polymorphism in dementia patients for the differential diagnosis of Alzheimer&#39;s disease, and of Factor V Leiden testing for predisposition to deep venous thrombosis. More importantly, in the treatment of cancer, diagnosis of genetic variants in tumor cells is used for the selection of the most appropriate treatment regime for the individual patient. In breast cancer, genetic variation in estrogen receptor expression or heregulin type 2 (Her2) receptor tyrosine kinase expression determine if anti-estrogenic drugs (tamoxifen) or anti-Her2 antibody (Herceptin) will be incorporated into the treatment plan. In chronic myeloid leukemia (CML) diagnosis of the Philadelphia chromosome genetic translocation fusing the genes encoding the Bcr and Ab1 receptor tyrosine kinases indicates that Gleevec (ST1571), a specific inhibitor of the Bcr-Ab1 kinase should be used for treatment of the cancer. For CML patients with such a genetic alteration, inhibition of the Bcr-Ab1 kinase leads to rapid elimination of the tumor cells and remission from leukemia.  
      Many general inflammatory markers predict risk of coronary heart disease, although these markers are not specific to atherosclerosis. For example, Stein (Stein, S.,  Am J Cardiol,  87 (suppl):21A-26A (2001)) discusses the use of any one of the following serum inflammatory markers as surrogates for predicting risk of coronary heart disease including C-reactive protein (CRP), serum amyloid A, fibrinogen, interleukin-6, tissue necrosis factor-alpha, soluble vascular cell adhesion molecules (sVCAM), soluble intervascular adhesion molecules (sICAM), E-selectin, matrix metalloprotease type-1, matrix metalloprotease type-2, matrix metalloprotease type-3, and matrix metalloprotease type-9. Elevation in one more of these serum inflammatory markers is not specific to coronary heart disease but also occurs with age or in association with cerebrovascular disease, peripheral vascular disease, non-insulin dependent diabetes, osteoarthritis, bacterial infection, and sepsis.  
      Serum C-reactive protein (CRP) is viewed as a convenient and sensitive marker of systemic inflammation. Generally CRP is measured in serum samples using commercially available enzyme-linked immunosorbent assays (EIA). Consistent across multiple published studies is the finding of a correlation between increased risk for coronary artery disease with increased serum CRP. For example, in the Women&#39;s Health Study, CRP was measured in 27,939 apparently healthy American women. The cut-off points for quintiles of serum CRP in women were: less than or equal to 0.49, more than 0.49 to 1.08, more than 1.08 to 2.09, more than 2.09 to 4.19, and more than 4.19 mg CRP per liter, see Ridker, P. M. et al.,  New England. J. Med.,  347: 1557-1565 (2001). In comparison to the lowest quintile, and even when adjusting for age, every quintile more than 0.49 mg CRP per liter was associated with increased risk for coronary heart disease with the highest relative risk of 4.5 seen for those women in the highest quintile of serum CRP (more than 4.19 mg CRP per liter). A similar correlation between increased serum CRP and increased risk for coronary heart disease in women has been reported (Ridker, P. M et al.,  New England. J. Med.,  342:836-843 (2000) and Bermudez, E. A. et. al.,  Arterioscler. Thromb. Vasc. Biol.,  22: 1668-1673 (2002)). Men also show a correlation between increased serum inflammatory markers such as CR and increased risk for coronary heart disease has been reported (Doggen, C. J. M. et al., J. Internal Med., 248:406-414 (2000) and Ridker, P. M. et al.,  New England. J. Med.,  336: 973-979 (1997)). Quintiles for serum CRP as reported by Doggen et al., were less than 0.65, more than 0.65 to 1.18, more than 1.18 to 2.07, more than 2.07 to 4.23, and more than 4.23 mg CRP per liter. Unlike women, elevated serum CRP correlates with increased relative risk for coronary heart disease only in the 4 th  and 5 th  quintiles of CRP (relative risk of 1.7x and 1.9x, respectively).  
      Serum CRP in women also has been measured in conjunction with lipid markers such as levels of serum low density lipoprotein-cholesterol (LDL-C). In the study by Ridker, P. M. et al. (2002), serum CRP and LDL-C are minimally correlated, screening for both serum markers provided better prognostic indication than either alone. Thus, women with serum CRP above median values (more than 1.52 mg CRP per liter) and also serum LDL-C above median values (more than 123.7 mg LDL-C per deciliter) were at highest risk for coronary heart disease.  
      Elevated CRP or other serum inflammatory markers is also prognostic for increased risk of a second myocardial infarct in patients with a previous myocardial infarct (Retterstol, L. et al.,  Atheroscler.,  160: 433-440 (2002)).  
      Since CRP is produced in the liver, there is no a priori mechanistic explanation for why elevation in CRP and other serum inflammatory markers should be prognostic for coronary artery disease. As discussed by Doggen, C. J. M., et al., one or more of the following factors were speculated to account for the correlation observed: (1) intrinsic inflammation and tissue damage within arterial lesions, (2) prior infection by  Helicobacter pylori  or by  Chlamydia pneumoniae,  (3) release of peptide cytokines including interleukin-6, or (4) activation of the complement system.  
      The end products of the leukotriene pathway are potent inflammatory lipid mediators derived from arachidonic acid. They can potentially contribute to development of atherosclerosis and destabilization of atherosclerotic plaques through lipid oxidation and/or proinflammatory effects. LTC4, LTD4, and LTE4, are known to induce vasoconstriction. Allen et al.,  Circulation,  97:2406-2413 (1998) described a novel mechanism in which atherosclerosis is associated with the appearance of a leukotriene receptor(s) capable of inducing hyperactivity of human epicardial coronary arteries in response to LTC4 and LTD4. LTB4, on the other hand, is a strong proinflammatory agent. Increased production of these end products, of the leukotriene pathway, could therefore serve as a risk factor for MI and atherosclerosis, whereas both inflammation and vasoconstriction/vasospasm have a well established role in the pathogenesis of MI and atherosclerosis. It has also been shown that a heterozygous deficiency of the 5-LO enzyme in a knockout mouse model decreases atherosclerotic lesion size in LDLR−/− mice by about 95%. (Mehrabian et al.,  Circulation Research.  91:120 (2002)). However, such genetic evidence for leukotriene involvement in MI or atherosclerosis in humans has not been reported. Mehrabian et al. did report a very small genetic association study looking for correlation between promoter polymorphisms of 5-LO and carotid intimal thickening in normal individuals. However, their data paradoxically suggest that a lower amount of leukotriene production correlates with carotid atherosclerosis.  
     SUMMARY OF THE INVENTION  
      As described herein, a gene on chromosome 13q12-13 has been identified as playing a major role in myocardial infarction (MI). This gene, herein after referred to as the MI gene, comprises nucleic acid that encodes 5-lipoxygenase activating protein (ALOX5AP or FLAP) herein after referred to as FLAP. The gene has also been shown to play a role in stroke and PAOD. In addition, a gene on chromosome 12q23 has been identified as playing a major role in myocardial infarction (MI). The gene comprises nucleic acid that encodes leukotriene A4 hydrolase, herein after referred to as LTA4H.  
      The invention pertains to methods of treatment (prophylactic and/or therapeutic) for certain diseases and conditions (e.g., MI, ACS, atherosclerosis, stroke, PAOD) associated with FLAP or with other members of the leukotriene pathway (e.g., biosynthetic enzymes or proteins such as FLAP, arachidonate 4-lipoxygenase (5-LO), leukotriene C4 synthase (LTC4S), leukotriene A4 hydrolase (LTA4H), leukotriene B4 12-hydroxydehydrogenase (LTB4DH)); receptors and/or binding agents of the enzymes; and receptors for the leukotrienes LTA4, LTB4, LTC4, LTD4, LTE4, Cys LT1, Cys LT2, including leukotriene B4 receptor 1 (BLT1), leukotriene B4 receptor 2 (BLT2), cysteinyl leukotriene receptor 1 (CysLTR1), cysteinyl leukotriene receptor 2 (CysLTR2). The methods include the following: methods of treatment for myocardial infarction or susceptibility to myocardial infarction; methods of phophylaxis therapy for myocardial infarction; methods of treatment for transient ischemic attack, transient monocular blindness or stroke, or susceptibility to stroke; methods of treatment for claudication, PAOD or susceptibility to PAOD; methods of treatment for acute coronary syndrome (e.g., unstable angina, non-ST-elevation myocardial infarction (NSTEMI) or ST-elevation myocardial infarction (STEMI)); methods for reducing risk of MI, stroke or PAOD in persons with asymptomatic ankle/brachial index less than 0.9; methods for decreasing risk of a second myocardial infarction or stroke; methods of treatment for atherosclerosis, such as for patients requiring treatment (e.g., angioplasty, stents, revascularization procedure) to restore blood flow in arteries (e.g., coronary, carotid, and/or femoral arteries); methods of treatment for asymptomatic ankle/brachial index of less than 0.9; and/or methods for decreasing leukotriene synthesis (e.g., for treatment of myocardial infarction, stroke or PAOD).  
      The invention provides for methods of prophylaxis therapy for myocardial infarction (MI). These methods comprise selecting a human subject susceptible to MI, administering to the subject a composition comprising a therapeutically effective amount of an MI therapeutic agent that inhibits leukotriene synthesis in vivo, wherein the MI therapeutic agent inhibits leukotriene synthesis by inhibiting the activity of at least one protein selected from 5-Lipoxygenase activating protein (FLAP) and 5-lipoxygenase (5-LO). The methods also comprise monitoring myeloperoxidase level before and during the prophylaxis treatement, wherein the MI therapeutic agent is administered in an amount effective to reduce MPO levels in a subject. These methods may further comprise monitoring at least one additional inflammatory marker, such as C-reactive protein, in the human subject before and during the prophylaxis therapy.  
      In the methods of the invention, a leukotriene synthesis inhibitor is administered to an individual in a therapeutically effective amount. The leukotriene synthesis inhibitor can be an agent that inhibits or antagonizes a member of the leukotriene synthesis pathway (e.g., FLAP, 5-LO, LTC4S, LTA4H, and LTB4DH). For example, the leukotriene synthesis inhibitor can be an agent that inhibits or antagonizes FLAP polypeptide activity (e.g., a FLAP inhibitor) and/or FLAP nucleic acid expression, as described herein (e.g., a FLAP nucleic acid antagonist). In another embodiment, the leukotriene synthesis inhibitor is an agent that inhibits or antagonizes polypeptide activity and/or nucleic acid expression of another member of the leukotriene biosynthetic pathway (e.g., LTC4S, LTA4H) or that increases breakdown of leukotrienes (e.g., LTB4DH). In preferred embodiments, the agent alters activity and/or nucleic acid expression of FLAP or of 5-LO. Preferred agents include those set forth in the Agent Table I herein. In another embodiment, preferred agents can be: 1-((4-chlorophenyl)methyl)-3-((1,1-dimethylethyl)thio)-alpha,alpha-dimethyl-5-(2-quinolinylmethoxy)-1H-Indole-2-propanoic acid otherwise known as MK-0591, (R)-(+)-alpha-cyclopentyl-4-(2-quinolinylmethoxy)-Benzeneacetic acid otherwise known as BAY-x-1005, 3-(3-(1,1-dimethylethylthio-5-(quinoline-2-ylmethoxy)-1-(4-chloromethylphenyl)indole-2-yl)-2,2-dimethylpropionaldehyde oxime-0-2-acetic acid otherwise known as A-81834, optically pure enantiomers, salts, chemical derivatives, and analogues; or can be zileuton, atreleuton, 6-((3-fluoro-5-(tetrahydro-4-methoxy-2H-pyran-4yl)phenoxy)methyl)-1-methyl-2(1H)-quinlolinone otherwise known as ZD-2138, 1-((4-chlorophenyl)methyl)-3-((1,1dimethylethyl)thio)-alpha,alpha-dimethyl-5-(2-quinolinylmethoxy)-1H-Indole-2-propanoic acid otherwise known as MK-886, 4-(3-(4-(2-Methyl-imidazol-1-yl)-phenylsulfanyl)-phenyl)-tetrahydro-pyran-4-carboxylic acid amide otherwise known as CJ-13610, their optically pure enantiomers, salts, chemical derivatives, and analogues. In another embodiment, the agent alters metabolism or activity of a leukotriene (e.g., LTA4, LTB4, LTC4, LTD4, LTE4, Cys LT1, Cys LT2), such as leukotriene antagonists or antibodies to leukotrienes, as well as agents which alter activity of a leukotriene receptor (e.g., BLT1, BLT2, CysLTR1, and CysLTR2).  
      In another preferred embodiments, the agent alters activity and/or nucleic acid expression of LTA4H. Preferred agents include those set forth in the Agent Table and in the Additional LTA4H Agent List herein. In another embodiment, preferred agents can be: ethyl-1-[2-[4-(phenylmethyl)phenoxy]ethyl]-4-piperidine-carboxylate, otherwise known as SC-56938; [4-[5-(3-Phenyl-propyl)thiophen-2-yl]butoxy]acetic acid, otherwise known as RP64966; (R)-S-[[4-(dimethylamino)phenyl]methyl]-N-(3-mercapto-2methyl-1-oxopropyl-L-cycteine, otherwise known as SA6541; optically pure enantiomers, salts, chemical derivatives, and analogues.  
      The results in Example 10 demonstrate that in patients with the at-risk FLAP and LTA 4  haplotypes, a FLAP inhibitor (DG-031 also known as Bay-X-1005) had a highly significant and dose-dependent effect at the cellular, whole bood and urinary metabolite level including a 26% reduction in leukotriene B 4  production by activated neutrophils, a 13% reduction of myeloperoxidase in whole blood, and a 27% increase in urinary leukotriene E 4 . Furthermore, there was evidence of a persistent effect, following discontinuation of the FLAP inhibitor, on high senstivity C-reactive protein and serum amyloid A. This reduction in CRP and serum amyloid A was observed on top of the beneficial effects that may have been acheived by statins taken by 85% of the study subjects.  
      The invention provides for compositions comprising a leukotriene synthesis inhbitor and a statin. The invention also provides for the use of a leukotriene synthesis inhibitor and a statin for the manufacture of a medicament for reducing CRP levels in a human subject. Such compositions are intended for human administration, and preferably further comprising a (at least one) pharmaceutically acceptable diluent, adjuvant, excipient, or carrier. Materials and methods for formulation and co-formulation are well known, and many are described herein in greater detail. In one variation, formulation of the composition into convenient unit dose formulations, such as pills or capsules for oral administration, including sustained release formulations, is specifically contemplated. In another variation, co-administration transdermally, e.g., through a skin patch, is contemplated. In still another variation, administration of one or both agents through a drug eluting stent is specifically contemplated. In particular, the compositions may comprises a leukotriene synthesis inhibitor that inhibits the activity of a member of the leukotriene synthesis pathway such as 5-lipoxygenase, 5-lipoxygenase activating protein (FLAP), leutokriene C4 synthase, leukriene A4 hydolase, arachidonate 4-lipoxygenase, leukotriene B4 12-hydroxydehydrogenase, leukotriene A4 receptor, leukotriene B4 receptor, leukotriene C4 receptor, leukotriene D4 receptor, leukotriene E4 receptor, leukotriene B4 receptor 1, leukotriene B4 receptor 2, cysteinyl leukotriene receptor 1 and cysteinyl leukotriene receptor 2. Any LT inhibitor is suitable for practice of the invention, and several LT inhibitors are described herein. To help minimize side effects, an LT inhibitor that is specific for a member of the LT synthesis pathway is preferred. Exemplary inhibitors include both small molecules, biological inhibitors of proteins, (e.g., antibody substances, peptides), and biological inhibitors that operate at the nucleic acid level (e.g., antisense nucleic acids and interfering RNA nucleic acids and zinc finger proteins).  
      Preferred agents that inhibit the activity of a member of the leukotriene pathway are listed in the Agent Table I herein, including the following agents: 1-((4-chlorophenyl)methyl)-3-((1,1-dimethylethyl)thio)-alpha,alpha-dimethyl-5-(2-quinolinylmethoxy)-1H-Indole-2-propanoic acid, (R)-(+)-alpha-cyclopentyl-4-(2-quinolinylmethoxy)-Benzeneacetic acid, 3-(3-(1,1-dimethylethylthio-5-(quinoline-2-ylmethoxy)-1-(4-chloromethylphenyl)indole-2-yl)-2,2-dimethylpropionaldehyde oxime-0-2-acetic acid, zileuton, atreleuton, 6-((3-fluoro-5-(tetrahydro-4-methoxy-2H-pyran-4yl)phenoxy)methyl)-1-methyl-2(1H)-quinlolinone, 1-((4-chlorophenyl)methyl)-3-((1,1 dimethylethyl)thio)-alpha,alpha-dimethyl-5-(2-quinolinylmethoxy)-1H-Indole-2-propanoic acid and 4-(3-(4-(2-Methyl-imidazol-1-yl)-phenylsulfanyl)-phenyl)-tetrahydro-pyran-4-carboxylic acid amide. In one variation, the LT inhibitor is an inhibitor of FLAP. One preferred group of compounds are described herein as BAY X1005 (also known as DG-031) as well as related compounds described in Mohrs et al., U.S. Pat. No. 4,970,215, incorporated herein by reference in its entirety. In another variation, the LT inhibitor is a LTA4H inhibitor. Other preferred agents include those set forth in the Agent Table II and the LTA4H Agent list set out herein. Additional preferred agents include those described in Penning et al.,  Med Chem.  2002 45(16):3482-90, Penning,  Curr Pharm Des.  2001, 7(3):163-79 and Penning et al.,  J Med Chem.  2000 43(4):721-35.  
                              Agent Table II                                 Compound               Target   ID   Chemical Name   Patent/Reference               LTA4H   SC-57461A   3-[methyl[3-[4-   Penning, T. D. et. al. Bioorg Med.       Inhibitor       (phenylmethyl)phenoxy]-   Chem. Letters (2003), 13, 1137-1139.               propyl]amino]propionic   ibid, (2002), 12, 3383-3386               acid       LTA4H   SC-56938   Ethyl-1-[2-[4-   Penning, T. D. et. al. Bioorg Med.       Inhibitor       (phenylmethyl)phenoxy]   Chem. Letters (2003), 13, 1137-1139;               ethyl]-4-piperidine-   ibid, (2002), 12, 3383-3386.               carboxylate   US6506876A1       LTA4H   RP 64966   [4-[5-(3-Phenyl-   WO9627585       Inhibitor       propyl)thiophen-2-               yl]butoxy]acetic acid       LTA4H   SA 6541   (R)-S-[[4-   WO9809943       Inhibitor       (dimethylamino)phenyl]               methyl]-N-(3-mercapto-               2methyl-1-oxopropyl-L-               cycteine       LTB4   Amelubant/   Carbamic acid,((4-((3-   U.S. Pat. No. 6,576,669       Receptor   BIIL-284   ((4-(1-(4-       Antagonist       hydroxyphenyl)-1-               methylethyl)phenoxy)methyl)               phenyl)methoxy)               phenyl)iminomethyl)-               ethyl ester       LTB4   BIRZ-227   5-Chloro-2-[3-(4-   Journal of Organic Chemistry       Receptor       methoxy-phenyl)-2-   1998, 63: 2(326-330).       Antagonist       pyridin-2-yl-pyrrolidin-               1-yl]-benzooxazole       LTB4   CP 195543   2-[(3S,4R)-3,4-dihydro-   Process: WO 98/11085 1998,       Receptor       4-hydroxy-3-   priority US 60/26372 1996; J.       Antagonist       (phenylmethyl)-2H-1-   Pharamacology and Expert.               benzopyran-7-yl]-4-   Therapy, 1998, 285: 946-54               (trifluoromethyl)benzoic               acid       LTB4   Ebselen   2-Phenyl-   Journal of Cerebral Blood Flow and       Receptor       benzo[d]isoselenazol-3-   Metabolism 1995, July 2-6 (S162);       Antagonist       one   Drugs of the Future 1995, 20: 10                   (1057)       LTB4   LTB 019;   4-[5-(4-Carbamimidoyl-   ACS Meeting 1994, 207th: San       Receptor   CGS-   phenoxy)-pentyloxy]-   Diego (MEDI 003); International       Antagonist   25019C   N,N-diisopropyl-3-   Congress of the Inflammation               methoxy-benzamide   Research Association 1994,               maleate   7th: White Haven (Abs W23)       LTB4   LY 210073   5-(2-Carboxy-ethyl)-6-   J Med Chem 1993 36 (12) 1726-1734       Receptor       [6-(4-methoxy-phenyl)-       Antagonist       hex-5-enyloxy]-9-oxo-               9H-xanthene-2-               carboxylic acid       LTB4   LY 213024   5-(3-carboxybenzoyl)-2-   J Med Chem 1993 36 (12) 1726-1734       Receptor       (decyloxy)benzenepropanoic       Antagonist       acid       LTB4   LY 255283   1-[5-ethyl-2-hydroxy-4-   EP 276064 B 1990, priority US       Receptor       [[6-methyl-6-(1H-   2479 1987       Antagonist       tetrazol-5-               yl)heptyl]oxy]phenyl]               ethanone       LTB4   LY 264086   7-carboxy-3-(decyloxy)-   U.S. Pat. No. 4996230 1991, priority US       Receptor       9-oxo-9H-xanthene-4-   481413 1990       Antagonist       propanoic acid       LTB4   LY 292728   7-carboxy-3-[3-[(5-   EP 743064 A 1996, priority US       Receptor       ethyl-4′-fluoro-2-   443179 1995       Antagonist       hydroxy[1,1′-biphenyl]-               4-yl)oxy]propoxy]-9-               oxo-9H-xanthene-4-               propanoic acid disodium               salt       LTB4   LY-293111   Benzoic acid,2-(3-(3-((5-   Proceedings of the American       Receptor   (VML-295)   ethyl-4′-fluoro-2-   Society for Clinical Oncology       Antagonist       hydroxy(1,1′-biphenyl)-   2002, 21: 1 (Abs 343) [LY-293111               4-yl)oxy)propoxy)-2-   for Cancer]               propylphenoxy)-   SCRIP World Pharmaceutical News                   1997, 2272 (13) [for VML-295]       LTB4   ONO 4057;   (E)-2-(4-   EP 405116 A 1991       Receptor   LB 457   carboxybutoxy)-6-[[6-       Antagonist       (4-methoxyphenyl)-5-               hexenyl]oxy]benzenepropanoic               acid       LTB4   PF 10042   1-[5-hydroxy-5-[8-(1-   EP 422329 B 1995, priority US       Receptor       hydroxy-2-phenylethyl)-   409630 1989       Antagonist       2-dibenzofuranyl]-1-oxo               pentyl]pyrrolidine       LTB4   RG-14893   8-Benzyloxy-4-[(methyl-   SCRIP World Pharmaceutical News       Receptor       phenethyl-carbamoyl)-   1996, 2168 (20)       Antagonist       methyl]-naphthalene-2-               carboxylic acid       LTB4   SB-201993   3-{6-(2-Carboxy-vinyl)-   WO-09500487       Receptor       5-[8-(4-methoxy-       Antagonist       phenyl)-octyloxy]-               pyridin-2-               ylmethylsulfanylmethyl}-               benzoic acid       LTB4   SC-52798   7-[3-(2-   Bioorganic and Medicinal       Receptor       Cyclopropylmethyl-3-   Chemistry Letters 1994, 4: 6 (811-816);       Antagonist       methoxy-4-thiazol-4-yl-   Journal of Medicinal               phenoxy)-propoxy]-8-   Chemistry 1995, 38: 6 (858-868)               propyl-chroman-2-               carboxylic acid       LTB4   SC-53228   3-{7-[3-(2-   International Congress of the       Receptor       Cyclopropylmethyl-3-   Inflammation Research Association       Antagonist       methoxy-4-   1994, 7th: White Haven (Abs W5)               methylcarbamoyl-               phenoxy)-propoxy]-8-               propyl-chroman-2-yl}-               propionic acid       LTB4   WAY   3-fluoro-4′-(2-   Drugs under Experimental and       Receptor   121006   quinolinylmethoxy)-   Clinical research 1991, 17: 8 (381-387)       Antagonist       [1,1′-biphenyl]-4-acetic               acid       LTB4   ZD-2138   3-Amino-3-(4-methoxy-   International Symposium on       Receptor       tetrahydro-pyran-4-yl)-   Medicinal Chemistry       Antagonist       acrylic acid 1-methyl-2-   1994, 13th: Paris (P 197)               oxo-1,2-dihydro-               quinolin-6-ylmethyl               ester                  
 
      In addition the following LTA4H inhibitors are described in USP2003/0004101A1, the teachings of which are incorporated herein by reference in their entirety:  
     Additional LTA4H Agent List  
      1. 1-[2-[4-(phenylmethyl)phenoxy]ethyl]-2-methyl-4-tetrazolylpieridine  
      2. 1-[2-[4-(4-oxazolyl)phenoxy)phenoxy]ethyl]pyrrolidine  
      3. 3-[methyl[3-[4-(2-thienylmethyl)phenoxy]propyl]amino]propionic acid  
      4. methyl 3-[methyl[3-[4-(2-thienylmethyl)phenoxy]propyl]amino]propionate  
      5. 3-[methyl[3-[4-(3-thienylmethyl)phenoxy]propyl]amino]propionic acid  
      6. methyl-3-[methyl[3-4-(3-theinylmethyl)phenoxy]propyl]amino]propionate  
      7. 3-[methyl[3-[4-(4-fluorophenoxy)phenoxy]propyl]amino]propionic acid  
      8. 3-[methyl[3-[4-(4-biphenyloxy)phenoxy]propyl]amino]propionic acid  
      9. N-[3-[[3-[4-(phenylmethyl)phenoxy]propyl]methylamino]propionyl]benzenesulfonamide  
      10. 1-[2-[4-(phenylmethyl)phenoxy]ethyl]-2-methyl-4-(1H-tetrazol-5-yl)piperidine  
      11. 1-[2-[4-(phenylmethyl)phenoxy]ethyl]-4-(1H-tetrazol-5-yl)piperidine  
      In some embodiments, compositions of the invention comprise a statin, and methods of the invention comprise administration of a statin. In this context, the term “statin” should be understood to refer to any of the class of inhibitors of 3-hydroxy-3-methylglutarlcoenzyme A (HMG-CoA) reductase, the enzyme that converts HMG-CoA to the cholesterol precursor mevalonic acid. Numerous compounds with high specificity for this enzyme have been developed and approved for human therapy. Compositions of the invention may comprise a statin that is listed in Agent Table III herein, such as rovuvastatin (also known visastatin), fluvastatin, atorvastatin, lovastatin (also known as mevolin), simvastatin, pravastatin, pitavastatin, mevastatin, crevastatin, ML-236A, ML-236B, MBV-530A and MB-530B.  
      References to agents should be understood to include pharmaceutically acceptable salts, acids, bases, esters, pro-drugs, metabolites, and other common formulation variants of the agents.  
      An increasing body of emerging evidence identifies serum CRP as a marker for cardiovascular morbidity/mortality, and correlates reductions in serum CRP to better clinical outcomes. (See, e.g., Ridker et al., N. Engl. J. Med. 352(1): 20-28 (2005); Nissen et al., N. Engl. J. Med. 352(1): 29-38 (2005); and Pearson et al., Circulation 107: 499-511 (2003).) Serum CRP in excess of 3.0 mg/L is considered high risk; from 1.0 to 3.0 average risk; and below 1 mg/L low risk. (Pearson et al.) Compositions and methods of the invention provide tools for reducing serum CRP. Reductions in CRP can be measured on a concentration basis, where compositions and methods that achieve CRP below 3.0 mg/L are preferred; with still more preferred targets of 2.75 mg/L, 2.5 mg/L, 2.25 mg/L, 2.0 mg/L, 1.75 mg/L, 1.5 mg/L, 1.25 mg/L, 1.0 mg/L, 0.75 mg/L, and 0.5 mg/L. Reductions in CRP also can be measured on a percentage basis, where clinical effectiveness is evaluated as a percentage reduction in CRP in a patient compared to no drug therapy or compared to single drug therapy. Depending on the initial CRP measurement, compositions and methods that reduce CRP anywhere from 10%-90% or more are contemplated, e.g., reductions of 10%, 20%, 25%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, or any target in between these values.  
      The invention also contemplates methods of reducing MPO and method of monitoring MPO levels. Reductions in MPO can be measured on a concentration basis, where compositions and methods that reduce MPO level relative to the quartile distribution of MPO in the normal population (i.e., from 4 th  quartile to 3 rd  or from 3 rd  to 2 nd ) are preferred. Reductions in MPO also can be measured on a percentage basis, where clinical effectiveness is evaluated as a percentage reduction in MPO in a patient compared to no drug therapy or compared to single drug therapy. Depending on the initial MPO measurement, compositions and methods that reduce MPO anywhere from 10%-90% or more are contemplated, e.g., reductions of 10%, 20%, 25%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, or any target in between these values.  
      In some variations of the invention, the composition of the invention includes the leukotriene synthesis inhibitor in an amount effective to reduce serum C-reactive protein (CRP) in a human subject. In some variations, the composition of the invention includes the statin in an amount effective to reduce serum low density lipoprotein cholesterol (LDL) and reduce serum CRP in a human subject. In at least one preliminary and short term study desribed herein, human subjects that already enjoyed the CRP-lowering benefits of statin therapy were administered the LT inhibitor BAY-X1005, and significant further reductions in CRP were detected. Combination therapy of a longer duration may result in further CRP reduction than the 20-30% effect observed in the short term study.  
      In an embodiment of the invention, the compositions comprise a leukotriene synthesis inhibitor in an amount effective to reduce serum CRP in a human subject and a statin. In another embodiment, the compositions comprise a statin in an amout effective to reduce serum LDL-C in a human subject and a leukotriene synthesis inhibitor. The invention also encompasses compositions comprising a leukotriene synthesis inhbitor and a statin in amounts effective to synergistically reduce CRP in a human subject.  
      In one variation, the leukotriene inhibitor and the statin are included in the composition of the invention in amounts effective to synergistically reduce serum C-reactive protein in a human subject.  
      For practice of the invention with BAY-X1005, doses of 50-750 mg per day for adult human patients are contemplated. Doses of 100-500 mg, from one to five times per day, is contemplated. Doses of 250-375 mg, from one to three times per day, is preferred.  
      Dosing for clinically approved statins have been developed and published by the manufacturers. In a preferred embodiment, the statin is co-formulated with the LT inhibitor in a pill or capsule for administrations 1-4 times per day.  
      The invention provides for methods of using these compositions to reduce risk factors for cardiovascular diseases such as for MI, ACS, stroke, or PAOD. In one method, a composition comprising a leukotriene synthesis inhibitor and a statin is administered to a human subject exhibiting one or more risk factors for MI, ACS, stroke or PAOD, wherein the composition is administered in an amount effective to reduce at least one risk factor for MI, ACS, stroke or PAOD. Preferably, the risk factor is elevated serum LDL-C or an elevated inflammatory marker such as CRP or serum amyloid A. In a highly preferred embodiment, LDL-C and CRP are both reduced clinically significant amounts, where a clinically significant amount is an amount that correlates with a statistically significant measurable reduction in risk for an adverse cardiovascular event, when analyzed in a population, e.g., in a clinical study.  
      The invention also provides for method of using these compounds to reduce CRP in human subject. In one variation, the invention is a method of reducing C reactive protein (CRP) in a human subject, comprising administering to a human in need of treatment to reduce CRP a composition of the invention containing the LT inhibitor and the statin as described above, in an amount effective to reduce serum C reactive protein in the human subject. The identification of a human in need of treatment for CRP reduction can be based on a variety of factors described herein, including genetic factors, CRP measurements, measurements of other inflammatory markers, and measurements of non-genetic and non-inflammatory markers for risk of MI. In one variation, the method includes selecting for the administering step a human subject at risk for a disease or condition selected from the group consisting of myocardial infarction, acute coronary syndrome, stroke, or peripheral arterial occlusive disease. Thus, the invention provides a method that comprises selecting a human subject at risk for MI, ACS, stroke or PAOD and administering to the subject a composition comprising a leukotriene synthesis inhibitor and a statin wherein the composition is in an amount effective to reduce serum CRP in a human subject. The method may further comprise the step of measuring serum CRP in the human subject to monitor therapeutic efficacy of the composition, wherein a decrease in serum CRP following the administering of the composition indicates therapeutic efficacy.  
      In still another variation, the monitoring of risk factors and/or toxicity is used to adjust dose or dosing. For example, dose or dosing of a statin or a leukotriene synthesis inhibitor is increased if serum CRP and/or LDL and/or serum or urinary leukotriene measurements do not decrease to a target level, such as a level equivalent to the bottom 50 percentile, 40 percentile, 30 percentile, 20 percentile, 10 percentile, 1 percentile of a population, or other target percentile in between these exemplary targets. As described above, monitoring also can be used to adjust dosing to achieve a target level of serum CRP, or to achieve a target percentage reduction in CRP for a particular human subject.  
      The monitoring may involve parameters in addition to CRP. A benefit of the statin for many human subjects will be the reduction in serum LDL, and methods of the invention include administering the composition of the invention in an amount effective to reduce serum LDL and serum leukotrienes in the human subject. In this embodiment, serum LDL may be monitored. Other markers described herein, including serum amyloid A nad myeloperoxidase, may be monitored.  
      In certain embodiments of the invention, the individual or human subject selected for treatment is an individual who has at least one risk factor, such as an at-risk haplotype for myocardial infarction, stroke or PAOD; an at-risk haplotype in the FLAP gene; a polymorphism in a FLAP nucleic acid; an at-risk polymorphism in the 5-LO gene promoter. The invention provides for methods of selecting a human subject susceptible to MI comprising determining a FLAP genotype or haplotype of a human subject, and selecting for treatment a human subject with a FLAP genotype or haplotype that correlates with an increased risk of MI. The methods of the invention include selecting a human subject with the presence of at least one at-risk haplotype within or near the FLAP gene such as a haplotype shown in Table 14; a haplotype shown in Table 15; a haplotype shown in Table 19; haplotype B4; haplotype B5; haplotype B6; haplotype A4; haplotype A5; haplotype HapB, haplotype HapC1, haplotype HapC2, haplotype HapC3, haplotype HapC4-A and haplotype HapC4-B.  
      The methods of the invention also include selecting a human subject for treatment, wherein the presence in said subject of a haplotype comprising marker SG13S106 (SNP DG00AAHII) (SEQ ID NO: 1, position 176579), allele G, identifies the subject as having a susceptibility to MI; the presence of a haplotype comprised of markers SG13S99 (DG00AAFIU), allele T (SEQ ID NO: 1, position 138551); SG13S377 (DG00AAJFF) (SEQ ID NO: 1, position 169965), allele G; SG13S106 [SNP DG00AAHII] (SEQ ID NO: 1, position 176579), allele G; SG13S32 (SEQ ID NO: 1, position 198547), allele A; and SG13S35 (SEQ ID NO: 1, position 206117), allele G identifies the subject as having a susceptibility to MI; the presence in said subject of a haplotype comprised of markers: SG13S375 (SEQ ID NO: 1, position 164874), allele T; SG13S25 (SEQ ID NO: 1, position 165553), allele G; SG13S32 (SEQ ID NO: 1, position 176579), allele A; and SG13S106 (SEQ ID NO: 1, position 198547), allele G or A identifies the subject as having a susceptibility to MI, the presence in said subject of a haplotype comprised of marker SG13S375(SNP DG00AAJFC) (SEQ ID NO: 1, position 164874), allele T; and SG13S25 (SEQ ID NO: 1, position 165553), allele G, identified the subject as having a susceptibility to MI; the presence in said subject of a haplotype comprised of marker SG13S375(SNP DG00AAJFC) (SEQ ID NO: 1, position 164874), allele T; and SG13S25 (SEQ ID NO: 1, position 165553), allele G, and SG13S32 (SEQ ID NO: 1, position 198547) identified the subject as having a susceptibility to MI, the presence in said subject of a haplotype comprised of marker SG13S106 (SNP DG00AAHII) (SEQ ID NO: 1, position 176579), allele G, SG13S30 (SEQ ID NO: 1, position 193840), allele G; and SG13S42 (SEQ ID NO: 1, position 203877), allele A, identifies the subject as having a susceptibility to MI, the presence in said subject of a haplotype comprised of markers: SG13S377 (SEQ ID NO: 1, position 169965), allele A; SG13S114 (SEQ ID NO: 1, position 178096), allele A; SG13S41 (SEQ ID NO: 1, position 202045), allele A; and SG13S35 (SEQ ID NO: 1, position 206117), allele G, identifies the subject as having a susceptibility to MI.  
      In another embodiment, the invention provides for a method of selecting a human subject susceptible to MI comprising analyzing nucleic acid of a human subject for the presenece or absence of at least one FLAP polymorphism that correlates with a susceptibility to MI. FLAP polymorphisms that that correlate to susceptibility to MI inlcude SG13S377 (SEQ ID NO: 1, position 169965), allele A; SG13S114 (SEQ ID NO: 1, position 178096), allele A; SG13S41 (SEQ ID NO: 1, position 202045), allele A; and SG13S35 (SEQ ID NO: 1, position 206117), allele G. Additional FLAP polymorphisms that that correlate to a susceptibility to MI include SG13S375 (SEQ ID NO: 1, position 164874), allele T, SG13S25 (SEQ ID NO: 1, position 165553), allele G; SG13S32 (SEQ ID NO: 1, position 176579), allele A; and SG13S106 (SEQ ID NO: 1, position 198547), allele G or A. The methods may further comprise selecting a subject with the presence of at least one FLAP polymorphism and with the presence of elevated CRP or MPO.  
      An another embodiment, the invention provides for methods of prophylaxis therapy for myocardial infarction (MI) comprising analyzing nucleic acid of a human subject for the presence and absence of a FLAP haplotype, wherein the haplotype is comprised of markers: SG13S377 (SEQ ID NO: 1, position 169965), allele A; SG13S114 (SEQ ID NO: 1, position 178096), allele A; SG13S41 (SEQ ID NO: 1, position 202045), allele A; and SG13S35 (SEQ ID NO: 1, position 206117), allele G, and selecting for treatment a human subject having nucleic acid with the presence of the FLAP haplotype. This method futher comprises administering to the subject a composition comprising a therapeutically effective amount of an MI therapeutic agent that inhibits leukotriene synthesis in vivo, wherein the MI therapeutic agent inhibits leukotriene synthesis by inhibiting the activity of at least one protein selected from 5-Lipoxygenase activating protein (FLAP) and 5-lipoxygenase (5-LO).  
      In one embodiment, the invention provides for methods of decreasing risk of a subsequent myocardial infarction in an individual who has had at least one myocardial infarction, comprising administering a therapeutically effective amount of an MI therapeutic agent to the individual, wherein the MI therapeutic agent inhibits leukotriene synthesis by inhibiting the activity of at least one protein selected from 5-Lipoxygenase activating protein (FLAP) and 5-lipoxygenase (5-LO) and monitoring myeloperoxidase (MPO) in the individual before and during administration of the therapeutic agent, wherein the therapeutic agent is administered in an amount effective to reduce the leukotriene level in a subject.  
      In another embodiment, the invention provides for methods of screening a human subject for susceptibility for MI comprising analyzing nucleic acid of a human subject for the presence and absence of the FLAP haplotype comprised of markers: SG13S377 (SEQ ID NO: 1, position 169965), allele A; SG13S114 (SEQ ID NO: 1, position 178096), allele A; SG13S41 (SEQ ID NO: 1, position 202045), allele A; and SG13S35 (SEQ ID NO: 1, position 206117), allele G, and identifying the subject as having a susceptibility to MI, wherein the presence of the FLAP haplotype correlates with an increased risk of MI.  
      The individuals or human subjects selected for treatment may have at least one family or medical history risk factor such as diabetes; hypertension; hypercholesterolemia; elevated triglycerides; elevated lp(a); obesity; ankle/brachial index (ABI) less than 0.9; a past or current smoker; transient ischemic attack; transient monocular blindness; carotid endarterectomy; asymptomatic carotid stenosis; claudicatioin; limb ischemia leading to gangrene, ulceration or amputation; a vascular or peripheral artery revascularization graft; increased serum LDL cholesterol and/or decreased HDL cholesterol; serum total cholesterol &gt;200 mg/dl, increased leukotriene synthesis; and/or at least one previous myocardial infarction, ACS, stable angina, previous transient ischemic attack, transient monocular blindness, or stroke, asymptomatic carotid stenosis or carotid endarterectomy, atherosclerosis, requires treatment for restoration of coronary artery blood flow (e.g., angioplasty, stent, revascularization procedure).  
      In addition, the individuals or human subjects selected for treatment may have an elevated inflammatory marker, e.g., a marker such as C-reactive protein (CRP), serum amyloid A, fibrinogen, a leukotriene, a leukotriene metabolite, interleukin-6, tissue necrosis factor-alpha, a soluble vascular cell adhesion molecule (sVCAM), a soluble intervascular adhesion molecule (sICAM), E-selectin, matrix metalloprotease type-1, matrix metalloprotease type-2, matrix metalloprotease type-3, matrix metalloprotease type-9, myeloperoxidase (MPO), and N-tyrosine). The invention provides for methods of prophylaxis therapy for MI comprising administering a MI therapeutic agent in an amount effective to reduce the elevated serum level of at least one elevated inflammatory markers.  
      In a particular embodiment, the invention provides for methods of prophylaxis for myocardial infarction (MI) comprising administering to a subject in need of prophylaxis for myocardial infraction a composition comprising a therapeutically effective amount of an MI therapeutic agent that inhibits leukotriene synthesis in vivo, and monitoring myeloperoxidase (MPO) level in the human subject before and during the prophylaxis treatment, wherein the MI therapeutic agent is administered in an amount effective to reduce the MPO level in a subject.  
      The invention also provides for methods of screening a human subject for risk of developing myocardial infarction, comprising contacting a blood sample from the human subject with a calcium ionophore to stimulate production of a leukotriene; and measuring production of a leukotriene in the blood sample after the contacting step, wherein elevated leukotriene production compared to a control correlates with increased risk of developing myocardial infarction (MI). The control in these methods may be a human of the same sex as the subject selected for treatment or may be a human age matched to the subject selected for treatment.  
      Human subjects that already are treated with statins can enjoy the benefit of the present invention if the subjects therapy is modified to include an LT antagonist. Thus, in still another embodiment, the invention is a method of reducing C reactive protein (CRP) in a human subject, comprising: selecting a human subject that receives statin therapy to reduce serum LDL, wherein the statin therapy optionally reduces serum CRP in the human subject; and administering to the human subject a leukotriene synthesis antagonist, in an amount effective to further reduce CRP in the human subject.  
      In still another embodiment, the invention is a method of reducing C reactive protein (CRP) in a human subject, comprising: identifying a human subject in need of treatment to reduce serum CRP; administering to the human subject a composition comprising a statin; and administering to the human subject a composition comprising a leukotriene synthesis inhibitor, wherein the statin and the leukotrience synthesis inhibitor are administered in amounts effective to reduce serum CRP in the human subject. The statin and the LT inhibitor can be simultaneously administered as a single composition, as described above; can be simultaneously administered as separate compositions; or can be sequentially administered. Depending on the dosing schedule, the daily administration regimen may include simultaneous administration at some times and separate administration at other times, e.g., if one agent is administered twice daily and another three times daily.  
      In certain embodiments of the invention, the individual or human subject selected for treatment is an individual who has at least one risk factor, such as an at-risk haplotype for myocardial infarction, stroke or PAOD; an at-risk haplotype in the FLAP gene; a polymorphism in a FLAP nucleic acid; an at-risk polymorphism in the 5-LO gene promoter, diabetes; hypertension; hypercholesterolemia; elevated triglycerides; elevated lp(a); obesity; ankle/brachial index (ABI) less than 0.9; a past or current smoker; transient ischemic attack; transient monocular blindness; carotid endarterectomy; asymptomatic carotid stenosis; claudicatioin; limb ischemia leading to gangrene, ulceration or amputation; a vascular or peripheral artery revascularization graft; an elevated inflammatory marker (e.g., a marker such as C-reactive protein (CRP), serum amyloid A, fibrinogen, a leukotriene, a leukotriene metabolite, interleukin-6, tissue necrosis factor-alpha, a soluble vascular cell adhesion molecule (sVCAM), a soluble intervascular adhesion molecule (sICAM), E-selectin, matrix metalloprotease type-1, matrix metalloprotease type-2, matrix metalloprotease type-3, matrix metalloprotease type-9, myeloperoxidase (MPO), and N-tyrosine); increased LDL cholesterol and/or decreased HDL cholesterol; increased leukotriene synthesis; and/or at least one previous myocardial infarction, ACS, stable angina, previous transient ischemic attack, transient monocular blindness, or stroke, asymptomatic carotid stenosis or carotid endarterectomy, atherosclerosis, requires treatment for restoration of coronary artery blood flow (e.g., angioplasty, stent, revascularization procedure).  
      The invention additionally pertains to methods of assessing an individual for an increased risk of MI, ACS, atherosclerosis, stroke, or PAOD, by assessing or monitoring a level of a leukotriene metabolite (e.g., LTE4, LTD4, LTB4) in the individual (e.g., in a sample of blood, serum, plasma or urine). An individual or human subject selected for treatment may have an elevated measurement of a leukotriene or leukotriene metabolite, such as LTC4, LTD4, LTB4 and LTE4. The level of leukotrienes and leukotriene metabolites may be measured in serum, plasma, blood or urine in the individual. An increased level of leukotriene metabolite is indicative of an increased risk. The invention also encompasses methods of assessing an individual for an increased risk of MI, ACS, atherosclerosis, stroke, transient ischemic attack, transient monocular blindness, asymptomatic carotid stenosis, PAOD, claudication, or limb ischemia, by stimulating production of a leukotriene or a leukotriene metabolite in a test sample from the individual (e.g., a sample comprising neutrophils), using a calcium ionophore, and comparing the level of the leukotriene or leukotriene metabolite with a control level. A level of production of the leukotriene or leukotriene metabolite that is significantly greater than the control level, is indicative of increased risk.  
      The invention further pertains to methods of assessing response to treatment with a leukotriene synthesis inhibitor, by assessing or monitoring a level of a leukotriene or leukotriene metabolite in the individual before treatment, and comparing the level to a level of the leukotriene or leukotriene metabolite assessed during or after treatment. A level that is significantly lower during or after treatment, than before treatment, is indicative of efficacy of the treatment with the leukotriene synthesis inhibitor. The level of leukotriene may be monitored in serum, plasma, blood or urine collected from the subject before, during and after treatment. The invention additionally pertains to methods of assessing response to treatment with a leukotriene synthesis inhibitor, by stimulating production of a leukotriene or a leukotriene metabolite in a first test sample from the individual (e.g., a sample comprising neutrophils) before treatment, using a calcium ionophore, and comparing the level of the leukotriene or leukotriene metabolite with a level of production of the leukotriene or leukotriene in a second test sample from the individual, during or after treatment. A level of production of the leukotriene or leukotriene metabolite in the second test sample that is significantly lower than the level in the first test sample, is indicative of efficacy of the treatment, for example, the treatment or therapeutic agent reduces the leukotriene level in the subject to the medial level of leukotrienes in human subjects in the general population or lower than that medial level.  
      Similarly, the invention encompasses methods of assessing response to treatment with a leukotriene synthesis inhibitor, by assessing or monitoring a level of an inflammatory marker in the individual before treatment, and during or after treatment. A level of the inflammatory marker during or after treatment, that is significantly lower than the level of inflammatory marker before treatment, is indicative of efficacy of the treatment.  
      To determine the effectiveness of compositions of the present invention comprising a statin, total cholesterol, LDL-C and/or triglycerides may be assessed from measurements of risk factor markers in the serum of a human subject administered the composition. A level of serum total cholesterol, LDL-C and/or triglycerides during or after treatment, that is significantly lower than the level of total cholesterol, LDL-C and/or triglycerides before treatment is indicative of the efficacy of the treatment.  
      The invention also pertains to use of leukotriene synthesis inhibitors for the manufacture of a medicament for the treatment of MI, ACS, stroke, PAOD, and/or atherosclerosis, as described herein, as well as for the manufacture of a medicament for the reduction of leukotriene synthesis. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
      The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention.  
       FIG. 1  shows the results from a haplotype association case-control analysis of 437 female MI patients versus 721 controls using combinations 4 and 5 microsatellite markers to define the test haplotypes. The p-value of the association is plotted on the y-axis and position of markers on the x-axis. Only haplotypes that show association with a p-value &lt;10 −5  are shown in the figure. The most significant microsatellite marker haplotype association is found using markers DG13S1103, DG13S166, DG13S1287, DG13S1061 and DG13S301, with alleles 4, 0, 2, 14 and 3, respectively (p-value of 1.02×10 −7 ). Carrier frequency of the haplotype is 7.3% in female MI patients and 0.3% in controls. The segment that is common to all the haplotypes shown in the figure includes only one gene, FLAP.  
       FIG. 2  shows the alleles of the markers defining the most significant microsatellite marker haplotypes. The segment defined with a black square is common to all the of most significantly associated haplotypes. The FLAP nucleic acid is located between makers DG13S166 and D13S1238. Two marker haplotype involving alleles 0 and −2 for markers DG13S166 and D13S1238, respectively, is found in excess in patients. Carrier frequency of this haploype is 27% in patients and 15.4% in controls (p-value 1×10 −3 ). Therefore, association analysis confirms that the most tightly MI-associated gene within the linkage peak is FLAP.  
       FIG. 3  shows the relative location of key SNPs and exons of the ALOX5AP/FLAP gene (exons shown in vertical rectangles). Haplotype length varies between 33 to 68 kb.  
       FIG. 4  shows a significant positive correlation between serum LTE4 levels and serum CRP levels.  
       FIG. 5  depicts LTB4 production of ionomycin stimulated neutrophils from MI patients (n=41) and controls (n=35). The log-transformed (mean+SD) values measured at 15 and 30 minutes of stimulated cells are shown. (7.1) LTB4 production in MI patients and controls. The difference in the mean values between patients and the controls is tested using a two-sample t-test of the log-transformed values. (7.2) LTB4 production in MI male carriers and non-carriers of haplotype A4. Mean values of controls are included for comparison. Of note, males with the haplotype A4 produce the highest amounts of LTB4 (p&lt;0.005 compared to controls). (7.3). Schematic representation of the 5-LO pathway with leukotriene bioactive products.  
       FIG. 6  shows a schematic view of the chromosome 13 linkage region showing the FLAP gene. (9.1) The linkage scan for female MI patients and the one LOD drop region that includes the FLAP gene; (9.2) Microsatellite association for all MI patients: single marker association and two, three, four and five marker haplotype association. The arrows indicate the location of the most significant haplotype association across the FLAP gene in males and females. (9.3) The FLAP gene structure, with exons shown as cylinders, and the location of all the SNPs typed in the region (vertical lines). The vertical lines indicate the position of the microsatellites (shown in 9.2) and SNPs (shown in 9.3) used in the analysis.  
       FIG. 7  shows a linkage scan using framework microsatellite markers on chromosome 13 for male patients with ischemic stroke or TIA (n=342 in 164 families at 6 meiosis). The LOD score is expressed on the y axis and the distance from the pter in Kosambi cM on the x axis.  
       FIG. 8  shows a pairwise linkage disequilibrium (LD) between SNPs in a 60 kb region encompassing FLAP. The markers are plotted equidistantly. Two measures of LD are shown: D′ in the upper left triangle and P values in the lower right triangle. Shaded lines indicate the positions of the exons of FLAP and the stars indicate the location of the markers of the at-risk haplotype A4. Scales for the LD strength are provided for both measures to the right.  
       FIG. 9  provides a schematic of the clinical trial schedule. This figure shows that at Visit 2 (on Day 1 of study) subjects were randomised into each of the three arms and to either placebo or active drug within each arm. A 2-week washout period separated the 4-week treatment periods. Cross-over was performed at week 6.  
       FIG. 10  shows the analysis of carry-over effect for CRP and SAA (on log-scale).  
       FIG. 11  shows the results of the first step of the linkage analysis: multipoint non-parametric LOD scores for a framework marker map on chromosome 12. A LOD score suggestive of linkage of 1.95 was found at marker D12S2081.  
       FIG. 12  shows the results of the second step of the linkage analysis: multipoint non-parametric LOD scores for the families after adding 20 fine mapping markers to the candidate region. The inclusion of additional microsatellite markers increased the information on sharing by decent from 0.8 to 0.9, around the markers that gave the highest LOD scores.  
       FIG. 13  shows LD and haplotypes in the LTA4H region.  FIG. 13   a  shows a pairwise LD between the 10 genotyped SNPs in a 48-kb region encompassing LTA4H. The markers are plotted equidistantly. Two measures of LD are shown: D′ in the upper left triangle and R 2 -values in the lower right triangle. Scales for both measures of LD are shown on the right. Colored lines indicate the positions of the first exon and the last exon of LTA4H (NM — 000895).  FIG. 1  depicts are all haplotypes found in Icelandic population controls that have allelic frequency greater than 1%. The haplotype showing strongest association to MI in Iceland (HapK) is bold. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
      Extensive genealogical information has been combined with powerful gene sharing methods to map a gene on chromosome 13q12-13 that is associated with myocardial infarction. A genome wide search for susceptibility genes for MI, using a framework map of 1000 microsatellite markers, revealed a locus suggestive of linkage on 13q12-13. Sixty families with 159 female MI patients that clustered within and including 6 meiotic events were used in linkage analysis. At first, only female MI patients were used in the linkage analysis in an effort to enrich for patients with stronger genetic factors contributing to their risk for MI. The epidemiological study of a population-based sample of Icelandic MI patients had previously suggested that the genetic factors for MI might be stronger for females than males, as the relative risk for siblings of female MI patients was significantly higher than the relative risk for siblings of male probands (1.59 (CI 1.47-1.73) vs. 1.35 (CI 1.28-1.42)) (unpublished data). The highest LOD score (2.5) was found at marker D13S289. The LOD score results for the families remained the same after adding 14 microsatellite markers to the candidate region. The inclusion of the additional markers increased the information on sharing by descent from 0.7 to 0.8, around the markers that gave the highest LOD scores. This linkage analysis mapped a gene contributing to MI to chromosome 13q12-13.  
      The candidate MI locus on chromosome 13q12-13 was then finely mapped with microsatellite markers. Patients with myocardial infarction and controls were initially genotyped with microsatellite markers with an average spacing between markers of less than 100 kb over the 12 Mb candidate region. Initial haplotype association analysis that included all genotyped microsatellite markers across the MI candidate locus, resulted in several extended haplotypes composed of 4 and 5 microsatellite markers that were significantly associated with female MI (see, e.g., Tables 14 and 15 below). A region common to all these extended haplotypes, is defined by markers DG13S166 and D13S1238. This region includes only one gene, the FLAP nucleic acid sequence. The two marker haplotype involving alleles 0 and −2 for markers DG13S166 and D13S1238, respectively, was found in excess in patients. Specific variants of the gene were then sought that were associated with MI.  
      In order to screen for SNPs in the FLAP gene, the whole gene was sequenced, both exons and introns. Initially, 9 SNPs identified within the gene were genotyped in patients and controls. Additional microsatellite markers close to or within the FLAP gene were also genotyped in all patients and controls. Five publicly known SNPs that are located within a 200 kb distance 5′ to the FLAP gene were also genotyped in patients and controls. Haplotype association analysis in this case-control study including these additional markers showed several different variants of the same haplotype that were all significantly associated with female MI (see, e.g., Table 8). Table 9 shows two haplotypes that are representative of these female MI risk haplotypes which are referred to herein as the female MI “at risk” haplotypes. The relative risk for male MI patients that had the female MI-“at risk” haplotype was increased (see, e.g., Table 9), indicating that the female MI-“at risk” haplotype also increased the risk of having an MI in males. These results further strengthened the hypothesis that the FLAP gene was an MI susceptibility gene.  
     SNP Haplotype Association to MI, and Subsequently to Stroke and PAOD  
      In an effort to identify haplotypes involving only SNP markers that associate with MI, additional SNPs were identified by sequencing the FLAP gene and the region flanking the gene. Currently, a total of 45 SNPs in 1343 patients and 624 unrelated controls have been genotyped. Two correlated series of SNP haplotypes have been observed in excess in patients, denoted as A and B in Table 7. The length of the haplotypes varies between 33 and 69 kb, and the haplotypes cover one or two blocks of linkage disequilibrium. Both series of haplotypes (HapA and HapB) contain the common allele G of the SNP SG13S25. HapC2, identified in the analysis of the North American cohort (see Example 13), also contains the allele G of the SNP SG13S25. All haplotypes in the A series contain the SNP SG13S114, while all haplotypes in the B series contain the SNP SG13S106. In the B series, the haplotypes B4, B5, and B6 have a relative risk (RR) greater than 2 and with allelic frequencies above 10%. The haplotypes in the A series have slightly lower RR and lower p-values, but higher frequency (15-16%). The haplotypes in series B and A are strongly correlated, i.e., the haplotypes in B define a subset of the haplotypes in A. Hence, haplotypes in series B are more specific than A. However, haplotypes in series A are more sensitive, i.e., they capture more individuals with the putative mutation, as is observed in the population attributable risk which is less for B than for A. Furthermore, these haplotypes show similar risk ratios and allelic frequencies for early-onset patients (defined as onset of first MI before the age of 55) and for both genders. In addition, analyzing various groups of patients with known risk factors, such as hypertension, high cholesterol, smoking and diabetes, does not reveal any significant correlation with these haplotypes, suggesting that the haplotypes in the FLAP gene represent an independent genetic susceptibility factor for MI.  
      Analysis of the North American cohort (described in Example 12) identified another haplotype C which is associated with MI as demonstrated in Table 33 (Example 13). HapC is defined by the T allele of marker SG13S375. There are 4 additional variations of the HapC haplotype which comprise SNPs in addition to the T allele of SG13S375. HapC2 is defined by allele T of the SNPs SG13S375 and allele G of the SNP SG13S25. HapC3 is defined by allele T of the SNPs SG13S375 and allele G of the SNP SG13S25 and allele A of SNP SG13S32. HapC4-A is defined by allele A of the SNP SG13S106 in addition to allele T of the SNPs SG13S375, allele G of the SNP SG13S25 and allele A of SNP SG13S32. HapC4-B is defined by allele G of the SNP SG13S106 in addition to allele T of the SNPs SG13S375, allele G of the SNP SG13S25 and allele A of SNP SG13S32. HapC4-A and HapC-4B correlate with HapA and HapB.  
      Because stroke and PAOD are diseases that are closely related to MI (all occur on the basis of atherosclerosis), the SNP haplotype in the FLAP gene that confers risk to MI was assessed to determine whether it also conferred risk of stroke and/or PAOD. Table 20 shows that haplotype A4 increases the risk of having a stroke to a similar extent as it increases the risk of having an MI. Table 34 demonstrates that HapA is associated with risk of stroke in a Scottish chort (Example 14). Although not as significantly, haplotype A4 also confers risk of developing PAOD.  
      The FLAP nucleic acid encodes a 5-lipoxygenase activating protein, which, in combination with 5-lipoxygenase (5-LO), is required for leukotriene synthesis. FLAP acts coordinately with 5-LO to catalyze the first step in the synthesis of leukotrienes from arachidonic acid. It catalyzes the conversion of arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE), and further to the allylic epoxide 5 (S)-trans7,9 trans 11,14-cis-eicosatetraenoic acid (leukotriene A4, LTA4).  
      The leukotrienes are a family of highly potent biological mediators of inflammatory processes produced primarily by bone marrow derived leukocytes such as monocytes, macrophages, and neurophils. Both FLAP and 5-LO are detected within atherosclerosis lesions (Proc Natl Acad Sci U S A. 2003 Feb. 4;100(3):1238-43.), indicating that the vessel itself can be a source of leukotrienes. It was found at first that the MI-risk FLAP haplotype was associated with higher serum leukotriene levels. Increased production of leukotriene in individuals with pre-existing atherosclerosis lesions may lead to plaque instability or friability of the fibrous cap leading to local thrombotic events. If this occurs in coronary artery arteries it leads to MI or unstable angina. If it occurs in the cerebrovasculature it leads to stroke or transient ischemic attack. If it occurs in large arteries to the limbs, it causes or exacerbates limb ischemia in persons with peripheral arterial occlusive disease (PAOD). Therefore, those with genetically influenced predisposition to produce higher leukotriene levels have higher risk for events due to pre-existing atherosclerosis such as MI.  
      Inhibitors of FLAP function impede translocation of 5-LO from the cytoplasm to the cell membrane and inhibit activation of 5-LO and thereby decrease leukotriene synthesis.  
      As a result of these discoveries, methods are now available for the treatment of myocardial infarction (MI) and acute coronary syndrome (ACS), as well as stroke and PAOD, through the use of leukotriene inhibitors, such as agents that inhibit leukotriene biosynthesis or antagonize signaling through leukotriene receptors. The term, “treatment” as used herein, refers not only to ameliorating symptoms associated with the disease or condition, but also preventing or delaying the onset of the disease or condition; preventing or delaying the occurrence of a second episode of the disease or condition; and/or also lessening the severity or frequency of symptoms of the disease or condition. In the case of atherosclerosis, “treatment” also refers to a minimization or reversal of the development of plaques. Methods are additionally available for assessing an individual&#39;s risk for MI, ACS, stroke or PAOD. In a preferred embodiment, the individual to be treated is an individual who is susceptible (at increased risk) for MI, ACS, stroke or PAOD, such as an individual who is in one of the representative target populations described herein.  
     Representative Target Populations  
      In one embodiment of the invention, an individual who is at risk for MI, ACS, stroke or PAOD is an individual who has an at-risk haplotype in FLAP, as described herein. In one embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S99, SG13S25, SG13S377, SG13S106, SG13S32 and SG13S35 at the 13q12-13 locus. In another embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S99, SG13S25, SG13S106, SG13S30 and SG13S42 at the 13q12 locus. In a third embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S25, SG13S106, SG13S30 and SG13S42 at the 13q12-13 locus. In a fourth embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S99, SG13S25, SG13S114, SG13S89 and SG13S32 at the 13q12-13 locus. In a fifth embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S25, SG13S114, SG13S89 and SG13S32 at the 13q12-13 locus. In another embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises marker SG13S375 at the 13q12-13 locus. In another embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S25 and SG13S375 at the 13q12-13 locus. In another embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S25, SG13S375 and SG13S32 at the 13q12-13 locus. In an additional embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S25, SG13S375, SG13S32 and SG13S106 at the 13q12-13 locus. Additional haplotypes associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD include the haplotypes shown in Tables 4, 8, 9, 14, 15, 17 and 19, as well as haplotypes comprising markers shown in Table 13.  
      Increased risk for MI, ACS, stroke or PAOD in individuals with a FLAP at-risk haplotype is logically conferred by increased production of leukotrienes in the arterial vessel wall or in bone-marrow derived inflammatory cells within the blood and/or arterial vessel wall. It is shown herein that FLAP at-risk haplotypes are associated with higher production of LTB4 ex vivo. It is further shown herein that serum leukotriene levels (specifically, leukotriene E4) correlate with serum CRP levels in myocardial infarction patients. FLAP genetic variation may drive high leukotriene levels (within the blood vessel and/or systemically), which in turn may drive higher CRP levels which has been shown as a risk factor for MI. Accordingly, individuals with a FLAP at-risk haplotype are likely to have elevated serum CRP as well as other serum inflammatory markers. The level of serum CRP or other serum inflammatory markers can be used as a surrogate for the level of arterial wall inflammation initiated by lipid deposition and atherogenesis conferred by the presence of the at-risk FLAP haplotype.  
      In another embodiment of the invention, an individual who is at risk for MI, ACS, stroke or PAOD is an individual who has a polymorphism in a FLAP gene, in which the presence of the polymorphism is indicative of a susceptibility to MI, ACS, stroke or PAOD. The term “gene,” as used herein, refers to not only the sequence of nucleic acids encoding a polypeptide, but also the promoter regions, transcription enhancement elements, splice donor/acceptor sites, and other non-transcribed nucleic acid elements. Representative polymorphisms include those presented in Table 13, below.  
      In a further embodiment of the invention, an individual who is at risk for MI, ACS, stroke or PAOD is an individual who has an at-risk polymorphism in the 5-LO gene in the promoter region, as described herein.  
      In one embodiment of the invention, an individual who is at risk for MI or ACS is an individual who has an at-risk haplotype in LTA4H, as described herein. In one embodiment, the haplotype can comprise alleles 0, T, 0, and A, of markers DG12S1664, SG12S26, DG12S1666, and SG12S144, respectively, at the 12q23 locus. This LTA4H “at-risk” haplotype is detected in over 76% of male patients who have previously had an MI, conferring an increased relative risk of 1.4 fold and in 72% of female MI patients with a relative risk of 1.2. Increased risk for MI or ACS in individuals with an LTA4H at-risk haplotype is logically conferred by increased production of leukotrienes in the arterial vessel wall or in bone-marrow derived inflammatory cells within the blood and/or arterial vessel wall. In another embodiment of the invention, an individual who is at risk for MI or ACS is an individual who has a polymorphism in an LTA4H gene, in which the presence of the polymorphism is indicative of a susceptibility to MI or ACS. The term “gene,” as used herein, refers to not only the sequence of nucleic acids encoding a polypeptide, but also the promoter regions, transcription enhancement elements, splice donor/acceptor sites, and other non-transcribed nucleic acid elements. Representative polymorphisms include those presented in Table 37. Along the same lines, certain variants in the FLAP gene and other members of the leukotriene biosynthetic and response pathway (see, U.S. Provisional Application No. 60/419,432, filed on Oct. 17, 2002; U.S. patent application Ser. No. 10/829,674, filed on Apr. 22, 2004) may indicate one&#39;s increased risk for MI and ACS. Other representatibe at-risk haplotypes are shown in Table 38 and Table 39. Additional “at-risk” haplotypes can be determined using linkage disequilibrium and/or haplotype blocks.  
      In an embodiment, an individual who is at risk for MI, ACS, stroke or PAOD is an individual who has an elevated inflammatory marker. An “elevated inflammatory marker,” as used herein, is the presence of an amount of an inflammatory marker that is greater, by an amount that is statistically significant, than the amount that is typically found in control individual(s) or by comparison of disease risk in a population associated with the lowest band of measurement (e.g., below the mean or median, the lowest quartile or the lowest quintile) compared to higher bands of measurement (e.g., above the mean or median, the second, third or fourth quartile; the second, third, fourth or fifth quintile). An “inflammatory marker” refers to a molecule that is indicative of the presence of inflammation in an individual, for example, C-reactive protein (CRP), serum amyloid A, fibrinogen, leukotriene levels (e.g., leukotriene B4, leukotriene C4), leukotriene metabolites (e.g., leukotriene E4), interleukin-6, tissue necrosis factor-alpha, soluble vasculare cell adhesion molecules (sVCAM), soluble intervascular adhesion molecules (sICAM), E-selectin, matrix metalloprotease type-1, matrix metalloprotease type-2, matrix metalloprotease type-3, matrix metalloprotease type-9, myeloperoxidase (MPO), N-tyrosine) or other markers (see, e.g., Doggen, C. J. M. et al.,  J. Internal Med.,  248:406-414 (2000); Ridker, P. M. et al.,  New Englnd. J. Med.  1997: 336: 973-979, Rettersol, L. et al., 2002: 160:433-440; Ridker, P. M. et. al.,  New England. J. Med.,  2002: 347: 1557-1565; Bermudez, E. A. et. al.,  Arterioscler. Thromb. Vasc. Biol.,  2002: 22:1668-1673). In certain embodiments, the presence of such inflammatory markers can be measured in serum or urine.  
      In an embodiment, an individual who is at risk for MI, ACS, stroke or PAOD is an individual who has increased LDL cholesterol and/or decreased HDL cholesterol levels. For example, the American Heart Association indicates that an LDL cholesterol level of less than 100 mg/dL is optimal; from 100-129 mg/dL is near/above optimal; from 130-159 mg/dL is borderline high; from 160-189 is high; and from 190 and up is very high. Therefore, an individual who is at risk for MI, ACS, stroke or PAOD because of an increased LDL cholesterol level is, for example, an individual who has more than 100 mg/dL cholesterol, such as an individual who has a near/above optimal level, a borderline high level, a high level or a very high level. Similarly, the American Heart Association indicates that an HDL cholesterol level of less than 40 mg/dL is a major risk factor for heart disease; and an HDL cholesterol level of 60 mg/dL or more is protective against heart disease. Thus, an individual who is at risk for MI, ACS, stroke or PAOD because of a decreased HDL cholesterol level is, for example, an individual who has less than 60 mg/dL HDL cholesterol, such as an individual who has less than 40 mg/dL HDL cholesterol.  
      In another embodiment, an individual who is at risk for MI, ACS, stroke or PAOD is an individual who has increased leukotriene synthesis. “Increased leukotriene synthesis,” as used herein, indicates an amount of production of leukotrienes that is greater, by an amount that is statistically significant, than the amount of production of leukotrienes that is typically found in control individual(s) or by comparison of leukotriene production in a population associated with the lowest band of measurement (e.g., below the mean or median, the lowest quartile or the lowest quintile) compared to higher bands of measurement (e.g., above the mean or median, the second, third or fourth quartile; the second, third, fourth or fifth quintile). For example, the FLAP at-risk haplotypes correlate with increased serum leukotriene synthesis levels, and with increased production of leukotrienes ex vivo. An individual can be assessed for the presence of increased leukotriene synthesis by a variety of methods. For example, an individual can be assessed for an increased risk of MI, ACS, stroke, PAOD or atherosclerosis, by assessing the level of a leukotriene metabolite (e.g., LTE4) in a sample (e.g., serum, plasma or urine) from the individual. Samples containing blood, cells, or tissue can also be obtained from an individual and used to assess leukotriene or leukotriene metabolite production ex vivo under appropriate assay conditions. An increased level of leukotriene metabolites, and/or an increased level of leukotriene production ex vivo, is indicative of increased production of leukotrienes in the individual, and of an increased risk of MI, ACS, stroke, PAOD or atherosclerosis.  
      In a further embodiment, an individual who is at risk for MI, ACS, or stroke is an individual who has already experienced at least one MI, ACS event or stroke, or who has stable angina, and is therefore at risk for a second MI, ACS event or stroke. In another embodiment, an individual who is at risk for MI, ACS, stroke or PAOD is an individual who has atherosclerosis or who requires treatment (e.g., angioplasty, stents, revascularization procedure) to restore blood flow in arteries.  
      In further embodiments, an individual who is at risk for MI, stroke or PAOD is an individual having asymptomatic ankle/brachial index of less than 0.9; an individual who is at risk for stroke, is an individual who has had one or more transient ischemic attacks; who has had transient monocular blindness; has had a carotid endarterectomy; or has asymptomatic carotid stenosis; an individual who is at risk for PAOD, is an individual who has (or had) claudication, limb ischemia leading to gangrene, ulceration or amputation, or has had a revascularization procedure.  
      In additional embodiments, an individual who is at risk for MI, ACS, stroke or PAOD is an individual who has diabetes; hypertension; hypercholesterolemia; elevated triglycerides (e.g., &gt;200 mg/dl); elevated lp(a); obesity; ankle/brachial index (ABI) less than 0.9; and/or is a past or current smoker.  
      Individuals at risk for MI, ACS, stroke or PAOD may fall into more than one of these representative target populations. For example, an individual may have experienced at least one MI, ACS event, transient ischemic attack, transient monocular blindness, or stroke, and may also have an increased level of an inflammatory marker. As used therein, the term “individual in a target population” refers to an individual who is at risk for MI, ACS, stroke or PAOD who falls into at least one of the representative target populations described above.  
     Assessment For At-Risk Haplotypes  
      A “haplotype,” as described herein, refers to a combination of genetic markers (“alleles”), such as those set forth in Table 13. In a certain embodiment, the haplotype can comprise one or more alleles (e.g., a haplotype containing a single SNP), two or more alleles, three or more alleles, four or more alleles, or five or more alleles. The genetic markers are particular “alleles” at “polymorphic sites” associated with FLAP. A nucleotide position at which more than one sequence is possible in a population (either a natural population or a synthetic population, e.g., a library of synthetic molecules), is referred to herein as a “polymorphic site”. Where a polymorphic site is a single nucleotide in length, the site is referred to as a single nucleotide polymorphism (“SNP”). For example, if at a particular chromosomal location, one member of a population has an adenine and another member of the population has a thymine at the same position, then this position is a polymorphic site, and, more specifically, the polymorphic site is a SNP. Polymorphic sites can allow for differences in sequences based on substitutions, insertions or deletions. Each version of the sequence with respect to the polymorphic site is referred to herein as an “allele” of the polymorphic site. Thus, in the previous example, the SNP allows for both an adenine allele and a thymine allele.  
      Typically, a reference sequence is referred to for a particular sequence. Alleles that differ from the reference are referred to as “variant” alleles. For example, the reference FLAP sequence is described herein by SEQ ID NO: 1. The term, “variant FLAP”, as used herein, refers to a sequence that differs from SEQ ID NO: 1, but is otherwise substantially similar. The genetic markers that make up the haplotypes described herein are FLAP variants.  
      Additional variants can include changes that affect a polypeptide, e.g., the FLAP polypeptide. These sequence differences, when compared to a reference nucleotide sequence, can include the insertion or deletion of a single nucleotide, or of more than one nucleotide, resulting in a frame shift; the change of at least one nucleotide, resulting in a change in the encoded amino acid; the change of at least one nucleotide, resulting in the generation of a premature stop codon; the deletion of several nucleotides, resulting in a deletion of one or more amino acids encoded by the nucleotides; the insertion of one or several nucleotides, such as by unequal recombination or gene conversion, resulting in an interruption of the coding sequence of a reading frame; duplication of all or a part of a sequence; transposition; or a rearrangement of a nucleotide sequence, as described in detail above. Such sequence changes alter the polypeptide encoded by a FLAP nucleic acid. For example, if the change in the nucleic acid sequence causes a frame shift, the frame shift can result in a change in the encoded amino acids, and/or can result in the generation of a premature stop codon, causing generation of a truncated polypeptide. Alternatively, a polymorphism associated with a susceptibility to MI, ACS, stroke or PAOD can be a synonymous change in one or more nucleotides (i.e., a change that does not result in a change in the amino acid sequence). Such a polymorphism can, for example, alter splice sites, affect the stability or transport of mRNA, or otherwise affect the transcription or translation of the polypeptide. The polypeptide encoded by the reference nucleotide sequence is the “reference” polypeptide with a particular reference amino acid sequence, and polypeptides encoded by variant alleles are referred to as “variant” polypeptides with variant amino acid sequences.  
      Haplotypes are a combination of genetic markers, e.g., particular alleles at polymorphic sites. The haplotypes described herein, e.g., having markers such as those shown in Table 13, are found more frequently in individuals with MI, ACS, stroke or PAOD than in individuals without MI, ACS, stroke or PAOD. Therefore, these haplotypes have predictive value for detecting a susceptibility to MI, ACS, stroke or PAOD in an individual. The haplotypes described herein are in some cases a combination of various genetic markers, e.g., SNPs and microsatellites. Therefore, detecting haplotypes can be accomplished by methods known in the art for detecting sequences at polymorphic sites, such as the methods described above.  
      In certain methods described herein, an individual who is at risk for MI, ACS, stroke or PAOD is an individual in whom an at-risk haplotype is identified. In one embodiment, the at-risk haplotype is one that confers a significant risk of MI, ACS, stroke or PAOD. In one embodiment, significance associated with a haplotype is measured by an odds ratio. In a further embodiment, the significance is measured by a percentage. In one embodiment, a significant risk is measured as an odds ratio of at least about 1.2, including by not limited to: 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. In a further embodiment, an odds ratio of at least 1.2 is significant. In a further embodiment, an odds ratio of at least about 1.5 is significant. In a further embodiment, a significant increase in risk is at least about 1.7 is significant. In a further embodiment, a significant increase in risk is at least about 20%, including but not limited to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 98%. In a further embodiment, a significant increase in risk is at least about 50%. In yet another embodiment, an at-risk haplotype has a p value &lt;0.05. It is understood however, that identifying whether a risk is medically significant may also depend on a variety of factors, including the specific disease, the haplotype, and often, environmental factors.  
      An at-risk haplotype in, or comprising portions of, the FLAP gene, in one where the haplotype is more frequently present in an individual at risk for MI, ACS, stroke or PAOD (affected), compared to the frequency of its presence in a healthy individual (control), and wherein the presence of the haplotype is indicative of susceptibility to MI, ACS, stroke or PAOD. As an example of a simple test for correlation would be a Fisher-exact test on a two by two table. Given a cohort of chromosomes the two by two table is constructed out of the number of chromosomes that include both of the haplotypes, one of the haplotype but not the other and neither of the haplotypes.  
      In certain embodiments, an at-risk haplotype is an at-risk haplotype within or near FLAP that significantly correlates with a haplotype such as a halotype shown in Table 14; a haplotype shown in Table 15; a haplotype shown in Table 19; haplotype B4; haplotype B5; haplotype B6; haplotype A4; haplotype A5; or haplotype HapB. In other embodiments, an at-risk haplotype comprises an at-risk haplotype within or near FLAP that significantly correlates with susceptibility to myocardial infarction or stroke. In a particular embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S99, SG13S25, SG13S377, SG13S106, SG13S32 and SG13S35 at the 13q12-13 locus. In another embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S99, SG13S25, SG13S106, SG13S30 and SG13S42 at the 13q12-13 locus. In a third embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S25, SG13S106, SG13S30 and SG13S42 at the 13q12-13 locus. In a fourth embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S99, SG13S25, SG13S114, SG13S89 and SG13S32 at the 13q12-13 locus. In another embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises marker SG13S375 at the 13q12-13 locus. In another embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S25 and SG13S375 at the 13q12-13 locus. In another embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S25, SG13S375 and SG13S32 at the 13q12-13 locus. In an additional embodiment, a -haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S25, SG13S375, SG13S32 and SG13S106. In other embodiments, the at-risk haplotype is selected from the group consisting of: haplotype B4, B5, B6, A4, A5, C1, C2, C3, C4-A and C4-B. The at-risk haplotype can also comprise a combination of the markers in the haplotypes B4, B5, B6, A4, A5, C1, C2, C3, C4-A and/or C4-B. In further embodiments, the at-risk haplotype can be haplotype HapB. In other embodiments, the at-risk haplotype comprises a polymorphism shown in Table 13.  
      Standard techniques for genotyping for the presence of SNPs and/or microsatellite markers can be used, such as fluorescent based techniques (Chen, et al.,  Genome Res.  9, 492 (1999)), PCR, LCR, Nested PCR and other techniques for nucleic acid amplification. In a preferred embodiment, the method comprises assessing in an individual the presence or frequency of SNPs and/or microsatellites in, comprising portions of, the FLAP gene, wherein an excess or higher frequency of the SNPs and/or microsatellites compared to a healthy control individual is indicative that the individual is susceptible to MI, ACS, stroke or PAOD. See, for example, Table 13 (below) for SNPs and markers that can form haplotypes that can be used as screening tools. These markers and SNPs can be identified in at-risk haploptypes. For example, an at-risk haplotype can include microsatellite markers and/or SNPs such as those set forth in Table 13. The presence of the haplotype is indicative of a susceptibility to MI, ACS, stroke or PAOD, and therefore is indicative of an individual who falls within a target population for the treatment methods described herein.  
      Haplotype analysis involves defining a candidate susceptibility locus using LOD scores. The defined regions are then ultra-fine mapped with microsatellite markers with an average spacing between markers of less than 100 kb. All usable microsatellite markers that are found in public databases and mapped within that region can be used. In addition, microsatellite markers identified within the deCODE genetics sequence assembly of the human genome can be used. The frequencies of haplotypes in the patient and the control groups can be estimated using an expectation-maximization algorithm (Dempster A. et al., 1977.  J. R. Stat. Soc. B,  39:1-389). An implementation of this algorithm that can handle missing genotypes and uncertainty with the phase can be used. Under the null hypothesis, the patients and the controls are assumed to have identical frequencies. Using a likelihood approach, an alternative hypothesis is tested, where a candidate at-risk-haplotype, which can include the markers described herein, is allowed to have a higher frequency in patients than controls, while the ratios of the frequencies of other haplotypes are assumed to be the same in both groups. Likelihoods are maximized separately under both hypotheses and a corresponding l-df likelihood ratio statistic is used to evaluate the statistic significance.  
      To look for at-risk-haplotypes in the 1-lod drop, for example, association of all possible combinations of genotyped markers is studied, provided those markers span a practical region. The combined patient and control groups can be randomly divided into two sets, equal in size to the original group of patients and controls. The haplotype analysis is then repeated and the most significant p-value registered is determined. This randomization scheme can be repeated, for example, over 100 times to construct an empirical distribution of p-values. In a preferred embodiment, a p-value of &lt;0.05 is indicative of an at-risk haplotype.  
      A detailed discussion of haplotype analysis is described in International Application No. PCT/US03/32556, filed on Oct. 16, 2003, which is incorporated by reference herein in its entirety.  
     Methods of Treatment  
      The present invention encompasses methods of treatment (prophylactic and/or therapeutic, as described above) for MI, ACS, stroke or PAOD in individuals, such as individuals in the target populations described above, as well as for other diseases and conditions associated with FLAP or with other members of the leukotriene pathway (e.g., for atherosclerosis). Members of the “leukotriene pathway,” as used herein, include polypeptides (e.g., enzymes, receptors) and other molecules that are associated with production of leukotrienes: for example, proteins or enzymes such as FLAP, 5-LO, other leukotriene biosynthetic enzymes (e.g., leukotriene C4 synthase, leukotriene A4.hydrolase); receptors or binding agents of the enzymes; leukotrienes such as LTA4, LTB4, LTC4, LTD4, LTE4; and receptors of leukotrienes (e.g., leukotriene B4 receptor 1 (BLT1), leukotriene B4 receptor 2 (BLT2), cysteinyl leukotriene receptor 1 (CysLTR1), cysteinyl leukotriene receptor 2 (CysLTR2)).  
      In particular, the invention relates to methods of treatment for myocardial infarction or susceptibility to myocardial infarction (for example, for individuals in an at-risk population such as those described above); as well as methods of treatment for acute coronary syndrome (e.g., unstable angina, non-ST-elevation myocardial infarction (NSTEMI) or ST-elevation myocardial infarction (STEMI)); methods for reducing risk of MI, stroke or PAOD in persons with asymptomatic ankle/brachial index less than 0.9; for decreasing risk of a second myocardial infarction; for stroke or susceptibility to stroke; for transient ischemic attack; for transient monocular blindness; for decreasing risk of a second stroke; for PAOD or susceptibility to PAOD; for ABI less than 0.9; for claudication or limb ischemia; for atherosclerosis, such as for patients requiring treatment (e.g., angioplasty, stents, revascularization procedure) to restore blood flow in arteries (e.g., coronary, carotid, and/or femoral arteries); for treatment of asymptomatic ankle/brachial index of less than 0.9; and/or for decreasing leukotriene synthesis (e.g., for treatment of MI, ACS, stroke or PAOD). The invention additionally pertains to use of one or more leukotriene synthesis inhibitors, as described herein, for the manufacture of a medicament for the treatment of MI, ACS, stroke, PAOD and/or atherosclerosis, e.g., using the methods described herein. The invention also provides for the use of one or more leukotriene synthesis inhibitors, as described herein, for the manufacture of a medicament for reducing the risk for MI, ACS, PAOD, stroke and/or. artherosclerosis using the methods described herein. These medicaments may comprise a leukotriene synthesis inhibitor alone or in combination with a statin, as described herein.  
      In the methods of the invention, a “leukotriene synthesis inhibitor” is used. In one embodiment, a “leukotriene synthesis inhibitor” is an agent that inhibits FLAP polypeptide activity and/or FLAP nucleic acid expression, as described herein (e.g., a nucleic acid antagonist). In another embodiment, a leukotriene synthesis inhibitor is an agent that inhibits polypeptide activity and/or nucleic acid expression of another member of the leukotriene biosynthetic pathway (e.g., 5-LO; LTC4S; LTA4H; LTB4DH). In still another embodiment, a leukotriene synthesis inhibitor is an agent that alters activity or metabolism of a leukotriene (e.g., an antagonist of a leukotriene; an antagonist of a leukotriene receptor). In preferred embodiments, the leukotriene synthesis inhibitor alters activity and/or nucleic acid expression of FLAP or of 5-LO, or alters interaction between FLAP and 5-LO.  
      Leukotriene synthesis inhibitors can alter polypeptide activity or nucleic acid expression of a member of the leukotriene pathway by a variety of means, such as, for example, by catalytically degrading, downregulating or interfering with the expression, transcription or translation of a nucleic acid encoding the member of the leukotriene pathway; by altering posttranslational processing of the polypeptide; by altering transcription of splicing variants; or by interfering with polypeptide activity (e.g., by binding to the polypeptide, or by binding to another polypeptide that interacts with that member of the leukotriene pathway, such as a FLAP binding agent as described herein or some other binding agent of a member of the leukotriene pathway; by altering interaction among two or more members of the leukotriene pathway (e.g., interaction between FLAP and 5-LO); or by antagonizing activity of a member of the leukotriene pathway.  
      Representative leukotriene synthesis inhibitors include the following: agents that inhibit activity of a member of the leukotriene biosynthetic pathway (e.g., FLAP, 5-LO), LTC4S, LTA4H, such as the agents presented in the Agent Tables I and II herein; agents that inhibit activity of receptors of members of the leukotriene pathway, such as FLAP receptors, LTA4 receptors, LTB4 receptors (e.g. BLT1, BLT2), LTC4 receptors, LTD4 receptors, LTE4 receptors, Cys LT1 receptors, Cys LT2 receptors, 5-LO receptors; BLT1; BLT2; CysLTR1; CysLTR2; agents that bind to the members of the leukotriene pathway, such as FLAP binding agents (e.g., 5-LO) or agents that bind to receptors of members of the leukotriene pathway (e.g., leukotriene receptor antagonists); agents that bind to a leukotriene (e.g., to LTA4, LTB4, LTC4, LTD4, LTE4, Cys LT1, Cys LT2); agents that increase breakdown of leukotrienes (e.g., LTB4DH); or other agents that otherwise affect (e.g., increase or decrease) activity of the leukotriene;  
      antibodies to leukotrienes;  
      antisense nucleic acids or small double-stranded interfering RNA, to nucleic acids encoding FLAP, 5-LO, LTA4H, or a leukotriene synthetase or other member of the leukotriene pathway, or fragments or derivatives thereof, including antisense nucleic acids to nucleic acids encoding the FLAP, 5-LO or leukotriene synthetase polypeptides, and vectors comprising such antisense nucleic acids (e.g., nucleic acid, cDNA, and/or mRNA, double-stranded interfering RNA, or a nucleic acid encoding an active fragment or derivative thereof, or an oligonucleotide; for example, the complement of one of SEQ ID Nos. 1, 3, 718 or 719, or a nucleic acid complementary to the nucleic acid encoding SEQ ID NO: 2 or 718, or fragments or derivatives thereof);  
      peptidomimetics; fusion proteins or prodrugs thereof; ribozymes; other small molecules; and  
      other agents that alter (e.g., inhibit or antagonize) expression of a member of the leukotriene pathway, such as FLAP, 5-LO or LTA4H nucleic acid expression or polypeptide activity, or that regulate transcription of FLAP splicing variants, 5-LO splicing variants or LTA4H splicing variants(e.g., agents that affect which splicing variants are expressed, or that affect the amount of each splicing variant that is expressed).  
      More than one leukotriene synthesis inhibitor can be used concurrently, if desired.  
      The therapy is designed to alter activity of a FLAP polypeptide, a 5-LO polypeptide, or another member of the leukotriene pathway in an individual, such as by inhibiting or antagonizing activity. For example, a leukotriene synthesis inhibitor can be administered in order to decrease synthesis of leukotrienes within the individual, or to downregulate or decrease the expression or availability of the FLAP nucleic acid or specific splicing variants of the FLAP nucleic acid. Downregulation or decreasing expression or availability of a native FLAP nucleic acid or of a particular splicing variant could minimize the expression or activity of a defective nucleic acid or the particular splicing variant and thereby minimize the impact of the defective nucleic acid or the particular splicing variant. Similarly, for example, a leukotriene synthesis inhibitor can be administered in order to downregulate or decrease the expression or availability of the nucleic acid encoding 5-LO or specific splicing variants of the nucleic acid encoding 5-LO.  
      The leukotriene synthesis inhibitor(s) are administered in a therapeutically effective amount (i.e., an amount that is sufficient to treat the disease or condition, such as by ameliorating symptoms associated with the disease or condition, preventing or delaying the onset of the disease or condition, and/or also lessening the severity or frequency of symptoms of the disease or condition). The amount which will be therapeutically effective in the treatment of a particular individual&#39;s disease or condition will depend on the symptoms and severity of the disease, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of a practitioner and each patient&#39;s circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.  
      In preferred embodiments of the invention, the leukotriene synthesis inhibitor agent is an agent that inhibits activity of FLAP and/or of 5-LO. Preferred agents include the following, as set forth in Agent Table I below:  
                                                                           Date Patent               Product_Name               Issued/Applica-       Company   (Code)   Structure   Chemical Name   Patent Ref   tion Published   MOA                                                  Abbott   atreleuton (ABT-761)                         (R)-(+)-N-[3[5-[(4-fluoro- phenyl)methyl]-2thienyl]-1meth- yl-2-propynyl]-N-hy- droxurea   U.S Pat. No. 5288751, U.S. Pat. No. 5288743, U.S. Pat. No. 5616596   2/22/94 04/01/97   5-LPO Inhibitor               Abbott   A-81834                         3-(3-(1,1-dimethylethylthio-5-(quino- line-2-ylmethoxy)-1-(4-chloro- methylphenyl)indole-2-yl)-2,2-di- methylpropionaldehyde oxime-0-2-acetic acid   WO9203132, U.S. Pat. No. 5459150   3/5/1992, 10/17/95   FLAP inhibitor               Abbott   A-86886                         3-(3-(1,1-dimethylethylthio-5-(py- ridin-2-ylmethoxy)-1-(4-chloro- methylphenyl)indole-2-yl)-2,2-di- methylpropionaldehyde oxime-0-2-acetic acid   WO9203132, U.S. Pat. No. 5459150   3/5/1992, 10/17/95   5-LPO inhibitor               Abbott   A-93178                                     FLAP inhibitor               Astra- Zeneca   AZD-4407                             EP 623614   09/11/94   5-LPO inhibitor               Astra- Zeneca   ZD-2138                         6-((3-fluoro-5-(tetra- hydro-4-methoxy-2H-py- ran-4yl)phenoxy)methyl)1-meth- yl-2(1H)-quino- linone (alternatively NH can be N-methyl)   EP 466452       5-LPO inhibitor               Bayer   BAY-X-1005                         (R)-(+)-alpha-cyclopentyl 4-(2-quinolinylmethoxy)-Benzene- acetic acid   U.S. Pat. No. 4970215 EP 344519, DE 19880531       FLAP inhibitor               Merck   MK-0591                         1-((4-chloro- phenyl)methyl)-3-((1,1-di- methylethyl)thio)alpha, alph-dimethyl-5-(2-quino- linylmethoxy)-1H-In- dole-2-proanoic acid   EP 419049, U.S. Pat. No. 19890822       FLAP inhibitor               Merck   MK-866       (3[3-)4-chlorobenzyl)-3-t-butyl-thi- o-5-isopropylindol-2-yl]2,2-di- methyl-propanoic acid           5-LPO inhibitor               Merck   MK-886                         1-((4-chloro- phenyl)methyl)-3-((1,1di- methylethyl)thio)-alpha, alpha-dimethyl-5-(2-quino- linylmethoxy)-1H-In- dole-2-propanoic acid   EP 419049, U.S. Pat. No. 19890822       5-LPO inhibitor               Pfizer   CJ-13610       4-(3-(4-(2-Methyl-imi- dazol-1-yl)-phenyl- sulfanyl)-phenyl)-tetra- hydro-pyran-4-carboxylic acid amide           5-LPO inhibitor                  
 
      In preferred methods of the invention, the agents set forth in the Agent Table III can be used for prophylactic and/or therapeutic treatment for diseases and conditions associated with FLAP or with other members of the leukotriene pathway, or with increased leukotriene synthesis. In particular, they can be used for treatment for myocardial infarction or susceptibility to myocardial infarction, such as for individuals in an at-risk population as described above, (e.g., based on identified risk factors such as elevated cholesterol, elevated C-reactive protein, and/or genotype); for individuals suffering from acute coronary syndrome, such as unstable angina, non-ST-elevation myocardial infarction (NSTEMI) or ST-elevation myocardial infarction (STEMI); methods for reducing risk of MI, stroke or PAOD in persons with asymptomatic ankle/brachial index less than 0.9; for decreasing risk of a subsequent myocardial infarction, such as in individuals who have already had one or more myocardial infarctions; for stroke or susceptibility to stroke; for decreasing risk of a second stroke; for PAOD or susceptibility to PAOD; for treatment of atherosclerosis, such as in patients requiring treatment (e.g., angioplasty, stents, revascularization procedure) to restore blood flow in arteries (e.g., coronary, carotid, and/or femoral arteries); for treatment of asymptomatic ankle/brachial index of less than 0.9; and/or for decreasing leukotriene synthesis (e.g., for treatment of myocardial infarction, ACS, stroke or PAOD  
      In one preferred embodiment of the invention, the leukotriene synthesis inhibitor is an inhibitor of FLAP such as 1-((4-chlorophenyl)methyl)-3-((1,1-dimethylethyl)thio)-alpha,alpha-dimethyl-5-(2-quinolinylmethoxy)-1H-Indole-2-propanoic acid otherwise known as MK-0591, (R)-(+)-alpha-cyclopentyl-4-(2-quinolinylmethoxy)-Benzeneacetic acid otherwise known as BAY-x-1005, 3-(3-(1,1-dimethylethylthio-5-(quinoline-2-ylmethoxy)-1-(4-chloromethylphenyl)indole-2-yl)-2,2-dimethylpropionaldehyde oxime-0-2-acetic acid otherwise known as A-81834, their optically pure enantiomers, salts, chemical derivatives, analogues, or other compounds inhibiting FLAP that effectively decrease leukotriene biosynthesis when administered to humans.  
      In another preferred embodiment of the invention, the leukotriene synthesis inhibitor is an inhibitor of 5LO such as zileuton, atreleuton, 6-((3-fluoro-5-(tetrahydro-4-methoxy-2H-pyran-4yl)phenoxy)methyl)-1-methyl-2(1H)-quinlolinone otherwise known as ZD-2138, 1-((4-chlorophenyl)methyl)-3-((1,1dimethylethyl)thio)-alpha,alpha-dimethyl-5-(2-quinolinylmethoxy)-1H-Indole-2-propanoic acid otherwise known as MK-886, 4-(3-(4-(2-Methyl-imidazol-1-yl)-phenylsulfanyl)-phenyl)-tetrahydro-pyran-4-carboxylic acid amide otherwise known as CJ-13610, their optically pure enantiomers, salts, chemical derivatives, analogues or other compounds inhibiting 5-LO that effectively decrease leukotriene biosynthesis when administered to humans.  
      The compound can be represented by the following formula:  
                 
 
      in M is selected from the group consisting of hydrogen, a pharmaceutically acceptable cation, and a pharmaceutically acceptable metabolically cleavable group; B is a straight or branched divalent alkylene group of from one to twelve carbon atoms; Z is thiazolyl, optionally substituted with alkyl of from one to six carbon atoms or haloalkyl of from one to six carbon atoms; L is selected from the group consisting of (a) alkylene of from 1-6 carbon atoms, (b) alkenylene of from 2-6 carbon atoms, (c) alkynylene of from 2-6 carbon atoms, (d) hydroxyalkyl of 1-6 carbon atoms, (e) &gt;C═O, (f) &gt;C═N—OR 1 , where R 1  is hydrogen or C 1 -C 6  alkyl, (g) —(CHR1) n  (CO)(CHR 2 ) m , where n and m are independently selected from an integer from one to six and R 1  and R 2  are independently selected from hydrogen and C 1 -C 6 -alkyl, (h) —(CHR1) n  C═NOR 2 , where R 1 , R 2  and n are as defined above; (i) —(CHR 1 ) n  ON═CR 2 , where R 1 , R 2  and n are as: defined above; (j) —(CHR 1 ) n  —O—(CHR 2 ) m —, where R 1 , R 2 , n and m are as defined above, (k) —(CHR 1 ) n  —NR 2  (CHR 3 ) m —, where R 1 , R 2 , n and m are as defined above and R 3  is selected from hydrogen and C 1 -C 6 -alkyl; (l) —(CHR 1 ) n  —S—CHR 2 ) m —, where R 1 , R 2 , n and m are as defined above; and (m) —(CHR 1 )N —(SO 2 )—(CHR 2 ) m —, where R 1 , R 2 , n and m are as defined above; A is carbocyclic aryl optionally substituted with alkyl of from one to six carbon atoms, haloalkyl of from one to six carbon atoms, hydroxyalkyl of from one to six carbon atoms, alkoxy of from one to twelve carbon atoms, alkoxyalkoxyl in which the two alkoxy portions may each independently contain from one to six carbon atoms, alkylthio of from one to six carbon atoms, hydroxy, halogen, cyano, amino, alkylamino of from one to six carbon atoms, dialkylamino in which the two alkyl groups may independently contain from one to six carbon atoms, alkanoylamino of from two to eight carbon atoms, N-alkanoyl-N-alkylamino in which the alkanoyl is of from two to eight carbon atoms and the alkyl group is of from one to six carbon atoms, alkylaminocarbonyl of from two to eight carbon atoms, dialkylaminocarbonyl in which the two alkyl groups are independently of from one to six carbon atoms, carboxyl, alkoxycarbonyl or from two to eight carbon atoms, phenyl, optionally substituted with alkyl of from one to six carbon atoms, haloalkyl of from one to six carbon atoms, alkoxy of from one to six carbon atoms, hydroxy or halogen, phenoxy, optionally substituted with alkyl of from one to six carbon atoms, haloalkyl of from one to six carbon atoms, alkoxy of from one to six carbon atoms, hydroxy or halogen, and phenylthio, optionally substituted with alkyl of from one to six carbon atoms, haloalkyl of from one to six carbon atoms, alkoxy of from one to six carbon atoms, hydroxy or halogen. Preferably, the compound is a compound or pharmaceutically acceptable salt thereof having the name (R)-N-{3-[-5-(4-fluorophenylmethyl)thiazo-2-yl]-1methyl-2-propynyl}-N-hydroxyurea. See U.S. Pat. No. 4,615,596, incorporated herein by reference.  
      The compound is represented by the following formula:  
                 
 
      or a pharmaceutically acceptable salt thereof, wherein A is selected from the group consisting of straight or branched divalent alkylene of from one to twelve carbon atoms and divalent cycloalkylene of from three to eight carbon atoms; R 1  is selected from the group consisting of hydrogen, alkylthio of from one to six carbon atoms, phenylthio, optionally substituted with alkyl of from one to six carbon atoms, alkoxy of from one to six carbon atoms, or halogen, phenylalkylthio in which the alkyl portion contains from one to six carbon atoms, and the phenyl group is optionally substituted with alkyl of from one to six carbon atoms, alkoxy of from one to six carbon atoms, or halogen, R 2  is selected from the group consisting of —COOB wherein B is selected from hydrogen, a pharmaceutically acceptable cation, or a metabolically cleavable group, —COOalkyl where the alkyl portion contains from one to six carbon atoms, —COOalkylcarbocyclicaryl where the alkyl portion contains from one to six carbon atoms and the aryl portion is optionally substituted with alkyl of from one to six carbon atoms, alkoxy of from one to six carbon atoms, or halogen, —CONR 5  R 6  wherein R 5  is selected from the group consisting of hydrogen, hydroxyl, alkyl of from one to six carbon atoms, and alkoxy of from one to six carbon atoms, and R 6  is selected from the group consisting of hydrogen and alkyl of from one to six carbon atoms, —COR 6 , and —OH; R 3  is selected from the group consisting of phenylalkyl in which the alkyl portion contains from one to six carbon atoms, and the phenyl group is optionally substituted with alkyl of from one to six carbon atoms, alkoxy of from one to six carbon atoms, or halogen, R 4  is selected from the group consisting of thiazolylalkyloxy in which the alkyl portion contains from one to six carbon atoms, and the heteroaryl portion is optionally substituted with alkyl of from one to six carbon atoms, alkoxy of from one to six carbon atoms, or halogen, and thiazolyloxy optionally substituted with alkyl of from one to six carbon atoms, alkoxy of from one to six carbon atoms, or halogen. See U.S. Pat. No. 5,288,743, incorporated herein by reference.  
      The compound can be represented by the formula:  
                 
 
      or a pharmaceutically acceptable salt thereof, wherein M is selected from the group consisting of hydrogen, and a pharmaceutically acceptable cation; B is a straight or branched divalent alkylene group of from one to twelve carbon atoms; Z is selected from the group consisting of: (a) furyl, optionally substituted with alkyl of from one to six carbon atoms, or haloalkyl of from one to six carbon atoms, and (b) thienyl, optionally substituted with alkyl of from one to six carbon atoms, or haloalkyl of from one to six carbon atoms; and L is alkylene of from 1-6 carbon atoms; A is phenyl optionally substituted with alkyl of from one to six carbon atoms, haloalkyl of from one to six carbon atoms, hydroxyalkyl of from one to six carbon atoms, alkoxy of from one to twelve carbon atoms, alkoxyalkoxyl in which the two alkoxy portions may each independently contain from one to six carbon atoms, alkylthio of from one to six carbon atoms, hydroxy, halogen, cyano, amino, alkylamino of from one to six carbon atoms, dialkylamino in which the two alkyl groups may independently contain from one to six carbon atoms, alkanoylamino of from two to eight carbon atoms, N-alkanoyl-N-alkylamino in which the alkanoyl is of from two to eight carbon atoms and the alkyl group is of from one to six carbon atoms, alkylaminocarbonyl of from two to eight carbon atoms, dialkylaminocarbonyl in which the two alkyl groups are independently of from one to six carbon atoms, carboxyl, alkoxycarbonyl of from two to eight carbon atoms, phenyl, optionally substituted with alkyl of from one to six carbon atoms, haloalkyl of from one to six carbon atoms, alkoxy of from one to six carbon atoms, hydroxy or halogen, phenoxy, optionally substituted with alkyl of from one to six carbon atoms, haloalkyl of from one to six carbon atoms, alkoxy of from one to six carbon atoms, hydroxy or halogen, or phenylthio, optionally substituted with alkyl of from one to six carbon atoms, haloalkyl of from one to six carbon atoms, alkoxy of from one to six carbon atoms, hydroxy or halogen. Preferably, the compound is a compound or a pharmaceutically acceptable salt thereof selected from the group consisting of: N-{3-(5-(4-fluorophenylmethyl)fur-2-yl)-3-butyn-2-yl}-N-hydroxyurea; N-{3-(5-(4-fluorophenylmethyl)-2-thienyl)-1-methyl-2-propynyl}-N-hydroxyurea; (R)—N-{3-(5-(4-fluorophenylmethyl)-2-thienyl)-1-methyl-2-propynyl}-N-hydroxyurea; and (R)—N-{3-(5-(4-chlorophenylmethyl)-2-thienyl)-1-methyl-2-propynyl}-N-hydroxyurea; (S)—N-{3-[5-(4-fluorophenylmethyl)-2-thienyl]-1-methyl-2-propynyl}-N-hydroxyurea. See U.S. Pat. No. 5,288,751, incorporated by reference herein.  
      The compound can be represented by the formula:  
                 
 
      or a pharmaceutically acceptable salt thereof, wherein A is selected from the group consisting of straight or branched divalent alkylene of one to twelve carbon atoms, straight or branched divalent alkenylene of two to twelve carbon atoms, and divalent cycloalkylene of three to eight carbon atoms; R 1  is alkylthio of one to six carbon atoms; R 6  is selected from the group consisting of hydrogen and alkyl of one to six carbon atoms; R 7  is selected from the group consisting of (carboxyl)alkyl in which the alkyl portion is of one to six carbon atoms, (alkoxycarbonyl)alkyl in which the alkoxycarbonyl portion is of two to six carbon atoms and the alkyl portion is of one to six carbon atoms, (aminocarbonyl)alkyl in which the alkyl portion is of one to six carbon atoms, ((alkylamino)carbonyl)alkyl in which each alkyl portion independently is of one to six carbon atoms, and ((dialkylamino)carbonyl)alkyl in which each alkyl portion independently is of one to six carbon atoms; R 3  is phenylalkyl in which the alkyl portion is of one to six carbon atoms; R 4  is 2-, 3- or 6-quinolylmethoxy, optionally substituted with alkyl of one to six carbon atoms, haloalkyl of one to six carbon atoms, alkoxy of one to twelve carbon atoms, halogen, or hydroxy. Preferably, the compound is selected from the group consisting of: 3-(3-1,1-dimethylethylthio)-5-(quinolin-2-ylmethoxy-1-(4-chlorophenylmethyl )-indol-2-yl)-2,2-dimethylpropionaldehyde oxime-O-2 acetic acid; 3-(3-(1,1-dimethylethylthio)-5-(quinolin-2-ylmethoxy)-1-(4-chloro-phenylmethyl) indol-2-yl)-2,2-dimethylpropionaldehyde oxime-O-2-(3-methyl)butyric acid; 3-(3-(1,1-dimethylethylthio)-5-(6,7-dichloroquinolin-2-ylmethoxy)-1-(4-chlorophenylmethyl)indol-2-yl)-2,2-dimethylpropionaldehyde oxime-O-2-acetic acid; and 3-(3-(1,1-dimethylethylthio)-5-(6-fluoroquinolin-2-ylmethoxy)-1-(4chlorophenylmethyl) indol-2-yl)-2,2-dimethylpropionaldehyde oxime-O-2-propionic acid; or a pharmaceutically acceptable salt or ester thereof. See U.S. Pat. No. 5,459,150, incorporated by reference herein.  
      The compound can be represented by the formula: 
 
Q 1 —X—Ar—Q 2  
 
      or pharmaceutically acceptable salts thereof, wherein Q is a 9-, 10- or 11-membered bicyclic heterocyclic moiety containing one or two nitrogen heteroatoms and optionally containing a further heteroatom selected from nitrogen, oxygen and sulphur, and Q may optionally bear up to four substituents selected from halogeno, hydroxy, cyano, formyl, oxo, thioxo, (1-4C)alkyl, (3-4C)alkenyl, (3-4C)alkynyl, (1-4C)alkoxy, fluoro-(1-4C)alkyl, hydroxy-(1-4C)alkyl, (2-5C)alkanoyl, phenyl, benzoyl and benzyl, and wherein said phenyl, benzoyl and benzyl substituents may optionally bear one or two substituents selected from halogeno, (1-4C)alkyl and (1-4C)alkoxy; X is oxy, thio, sulphinyl or sulphonyl; Ar is phenylene, pyridinediyl, pyrimidinediyl, thiophenediyl, furandiyl, thiazolediyl, oxazolediyl, thiadiazolediyl or oxadiazolediyl which may optionally bear one or two substituents selected from halogeno, cyano, trifluoromethyl, hydroxy, amino, (1-4C)alkyl, (1-4C)alkoxy, (1-4C)alkylamino and di-(1-4C)alkylamino; and Q is selected from the groups of the formulae II and III:  
                 
 
      wherein R is hydrogen, (2-5C)alkanoyl or benzoyl, and wherein said benzoyl group may optionally bear one or two substituents selected from halogeno, (1-4C)alkyl and (1-4C)alkoxy; R is (1-4C)alkyl; and R is hydrogen or (1-4C)alkyl; or R and R are linked to form a methylene, vinylene, ethylene or trimethylene group. Preferably, the compound is selected from the group consisting of: (2S,4R)-4-[5-fluoro-3-(1-methyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-ylthio)phenyl]-4-hydroxy-2-ethyltetrahydropyran, (2S,4R)-4-[5-fluoro-3-(1-methyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-ylsulphonyl)phenyl]-4-hydroxy-2-methyltetrahydropyran, (2S,4R)-4-hydroxy-2-methyl-4-[2-(1-methyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-ylthio)thiazol-5-yl]tetrahydropyran, (2S,4R)-4-hydroxy-2-methyl-4-[2-(1-methyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-ylsulphonyl)thiazol-5-yl]tetrahydropyran, (2S,4R)-4-[2-(7-fluoro-1-methyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-ylthio)thiazol-5-yl]-4-hydroxy-2-methyltetrahydropyran, (2S,4R)-4-hydroxy-2-methyl-4-[2-(1-methyl-2-oxoindolin-5-ylthio)thiazol-5-yl]tetrahydropyran, (2S,4R)-4-hydroxy-2-methyl-4-[2-(1-methyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-ylthio)thien-4-yl]tetrahydropyran, (2S,4R)-4-hydroxy-2-methyl-4-[2-(1-methyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-ylsulphonyl)thien-4-yl]tetrahydropyran, (2S,4R)-4-hydroxy-2-methyl-4-[2-(1-methyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-ylthio)thien-5-yl]tetrahydropyran, (2S,4R)-4-hydroxy-2-methyl-4-[2-(1-methyl-2-oxo-1,2-dihydroquinolin-6-ylthio)thien-4-yl]tetrahydropyran, (2S,4R)-4-hydroxy-2-methyl-4-[2-(1,8-dimethyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-ylthio)thien-4-yl]tetrahydropyran, 4-[2-(8-fluoro-1-methyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-ylthio)thien-4-yl]-4-hydroxy-2-methyltetrahydropyran, 4-[2-(7-fluoro-1-methyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-ylthio)thien-4-yl]-4-hydroxy-2-methyltetrahydropyran, (2S,4R)-4-hydroxy-2-methyl-4-[2-(1-methyl-2-oxoindolin-5-ylthio)thien-4-yl]tetrahydropyran, (2S,4R)-4-hydroxy-2-methyl-4-[3-(1-methyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-ylthio)phenyl]tetrahydropyran, (2S,4R)-4-hydroxy-2-methyl-4-[3-(1-methyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-ylsulphonyl)phenyl]tetrahydropyran, (2S,4R)-4-[3-(1-ethyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-ylthio)phenyl]-4-hydroxy-2-methyltetrahydropyran, (2S,4R)-4-[3-(7-fluoro-1-methyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-ylthio)phenyl]-4-hydroxy-2-methyltetrahydropyran, (2S,4R)-4-hydroxy-2-methyl-4-[3-(1-methyl-2-oxo-1,2-dihydroquinolin-6-ylthio)phenyl]tetrahydropyran, (2S,4R)-4-[3-(8-chloro-1-methyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-ylthio)phenyl]-4-hydroxy-2-methyltetrahydropyran and (2S,4R)-4-hydroxy-2-methyl-4-[3-(1-methyl-2-oxoindolin-5-ylthio)phenyl]tetrahydropyran. See EP 623614 B1, incorporated herein by reference.  
      The compound can be represented by the formula:  
                 
 
      wherein Q is a 10-membered bicyclic heterocyclic moiety containing one or two nitrogen heteroatoms which bears one or two thioxo substituents, and which heterocyclic moiety may optionally bear one, two or three further substituents selected from halogeno, hydroxy, cyano, amino, (1-4C)alkyl, (1-4C)alkoxy, fluoro-(1-4C)alkyl, (1-4C)alkylamino, di-[(1-4C)alkyl]amino, amino-(1-4C)alkyl, (1-4C)alkylamino-(1-4C)alkyl, di-[(1-4C)alkyl]amino-(1-4C)alkyl, phenyl and phenyl-(1-4C)alkyl, and wherein said phenyl or phenyl-(1-4C)alkyl substituent may optionally bear a substituent selected from halogeno, (1-4C)alkyl and (1-4C)alkoxy; wherein A is a direct link to X or is (1-3C)alkylene; wherein X is oxy, thio, sulphinyl, sulphonyl or imino; wherein Ar is phenylene which may optionally bear one or two substituents selected from halogeno, hydroxy, amino, nitro, cyano, carbamoyl, ureido, (1-4C)alkyl, (1-4C)alkoxy, (1-4C)alkylamino, di-[(1-4C)alkyl]amino, fluoro-(1-4C)alkyl and (2-4C)alkanoylamino; or Ar is pyridylene; wherein R is (1-4C)alkyl, (3-4C)alkenyl or (3-4C)alkynyl; and wherein R and R together form a group of the formula -A-X-A- which, together with the carbon atom to which A and A are attached, defines a ring having 5 to 7 ring atoms, wherein A and A, which may be the same or different, each is (1-3C)alkylene and X is oxy, thio, sulphinyl or sulphonyl, and which ring may bear one, two or three substituents, which may be the same or different, selected from hydroxy, (1-4C)alkyl and (1-4C)alkoxy; or wherein R and R together form a group of the formula -A-X-A- which, together with the oxygen atom to which A is attached and with the carbon atom to which A is attached, defines a ring having 5 to 7 ring atoms, wherein A and A, which may be the same or different, each is (1-3C)alkylene and X is oxy, thio, sulphinyl or sulphonyl, and which ring may bear one, two or three (1-4C)alkyl substituents, and wherein R is (1-4C)alkyl, (2-4C)alkenyl or (2-4C)alkynyl; or a pharmaceutically-acceptable salt thereof. Preferably, the compound is selected from the group consisting of: 4-(5-fluoro-3-(1-methyl-2-thioxo-1,2-dihydroquinolin-6-ylmethoxy)phenyl]-4-ethoxytetrahydropyran and 4-(5-fluoro-3-(1-methyl-2-thioxo-1,2,3,4-tetrahydroquinolin-6-1methoxy)phenyl]-4-methoxytetrahydropyran, 4-(5-fluoro-3-(1-methyl-2-thioxo-1,2,3,4-tetrahydroquinolin-6-ylthio)phenyl]-4-methoxytetrahydropyran and pharmaceutically-acceptable salt thereof. See EP 466452 B1, incorporated herein by reference.  
      The compound can be a substituted 4-(quinolin-2-61-methoxy)phenylacetic acid derivative represented by the following formula:  
                 
 
      or pharmaceutically acceptable salt thereof, wherein R 1  represents a group of the formula:  
                 
 
      R 2  and R 3  are identical or different and represent hydrogen, lower alkyl, phenyl, benzyl or a group of the formula:  
                 
 
      R 4  represents hydrogen, lower alkyl, phenyl or benzyl, which can optionally be substituted by hydroxyl, carboxyl, lower alkoxycarbonyl, lower alkylthio, heteroaryl or carbamoyl, R 5  represents hydrogen, lower alkyl, phenyl or benzyl, R 6  represents a group of the formula —COR 5  or —CO 2  R 5 , R 7  represents hydrogen, lower alkyl or phenyl, Y represents a group of the formula:  
                 
 
      wherein R 8  represents hydrogen, lower alkyl or phenyl and n denotes a number of 0 to 5, Z represents norbornyl, or represents a group of the formula:  
                 
 
      wherein R 9  and R 10  are identical or different and denote hydrogen, lower alkyl or phenyl, or R 9  and R 10  can together form a saturated carbocyclic ring having up to 6 carbon atoms and m denotes a number from 1 to 6, and A and B are identical or different and denote hydrogen, lower alkyl or halogen, or a pharmaceutically acceptable salt thereof. Preferably the compounds are selected from the group consisting of: 2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentylacetic acid, 2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclohexylacetic acid, and 2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cycloheptylacetic acid, (+)-enantiomer of 2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentylacetic acid, (−)-enantiomer of 2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentylacetic acid and pharmaceutically acceptable salts thereof. See U.S. Pat. No. 4,970,215, incorporated herein by reference.  
      The compound can be represented by the formula:  
                 
 
      wherein R, R, R, R and R are independently hydrogen, halogen, lower alkyl, lower alkenyl, lower alkynyl, —CF3, —CN, —NO2, —N3, —C(OH)RR, —CO2R, —SR,  
      —S(O)R, —S(O)2R, —S(O)2NRR, —OR, —NRR, —C(O)R or —(CH2)tR; R is hydrogen,  
      —CH3, —CF3, —C(O)H, X—R or X—R; R and R are independently: alkyl, —(CH2)uPh(R)2 or —(CH2)uTh(R)2; R is —CF3 or R; R is hydrogen or X—R; each R is independently hydrogen or lower alkyl, or two R&#39;s on same carbon atom are joined to form a cycloalkyl ring of 3 to 6 carbon atoms; R is hydrogen, lower alkyl or —CH2R; R is lower alkyl or —(CH2)rR; R is —CF3 or R; R is hydrogen, —C(O)R, R, or two R&#39;s on the same nitrogen may be joined to form a monocyclic heterocyclic ring of 4 to 6 atoms containing up to 2 heteroatoms chosen from O, S or N; R is hydrogen, —CF3, lower alkyl, lower alkenyl, lower alkynyl or —(CH2)rR; R is —(CH2)s-C(RR)—(CH2)s-R or —CH2C(O)NRR; R is hydrogen or lower alkyl; R is a) a monocyclic or bicyclic heterocyclic ring containing from 3 to 9 nuclear carbon atoms and 1 or 2 nuclear hetero-atoms selected from N, S or O and with each ring in the heterocyclic radical being formed of 5 or 6 atoms, or b) the radical W—R; R is alkyl or C(O)R; R is phenyl substituted with 1 or 2 R groups; R is hydrogen, halogen, lower alxyl, lower alkoxy, lower alkylthio, lower alkylsulfonyl, lower alkylcarbonyl, —CF3, —CN,  
      —NO2 or —N3; R is alkyl, cycloalkyl, monocyclic monoheterocyclic ring;  
      R is the residual structure of a standard amino acid, or R and R attached to the same N can cyclize to form a proline residue; m is 0 to 1; n is 0 to 3; p is 1 to 3 when m is 1; p is 0 to 3 when m is 0; r is 0 to 2; s is 0 to 3; t is 0 to 2; u is 0 to 3; v is 0 or 1;  
      W is 0, S or NR; X is 0, or NR; X is C(O), CRR, S, S(O) or S(O)2; X is C(O), CRR, S(O)2 or a bond; Y is X or X; Q is —CO2R, —C(O)NHS(O)2R, —NHS(O)2R,  
      —S(O)2NHR —C(O)NRR, —CO2R, —C(O)NRR, —CH2OH, or 1H— or 2H-tetrazol-5-yl; and the pharmaceutically acceptable salts thereof. Preferred embodiments of the compounds are selected from the following and pharmaceutically acceptable salts thereof:  
      3-[N-(p-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid; 3-[N-(p-chlorobenzyl)-3-methyl-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid; 3-[N-(p-t-butylthiobenzyl)-3-(t-butylthio)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid; 3-[N-(p-chlorobenzyl)-3-(phenylthio)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid; 3-[N-(p-chlorobenzyl)-3-(phenylsulfonyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethyl propanoic acid, N-oxide; 3-[N-(p-chlorobenzyl)-3-(phenylsulfonyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid; 3-[N-(p-chlorobenzyl)-3-(phenylsulfinyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid; 3-[N-(p-chlorobenzyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid; 3-[N-(p-chlorobenzyl)-3-benzoyl-5-(quinolin2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid; 3-[N-(p-chlorobenzyl)-3-benzyl-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid; 3-[N-(p-chlorobenzyl)-3-(3,3-dimethyl-1-oxo-1-butyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid; 2-[N-(p-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-ylmethoxy)indol-2-yl]ethoxyethanoic acid; 3-[N-(p-chlorobenzyl)-3-(3,3-dimethyl-1-butyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid; 3-[N-(p-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2-dimethylpropanoic acid; 3-[N-(p-chlorobenzyl)-3-methyl-5-(6,7-dichloroquinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid; 3-[N-(p-chlorobenzyl)-3-methyl-5-(7-chloroquinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid; 3-[N-(p-chlorobenzyl)-4-allyl-5-(quinolin-2-ylmethoxy)-3-(t-butylthio)indol-2-yl]-2,2-dimethylpropanoic acid; 3-[N-(p-chlorobenzyl)-4-allyl-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid; 3-[N-(p-chlorobenzyl)-6-(quinolin-2-ylmethoxy)-3-(t-butylthio)indol-2-yl]-2,2-dimethylpropanoic acid; 3-[N-(p-chlorobenzyl)-4-(quinolin-2-ylmethoxy)-3-(t-butylthio)indol-2-yl]-2,2-dimethylpropanoic acid; 3-[N-(p-chlorobenzyl)-7-(quinolin-2-ylmethoxy)-3-(t-butylthio)indol-2-yl]-2,2-dimethylpropanoic acid; 2-[2-[N-(p-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-ylmethoxy)indol-2-yl]ethoxy]propanoic acid; 3-[N-(p-chlorobenzyl)-4-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid; 3-[N-methyl-3-(p-chlorobenzoyl)-6-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-methyl-3-(p-chlorobenzyl)-6-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-i-propoxy-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)indol-2-yl]-2-ethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-trifluoroacetyl-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-(3,3-dimethyl-1-oxo-1-butyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2-methylpropanoic acid, 3-[3-(3,3-dimethyl-1-oxo-1-butyl-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-triflouromethylbenzyl)-3-(3,3-dimethyl-1-oxo-1-butyl)-5-(quinolin-2-yl-methoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-benzyl-3-(3,3-dimethyl-1-oxo-1-butyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(3-methoxybenzyl)-3-(3,3-dimethyl-1-oxo-1-butyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-allyl-3-(3,3-dimethyl-1-oxo-1-butyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-methoxybenzyl)-3-(3,3-dimethyl-1-oxo-1-butyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-methyl-3-(3,3-dimethyl-1-oxo-3-butyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[3-(4-chlorobenzyl)-6-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid. 3-[N-(phenylsulfonyl)-3-(4-chlorobenzyl)-6-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-benzyl-3-(4-chlorobenzyl)-6-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-(t-butylsulfonyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-(t-butylsulfinyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-allyl-3-(4-chlorobenzyl)-6-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(n-propyl)-3-(4-chlorobenzyl)-6-(quinoline-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-ethyl-3-(4-chlorobenzyl)-6-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-(4-t-butylbenzoyl)-5-(quinolin-2-yl-methoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-(4-chlorobenzoyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-(1,1-dimethylethyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-acetyl-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid 3-[N-(4-chlorobenzyl)-3-cyclopropanecarbonyl-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-(3-cyclopentylpropanoyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-(3-methylbutanoyl)-5-(quinolin-2-yl-methoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-propanoyl-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-(2-methylpropanoyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-trimethylacetyl-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-phenylacetyl-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-fluorobenzyl)-3-(3,3-dimethyl-1-oxo-1-butyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-bromobenzyl)-3-(3,3-dimethyl-1-oxo-1-butyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-iodobenzyl)-3-(3,3-dimethyl-1-oxo-1-butyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-(1,1-dimethylbutyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-(1,1-dimethylpropyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(3-fluorobenzyl)-3-(1,1-dimethylethyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-(3-methylethyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-cyclopropyl-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-(1-methyl-1-cyclopropyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-cyclopentyl-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-cyclohexyl-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-(alpha, alpha-dimethylbenzyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-(2-{4-chloro-alpha, alpha-dimethylbenzyl}-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-(1-adamantyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-((1-adamantyl)methyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(1,1-dimethylethyl)-3-(4-chlorobenzyl)-6-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(1,1-dimethylpropyl)-3-(4-chlorobenzyl)-6-(quinoline-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid, 3-[N-(4-chlorobenzyl)-3-(3,3-dimethyl-1-oxo-1-butyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-diethylpropanoic acid, methyl 3-[N-(4-chlorobenzyl)-3,6-bis(acetyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2 dimethyl propanoate or methyl 3-[N-(4-chlorobenzyl)-3,6-bis(cyclopropanecarbonyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethyl propanoate. See EP 419049 B1, incorporated herein by reference.  
      The term “alkyl” refers to a monovalent group derived from a straight or branched chain saturated hydrocarbon by the removal of a single hydrogen atom. Alkyl groups are exemplified by methyl, ethyl, n- and iso-propyl, n-, sec-, iso- and tert-butyl, and the like. The term “hydroxyalkyl” represents an alkyl group, as defined above, substituted by one to three hydroxyl groups with the proviso that no more than one hydroxy group may be attached to a single carbon atom of the alkyl group. The term “alkylamino” refers to a group having the structure —NHR′ wherein R′ is alkyl, as previously defined, examples of alkylamino include methylamino, ethylamino, iso-propylamino and the like. The term “alkylaminocarbonyl” refers to an alkylamino group, as previously defined, attached to the parent molecular moiety through a carbonyl group. Examples of alkylaminocarbonyl include methylamino-carbonyl, ethylaminocarbonyl, iso-propylaminocarbonyl and the like. The term “alkylthio” refers to an alkyl group, as defined above, attached to the parent molecular moiety through a sulfur atom and includes such examples as methylthio, ethylthio, propylthio, n-, sec- and tert-butylthio and the like. The term “alkanoyl” represents an alkyl group, as defined above, attached to the parent molecular moiety through a carbonyl group. Alkanoyl groups are exemplified by formyl, acetyl, propionyl, butanoyl and the like. The term “alkanoylamino” refers to an alkanoyl group, as previously defined, attached to the parent molecular moiety through a nitrogen atom. Examples of alkanoylamino include formamido, acetamido, and the like. The term “N-alkanoyl-N-alkylamino” refers to an alkanoyl group, as previously defined, attached to the parent molecular moiety through an aminoalkyl group. Examples of N-alkanoyl-N-alkylamino include N-methylformamido, N-methyl-acetamido, and the like. The terms “alkoxy” or “alkoxyl” denote an alkyl group, as defined above, attached to the parent molecular moiety through an oxygen atom. Representative alkoxy groups include methoxyl, ethoxyl, propoxyl, butoxyl, and the like. The term “alkoxyalkoxyl” refers to an alkyl group, as defined above, attached through an oxygen to an alkyl group, as defined above, attached in turn through an oxygen to the parent molecular moiety. Examples of alkoxyalkoxyl include methoxymethoxyl, methoxyethyoxyl, ethoxyethoxyl and the like. The term “alkoxyalkyl” refers to an alkoxy group, as defined above, attached through an alkylene group to the parent molecular moiety. The term “alkoxycarbonyl” represents an ester group; i.e., an alkoxy group, attached to the parent molecular moiety through a carbonyl group such as methoxycarbonyl, ethoxycarbonyl, and the like. The term “alkenyl” denotes a monovalent group derived from a hydrocarbon containing at least one carbon-carbon double bond by the removal of a single hydrogen atom. Alkenyl groups include, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl and the like. The term “alkylene” denotes a divalent group derived from a straight or branched chain saturated hydrocarbon by the removal of two hydrogen atoms, for example methylene, 1,2-ethylene, 1,1-ethylene, 1,3-propylene, 2,2-dimethylpropylene, and the like. The term “alkenylene” denotes a divalent group derived from a straight or branched chain hydrocarbon containing at least one carbon-carbon double bond. Examples of alkenylene include —CH═CH—, —CH 2  CH═CH—, —C(CH 3 )═CH—, —CH 2  CH═CHCH 2 —, and the like. The term “cycloalkylene” refers to a divalent group derived from a saturated carbocyclic hydrocarbon by the removal of two hydrogen atoms, for example cyclopentylene, cyclohexylene, and the like. The term “cycloalkyl” denotes a monovalent group derived from a monocyclic or bicyclic saturated carbocyclic ring compound by the removal of a single hydrogen atom. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptanyl, and bicyclo[2.2.2]octanyl. The term “alkynylene” refers to a divalent group derived by the removal of two hydrogen atoms from a straight or branched chain acyclic hydrocarbon group containing a carbon-carbon triple bond. Examples of alkynylene include —CH≡CH—, —CH≡CH—CH 2 —, —CH≡CH—CH(CH 3 )—, and the like. The term “carbocyclic aryl” denotes a monovalent carbocyclic ring group derived by the removal of a single hydrogen atom from a monocyclic or bicyclic fused or non-fused ring system obeying the “4n+2 p electron” or Huckel aromaticity rule. Examples of carbocyclic aryl groups include phenyl, 1- and 2-naphthyl, biphenylyl, fluorenyl, and the like. The term “(carbocyclic aryl)alkyl” refers to a carbocyclic aryl ring group as defined above, attached to the parent molecular moiety through an alkylene group. Representative (carbocyclic aryl)alkyl groups include phenylmethyl, phenylethyl, phenylpropyl, 1-naphthylmethyl, and the like. The term “carbocyclicarylalkoxy” refers to a carbocyclicaryl alkyl group, as defined above, attached to the parent molecular moiety through an oxygen atom. The term “carbocyclic aryloxyalkyl” refers to a carbocyclic aryl group, as defined above, attached to the parent molecular moiety through an oxygen atom and thence through an alkylene group. Such groups are exemplified by phenoxymethyl, 1- and 2-naphthyloxymethyl, phenoxyethyl and the like. The term “(carbocyclic aryl)alkoxyalkyl” denotes a carbocyclic aryl group as defined above, attached to the parent molecular moiety through an alkoxyalkyl group. Representative (carbocyclic aryl)alkoxyalkyl groups include phenylmethoxymethyl, phenylethoxymethyl, 1- and 2-naphthylmethoxyethyl, and the like. “Carbocyclic arylthioalkyl” represents a carbocyclic aryl group as defined above, attached to the parent molecular moeity through a sulfur atom and thence through an alklyene group and are typified by phenylthiomethyl, 1- and 2-naphthylthioethyl and the like. The term “dialkylamino” refers to a group having the structure —NR′R″ wherein R′ and R″ are independently selected from alkyl, as previously defined. Additionally, R′ and R″ taken together may optionally be —(CH 2 ) kk — where kk is an integer of from 2 to 6. Examples of dialkylamino include, dimethylamino, diethylaminocarbonyl, methylethylamino, piperidino, and the like. The term “halo or halogen” denotes fluorine, chlorine, bromine or iodine. The term “haloalkyl” denotes an alkyl group, as defined above, having one, two, or three halogen atoms attached thereto and is exemplified by such groups as chloromethyl, bromoethyl, trifluoromethyl, and the like. The term “hydroxyalkyl” represents an alkyl group, as defined above, substituted by one to three hydroxyl groups with the proviso that no more than one hydroxy group may be attached to a single carbon atom of the alkyl group. The term “phenoxy” refers to a phenyl group attached to the parent molecular moiety through an oxygen atom. The term “phenylthio” refers to a phenyl group attached to the parent molecular moiety through a sulfur atom. The term “pyridyloxy” refers to a pyridyl group attached to the parent molecular moiety through an oxygen atom. The terms “heteroaryl” or “heterocyclic aryl” as used herein refers to substituted or unsubstituted 5- or 6-membered ring aromatic groups containing one oxygen atom, one, two, three, or four nitrogen atoms, one nitrogen and one sulfur atom, or one nitrogen and one oxygen atom. The term heteroaryl also includes bi-or tricyclic groups in which the aromatic heterocyclic ring is fused to one or two benzene rings. Representative heteroaryl groups are pyridyl, thienyl, indolyl, pyrazinyl, isoquinolyl, pyrrolyl, pyrimidyl, benzothienyl, furyl, benzo[b]furyl, imidazolyl, thiazolyl, carbazolyl, and the like. The term “heteroarylalkyl” denotes a heteroaryl group, as defined above, attached to the parent molecular moiety through an alkylene group. The term “heteroaryloxy” denotes a heteroaryl group, as defined above, attached to the parent molecular moiety through an oxygen atom. The term “heteroarylalkoxy” denotes a heteroarylalkyl group, as defined above, attached to the parent molecular moiety through an oxygen atom.  
     Method of Reducing Risk Factors For Cardiovascular Disease  
      The present invention encompasses compositions and methods for reducing risk factors for MI, ACS, stroke, and/or PAOD. The method of reducing risk factors comprise administering a composition comprising a leukotriene synthesis inhibitor, described in detail herein alone, or in combination with a statin, to an individuals at risk for any of these conditions. Individuals at risk include the target population described herein, especially individuals with elevated CRP, and those at risk for other diseases and conditions associated with FLAP and/or other members of the leukotriene pathway. In particular, the invention encompasses methods of reducing plasma CRP levels or plasma serum amyloid A levels comprising administering an effective amount of leukotriene inhibitor alone or in combination with a statin.  
      Statins are competitive inhibitors of 3-hydroxy-3-methylglutarlcoenzyme A (HMG-CoA) reductase, the enzyme that converts HMG-CoA to the cholesterol precursor mevalonic acid. Upon binding to the active site of HMG-CoA reducatase, statins alter the conformation of the enzyme, thereby preventing it from attaining a functional structure. The conformational change of the HMG-CoA reducatase active site makes statin drugs very effective and specific. Inhibition of HMG-CoA reducatase reduces intracellular cholesterol synthesis in hepatocytes. The reduction of intracellular cholesterol results in an increase in hepatic LDL receptors on the cell surface, which in turn reduces the level of circulating LDL and its precursors, intermediate density lipoproteins (IDL) and very low density lipoproteins (VLDL). In addition, statins inhibit hepatic synthesis of apolipoprotein B-100, which results in a decrease in the synthesis and secretion of triglyercide rich lipoproteins. Additional beneficial effects of statins on lipid biosynthesis include inhibition of LDL oxidation, and inhibition of the expression of scavenger receptors. Statins also reduce the accumulation of esterified cholersterol into macrophages, increase endothelial cell nitric oxide synthesis, reduce inflammatory processes, increase the stability of artherosclerotic plaques, and restore platelet activity and the coagulation process.  
      Because of their beneficial effects and high specificity, statins have become some of the most prescribed medicines in the Western industrialized world. In preferred embodiments of the invention, the statin is one of the following agents: rovuvastatin, fluvastatin, atorvastatin, lovastatin, simvastatin, pravastatin or pitavastatin. These agents are described in detail in the Statin Agent Table III below.  
                                                                                       (Infor from           Approved                       PDR)           St                       Dosages       Resigeeter   Marketed                       (tablet       to   as   Stain Name   Chemical   Structure   U.S. Pat. No.   Patent Expiration   sizes)                                                      Astra- Zeneca   CREST- OR   ROVU- VASTATIN   bis(E)-7-[4-(4-fluoro- phenyl)-6-isopropyl-2-[meth- yl(methylsulfonyl)amino]py- rimidin-5-yl](3R,5S)-3,5-di- hydroxyhept-6-enoic acid]calcium salt                         6316460 6589959 RE37314   AUG. 04, 2020 DEC. 23, 2019 JUN. 12, 2012   5 mg,        #10 mg, 20 mg and 40 mg               Novartis   LESCOL   FLUVASTATIN   [R*,S*-(E)]-(±)-7-[3-(4-fluoro- phenyl)-1-(1-meth- ylethyl)-1H-indol-2-yl]-3,5-di- hydroxy-6-heptenoic acid, monosodium salt                         5354772 5354772 5356896   OCT. 11, 2011 OCT. 22, 2011 DEC. 12, 2011   EQ 20 mg and EQ 40 mg               Pfizer   LIPITOR   ATOR- VASTATIN   [R-(R*,R*)]-2-(4-fluorophenyl)-(beta), [dgr]-dihy- droxy-5-(1-methylethyl)-3-phe- nyl-4-[(phenylamino)carbonyl]-1H-pyr- role-1-heptanoic acid, calcium salt (2:1)trihydrate                         4681893 4681893*PED 5273995 5273995*PED 5686104 5686104*PED        #5969156 5969156*PED 6126971 6126971*PED   SEP. 24, 2009 MAR. 24, 2010 DEC. 28, 2010 JUN 28, 2011 NOV. 11, 2014 MAY 11, 2015 JUL. 08, 2016 JAN. 08, 2017 JAN. 19, 2013 JUL. 19, 2013   EQ 10 mg, EQ 20 mg, EQ 40 mg and EQ 40 mg                                                                         MERCK   MEVA- COR   LOVASTATIN   [1,S-[1(alpha)(R*),3(al- pha),7(beta),8(beta)(2S*,4S*),8a(bet- a)]]-1,2,3,7,8,8a-hexa- hydro-3,7-di- methyl-8-[2-(tetra- hydro-4-hydroxy-6-ox- o-2H-pyran-2-yl)ethyl]-1-naph- thalenyl2-methylbutanoate                         There are no unexpired patents for this product in the Orange Book        #Database.   10 mg, 20 mg and 40 mg                                                                                 MERCK   ZOCOR   SIMVASTATIN   butanoic acid, 2,2-dimethyl-,1,2,3,7,8,8a-hexa- hydro-3,7-di- methyl-8-[2-(tetrahydro-4-hy- droxy-6-oxo-2H-py- ran-2-yl)-ethyl]-1-naph- thalenyl ester,[1S-[1(al- pha),3(alpha),7(bet- a),8(beta)(2S*,4S*),-8a(beta)]].                         4444784 4444784*PED   DEC. 23, 2005 JUN. 23, 2006   5 mg, 10 mg, 20 mg,        #40 mg and 80 mg               BMS   PRAVA- COL   PRAVASTATIN   1-Naphthalene-heptanoic acid, 1,2,6,7,8,8a-hexa- hydro-(beta),(delta),6-tri- hydroxy-2-methyl-8-(2-methyl-1-oxo- butoxy)-,monosodium salt,[1S-[1(al- pha)((beta)S*,(delta)S*),2(alpha),6(al- pha),8(beta)(R*),8a(alpha)]]-.                         4346227 4346227*PED 5030447 5030447*PED        #5180589 5180589*PED 5622985 5622985*PED   OCT. 20, 2005 APR. 20, 2006 JUL. 09, 2008 JAN. 09, 2009 JUL. 09, 2008 JAN. 09, 2009 APR. 22, 2014 OCT. 22, 2014   10 mg, 20 mg, 40 mg and 80 mg                   Livalo   Pitavastatin                                                           Astra- Zeneca   CREST- OR   ROVU- VASTATIN   bis[(E)-7-[4-(4-fluoro- phenyl)-6-isopropyl 2-[methyl)(methylsulfonyl)amino]py- rimidin-5-yl](3R,5S)-di- hydroxyhept-6-enoic acid]calcium salt                         6316460 6589959 RE37314   AUG. 04, 2020 DEC. 23, 2019 JUN. 12, 2012   5 mg, 10        #mg, 20 mg and 40 mg               Novartis   LESCOL   FLUVASTATIN   [R*,S*-(E)]-(±)-7-[3-(4-fluoro- phenyl)-1-(1-meth- ylethyl)-1H-indol-2-yl]-3,5-di- hydroxy-6-heptenoic acid, monosodium salt                         5354772 5354772 5356896   OCT. 11, 2011 OCT. 11, 2011 DEC. 12, 2011   EQ 20 mg and EQ 40 mg               Pfizer   LIPITOR   ATOR- VASTATIN   [R-(R*,R*)]-2-(4-fluorophenyl)-(beta), [dgr]-dihy- droxy-5-(1-methylethyl)-3-phe- nyl-4-[(phenylamino)carbonyl]-1H-pyr- role-1-heptanoic acid, calcium salt (2:1)trihydrate                         4861893 4861893*PED 5273995 5273995*PED 5686104 5686104*PED        #5969156 5969156*PED 6126971 6126971*PED   SEP. 24, 2009 MAR. 24, 2010 DEC. 28, 2010 JUN. 28, 2011 NOV. 11, 2014 MAY 11, 2015 JUL. 08, 2016 JAN. 08, 2017 JAN. 19, 2013 JUL. 19, 2013   EQ 10 mg, EQ 20 mg, EQ 40 mg and EQ 40 mg                                                                         MERCK   MEVA- COR   LOVASTATIN   [1S-[1(alpha)(R*),3(al- pha),7(beta),8(beta)(2S*,4S*),8a(bet- a)]]-1,2,3,7,8,8a-hexahydro-3,7-di- methyl-8-[2-(tetra- hydro-4-hydroxy-6-ox- o-2H-pyran-2-yl)ethyl]-1-naph- thalenyl2-methylbutanoate                         There are no unexpired patents for this product in the Orange Book        #Database.   10 mg, 20 mg and 40 mg                                                                                 MERCK   ZOCOR   SIMVASTATIN   butanoic acid,2,2-di- methyl-,1,2,3,7,8,8a-hexa- hydro-3,7-dimeth- yl-8-[2-(tetrahydro-4-hy- droxy-6-oxo-2H-py- ran-2-yl)-ethyl]-1-naph- thalenyl ester,[1S-[1(al- pha),3(alpha),7(bet- a),8(beta)(2S*,4S*),-8a(beta)]].                         4444784 4444784*PED   DEC. 23, 2005 JUN. 23, 2006   5 mg, 10 mg, 20 mg,        #40 mg and 80 mg               BMS   PRAVA- COL   PRAVASTATIN   1-Naphthelene-heptanoic acid, 1,2,6,7,8,8a-hexa- hydro-(beta),(delta),6-tri- hydroxy-2-methyl-8-(2-methyl-1-oxo- butoxy)-,monosodium salt,[1S-[1(al- pha)(beta)S*,(delta)S*),2(alpha),6(al- pha),8(beta)(R*),8a(alpha)]]-.                         4346227 4346227*PED 5030447 5030447*PED        #5180589 5180589*PED 5622985 5622985*PED   OCT. 20, 2005 APR. 20, 2006 JUL. 09, 2008 JAN. 09, 2009 JUL. 09, 2008 JAN. 09, 2009 APR. 22, 2014 OCT. 22, 2014   10 mg, 20 mg, 40 mg and 80 mg                   Livalo   Pitavastatin                                                              
 
      Mevastatin and related compounds are disclosed in U. S. Pat. No. 3,983,140. Lovastatin (mevinolin) and related compounds are disclosed in U.S. Pat. No. 4,231,938. Keto analogs of mevinolin (lovastatin) are disclosed in European Patent Application No. 0,142,146 A2, and quinoline and pyridine derivatives are disclosed in U.S. Pat. Nos. 5,506,219 and 5,691,322.  
      Pravastatin and related compounds are disclosed in U.S. Pat. No. 4,346,227. Simvastatin and related compounds are disclosed in U.S. Pat. Nos. 4,448,784 and 4,450,171.  
      Fluvastatin and related compounds are disclosed in U.S. Pat. No. 5,354,772. Cerivastatin and related compounds are disclosed in U.S. Pat. Nos. 5,006,530 and 5,177,080. Atorvastatin and related compounds are disclosed in U.S. Pat. Nos. 4,681,893; 5,273,995; 5,385,929 and 5,686,104.  
      Pitavastatin (nisvastatin (NK-104) or itavastatin) and related compounds are disclosed in U.S. Pat. No. 5,011,930. Rosuvastatin (visastatin (ZD-4522)) and related compounds are disclosed in U.S. Pat. No. 5,260,440.  
      Other possible HMG CoA reductase molecules are described in U.S. Pat. Nos. 5,753,675; 4,613,610; 4,686,237; 4,647,576; and 4,499,289; and British patent no. GB 2205837.  
      The patents cited in relation to statins or other agents identified herein describe how to make and use the statins/agents, as well as biochemically active homologs thereof, salts, pro-drugs, metabolites, and the like. Such patents are incorporated herein by reference in their entirety. Dosings for the statins also have been described in patent and trade literature (e.g., Physician&#39;s Desk Reference 2004, incorporated herein by reference) and by the manufacturers and clinical practitioners that prescribe them. Combination therapy using statin dosings similar to what is used when prescribing statins alone, or less, is specifically contemplated.  
      Compositions comprising a leukotriene synthesis inhibitor alone or in combination with a statin may comprises a leukotriene synthesis inhibitor in an amount effective to reduce a risk factor such as CRP or serum amyloid A. Effective daily doses of the leukotriene synthesis inhibitors are between 0.01 mg and 100 g, more preferably 0.1 mg to 1 g, and all individual doses within these ranges are specifically contemplated. Exemplary single adult doses include 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg and 750 mg, from one to four times daily. The compositions may comprise a statin in an amount effective to reduce total serum cholesterol, serum LDL, and/or serum CRP. Effective daily doses are between 0.01 mg and 100 g, more preferably 0.1 mg to 1 g, and all individual doses within these ranges are specifically contemplated. Exemplary individual doses include 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg 50 mg, 60 mg, and 80 mg, 100 mg, 150 mg, 200 mg, 250 mg, and 500 mg, from one to four times daily.  
      Emerging evidence suggests that elevated CRP is an independent risk factor for adverse clinical outcomes. See, e.g., Ridker et al., N. Engl. J. Med. 352: 1 (Jan. 6, 2005). In another variation, the invention provides compositions, unit doses, and methods of treatment where a leukotriene synthesis inhibitor and a statin are included or administered in amounts that synergistically act to reduce serum CRP levels. Synergistically effective amounts are amounts that either (a) achieve a greater percentage reduction in CRP than is achieved in an average patient using either type of agent alone, at a safe and effective amount, or (b) reduces CRP a comparable amount to single agent therapy, with fewer side effects; or (c) reduces CRP a comparable amount to single agent therapy, and also reduces at least one other cardiovascular-risk factor more effectively than single agent therapy alone.  
      In one variation, the invention provides a composition comprising a leukotriene synthesis inhibitor and a statin for simultaneous administration, e.g., in one dose. A composition in tablet, pill, or capsule form, including sustained release formulations, are specifically contemplated. In another variation, a unit dose comprising a single dose of the leukotriene synthesis inhibitor and a single dose of the statin, packaged together but not in admixture, is contemplated. In another variation, methods of the invention involve administering a composition comprising a leukotriene inhibitor and a composition comprising a statin at the same or different times, e.g., administering the leukotriene synthesis inhibitor before or after administration of a composition comprising a statin. Compositions for and methods of administering the agents to an individual continuously (e.g., through a patch or i.v.), one to twelve times a day, once a day, every other day, twice a week, weekly, or monthly for one or more weeks, months, or years, or for the entire life of a patient, depending on the level of risk for the individual, is specifically contemplated, to manage serum CRP and other cardiovascular risk factor levels. It is contemplated that these compositions will be used for treatment and lifestyle management plans for primary or secondary MI, ACS, stroke, or PAOD prevention.  
     Nucleic Acid Therapeutic Agents  
      In another embodiment, a nucleic acid of the invention; a nucleic acid complementary to a nucleic acid of the invention; or a portion of such a nucleic acid (e.g., an oligonucleotide as described below); or a nucleic acid encoding a member of the leukotriene pathway (e.g., 5-LO), can be used in “antisense” therapy, in which a nucleic acid (e.g., an oligonucleotide) which specifically hybridizes to the mRNA and/or genomic DNA of a nucleic acid is administered or generated in situ. The antisense nucleic acid that specifically hybridizes to the mRNA and/or DNA inhibits expression of the polypeptide encoded by that mRNA and/or DNA, e.g., by inhibiting translation and/or transcription. Binding of the antisense nucleic acid can be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interaction in the major groove of the double helix.  
      An antisense construct can be delivered, for example, as an expression plasmid as described above. When the plasmid is transcribed in the cell, it produces RNA that is complementary to a portion of the mRNA and/or DNA that encodes the polypeptide for the member of the leukotriene pathway (e.g., FLAP or 5-LO). Alternatively, the antisense construct can be an oligonucleotide probe that is generated ex vivo and introduced into cells; it then inhibits expression by hybridizing with the mRNA and/or genomic DNA of the polypeptide. In one embodiment, the oligonucleotide probes are modified oligonucleotides that are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, thereby rendering them stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996, 5,264,564 and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy are also described, for example, by Van der Krol et al. ( Biotechniques  6:958-976 (1988)); and Stein et al ( Cancer Res.  48:2659-2668 (1988)). With respect to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site are preferred.  
      To perform antisense therapy, oligonucleotides (mRNA, cDNA or DNA) are designed that are complementary to mRNA encoding the polypeptide. The antisense oligonucleotides bind to mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required. A sequence “complementary” to a portion of an RNA, as referred to herein, indicates that a sequence has sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid, as described in detail above. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures.  
      The oligonucleotides used in antisense therapy can be DNA, RNA, or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotides can include other appended groups such as peptides (e.g. for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al.,  Proc. Natl. Acad. Sci. USA  86:6553-6556 (1989); Lemaitre et al.,  Proc. Natl. Acad. Sci. USA  84:648-652 (1987); PCT International Publication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCT International Publication No. WO 89/10134), or hybridization-triggered cleavage agents (see, e.g., Krol et al.,  BioTechniques  6:958-976 (1988)) or intercalating agents. (See, e.g., Zon, Pharm. Res. 5: 539-549 (1988)). To this end, the oligonucleotide may be conjugated to another molecule (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent).  
      The antisense molecules are delivered to cells that express the member of the leukotriene pathway in vivo. A number of methods can be used for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systematically. Alternatively, in a preferred embodiment, a recombinant DNA construct is utilized in which the antisense oligonucleotide is placed under the control of a strong promoter (e.g., pol III or pol II). The use of such a construct to transfect target cells in the patient results in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous transcripts and thereby prevent translation of the mRNA. For example, a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art and described above. For example, a plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct that can be introduced directly into the tissue site. Alternatively, viral vectors can be used which selectively infect the desired tissue, in which case administration may be accomplished by another route (e.g., systemically).  
      In another embodiment of the invention, small double-stranded interfering RNA (RNA interference (RNAi)) can be used. RNAi is a post-transcription process, in which double-stranded RNA is introduced, and sequence-specific gene silencing results, though catalytic degradation of the targeted mRNA. See, e.g., Elbashir, S. M. et al.,  Nature  411:494-498 (2001); Lee, N. S.,  Nature Biotech.  19:500-505 (2002); Lee, S-K. et al.,  Nature Medicine  8(7):681-686 (2002); the entire teachings of these references are incorporated herein by reference. RNAi is used routinely to investigate gene function in a high throughput fashion or to modulate gene expression in human diseases (Chi et al.,  PNAS,  100 (11):6343-6346 (2003)). Introduction of long double standed RNA leads to sequence-specific degradation of homologous gene transcripts. The long double stranded RNA is metabolized to small 21-23 nucleotide siRNA (small interfering RNA). The siRNA then binds to protein complex RISC (RNA-induced silencing complex) with dual function helicase. The helicase has RNA as activity and is able to unwind the RNA. The unwound si RNA allows an antisense strand to bind to a target. This results in sequence dependent degradation of cognate mRNA. Aside from endogenous RNAi, exogenous RNAi, chemically synthesized or recombinantly produced can also be used. Using non-intronic portions of the FLAP gene, such as corresponding mRNA portions of SEQ ID NO. 1, or portions of SEQ ID NO: 3, target regions of the FLAP gene that are accessible for RNAi are targeted and silenced. With this technique it is possible to conduct a RNAi gene walk of the nucleic acids of the FLAP gene and determine the amount of inhibition of the protein product. Thus it is possible to design gene-specific therapeutics by directly targeting the mRNAs of the gene.  
      Endogenous expression of a member of the leukotriene pathway (e.g., FLAP, 5-LO) can also be reduced by inactivating or “knocking out” the gene or its promoter using targeted homologous recombination (e.g., see Smithies et al.,  Nature  317:230-234 (1985); Thomas &amp; Capecchi,  Cell  51:503-512 (1987); Thompson et al.,  Cell  5:313-321 (1989)). For example, an altered, non-functional gene of a member of the leukotriene pathway (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous gene (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the gene in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the gene. The recombinant DNA constructs can be directly administered or targeted to the required site in vivo using appropriate vectors, as described above. Alternatively, expression of non-altered genes can be increased using a similar method: targeted homologous recombination can be used to insert a DNA construct comprising a non-altered functional gene, or the complement thereof, or a portion thereof, in place of a gene in the cell, as described above. In another embodiment, targeted homologous recombination can be used to insert a DNA construct comprising a nucleic acid that encodes a polypeptide variant that differs from that present in the cell.  
      Alternatively, endogenous expression of a member of the leukotriene pathway can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the member of the leukotriene pathway (i.e., the promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells in the body. (See generally, Helene, C.,  Anticancer Drug Des.,  6(6):569-84 (1991); Helene, C. et al.,  Ann. N. Y. Acad. Sci.  660:27-36 (1992); and Maher, L. J.,  Bioassays  14(12):807-15 (1992)). Likewise, the antisense constructs described herein, by antagonizing the normal biological activity of one of the members of the leukotriene pathway, can be used in the manipulation of tissue, e.g., tissue differentiation, both in vivo and for ex vivo tissue cultures. Furthermore, the anti-sense techniques (e.g., microinjection of antisense molecules, or transfection with plasmids whose transcripts are anti-sense with regard to a nucleic acid RNA or nucleic acid sequence) can be used to investigate the role of one or more members of the leukotriene pathway in the development of disease-related conditions. Such techniques can be utilized in cell culture, but can also be used in the creation of transgenic animals.  
      The therapeutic agents as described herein can be delivered in a composition, as described above, or by themselves. They can be administered systemically, or can be targeted to a particular tissue. The therapeutic agents can be produced by a variety of means, including chemical synthesis; recombinant production; in vivo production (e.g., a transgenic animal, such as U.S. Pat. No. 4,873,316 to Meade et al.), for example, and can be isolated using standard means such as those described herein. In addition, a combination of any of the above methods of treatment (e.g., administration of non-altered polypeptide in conjunction with antisense therapy targeting altered mRNA for a member of the leukotriene pathway; administration of a first splicing variant in conjunction with antisense therapy targeting a second splicing variant) can also be used.  
      The invention additionally pertains to use of such therapeutic agents, as described herein, for the manufacture of a medicament for the treatment of MI, ACS, stroke, PAOD and/or atherosclerosis, e.g., using the methods described herein.  
     Monitoring Progress of Treatment  
      The current invention also pertains to methods of monitoring the response of an individual, such as an individual in one of the target populations described above, to treatment with a leukotriene synthesis inhibitor.  
      Because the level of inflammatory markers can be elevated in individuals who are in the target populations described above, an assessment of the level of inflammatory markers of the individual both before, and during, treatment with the leukotriene synthesis inhibitor will indicate whether the treatment has successfully decreased production of leukotrienes in the arterial vessel wall or in bone-marrow derived inflammatory cells. For example, in one embodiment of the invention, an individual who is a member of a target population as described above (e.g., an individual at risk for MI, ACS, stroke or PAOD, such as an individual who is at-risk due to a FLAP haplotype) can be assessed for response to treatment with a leukotriene synthesis inhibitor, by examining leukotriene levels or leukotriene metabolite levels in the individual. Blood, serum, plasma or urinary leukotrienes (e.g., leukotriene E4, cysteinyl leukotriene 1), or ex vivo production of leukotrienes (e.g., in blood samples stimulated with a calcium ionophore to produce leukotrienes), or leukotriene metabolites, can be measured before, and during or after treatment with the leukotriene synthesis inhibitor. The leukotriene or leukotriene metabolite level before treatment is compared with the leukotriene or leukotriene metabolite level during or after treatment. The efficacy of treatment is indicated by a decrease in leukotriene production: a level of leukotriene or leukotriene metabolite during or after treatment that is significantly lower than the level of leukotriene or leukotriene metabolite before treatment, is indicative of efficacy. A level that is lower during or after treatment can be shown, for example, by decreased serum or urinary leukotrienes, or decreased ex vivo production of leukotrienes, or decreased leukotriene metabolites. A level that is “significantly lower”, as used herein, is a level that is less than the amount that is typically found in control individual(s), or is less in a comparison of disease risk in a population associated with the other bands of measurement (e.g., the mean or median, the highest quartile or the highest quintile) compared to lower bands of measurement (e.g., the mean or median, the other quartiles; the other quintiles).  
      For example, in one embodiment of the invention, the level of a leukotriene or leukotriene metabolite is assessed in an individual before treatment with a leukotriene synthesis inhibitor; and during or after treatment with the leukotriene synthesis inhibitor, and the levels are compared. A level of the leukotriene or leukotriene metabolite during or after treatment that is significantly lower than the level of the leukotriene or leukotriene metabolite before treatment, is indicative of efficacy of treatment with the leukotriene synthesis inhibitor. In another embodiment, production of a leukotriene or a leukotriene metabolite is stimulated in a first test sample from the individual, using a calcium ionophore, before treatment with a leukotriene synthesis inhibitor, and is also stimulated in a second test sample from the individual, using a calcium ionophore, during or after treatment with the leukotriene synthesis inhibitor, and the level of production in the first test sample is compared with with the level of production of the leukotriene or leukotriene metabolite in the second test sample. A level of the leukotriene or leukotriene metabolite in the second test sample that is significantly lower than the level of the leukotriene or leukotriene metabolite in the first test sample, is indicative of efficacy of treatment with the leukotriene synthesis inhibitor.  
      In another embodiment of the invention, an individual who is a member of a target population of individuals at risk for MI, ACS, stroke or PAOD (e.g., an individual in a target population described above, such as an individual at-risk due to elevated C-reactive protein) can be assessed for response to treatment with a leukotriene synthesis inhibitor, by examining levels of inflammatory markers in the individual. For example, levels of an inflammatory marker in an appropriate test sample (e.g., serum, plasma or urine) can be measured before, and during or after treatment with the leukotriene synthesis inhibitor. The level of the inflammatory marker before treatment is compared with the level of the inflammatory marker during or after treatment. The efficacy of treatment is indicated by a decrease in the level of the inflammatory marker, that is, a level of the inflammatory marker during or after treatment that is significantly lower (e.g., significantly lower), than the level of inflammatory marker before treatment, is indicative of efficacy. Representative inflammatory markers include: C-reactive protein (CRP), serum amyloid A, fibrinogen, a leukotriene (e.g., LTB4, LTC4, LTD4, LTE4), a leukotriene metabolite, interleukin-6, tissue necrosis factor-alpha, soluble vascular cell adhesion molecules (sVCAM), soluble intervascular adhesion molecules (sICAM), E-selectin, matrix metalloprotease type-1, matrix metalloprotease type-2, matrix metalloprotease type-3, matrix metalloprotease type-9, myeloperoxidase (MPO), and N-tyrosine. In a preferred embodiment, the marker is CRP or MPO.  
      The efficacy of treatment of a leukotriene synthesis inhibitor may be monitored by measuring at-risk biomarkers in plasma, serum or urine. Clinical assays are available for the following biomarkers: CRP, serum amyloid A, IL-1β, IL-6, IL-8, IL-10, TNF-α, E-selectin, P-selectin and intracellular adhesion molecule-1, vascular cell ashesion molecule-1. The relative risk of a cardiovascular event predicted by CRP levels is low risk has less thatn 1 mg/L, average is 1.0-3.0 mg/L and high risk patients have greater than 3.0 mg/L. Thus, optimal therapeutic effect of a leukotriene synthesis inhibitor alone or in combination with a statin is reducing CRP level to 2.0 mg/L or lower.  
      The efficacy of treatment of a statin is monitored by measuring the level of total serum cholesterol, serum LDL and/or serum triglycerides. A level of serum total cholesterol, LDL-C and/or triglycerides during or after treatment, which is significantly lower than the level of total cholesterol, LDL-C and/or triglycerides before treatment is indicative of the efficacy of the treatment. For cholesterol management purposes, “high risk patients” have an LDL level of 130 mg/Dl or higher and optimally the statin treatment will reduce the LDL level to less than 100 mg/dL. “Moderately-high risk patients” are those individuals with two or more risk factors for coronary heart disease with a 10-20% risk of heart attack within ten years. Optimally, the statin treatment will keep the LDL level under 129 mg/dL. More recent studies show an additional benefit on morbidity and mortality when statin therapy decreased serum LDL-C to less than 70 mg/dL. (Ridker et al.,  N. Engl. J. Med.  352(1): 20-28, 2005; Nissen et al.,  N. Engl. J. Med.  352(1): 29-38, 2005). Thus optimal therapeutic effect of a statin would be to lower LDL-C levels to under 70 mg/dL. as described by Ridker et al.,  N. Engl. J. Med.  352(1): 20-28, 2005 and Nissen et al.,  N. Engl. J. Med.  352(1): 29-38, 2005, statin therapy may reduce CRP. CRP is an additional parameter that may be monitored in connection with statin therapy.  
     Assessment of Increased Risk  
      The present invention additionally pertains to methods for assessing an individual (e.g., an individual who is in a target population as described herein, such as an individual who is at risk for MI, ACS, stroke or PAOD), for an increased risk of MI, ACS, atherosclerosis, stroke, transient ischemic attack, transient monocular blindness, asymptomatic carotid stenosis, PAOD, claudication, or limb ischemia. The methods comprise assessing the level of a leukotriene metabolite (e.g., LTE4, LTD4, LTB4) in the individual, wherein an increased level of leukotriene metabolite is indicative of an increased risk. The level can be measured in any appropriate tissue or fluid sample, such as blood, serum, plasma, or urine. In one particular embodiment, the sample comprises neutrophils. The level of the leukotriene metabolite can be measured by standard methods, such as the methods described herein. For example, in one embodiment, production of a leukotriene metabolite is stimulated in a first test sample from the individual, using a calcium ionophore. The level of production is compared with a control level. The control level is a level that is typically found in control individual(s), such as individual who are not at risk for MI, ACS, stroke or PAOD; alternatively, a control level is the level that is found by comparison of disease risk in a population associated with the lowest band of measurement (e.g., below the mean or median, the lowest quartile or the lowest quintile) compared to higher bands of measurement (e.g., above the mean or median, the second, third or fourth quartile; the second, third, fourth or fifth quintile). A level of production of the leukotriene metabolite that is significantly greater than the control level, is indicative of an increased risk. Individuals at increased risk are candidates for treatments described herein.  
     Pharmaceutical Compositions  
      The present invention also pertains to pharmaceutical compositions comprising agents described herein, for example, an agent that is a leukotriene synthesis inhibitor as described herein. For instance, a leukotriene synthesis inhibitor can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition. The carrier and composition can be sterile. The formulation should suit the mode of administration.  
      The invention also provides for compositions comprising a leukotriene synthesis inhibit, as set out in Agent Table I, and a statin, as set out in the Agent Table III. The leukotriene synthesis inhibitor and the statin may be coformulated with a physiological acceptable carrier or expedient to prepare a pharmaceutical composition. This composition may be formulation to deliver the leukotriene synthesis inhibitor and statin in a single dose. The processes for the isolation and purification of statins and other HMG-CoA reductase inhibitors include different combinations of extraction, chromatography, lactonization and crystallization methods. Examples of formulations for statins, statin derivatives and statin salts are found in the following, all incorporated by reference in their entirety, U.S. Pat. Nos. 6,316,460, 6,589,959, RE37,314, 5,354,772, 5,356,896, 5,686,104, 5,969,156, 6,126,971, 5,030,447, 5,180,589, 5,622,985, 6,825,015, 6,838,566, 5,403,860, 5,763,653, and 5,763,646, International Patent Publications WO 86/03488, WO 86/07054, French Patent No. 2596393, European Patent Application No. 0221025, British Patent Nos. 2055100A and 2073199A and European Patent No. 65,835.  
      Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof. The pharmaceutical preparations can, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active agents.  
      The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.  
      Methods of introduction of these compositions include, but are not limited to, intradermal, intramuscular, intraperitoneal, intraocular, intravenous, subcutaneous, topical, oral and intranasal. Other suitable methods of introduction can also include gene therapy (as described below), rechargeable or biodegradable devices, particle acceleration devices (“gene guns”) and slow release polymeric devices. The pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents.  
      The composition can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings. For example, compositions for intravenous administration typically are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.  
      For topical application, nonsprayable forms, viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water, can be employed. Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, enemas, lotions, sols, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc. The agent may be incorporated into a cosmetic formulation. For topical application, also suitable are sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., pressurized air.  
      Agents described herein can be formulated as neutral or salt forms.  
      Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.  
      The agents are administered in a therapeutically effective amount. The amount of agents which will be therapeutically effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the symptoms, and should be decided according to the judgment of a practitioner and each patient&#39;s circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.  
      The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use of sale for human administration. The pack or kit can be labeled with information regarding mode of administration, sequence of drug administration (e.g., separately, sequentially or concurrently), or the like. The pack or kit may also include means for reminding the patient to take the therapy. The pack or kit can be a single unit dosage of the combination therapy or it can be a plurality of unit dosages. In particular, the agents can be separated, mixed together in any combination, present in a single vial or tablet. For example, a pack or kit of the invention may contain a single dose for delivery of both a leukotriene synthesis inhibitor and a statin concurrently, or contain two or more doses wherein one dose is to deliver a leukotriene synthesis inhibitor and one dose is to deliver a statin either in parallel or one following the other.  
      Agents assembled in a blister pack or other dispensing means is preferred. For the purpose of this invention, unit dosage is intended to mean a dosage that is dependent on the individual pharmacodynamics of each agent and administered in FDA approved dosages in standard time courses.  
     Nucleic Acids of the Invention  
     FLAP Nucleic Acids, Portions and Variants  
      In addition, the invention pertains to isolated nucleic acid molecules comprising a human FLAP nucleic acid. The term, “FLAP nucleic acid,” as used herein, refers to an isolated nucleic acid molecule encoding FLAP polypeptide. The FLAP nucleic acid molecules of the present invention can be RNA, for example, mRNA, or DNA, such as cDNA and genomic DNA. DNA molecules can be double-stranded or single-stranded; single stranded RNA or DNA can be either the coding, or sense strand or the non-coding, or antisense strand. The nucleic acid molecule can include all or a portion of the coding sequence of the gene or nucleic acid and can further comprise additional non-coding sequences such as introns and non-coding 3′ and 5′ sequences (including regulatory sequences, for example, as well as promoters, transcription enhancement elements, splice donor/acceptor sites, etc.).  
      For example, a FLAP nucleic acid can consist of SEQ ID NOs: 1 or 3 or the complement thereof, or to a portion or fragment of such an isolated nucleic acid molecule (e.g., cDNA or the nucleic acid) that encodes FLAP polypeptide (e.g., a polypeptide such as SEQ ID NO: 2). In a preferred embodiment, the isolated nucleic acid molecule comprises a nucleic acid molecule selected from the group consisting of SEQ ID NOs: 1 or 3, or their complement thereof.  
     LTA4H Nucleic Acids, Portions and Variant  
      In addition, the invention pertains to isolated nucleic acid molecules comprising a human LTA4H nucleic acid. The term, “LTA4H nucleic acid,” as used herein, refers to an isolated nucleic acid molecule encoding LTA4H polypeptide. The LTA4H nucleic acid molecules of the present invention can be RNA, for example, mRNA, or DNA, such as cDNA and genomic DNA. DNA molecules can be double-stranded or single-stranded; single stranded RNA or DNA can be either the coding, or sense strand or the non-coding, or antisense strand. The nucleic acid molecule can include all or a portion of the coding sequence of the gene or nucleic acid and can further comprise additional non-coding sequences such as introns and non-coding 3′ and 5′ sequences (including regulatory sequences, for example, as well as promoters, transcription enhancement elements, splice donor/acceptor sites, etc.).  
      For example, an LTA4H nucleic acid can consist of SEQ ID NOs: 718 or 719 or the complement thereof, or to a portion or fragment of such an isolated nucleic acid molecule (e.g., cDNA or the nucleic acid) that encodes LTA4H polypeptide (e.g., a polypeptide such as SEQ ID NO: 720). In a preferred embodiment, the isolated nucleic acid molecule comprises a nucleic acid molecule selected from the group consisting of SEQ ID NOs: 718 or 719, or their complement thereof.  
      Additionally, the nucleic acid molecules of the invention can be fused to a marker sequence, for example, a sequence that encodes a polypeptide to assist in isolation or purification of the polypeptide. Such sequences include, but are not limited to, those that encode a glutathione-S-transferase (GST) fusion protein and those that encode a hemagglutinin A (HA) polypeptide marker from influenza.  
      An “isolated” nucleic acid molecule, as used herein, is one that is separated from nucleic acids that normally flank the gene or nucleic acid sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in an RNA library). For example, an isolated nucleic acid of the invention may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. In some instances, the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix. In other circumstances, the material may be purified to essential homogeneity, for example as determined by PAGE or column chromatography such as HPLC. In certain embodiments, an isolated nucleic acid molecule comprises at least about 50, 80 or 90% (on a molar basis) of all macromolecular species present. With regard to genomic DNA, the term “isolated” also can refer to nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated. For example, the isolated nucleic acid molecule can contain less than about 5 kb, including but not limited to 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotides which flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule is derived.  
      The nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated. Thus, recombinant DNA contained in a vector is included in the definition of “isolated” as used herein. Also, isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells, as well as partially or substantially purified DNA molecules in solution. “Isolated” nucleic acid molecules also encompass in vivo and in vitro RNA transcripts of the DNA molecules of the present invention. An isolated nucleic acid molecule or nucleic acid sequence can include a nucleic acid molecule or nucleic acid sequence that is synthesized chemically or by recombinant means. Therefore, recombinant DNA contained in a vector is included in the definition of “isolated” as used herein. Also, isolated nucleotide sequences include recombinant DNA molecules in heterologous organisms, as well as partially or substantially purified DNA molecules in solution. In vivo and in vitro RNA transcripts of the DNA molecules of the present invention are also encompassed by “isolated” nucleotide sequences. Such isolated nucleotide sequences are useful in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g., from other mammalian species), for gene mapping (e.g., by in situ hybridization with chromosomes), or for detecting expression of the nucleic acid in tissue (e.g., human tissue), such as by Northern blot analysis.  
      The present invention also pertains to nucleic acid molecules which are not necessarily found in nature but which encode a FLAP polypeptide (e.g., a polypeptide having an amino acid sequence comprising an amino acid sequence of SEQ ID NOs: 2), or another splicing variant of a FLAP polypeptide or polymorphic variant thereof. Thus, for example, DNA molecules that comprise a sequence that is different from the naturally occurring nucleic acid sequence but which, due to the degeneracy of the genetic code, encode a FLAP polypeptide of the present invention are also the subjects of this invention. The invention also encompasses nucleotide sequences encoding portions (fragments), or encoding variant polypeptides such as analogues or derivatives of a FLAP polypeptide. Such variants can be naturally occurring, such as in the case of allelic variation or single nucleotide polymorphisms, or non-naturally-occurring, such as those induced by various mutagens and mutagenic processes. Intended variations include, but are not limited to, addition, deletion and substitution of one or more nucleotides that can result in conservative or non-conservative amino acid changes, including additions and deletions. Preferably the nucleotide (and/or resultant amino acid) changes are silent or conserved; that is, they do not alter the characteristics or activity of a FLAP polypeptide. In one preferred embodiment, the nucleotide sequences are fragments that comprise one or more polymorphic microsatellite markers. In another preferred embodiment, the nucleotide sequences are fragments that comprise one or more single nucleotide polymorphisms in a FLAP nucleic acid (e.g., the single nucleotide polymorphisms set forth in Table 13, below).  
      The present invention also pertains to nucleic acid molecules which are not necessarily found in nature but which encode a LTA4H polypeptide (e.g., a polypeptide having an amino acid sequence comprising an amino acid sequence of SEQ ID NO: 720), or another splicing variant of a LTA4H polypeptide or polymorphic variant thereof. Thus, for example, DNA molecules that comprise a sequence that is different from the naturally occurring nucleic acid sequence but which, due to the degeneracy of the genetic code, encode a LTA4H polypeptide of the present invention are also the subjects of this invention. The invention also encompasses nucleotide sequences encoding portions (fragments), or encoding variant polypeptides such as analogues or derivatives of a LTA4H polypeptide. Such variants can be naturally occurring, such as in the case of allelic variation or single nucleotide polymorphisms, or non-naturally-occurring, such as those induced by various mutagens and mutagenic processes. Intended variations include, but are not limited to, addition, deletion and substitution of one or more nucleotides that can result in conservative or non-conservative amino acid changes, including additions and deletions. Preferably the nucleotide (and/or resultant amino acid) changes are silent or conserved; that is, they do not alter the characteristics or activity of a LTA4H polypeptide. In one preferred embodiment, the nucleotide sequences are fragments that comprise one or more polymorphic microsatellite markers. In another preferred embodiment, the nucleotide sequences are fragments that comprise one or more single nucleotide polymorphisms in a LTA4H nucleic acid (e.g., the single nucleotide polymorphisms set forth in Table 37, below).  
      Other alterations of the nucleic acid molecules of the invention can include, for example, labeling, methylation, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates), charged linkages (e.g., phosphorothioates, phosphorodithioates), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids). Also included are synthetic molecules that mimic nucleic acid molecules in the ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.  
      The invention also pertains to nucleic acid molecules that hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleic acid sequence described herein (e.g., nucleic acid molecules which specifically hybridize to a nucleic acid sequence encoding polypeptides described herein, and, optionally, have an activity of the polypeptide). In one embodiment, the invention includes variants described herein which hybridize under high stringency hybridization conditions (e.g., for selective hybridization) to a nucleic acid sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 718 and 719 or the complement thereof. In another embodiment, the invention includes variants described herein which hybridize under high stringency hybridization conditions (e.g., for selective hybridization) to a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 720 or a polymorphic variant thereof. In a preferred embodiment, the variant that hybridizes under high stringency hybridizations has an activity of a FLAP. In another preferred embodiment, the variant that hybridizes under high stringency hybridizations has an activity of a LTA4H.  
      Such nucleic acid molecules can be detected and/or isolated by specific hybridization (e.g., under high stringency conditions). “Specific hybridization,” as used herein, refers to the ability of a first nucleic acid to hybridize to a second nucleic acid in a manner such that the first nucleic acid does not hybridize to any nucleic acid other than to the second nucleic acid (e.g., when the first nucleic acid has a higher similarity to the second nucleic acid than to any other nucleic acid in a sample wherein the hybridization is to be performed). “Stringency conditions” for hybridization is a term of art which refers to the incubation and wash conditions, e.g., conditions of temperature and buffer concentration,.which permit hybridization of a particular nucleic acid to a second nucleic acid; the first nucleic acid may be perfectly (i.e., 100%) complementary to the second, or the first and second may share some degree of complementarity that is less than perfect (e.g., 70%, 75%, 85%, 95%). For example, certain high stringency conditions can be used which distinguish perfectly complementary nucleic acids from those of less complementarity. “High stringency conditions”, “moderate stringency conditions” and “low stringency conditions” for nucleic acid hybridizations are explained on pages 2.10.1-2.10.16 and pages 6.3.1-6.3.6 in  Current Protocols in Molecular Biology  (Ausubel, F. M. et al., “ Current Protocols in Molecular Biology ”, John Wiley &amp; Sons, (1998), the entire teachings of which are incorporated by reference herein). The exact conditions which determine the stringency of hybridization depend not only on ionic strength (e.g., 0.2×SSC, 0.1×SSC), temperature (e.g., room temperature, 42° C., 68° C.) and the concentration of destabilizing agents such as formamide or denaturing agents such as SDS, but also on factors such as the length of the nucleic acid sequence, base composition, percent mismatch between hybridizing sequences and the frequency of occurrence of subsets of that sequence within other non-identical sequences. Thus, equivalent conditions can be determined by varying one or more of these parameters while maintaining a similar degree of identity or similarity between the two nucleic acid molecules. Typically, conditions are used such that sequences at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95% or more identical to each other remain hybridized to one another. By varying hybridization conditions from a level of stringency at which no hybridization occurs to a level at which hybridization is first observed, conditions which will allow a given sequence to hybridize (e.g., selectively) with the most similar sequences in the sample can be determined.  
      Exemplary conditions are described in Krause, M. H. and S. A. Aaronson,  Methods in Enzymology  200: 546-556 (1991), and in, Ausubel, et al., “ Current Protocols in Molecular Biology ”, John Wiley &amp; Sons, (1998), which describes the determination of washing conditions for moderate or low stringency conditions. Washing is the step in which conditions are usually set so as to determine a minimum level of complementarity of the hybrids. Generally, starting from the lowest temperature at which only homologous hybridization occurs, each ° C. by which the final wash temperature is reduced (holding SSC concentration constant) allows an increase by 1% in the maximum extent of mismatching among the sequences that hybridize. Generally, doubling the concentration of SSC results in an increase in T m  of −17° C. Using these guidelines, the washing temperature can be determined empirically for high, moderate or low stringency, depending on the level of mismatch sought.  
      For example, a low stringency wash can comprise washing in a solution containing 0.2×SSC/0.1% SDS for 10 minutes at room temperature; a moderate stringency wash can comprise washing in a prewarmed solution (42° C.) solution containing 0.2×SSC/0.1% SDS for 15 minutes at 42° C.; and a high stringency wash can comprise washing in prewarmed (68° C.) solution containing 0.1×SSC/0.1% SDS for 15 minutes at 68° C. Furthermore, washes can be performed repeatedly or sequentially to obtain a desired result as known in the art. Equivalent conditions can be determined by varying one or more of the parameters given as an example, as known in the art, while maintaining a similar degree of identity or similarity between the target nucleic acid molecule and the primer or probe used.  
      The percent homology or identity of two nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence for optimal alignment). The nucleotides or amino acids at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions×100). When a position in one sequence is occupied by the same nucleotide or amino acid residue as the corresponding position in the other sequence, then the molecules are homologous at that position. As used herein, nucleic acid or amino acid “homology” is equivalent to nucleic acid or amino acid “identity”. In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, for example, at least 40%, in certain embodiments at least 60%, and in other embodiments at least 70%, 80%, 90% or 95% of the length of the reference sequence. The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A preferred, non-limiting example of such a mathematical algorithm is described in Karlin et al.,  Proc. Natl. Acad. Sci. USA  90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) as described in Altschul et al.,  Nucleic Acids Res.  25:389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., NBLAST) can be used. In one embodiment, parameters for sequence comparison can be set at score=100, wordlength=12, or can be varied (e.g., W=5 or W=20).  
      Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller,  CABIOS  4(1): 11-17 (1988). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package (Accelrys, Cambridge, UK). When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti,  Comput. Appl. Biosci.  10:3-5 (1994); and FASTA described in Pearson and Lipman,  Proc. Natl. Acad. Sci. USA  85:2444-8 (1988).  
      In another embodiment, the percent identity between two amino acid sequences can be accomplished using the GAP program in the GCG software package using either a BLOSUM63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. In yet another embodiment, the percent identity between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package using a gap weight of 50 and a length weight of 3.  
      The present invention also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleic acid sequence comprising SEQ ID NO: 1 or 3 or the complement of SEQ ID NO: 1 or 3, and also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleic acid sequence encoding an amino acid sequence of the invention or polymorphic variant thereof. The nucleic acid fragments of the invention are at least about 15, for example, at least about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50, 100, 200 or more nucleotides in length. Longer fragments, for example, 30 or more nucleotides in length, encoding antigenic polypeptides described herein are particularly useful, such as for the generation of antibodies as described below.  
      Probes and Primers  
      In a related aspect, the nucleic acid fragments of the invention are used as probes or primers in assays such as those described herein. “Probes” or “primers” are oligonucleotides that hybridize in a base-specific manner to a complementary strand of nucleic acid molecules. Such probes and primers include polypeptide nucleic acids, as described in Nielsen et al., ( Science  254:1497-1500 (1991)).  
      A probe or primer comprises a region of nucleic acid that hybridizes to at least about 15, for example about 20-25, and in certain embodiments about 40, 50 or 75, consecutive nucleotides of a nucleic acid of the invention, such as a nucleic acid comprising a contiguous nucleic acid sequence of SEQ ID NOs: 1 or 3 or the complement of SEQ ID Nos: 1 or 3, or a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 2 or polymorphic variant thereof. In preferred embodiments, a probe or primer comprises 100 or fewer nucleotides, in certain embodiments, from 6 to 50 nucleotides, for example, from 12 to 30 nucleotides. In other embodiments, the probe or primer is at least 70% identical to the contiguous nucleic acid sequence or to the complement of the contiguous nucleotide sequence, for example, at least 80% identical, in certain embodiments at least 90% identical, and in other embodiments at least 95% identical, or even capable of selectively hybridizing to the contiguous nucleic acid sequence or to the complement of the contiguous nucleotide sequence. Often, the probe or primer further comprises a label, e.g., radioisotope, fluorescent compound, enzyme, or enzyme co-factor.  
      Particularly useful probes and primers of the invention are those which hybridize to marker locations (e.g. in the FLAP gene) and those that permit amplication (e.g. using PCR) of a small DNA fragment that include a marker of interest, especially markers that form haplotypes of the invention, Kits containing one or two or three or more of such probes and primers are contemplated as aspects of the invention.  
      The nucleic acid molecules of the invention such as those described above can be identified and isolated using standard molecular biology techniques and the sequence information provided herein. For example, nucleic acid molecules can be amplified and isolated using the polymerase chain reaction and synthetic oligonucleotide primers based on one or more of SEQ ID NOs: 1 or 3, or the complement thereof, or designed based on nucleotides based on sequences encoding one or more of the amino acid sequences provided herein. See generally  PCR Technology: Principles and Applications for DNA Amplification  (ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992);  PCR Protocols: A Guide to Methods and Applications  (Eds. Innis et al., Academic Press, San Diego, Calif., 1990); Mattila et al.,  Nucl. Acids Res.  19:4967 (1991); Eckert et al.,  PCR Methods and Applications  1:17 (1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. No. 4,683,202. The nucleic acid molecules can be amplified using cDNA, mRNA or genomic DNA as a template, cloned into an appropriate vector and characterized by DNA sequence analysis.  
      Other suitable amplification methods include the ligase chain reaction (LCR) (see Wu and Wallace,  Genomics  4:560 (1989), Landegren et al.,  Science  241:1077 (1988), transcription amplification (Kwoh et al.,  Proc. Natl. Acad. Sci. USA  86:1173 (1989)), and self-sustained sequence replication (Guatelli et al.,  Proc. Nat. Acad. Sci. USA  87:1874 (1990)) and nucleic acid based sequence amplification (NASBA). The latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.  
      The amplified DNA can be labeled, for example, radiolabeled, and used as a probe for screening a cDNA library derived from human cells, mRNA in zap express, ZIPLOX or other suitable vector. Corresponding clones can be isolated, DNA can obtained following in vivo excision, and the cloned insert can be sequenced in either or both orientations by art recognized methods to identify the correct reading frame encoding a polypeptide of the appropriate molecular weight. For example, the direct analysis of the nucleic acid molecules of the present invention can be accomplished using well-known methods that are commercially available. See, for example, Sambrook et al.,  Molecular Cloning, A Laboratory Manual  (2nd Ed., CSHP, New York 1989); Zyskind et al.,  Recombinant DNA Laboratory Manual,  (Acad. Press, 1988)). Using these or similar methods, the polypeptide and the DNA encoding the polypeptide can be isolated, sequenced and further characterized.  
      Antisense nucleic acid molecules of the invention can be designed using the nucleotide sequences of SEQ ID NOs: 1 or 3 and/or the complement of one or more of SEQ ID NOs: 1 or 3 and/or a portion of one or more of SEQ ID NOs: 1 or 3 or the complement of one or more of SEQ ID NOs: 1 or 3 and/or a sequence encoding the amino acid sequences of SEQ ID NOs: 2 or encoding a portion of one or more of SEQ ID NOs: 1 or 3 or their complement. They can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid molecule (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Alternatively, the antisense nucleic acid molecule can be produced biologically using an expression vector into which a nucleic acid molecule has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid molecule will be of an antisense orientation to a target nucleic acid of interest).  
      The nucleic acid sequences can also be used to compare with endogenous DNA sequences in patients to identify one or more of the disorders related to FLAP, and as probes, such as to hybridize and discover related DNA sequences or to subtract out known sequences from a sample. The nucleic acid sequences can further be used to derive primers for genetic fingerprinting, to raise anti-polypeptide antibodies using DNA immunization techniques, and as an antigen to raise anti-DNA antibodies or elicit immune responses. Portions or fragments of the nucleotide sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions or nucleic acid regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. Additionally, the nucleotide sequences of the invention can be used to identify and express recombinant polypeptides for analysis, characterization or therapeutic use, or as markers for tissues in which the corresponding polypeptide is expressed, either constitutively, during tissue differentiation, or in diseased states. The nucleic acid sequences can additionally be used as reagents in the screening and/or diagnostic assays described herein, and can also be included as components of kits (e.g., reagent kits) for use in the screening and/or diagnostic assays described herein.  
      Vectors  
      Another aspect of the invention pertains to nucleic acid constructs containing a nucleic acid molecule of SEQ ID NOs: 1, 3, 718 or 719 or the complement thereof (or a portion thereof). Yet another aspect of the invention pertains to nucleic acid constructs containing a nucleic acid molecule encoding an amino acid of SEQ ID NO: 2, SEQ ID NO: 720 or polymorphic variant thereof. The constructs comprise a vector (e.g., an expression vector) into which a sequence of the invention has been inserted in a sense or antisense orientation. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors, such as expression vectors, are capable of directing the expression of genes or nucleic acids to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) that serve equivalent functions.  
      Preferred recombinant expression vectors of the invention comprise a nucleic acid molecule of the invention in a form suitable for expression of the nucleic acid molecule in a host cell. This means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” or “operatively linked” is intended to mean that the nucleic acid sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleic acid sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, “Gene Expression Technology”,  Methods in Enzymology  185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleic acid sequence in many types of host cell and those which direct expression of the nucleic acid sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed and the level of expression of polypeptide desired. The expression vectors of the invention can be introduced into host cells to thereby produce polypeptides, including fusion polypeptides, encoded by nucleic acid molecules as described herein.  
      The recombinant expression vectors of the invention can be designed for expression of a polypeptide of the invention in prokaryotic or eukaryotic cells, e.g., bacterial cells such as  E. coli,  insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, supra. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.  
      Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or enviromnental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.  
      A host cell can be any prokaryotic or eukaryotic cell. For example, a nucleic acid molecule of the invention can be expressed in bacterial cells (e.g.,  E. coli ), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.  
      Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing a foreign nucleic acid molecule (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.  
      For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene or nucleic acid that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene or nucleic acid of interest. Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector as the nucleic acid molecule of the invention or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene or nucleic acid will survive, while the other cells die).  
      A host cell of the invention, such as a prokaryotic host cell or eukaryotic host cell in culture can be used to produce (i.e., express) a polypeptide of the invention. Accordingly, the invention further provides methods for producing a polypeptide using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is produced. In another embodiment, the method further comprises isolating the polypeptide from the medium or the host cell.  
      The host cells of the invention can also be used to produce nonhuman transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a nucleic acid molecule of the invention has been introduced (e.g., an exogenous FLAP nucleic acid, or an exogenous nucleic acid encoding a FLAP polypeptide). Such host cells can then be used to create non-human transgenic animals in which exogenous nucleotide sequences have been introduced into the genome or homologous recombinant animals in which endogenous nucleotide sequences have been altered. Such animals are useful for studying the function and/or activity of the nucleic acid sequence and polypeptide encoded by the sequence and for identifying and/or evaluating modulators of their activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal include a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens and amphibians. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a “homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.  
      Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191 and in Hogan,  Manipulating the Mouse Embryo  (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley,  Current Opinion in BioTechnology  2:823-829 (1991) and in PCT Publication Nos. WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169. Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al.,  Nature  385:810-813 (1997) and PCT Publication Nos. WO 97/07668 and WO 97/07669.  
     Polypeptides of the Invention  
      The present invention pertains to isolated polypeptides encoded by FLAP nucleic acids (“FLAP polypeptides”), and fragments and variants thereof, as well as polypeptides encoded by nucleotide sequences described herein (e.g., other splicing variants). The present invention also pertains to isolated polypeptides encoded by LTA4H nucleic acids (“LTA4H polypeptides”), and fragments and variants thereof, as well as polypeptides encoded by nucleotide sequences described herein (e.g., other splicing variants) The term “polypeptide” refers to a polymer of amino acids, and not to a specific length; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. As used herein, a polypeptide is said to be “isolated” or “purified” when it is substantially free of cellular material when it is isolated from recombinant and non-recombinant cells, or free of chemical precursors or other chemicals when it is chemically synthesized. A polypeptide, however, can be joined to another polypeptide with which it is not normally associated in a cell (e.g., in a “fusion protein”) and still be “isolated” or “purified.” A detailed discussion of the methods to make a polypeptide of the invention is provided in International Application No. PCT/US03/32556, filed on Oct. 16, 2003, which is incorporated by reference herein in its entirety.  
     Antibodies of the Invention  
      Polyclonal and/or monoclonal antibodies that specifically bind one form of the polypeptide or nucleic acid product (e.g., a polypeptide encoded by a nucleic acid having a SNP as set forth in Table 13), but not to another form of the polypeptide or nucleic acid product, are also provided. Antibodies are also provided which bind a portion of either polypeptide encoded by nucleic acids of the invention (e.g., SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 718 or SEQ ID NO: 719, or the complement thereof), or to a polypeptide encoded by nucleic acids of the invention that contain a polymorphic site or sites. The invention also provides antibodies to the polypeptides and polypeptide fragments of the invention, or a portion thereof, or having an amino acid sequence encoded by a nucleic acid molecule comprising all or a portion of SEQ ID NOs: 1, 3, 718 or 719, or the complement thereof, or another variant or portion thereof. A detailed discussion of the methods to make the antibodies of the invention is provided in International Application No. PCT/US03/32556, filed on Oct. 16, 2003, which is incorporated by reference herein in its entirety.  
     Diagnostic Assays  
      The nucleic acids, probes, primers, polypeptides and antibodies described herein can be used in methods of diagnosis of a susceptibility to MI, ACS, stroke or PAOD, or to another disease or condition associated with an MI gene, such as FLAP or LTA4H, as well as in kits useful for diagnosis of a susceptibility to MI, ACS, stroke or PAOD, or to another disease or condition associated with FLAP or LTA4H. In one embodiment, the kit useful for diagnosis of susceptibility to MI, ACS, stroke or PAOD, or to another disease or condition associated with FLAP or LTA4H comprises primers as described herein, wherein the primers contain one or more of the SNPs identified in Table 13 or Table 37.  
      In one embodiment of the invention, diagnosis of susceptibility to MI, ACS, stroke or PAOD (or diagnosis of susceptibility to another disease or condition associated with FLAP or LTA4H), is made by detecting a polymorphism in a FLAP or LTA4H nucleic acid as described herein. The polymorphism can be an alteration in a FLAP or LTA4H nucleic acid, such as the insertion or deletion of a single nucleotide, or of more than one nucleotide, resulting in a frame shift alteration; the change of at least one nucleotide, resulting in a change in the encoded amino acid; the change of at least one nucleotide, resulting in the generation of a premature stop codon; the deletion of several nucleotides, resulting in a deletion of one or more amino acids encoded by the nucleotides; the insertion of one or several nucleotides, such as by unequal recombination or gene conversion, resulting in an interruption of the coding sequence of the gene or nucleic acid; duplication of all or a part of the gene or nucleic acid; transposition of all or a part of the gene or nucleic acid; or rearrangement of all or a part of the gene or nucleic acid. More than one such alteration may be present in a single gene or nucleic acid. Such sequence changes cause an alteration in the polypeptide encoded by a FLAP or LTA4H nucleic acid. For example, if the alteration is a frame shift alteration, the frame shift can result in a change in the encoded amino acids, and/or can result in the generation of a premature stop codon, causing generation of a truncated polypeptide. Alternatively, a polymorphism associated with a disease or condition associated with a FLAP or LTA4H nucleic acid or a susceptibility to a disease or condition associated with a FLAP or LTA4H nucleic acid can be a synonymous alteration in one or more nucleotides (i.e., an alteration that does not result in a change in the polypeptide encoded by a FLAP nucleic acid or LTA4H nucleic acid). Such a polymorphism may alter splicing sites, affect the stability or transport of mRNA, or otherwise affect the transcription or translation of the nucleic acid. A FLAP nucleic acid or a LTA4H nucleic acid that has any of the alteration described above is referred to herein as an “altered nucleic acid.” 
      In a first method of diagnosing a susceptibility to MI, ACS, stroke or PAOD, hybridization methods, such as Southern analysis, Northern analysis, or in situ hybridizations, can be used (see  Current Protocols in Molecular Biology,  Ausubel, F. et al., eds., John Wiley &amp; Sons, including all supplements through 1999). For example, a biological sample from a test subject (a “test sample”) of genomic DNA, RNA, or cDNA, is obtained from an individual suspected of having, being susceptible to or predisposed for, or carrying a defect for, a susceptibility to a disease or condition associated with a FLAP nucleic acid or a disease or condition associated with a LTA4H nucleic acid (the “test individual”). The individual can be an adult, child, or fetus. The test sample can be from any source which contains genomic DNA, such as a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs. A test sample of DNA from fetal cells or tissue can be obtained by appropriate methods, such as by amniocentesis or chorionic villus sampling. The DNA, RNA, or cDNA sample is then examined to determine whether a polymorphism in an MI nucleic acid is present, and/or to determine which splicing variant(s) encoded by the FLAP or LTA4H is present. The presence of the polymorphism or splicing variant(s) can be indicated by hybridization of the nucleic acid in the genomic DNA, RNA, or cDNA to a nucleic acid probe. A “nucleic acid probe,” as used herein, can be a DNA probe or an RNA probe; the nucleic acid probe can contain at least one polymorphism in a FLAP nucleic acid, LTA4H nucleic acid or contains a nucleic acid encoding a particular splicing variant of a FLAP nucleic acid or a particular splicing variant of a LTA4H nucleic acid. The probe can be any of the nucleic acid molecules described above (e.g., the nucleic acid, a fragment, a vector comprising the nucleic acid, a probe or primer, etc.).  
      To diagnose a susceptibility to MI, ACS, stroke or PAOD (or another disease or condition associated with FLAP), the test sample containing a FLAP nucleic acid is contacted with at least one nucleic acid probe to form a hybridization sample. A preferred probe for detecting mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA sequences described herein. The nucleic acid probe can be, for example, a full-length nucleic acid molecule, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to appropriate mRNA or genomic DNA. For example, the nucleic acid probe can be all or a portion of one of SEQ ID NOs: 1, 3, 718 or 719 or the complement thereof or a portion thereof; or can be a nucleic acid encoding all or a portion of one of SEQ ID NO: 2 or 720. Other suitable probes for use in the diagnostic assays of the invention are described above (see e.g., probes and primers discussed under the heading, “Nucleic Acids of the Invention”).  
      The hybridization sample is maintained under conditions that are sufficient to allow specific hybridization of the nucleic acid probe to a FLAP nucleic acid or a LTA4H nucleic acid. “Specific hybridization,” as used herein, indicates exact hybridization (e.g., with no mismatches). Specific hybridization can be performed under high stringency conditions or moderate stringency conditions, for example, as described above. In a particularly preferred embodiment, the hybridization conditions for specific hybridization are high stringency.  
      Specific hybridization, if present, is then detected using standard methods. If specific hybridization occurs between the nucleic acid probe and FLAP nucleic acid in the test sample, then the FLAP has the polymorphism, or is the splicing variant, that is present in the nucleic acid probe. More than one nucleic acid probe can also be used concurrently in this method. Specific hybridization of any one of the nucleic acid probes is indicative of a polymorphism in the FLAP nucleic acid, or of the presence of a particular splicing variant encoding the FLAP nucleic acid, and is therefore diagnostic for a susceptibility to a disease or condition associated with FLAP (e.g., MI, ACS, stroke or PAOD).  
      In addition, if specific hybridization occurs between the nucleic acid probe and LTA4H nucleic acid in the test sample, then the LTA4H has the polymorphism, or is the splicing variant, that is present in the nucleic acid probe. More than one nucleic acid probe can also be used concurrently in this method. Specific hybridization of any one of the nucleic acid probes is indicative of a polymorphism in the LTA4H nucleic acid, or of the presence of a particular splicing variant encoding the LTA4H nucleic acid, and is therefore diagnostic for a susceptibility to a disease or condition associated with LTA4H (e.g., MI, ACS, stroke or PAOD).  
      In Northern analysis (see  Current Protocols in Molecular Biology,  Ausubel, F. et al., eds., John Wiley &amp; Sons, supra) the hybridization methods described above are used to identify the presence of a polymorphism or a particular splicing variant, associated with a susceptibility to a disease or condition associated with FLAP or LTA4H (e.g., MI, ACS, stroke or PAOD). For Northern analysis, a test sample of RNA is obtained from the individual by appropriate means. Specific hybridization of a nucleic acid probe, as described above, to RNA from the individual is indicative of a polymorphism in a FLAP nucleic acid, or of the presence of a particular splicing variant encoded by a FLAP nucleic acid, a polymorphism in a LTA4H nucleic acid, or of the presence of a particular splicing variant encoded by a LTA4H nucleic acid and is therefore diagnostic for susceptibility to a disease or condition associated with FLAP or LTA4H (e.g., MI, ACS, stroke or PAOD).  
      For representative examples of use of nucleic acid probes, see, for example, U.S. Pat. Nos. 5,288,611 and 4,851,330.  
      Alternatively, a peptide nucleic acid (PNA) probe can be used instead of a nucleic acid probe in the hybridization methods described above. PNA is a DNA mimic having a peptide-like, inorganic backbone, such as N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen via a methylene carbonyl linker (see, for example, Nielsen, P. E. et al.,  Bioconjugate Chemistry  5, American Chemical Society, p. 1 (1994). The PNA probe can be designed to specifically hybridize to a nucleic acid having a polymorphism associated with a susceptibility to a disease or condition associated with FLAP (e.g., MI). Hybridization of the PNA probe to a FLAP nucleic acid as described herein is diagnostic for the susceptibility to the disease or condition.  
      In another method of the invention, mutation analysis by restriction digestion can be used to detect an altered nucleic acid, or nucleic acids containing a polymorphism(s), if the mutation or polymorphism in the nucleic acid results in the creation or elimination of a restriction site. A test sample containing genomic DNA is obtained from the individual. Polymerase chain reaction (PCR) can be used to amplify a FLAP nucleic acid or a LTA4H nucleic acid (and, if necessary, the flanking sequences) in the test sample of genomic DNA from the test individual. RFLP analysis is conducted as described (see  Current Protocols in Molecular Biology,  supra). The digestion pattern of the relevant DNA fragment indicates the presence or absence of the alteration or polymorphism in the FLAP nucleic acid or LTA4H nucleic acid, and therefore indicates the presence or absence of the susceptibility to a disease or condition associated with FLAP or LTA4H (e.g., MI, ACS, stroke or PAOD).  
      Sequence analysis can also be used to detect specific polymorphisms in the FLAP nucleic acid. A test sample of DNA or RNA is obtained from the test individual. PCR or other appropriate methods can be used to amplify the nucleic acid, and/or its flanking sequences, if desired. The sequence of a FLAP nucleic acid, or a fragment of the nucleic acid, or cDNA, or fragment of the cDNA, or mRNA, or fragment of the mRNA, is determined, using standard methods. The sequence of the nucleic acid, nucleic acid fragment, cDNA, cDNA fragment, mRNA, or mRNA fragment is compared with the known nucleic acid sequence of the nucleic acid, cDNA (e.g., one or more of SEQ ID NOs: 1, 3, 718 or 719and/or the complement of SEQ ID NO: 1, 3, 718 or 719), or a nucleic acid sequence encoding SEQ ID NO: 2, SEQ ID NO: 720 or a fragment thereof) or mRNA, as appropriate. The presence of a polymorphism in the FLAP or LTA4H indicates that the individual has a susceptibility to a disease associated with FLAP or LTA4H (e.g., MI, ACS, stroke or PAOD).  
      Allele-specific oligonucleotides can also be used to detect the presence of polymorphism(s) in the FLAP nucleic acid or the LTA4H nucleic acid, through the use of dot-blot hybridization of amplified oligonucleotides with allele-specific oligonucleotide (ASO) probes (see, for example, Saiki, R. et al.,  Nature  324:163-166 (1986)). An “allele-specific oligonucleotide” (also referred to herein as an “allele-specific oligonucleotide probe”) is an oligonucleotide of approximately 10-50 base pairs, for example, approximately 15-30 base pairs, that specifically hybridizes to a FLAP nucleic acid, and that contains a polymorphism associated with a susceptibility to a disease or condition associated with FLAP or LTA4H (e.g., MI, ACS, stroke or PAOD). An allele-specific oligonucleotide probe that is specific for particular polymorphisms in a FLAP or LTA4H nucleic acid can be prepared, using standard methods (see  Current Protocols in Molecular Biology,  supra). To identify polymorphisms in the nucleic acid associated with susceptibility to disease, a test sample of DNA is obtained from the individual. PCR can be used to amplify all or a fragment of a FLAP nucleic acid or LTA4H nucleic acid, and their flanking sequences. The DNA containing the amplified FLAP or LTA4H nucleic acid (or fragment of the nucleic acid) is dot-blotted, using standard methods (see  Current Protocols in Molecular Biology,  supra), and the blot is contacted with the oligonucleotide probe. The presence of specific hybridization of the probe to the amplified FLAP or LTA4H is then detected. Specific hybridization of an allele-specific oligonucleotide probe to DNA from the individual is indicative of a polymorphism in the FLAP or a polymorphism in the LTA4H and is therefore indicative of a susceptibility to a disease or condition associated with FLAP or LTA4H respectively (e.g., MI, ACS, stroke or PAOD).  
      An allele-specific primer hybridizes to a site on target DNA overlapping a polymorphism and only primes amplification of an allelic form to which the primer exhibits perfect complementarity. See Gibbs,  Nucleic Acid Res.  17, 2427-2448 (1989). This primer is used in conjunction with a second primer which hybridizes at a distal site. Amplification proceeds from the two primers, resulting in a detectable product which indicates the particular allelic form is present. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polymorphic site and the other of which exhibits perfect complementarity to a distal site. The single-base mismatch prevents amplification and no detectable product is formed. The method works best when the mismatch is included in the 3′-most position of the oligonucleotide aligned with the polymorphism because this position is most destabilizing to elongation from the primer (see, e.g., WO 93/22456).  
      With the addition of such analogs as locked nucleic acids (LNAs), the size of primers and probes can be reduced to as few as 8 bases. LNAs are a novel class of bicyclic DNA analogs in which the 2′ and 4′ positions in the furanose ring are joined via an O-methylene (oxy-LNA), S-methylene (thio-LNA), or amino methylene (amino-LNA) moiety. Common to all of these LNA variants is an affinity toward complementary nucleic acids, which is by far the highest reported for a DNA analog. For example, particular all oxy-LNA nonamers have been shown to have melting temperatures of 64° C. and 74° C. when in complex with complementary DNA or RNA, respectively, as oposed to 28° C. for both DNA and RNA for the corresponding DNA nonamer. Substantial increases in T m  are also obtained when LNA monomers are used in combination with standard DNA or RNA monomers. For primers and probes, depending on where the LNA monomers are included (e.g., the 3′ end, the 5′end, or in the middle), the T m  could be increased considerably.  
      In another embodiment, arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual, can be used to identify polymorphisms in a FLAP nucleic acid or a LTA4H nucleic acid For example, in one embodiment, an oligonucleotide array can be used. Oligonucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. These oligonucleotide arrays, also described as “Genechips™,” have been generally described in the art, for example, U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and WO 92/10092. These arrays can generally be produced using mechanical synthesis methods or light directed synthesis methods that incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods. See Fodor et al.,  Science  251:767-777 (1991); Pirrung et al., U.S. Pat. No. 5,143,854; (see also PCT Application WO 90/15070); Fodor et al., PCT Publication WO 92/10092; and U.S. Pat. No. 5,424,186, the entire teachings of each of which are incorporated by reference herein. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. No. 5,384,261, the entire teachings of which are incorporated by reference herein. In another example, linear arrays can be utilized.  
      Once an oligonucleotide array is prepared, a nucleic acid of interest is hybridized with the array and scanned for polymorphisms. Hybridization and scanning are generally carried out by methods described herein and also in, e.g., published PCT Application Nos. WO 92/10092 and WO 95/11995, and U.S. Pat. No. 5,424,186, the entire teachings of which are incorporated by reference herein. In brief, a target nucleic acid sequence that includes one or more previously identified polymorphic markers is amplified using well-known amplification techniques, e.g., PCR. Typically, this involves the use of primer sequences that are complementary to the two strands of the target sequence both upstream and downstream from the polymorphism. Asymmetric PCR techniques may also be used. Amplified target, generally incorporating a label, is then hybridized with the array under appropriate conditions. Upon completion of hybridization and washing of the array, the array is scanned to determine the position on the array to which the target sequence hybridizes. The hybridization data obtained from the scan is typically in the form of fluorescence intensities as a function of location on the array. In a reverse method, a probe, containing a polymorphism, can be coupled to a solid surface and PCR amplicons are then added to hybridize to these probes.  
      Although primarily described in terms of a single detection block, e.g., detection of a single polymorphism arrays can include multiple detection blocks, and thus be capable of analyzing multiple, specific polymorphisms. It will generally be understood that detection blocks may be grouped within a single array or in multiple, separate arrays so that varying, optimal conditions may be used during the hybridization of the target to the array. For example, it may often be desirable to provide for the detection of those polymorphisms that fall within G-C rich stretches of a genomic sequence, separately from those falling in A-T rich segments. This allows for the separate optimization of hybridization conditions for each situation.  
      Additional uses of oligonucleotide arrays for detection of polymorphisms can be found, for example, in U.S. Pat. Nos. 5,858,659 and 5,837,832, the entire teachings of which are incorporated by reference herein. Other methods of nucleic acid analysis can be used to detect polymorphisms in a nucleic acid described herein, or variants encoded by a nucleic acid described herein. Representative methods include direct manual sequencing (Church and Gilbert,  Proc. Natl. Acad. Sci. USA  81:1991-1995 (1988); Sanger, F. et al.,  Proc. Natl. Acad. Sci., USA  74:5463-5467 (1977); Beavis et al. U.S. Pat. No. 5,288,644); automated fluorescent sequencing; single-stranded conformation polymorphism assays (SSCP); clamped denaturing gel electrophoresis (CDGE); denaturing gradient gel electrophoresis (DGGE) (Sheffield, V. C. et al.,  Proc. Natl. Acad. Sci. USA  86:232-236 (1989)), mobility shift analysis (Orita, M. et al.,  Proc. Natl. Acad. Sci. USA  86:2766-2770 (1989)), restriction enzyme analysis (Flavell et al.,  Cell  15:25 (1978); Geever, et al.,  Proc. Natl. Acad. Sci. USA  78:5081 (1981)); heteroduplex analysis; chemical mismatch cleavage (CMC) (Cotton et al.,  Proc. Natl. Acad. Sci. USA  85:4397-4401 (1985)); RNase protection assays (Myers, R. M. et al.,  Science  230:1242 (1985)); use of polypeptides which recognize nucleotide mismatches, such as  E. coli  mutS protein; allele-specific PCR, for example.  
      In one embodiment of the invention, diagnosis of a susceptibility to a disease or condition associated with FLAP or LTA4H (e.g., MI, ACS, stroke or PAOD) can also be made by expression analysis by quantitative PCR (kinetic thermal cycling). This technique utilizing TaqMan® can be used to allow the identification of polymorphisms and whether a patient is homozygous or heterozygous. The technique can assess the presence of an alteration in the expression or composition of the polypeptide encoded by a FLAP nucleic acid or splicing variants encoded by a FLAP nucleic acid. The technique can likewise assess the presence of an alteration in the expression or composition of the polypeptide encoded by a LTA4H nucleic acid or splicing variants encoded by a LTA4H nucleic acid. Further, the expression of the variants can be quantified as physically or functionally different.  
      In another embodiment of the invention, diagnosis of a susceptibility to MI, ACS, stroke or PAOD (or of another disease or condition associated with FLAP or LTA4H) can also be made by examining expression and/or composition of a FLAP polypeptide, by a variety of methods, including enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. A test sample from an individual is assessed for the presence of an alteration in the expression and/or an alteration in composition of the polypeptide encoded by a FLAP nucleic acid, or a polypeptide encoded by a LTA4H nucleic acid or for the presence of a particular variant encoded by a FLAP or LTA4H nucleic acid. An alteration in expression of a polypeptide encoded by a FLAP or LTA4H nucleic acid can be, for example, an alteration in the quantitative polypeptide expression (i.e., the amount of polypeptide produced); an alteration in the composition of a polypeptide encoded by a FLAP or LTA4H nucleic acid is an alteration in the qualitative polypeptide expression (e.g., expression of an altered FLAP polypeptide, LTA4H polypeptide or of a different splicing variant). In a preferred embodiment, diagnosis of a susceptibility to a disease or condition associated with FLAP is made by detecting a particular splicing variant encoded by that FLAP variant, or a particular pattern of splicing variants. In another preferred embodiment, diagnosis of a susceptibility to a disease or condition associated with LTA4H is made by detecting a particular splicing variant encoded by that LTA4H variant, or a particular pattern of splicing variants.  
      Both such alterations (quantitative and qualitative) can also be present. An “alteration” in the polypeptide expression or composition, refers to an alteration in expression or composition in a test sample, as compared with the expression or composition of polypeptide by a FLAP or LTA4H nucleic acid in a control sample. A control sample is a sample that corresponds to the test sample (e.g., is from the same type of cells), and is from an individual who is not affected by the disease or a susceptibility to a disease or condition associated with a FLAP or LTA4H nucleic acid. An alteration in the expression or composition of the polypeptide in the test sample, as compared with the control sample, is indicative of a susceptibility to a disease or condition associated with FLAP or LTA4H (e.g., MI, ACS, stroke or PAOD). Similarly, the presence of one or more different splicing variants in the test sample, or the presence of significantly different amounts of different splicing variants in the test sample, as compared with the control sample, is indicative of a susceptibility to a disease or condition associated with a FLAP or LTA4H nucleic acid. Various means of examining expression or composition of the polypeptide encoded by a FLAP or LTA4H nucleic acid can be used, including: spectroscopy, colorimetry, electrophoresis, isoelectric focusing and immunoassays (e.g., David et al., U.S. Pat. No. 4,376,110) such as immunoblotting (see also  Current Protocols in Molecular Biology,  particularly Chapter 10). For example, in one embodiment, an antibody capable of binding to the polypeptide (e.g., as described above), preferably an antibody with a detectable label, can be used. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′) 2 ) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.  
      Western blotting analysis, using an antibody as described above that specifically binds to a polypeptide encoded by an altered FLAP (e.g., by a FLAP having a SNP as shown in Table 13) or an antbody that specifically binds to a polypeptide encoded by an altered LTA4H (e.g. by a LTA4H having a SNP as shown in Table 37), or an antibody that specifically binds to a polypeptide encoded by a non-altered nucleic acid, or an antibody that specifically binds to a particular splicing variant encoded by a nucleic acid, can be used to identify the presence in a test sample of a particular splicing variant or of a polypeptide encoded by a polymorphic or altered FLAP or altered LTA4H, or the absence in a test sample of a particular splicing variant or of a polypeptide encoded by a non-polymorphic or non-altered nucleic acid. The presence of a polypeptide encoded by a polymorphic or altered nucleic acid, or the absence of a polypeptide encoded by a non-polymorphic or non-altered nucleic acid, is diagnostic for a susceptibility to a disease or condition associated with FLAP or LTA4H, as is the presence (or absence) of particular splicing variants encoded by the FLAP nucleic acid or the LTA4H nucleic acid.  
      In one embodiment of this method, the level or amount of polypeptide encoded by a FLAP nucleic acid in a test sample is compared with the level or amount of the polypeptide encoded by the FLAP in a control sample. A level or amount of the polypeptide in the test sample that is higher or lower than the level or amount of the polypeptide in the control sample, such that the difference is statistically significant, is indicative of an alteration in the expression of the polypeptide encoded by the FLAP, and is diagnostic for a susceptibility to a disease or condition associated with that FLAP. Alternatively, the composition of the polypeptide encoded by a FLAP nucleic acid in a test sample is compared with the composition of the polypeptide encoded by the FLAP in a control sample (e.g., the presence of different splicing variants). A difference in the composition of the polypeptide in the test sample, as compared with the composition of the polypeptide in the control sample, is diagnostic for a susceptibility to a disease or condition associated with that FLAP. In another embodiment, both the level or amount and the composition of the polypeptide can be assessed in the test sample and in the control sample. A difference in the amount or level of the polypeptide in the test sample, compared to the control sample; a difference in composition in the test sample, compared to the control sample; or both a difference in the amount or level, and a difference in the composition, is indicative of a susceptibility to a disease or condition associated with FLAP (e.g., MI).  
      The invention further pertains to a method for the diagnosis and identification of susceptibility to myocardial infarction, ACS, stroke or PAOD in an individual, by identifying an at-risk haplotype in FLAP or by identifying an at-risk haplotype in LTA4H. In one embodiment, the at-risk haplotype is one which confers a significant risk of MI, ACS, stroke or PAOD. In one embodiment, significance associated with a haplotype is measured by an odds ratio. In a further embodiment, the significance is measured by a percentage. In one embodiment, a significant risk is measured as an odds ratio of at least about 1.2, including by not limited to: 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. In a further embodiment, an odds ratio of at least 1.2 is significant. In a further embodiment, an odds ratio of at least about 1.5 is significant. In a further embodiment, a significant increase in risk is at least about 1.7 is significant. In a further embodiment, a significant increase in risk is at least about 20%, including but not limited to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95, and 98%. In a further embodiment, a significant increase in risk is at least about 50%. In yet another embodiment, an at-risk haplotype has a p value &lt;0.05. It is understood however, that identifying whether a risk is medically significant may also depend on a variety of factors, including the specific disease, the haplotype, and often, environmental factors.  
      The invention also pertains to methods of diagnosing a susceptibility to myocardial infarction, ACS, stroke or PAOD in an individual, comprising screening for an at-risk haplotype in the FLAP nucleic acid that is more frequently present in an individual susceptible to myocardial infarction (affected) or by screening for an at-risk haplotype in LTA4H, compared to the frequency of its presence in a healthy individual (control), wherein the presence of the haplotype is indicative of susceptibility to myocardial infarction. Standard techniques for genotyping for the presence of SNPs and/or microsatellite markers that are associated with myocardial infarction, ACS, stroke or PAOD can be used, such as fluorescent based techniques (Chen, et al.,  Genome Res.  9, 492 (1999), PCR, LCR, Nested PCR and other techniques for nucleic acid amplification. In a preferred embodiment, the method comprises assessing in an individual the presence or frequency of SNPs and/or microsatellites in the FLAP nucleic acid that are associated with myocardial infarction, ACS, stroke or PAOD, wherein an excess or higher frequency of the SNPs and/or microsatellites compared to a healthy control individual is indicative that the individual is susceptible to myocardial infarction, ACS, stroke or PAOD. See Table 7 for SNPs that comprise haplotypes that can be used as screening tools. See also Tables 13 and 37 that sets forth SNPs and markers for use as screening tools.  
      In one embodiment, the at-risk haplotype is characterized by the presence of polymorphism(s) represented in Table 13. For example, SG13S99, where the SNP can be a “C” or a “T”; SG13S25, where the SNP can be a “G” or an “A”; SG13S377, where the SNP can be a “G” or an “A”; SG13S106, where the SNP can be a “G” or an “A”; SG13S114, where the SNP can be a “T” or an “A”; SG13S89, where the SNP can be a “G” or an “A”; SG13S30, where the SNP can be a “G” or a “T”; SG13S32, where the SNP can be a “C” or an “A”; SG13S42, where the SNP can be a “G” or an “A”; and SG13S35, where the SNP can be a “G” or an “A”. In addition, SG13A375, where the SNP can be a “T”, SG13S32, where the SNP can be an “A”, aand SG13S106, where the SNP can be a “G” or an “A”.  
      Kits (e.g., reagent kits) useful in the methods of diagnosis comprise components useful in any of the methods described herein, including for example, hybridization probes or primers as described herein (e.g., labeled probes or primers), reagents for detection of labeled molecules, restriction enzymes (e.g., for RFLP analysis), allele-specific oligonucleotides, antibodies which bind to altered or to non-altered (native) FLAP polypeptide or a non-altered (native) LTA4H polypeptide, means for amplification of nucleic acids comprising a FLAP or LTA4H, or means for analyzing the nucleic acid sequence of a nucleic acid described herein, or for analyzing the amino acid sequence of a polypeptide as described herein, etc. In one embodiment, a kit for diagnosing susceptibility to MI, ACS, stroke or PAOD can comprise primers for nucleic acid amplification of a region in the FLAP nucleic acid or LTA4H nucleic acid comprising an at-risk haplotype that is more frequently present in an individual having MI, ACS, stroke or PAOD or susceptible to MI, ACS, stroke or PAOD. The primers can be designed using portions of the nucleic acids flanking SNPs that are indicative of MI. In a particularly preferred embodiment, the primers are designed to amplify regions of the LTA4H nucleic acid associated with an at-risk haplotype for MI, as shown in Table 38 or Table 39, or more particularly the haplotype defined by the microsatellite markers and SNPs at the locus on chromosome 12q23.  
      In another particularly preferred embodiment, the primers are designed to amplify regions of the FLAP nucleic acid associated with an at-risk haplotype for MI, ACS, stroke or PAOD, as shown in Table 7, or more particularly the haplotype defined by the following SNP markers: In one embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S99, SG13S25, SG13S377, SG13S106, SG13S32 and SG13S35 at the 13q12-13 locus. In one particular embodiment, the presence of the alleles T, G, G, G. A and G at SG13S99, SG13S25, SG13S377, SG13S106, SG13S32 and SG13S35, respectively (the B6 haplotype), is diagnostic of susceptibility to myocardial infarction, ACS, stroke or PAOD. In another embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S99, SG13S25, SG13S106, SG13S30 and SG13S42 at the 13q12-13 locus. In one particular embodiment, the presence of the alleles T, G, G, G and A at SG13S99, SG13S25, SG13S106, SG13S30 and SG13S42, respectively (the B5 haplotype), is diagnostic of susceptibility to myocardial infarction, ACS, stroke or PAOD. In a third embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S25, SG13S106, SG13S30 and SG13S42 at the 13q12-13 locus. In one particular embodiment, the presence of the alleles G, G, G and A at SG13S25, SG13S106, SG13S30 and SG13S42, respectively (the B4 haplotype), is diagnostic of susceptibility to myocardial infarction, ACS, stroke or PAOD. In a fourth embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S99, SG13S25, SG13S114, SG13S89 and SG13S32 at the 13q12-13 locus. In one particular embodiment, the presence of the alleles T, G, T, G and A at SG13S99, SG13S25, SG13S114, SG13S89 and SG13S32, respectively (the A5 haplotype), is diagnostic of susceptibility to myocardial infarction, ACS, stroke or PAOD. In a fifth embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S25, SG13S114, SG13S89 and SG13S32 at the 13q12-12 locus. In one particular-embodiment, the presence of the alleles G, T, G and A at SG13S25, SG13S 114, SG13S89 and SG13S32, respectively (the A4 haplotype), is diagnostic of susceptibility to myocardial infarction, ACS, stroke or PAOD. In another embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises marker SG13S375 at the 13q12-13 locus. In one particular embodiment, the presence of T at SG13S375, (the HapC1 haplotype) is diagnostic of susceptibility to to myocardial infarction, ACS, stroke or PAOD. In another embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S25 and SG13S375 at the 13q12-13 locus. In one particular embodiment, the presence of T and G at SG13S375 and SG13S25, respectively (the HapC2 haplotype) is diagnostic of susceptibility to to myocardial infarction, ACS, stroke or PAOD. In another embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S25, SG13S375 and SG13S32 at the 13q12-13 locus. In one particular embodiment, the presence of T, G and A at SG13S375, SG13S25 and SG13S32, respectively (the HapC3 haplotype) is diagnostic of susceptibility to to myocardial infarction, ACS, stroke or PAOD. In an additional embodiment, a haplotype associated with a susceptibility to myocardial infarction, ACS, stroke or PAOD comprises markers SG13S25, SG13S375, SG13S32 and SG13S106 at the 13q12-13 locus. In one particular embodiment, the presence of T, G, A and G at SG13S375, SG13S25, SG13S32 and SG13S106, respectively (the HapC4-A) haplotype) is diagnostic of susceptibility to to myocardial infarction, ACS, stroke or PAOD. In one particular embodiment, the presence of T, G, A and A at SG13S375, SG13S25, SG13S32 and SG13S106, respectively (the HapC4-B) haplotype) is diagnostic of susceptibility to to myocardial infarction, ACS, stroke or PAOD.  
     Screening Assays and Agents Identified Thereby  
      The invention provides methods (also referred to herein as “screening assays”) for identifying the presence of a nucleotide that hybridizes to a nucleic acid of the invention, as well as for identifying the presence of a polypeptide encoded by a nucleic acid of the invention. In one embodiment, the presence (or absence) of a nucleic acid molecule of interest (e.g., a nucleic acid that has significant homology with a nucleic acid of the invention) in a sample can be assessed by contacting the sample with a nucleic acid comprising a nucleic acid of the invention (e.g., a nucleic acid having the sequence of one of SEQ ID NOs: 1, 3, 718 or 719 or the complement thereof, or a nucleic acid encoding an amino acid having the sequence of SEQ ID NO: 2, SEQ ID NO: 720, or a fragment or variant of such nucleic acids), under stringent conditions as described above, and then assessing the sample for the presence (or absence) of hybridization. In a preferred embodiment, high stringency conditions are conditions appropriate for selective hybridization. In another embodiment, a sample containing a nucleic acid molecule of interest is contacted with a nucleic acid containing a contiguous nucleic acid sequence (e.g., a primer or a probe as described above) that is at least partially complementary to a part of the nucleic acid molecule of interest (e.g., a FLAP nucleic acid or a LTA4H nucleic acid), and the contacted sample is assessed for the presence or absence of hybridization. In a preferred embodiment, the nucleic acid containing a contiguous nucleic acid sequence is completely complementary to a part of the nucleic acid molecule of interest.  
      In any of these embodiments, all or a portion of the nucleic acid of interest can be subjected to amplification prior to performing the hybridization.  
      In another embodiment, the presence (or absence) of a polypeptide of interest, such as a polypeptide of the invention or a fragment or variant thereof, in a sample can be assessed by contacting the sample with an antibody that specifically hybridizes to the polypeptide of interest (e.g., an antibody such as those described above), and then assessing the sample for the presence (or absence) of binding of the antibody to the polypeptide of interest.  
      In another embodiment, the invention provides methods for identifying agents (e.g., fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes which alter (e.g., increase or decrease) the activity of the polypeptides described herein, or which otherwise interact with the polypeptides herein. For example, such agents can be agents which bind to polypeptides described herein (e.g., binding agent for members of the leukotriene pathway, such as FLAP binding agents or LTA4H binding agents); which have a stimulatory or inhibitory effect on, for example, activity of polypeptides of the invention; or which change (e.g., enhance or inhibit) the ability of the polypeptides of the invention to interact with members of the leukotriene pathway binding agents (e.g., receptors or other binding agents); or which alter posttranslational processing of the leukotriene pathway member polypeptide, such as a FLAP polypeptide or a LTA4H polypeptide (e.g., agents that alter proteolytic processing to direct the polypeptide from where it is normally synthesized to another location in the cell, such as the cell surface; agents that alter proteolytic processing such that more polypeptide is released from the cell, etc.)  
      In one embodiment, the invention provides assays for screening candidate or test agents that bind to or modulate the activity of polypeptides described herein (or biologically active portion(s) thereof), as well as agents identifiable by the assays. Test agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the “one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K. S.,  Anticancer Drug Des.  12:145 (1997)).  
      In one embodiment, to identify agents which alter the activity of a FLAP polypeptide or a LTA4H polypeptide, a cell, cell lysate, or solution containing or expressing a the polypeptide (e.g., SEQ ID NO: 2, SEQ ID NO; 720 or another splicing variant thereof such as a nucleic acid comprising a SNP as shown in Table 13 or 37), or a fragment or derivative thereof (as described above), can be contacted with an agent to be tested; alternatively, the polypeptide can be contacted directly with the agent to be tested. The level (amount) of polypeptide activity is assessed (e.g., the level (amount) of polypeptide activity is measured, either directly or indirectly), and is compared with the level of activity in a control (i.e., the level of activity of the FLAP or LTA4H polypeptide or active fragment or derivative thereof in the absence of the agent to be tested). If the level of the activity in the presence of the agent differs, by an amount that is statistically significant, from the level of the activity in the absence of the agent, then the agent is an agent that alters the activity of the polypeptide. An increase in the level of polypeptide activity in the presence of the agent relative to the activity in the absence of the agent, indicates that the agent is an agent that enhances the activity. Similarly, a decrease in the level of polypeptide activity in the presence of the agent, relative to the activity in the absence of the agent, indicates that the agent is an agent that inhibits polypeptide activity. In another embodiment, the level of activity of a polypeptide or derivative or fragment thereof in the presence of the agent to be tested, is compared with a control level that has previously been established. A statistically significant difference in the level of the activity in the presence of the agent from the control level indicates that the agent alters polypeptide activity.  
      The present invention also relates to an assay for identifying agents which alter the expression of a FLAP nucleic acid or a LTA4H nucleic acid (e.g., antisense nucleic acids, fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes; which alter (e.g., increase or decrease) expression (e.g., transcription or translation) of the nucleic acid or which otherwise interact with the nucleic acids described herein, as well as agents identifiable by the assays. For example, a solution containing a nucleic acid encoding a FLAP polypeptide (e.g., a FLAP nucleic acid) or a nucleic acid encoding a LTA4H polypeptide ( e.g., a LTA4H nucleic acid) can be contacted with an agent to be tested. The solution can comprise, for example, cells containing the nucleic acid or cell lysate containing the nucleic acid; alternatively, the solution can be another solution that comprises elements necessary for transcription/translation of the nucleic acid. Cells not suspended in solution can also be employed, if desired. The level and/or pattern of FLAP or LTA4H expression (e.g., the level and/or pattern of mRNA or of protein expressed, such as the level and/or pattern of different splicing variants) is assessed, and is compared with the level and/or pattern of expression in a control (i.e., the level and/or pattern of the FLAP expression or LTA4H exression in the absence of the agent to be tested). If the level and/or pattern in the presence of the agent differ, by an amount or in a manner that is statistically significant, from the level and/or pattern in the absence of the agent, then the agent is an agent that alters the expression of the FLAP nucleic acid or alters the expression of the LTA4H nucleic acid Enhancement of FLAP expression or LTA4H expression indicates that the agent is an activator of FLAP or LTA4H activity. Similarly, inhibition of FLAP expression or LTA4H expression indicates that the agent is a repressor of FLAP or LTA4H activity.  
      In another embodiment, the level and/or pattern of FLAP polypeptide(s) or LTA4H polypeptide(s) (e.g., different splicing variants) in the presence of the agent to be tested, is compared with a control level and/or pattern that have previously been established. A level and/or pattern in the presence of the agent that differs from the control level and/or pattern by an amount or in a manner that is statistically significant indicates that the agent alters FLAP or LTA4H expression.  
      In another embodiment of the invention, agents which alter the expression of a FLAP nucleic acid or a LTA4H nucleic acid or which otherwise interact with the nucleic acids described herein, can be identified using a cell, cell lysate, or solution containing a nucleic acid encoding the promoter region of the FLAP or LTA4H nucleic acid operably linked to a reporter gene. After contact with an agent to be tested, the level of expression of the reporter gene (e.g., the level of mRNA or of protein expressed) is assessed, and is compared with the level of expression in a control (i.e., the level of the expression of the reporter gene in the absence of the agent to be tested). If the level in the presence of the agent differs, by an amount or in a manner that is statistically significant, from the level in the absence of the agent, then the agent is an agent that alters the expression of the FLAP or LTA4H nucleic acid, as indicated by its ability to alter expression of a nucleic acid that is operably linked to the FLAP or LTA4H nucleic acid promoter.  
      Enhancement of the expression of the reporter indicates that the agent is an activator of expression of the gene of interest. Similarly, inhibition of the expression of the reporter indicates that the agent is a repressor of expression of the gene of interest. In another embodiment, the level of expression of the reporter in the presence of the test agent, is compared with a control level that has previously been established. A level in the presence of the agent that differs from the control level by an amount or in a manner that is statistically significant indicates that the agent alters expression.  
      Agents which alter the amounts of different splicing variants encoded by a FLAP nucleic acid or a LTA4H nucleic acid (e.g., an agent which enhances expression of a first splicing variant, and which inhibits expression of a second splicing variant), as well as agents which stimulate activity of a first splicing variant and inhibit activity of a second splicing variant, can easily be identified using these methods described above.  
      In one mbodiments of the invention, assays can be used to assess the impact of a test agent on the activity of a polypeptide relative to a FLAP binding agent. For example, a cell that expresses a compound that interacts with a FLAP nucleic acid (herein referred to as a “FLAP binding agent”, which can be a polypeptide or other molecule that interacts with a FLAP nucleic acid, such as a receptor, or another molecule, such as 5-LO) is contacted with a FLAP in the presence of a test agent, and the ability of the test agent to alter the interaction between the FLAP and the FLAP binding agent is determined. Alternatively, a cell lysate or a solution containing the FLAP binding agent, can be used. An agent which binds to the FLAP or the FLAP binding agent can alter the interaction by interfering with, or enhancing the ability of the FLAP to bind to, associate with, or otherwise interact with the FLAP binding agent.  
      In other embodiments of the invention, assays can be used to assess the impact of a test agent on the activity of a polypeptide relative to a LTA4H binding agent. For example, a cell that expresses a compound that interacts with a LTA4H nucleic acid (herein referred to as a “LTA4H binding agent”, which can be a polypeptide or other molecule that interacts with a LTA4H nucleic acid, such as a receptor, or another molecule) is contacted with a LTA4H in the presence of a test agent, and the ability of the test agent to alter the interaction between the LTA4H and the LTA4H binding agent is determined. Alternatively, a cell lysate or a solution containing the LTA4H binding agent, can be used. An agent which binds to the LTA4H or the LTA4H binding agent can alter the interaction by interfering with, or enhancing the ability of the LTA4H to bind to, associate with, or otherwise interact with the LTA4H binding agent.  
      Determining the ability of the test agent to bind to a nucleic acid or a nucleic acid binding agent can be accomplished, for example, by coupling the test agent with a radioisotope or enzymatic label such that binding of the test agent to the polypeptide can be determined by detecting the labeled with  125 I,  35 S,  14 C or  3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, test agents can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. It is also within the scope of this invention to determine the ability of a test agent to interact with the polypeptide without the labeling of any of the interactants. For example, a microphysiometer can be used to detect the interaction of a test agent with a polypeptide or a polypeptide binding agent without the labeling of either the test agent, the polypeptide, or the polypeptide binding agent. McConnell, H. M. et al.,  Science  257:1906-1912 (1992). As used herein, a “microphysiometer” (e.g., Cytosensor™) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between ligand and polypeptide.  
      Thus, these receptors can be used to screen for compounds that are agonists for use in treating a disease or condition associated with FLAP or LTA4H or a susceptibility to a disease or condition associated with FLAP or LTA4H, or antagonists for studying a susceptibility to a disease or condition associated with FLAP (e.g., MI, ACS, stroke or PAOD). Drugs can be designed to regulate FLAP activation, that in turn can be used to regulate signaling pathways and transcription events of genes downstream or of proteins or polypeptides interacting with FLAP (e.g., 5-LO).  
      In another embodiment of the invention, assays can be used to identify polypeptides that interact with one or more polypeptides, as described herein. For example, a yeast two-hybrid system such as that described by Fields and Song (Fields, S. and Song, O.,  Nature  340:245-246 (1989)) can be used to identify polypeptides that interact with one or more FLAP polypeptides. In such a yeast two-hybrid system, vectors are constructed based on the flexibility of a transcription factor that has two functional domains (a DNA binding domain and a transcription activation domain). If the two domains are separated but fused to two different proteins that interact with one another, transcriptional activation can be achieved, and transcription of specific markers (e.g., nutritional markers such as His and Ade, or color markers such as lacZ) can be used to identify the presence of interaction and transcriptional activation. For example, in the methods of the invention, a first vector is used which includes a nucleic acid encoding a DNA binding domain and also a FLAP or LTA4H polypeptide of the invention, splicing variant, or fragment or derivative thereof, and a second vector is used which includes a nucleic acid encoding a transcription activation domain and also a nucleic acid encoding a polypeptide which potentially may interact with the FLAP or LTA4H polypeptide, splicing variant, or fragment or derivative thereof (e.g., a FLAP polypeptide binding agent or receptor, or a LTA4H polypeptide binding agent or receptor). Incubation of yeast containing the first vector and the second vector under appropriate conditions (e.g., mating conditions such as used in the Matchmaker™ system from Clontech (Palo Alto, Calif., USA)) allows identification of colonies that express the markers of interest. These colonies can be examined to identify the polypeptide(s) that interact with the polypeptide of interest or fragment or derivative thereof. Such polypeptides may be useful as agents that alter the activity or expression of a FLAP polypeptide or alter the activity or expression of a LTA4H polypeptide, as described above.  
      In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either the FLAP or LTA4H, the FLAP or LTA4H binding agent, or other components of the assay on a solid support, in order to facilitate separation of complexed from uncomplexed forms of one or both of the polypeptides, as well as to accommodate automation of the assay. Binding of a test agent to the polypeptide, or interaction of the polypeptide with a binding agent in the presence and absence of a test agent, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein (e.g., a glutathione-S-transferase fusion protein) can be provided which adds a domain that allows a FLAP or LTA4H nucleic acid or a FLAP or LTA4H binding agent to be bound to a matrix or other solid support.  
      In one embodiment, modulators of expression of nucleic acid molecules of the invention are identified in a method wherein a cell, cell lysate, or solution containing a nucleic acid encoding a FLAP polypeptide or a LTA4H polypeptide is contacted with a test agent and the expression of appropriate mRNA or polypeptide (e.g., splicing variant(s)) in the cell, cell lysate, or solution, is determined. The level of expression of appropriate mRNA or polypeptide(s) in the presence of the test agent is compared to the level of expression of mRNA or polypeptide(s) in the absence of the test agent. The test agent can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater (statistically significantly greater) in the presence of the test agent than in its absence, the test agent is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less (statistically significantly less) in the presence of the test agent than in its absence, the test agent is identified as an inhibitor of the mRNA or polypeptide expression. The level of mRNA or polypeptide expression in the cells can be determined by methods described herein for detecting mRNA or polypeptide.  
      In yet another embodiment, the invention provides methods for identifying agents (e.g., fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes) which alter (e.g., increase or decrease) the activity of a member of leukotriene pathway binding agent, such as a FLAP binding agent (e.g., 5-LO) or a LTA4H binding agent, as described herein. For example, such agents can be agents which have a stimulatory or inhibitory effect on, for example, the activity of a member of leukotriene pathway binding agent, such as a FLAP binding agent or a LTA4H binding agnet; which change (e.g., enhance or inhibit) the ability a member of leukotriene pathway binding agents, (e.g., receptors or other binding agents) to interact with the polypeptides of the invention; or which alter posttranslational processing of the member of leukotriene pathway binding agent, (e.g., agents that alter proteolytic processing to direct the member of the leukotriene pathway binding agent from where it is normally synthesized to another location in the cell, such as the cell surface; agents that alter proteolytic processing such that more active binding agent is released from the cell, etc.).  
      For example, the invention provides assays for screening candidate or test agents that bind to or modulate the activity of a member of the leukotriene pathway (or enzymatically active portion(s) thereof), as well as agents identifiable by the assays. As described above, test agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the “one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K. S.  Anticancer Drug Des.,  12:145 (1997)).  
      In one embodiment, to identify agents which alter the activity of a member of the leukotriene pathway (such as a FLAP binding agent, LTA4H binding agent or an agent which binds to a member of the leukotriene pathway (a “binding agent”)), a cell, cell lysate, or solution containing or expressing a binding agent (e.g., 5-LO, or a leukotriene pathway member receptor, or other binding agent), or a fragment (e.g., an enzymatically active fragment) or derivative thereof, can be contacted with an agent to be tested; alternatively, the binding agent (or fragment or derivative thereof) can be contacted directly with the agent to be tested. The level (amount) of binding agent activity is assessed (either directly or indirectly), and is compared with the level of activity in a control (i.e., the level of activity in the absence of the agent to be tested). If the level of the activity in the presence of the agent differs, by an amount that is statistically significant, from the level of the activity in the absence of the agent, then the agent is an agent that alters the activity of the member of the leukotriene pathway. An increase in the level of the activity relative to a control, indicates that the agent is an agent that enhances (is an agonist of) the activity. Similarly, a decrease in the level of activity relative to a control, indicates that the agent is an agent that inhibits (is an antagonist of) the activity. In another embodiment, the level of activity in the presence of the agent to be tested, is compared with a control level that has previously been established. A level of the activity in the presence of the agent that differs from the control level by an amount that is statistically significant indicates that the agent alters the activity.  
      This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a test agent that is a modulating agent, an antisense nucleic acid molecule, a specific antibody, or a polypeptide-binding agent) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.  
      Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein. In addition, an agent identified as described herein can be used to alter activity of a polypeptide encoded by a FLAP nucleic acid or a LTA4H nucleic acid, or to alter expression of a FLAP nucleic acid or LTA4H nucleic acid, by contacting the polypeptide or the nucleic acid (or contacting a cell comprising the polypeptide or the nucleic acid) with the agent identified as described herein.  
      The present invention is now illustrated by the following Examples, which are not intended to be limiting in any way. The teachings of all references cited are incorporated herein in their entirety.  
     EXAMPLE 1  
     Identification of Gene and Haplotypes Associated With MI  
      A genome wide scan of 296 multiplex Icelandic families with 713 MI patients was performed. Through the suggestive linkage to a locus on chromosome 13q12-13 for female MI patients and early onset MI patients, and haplotype association analysis, the gene encoding the 5-lipoxygenase activating protein (FLAP) was identified, and a 4-SNP haplotype within the gene was determined to confer a near 2-fold risk of MI. Male patients showed strongest association to the at-risk haplotype. Independent confirmation of FLAP association to MI was obtained in a British cohort of patients with sporadic MI. These findings support FLAP as the first specific gene isolated that confers substantial risk of the complex trait of MI.  
     Methods  
      Study Population  
      Patients entering the study were recruited from a registry that includes all MIs that occurred before the age of 75 (over 8,000 patients) in Iceland from 1981 to 2000. This registry is a part of the World Health Organization MONICA Project (The World Health Organization MONICA Project, WHO MONICA Project Principal Investigators,.  J Clin Epidemiol  41, 105-14 (1988)). Diagnoses of all patients in the registry followed strict diagnostic rules based on signs, symptoms, electrocardiograms, cardiac enzymes, and necropsy findings.  
      Genotypes from 713 MI patients and 1741 of their first-degree relatives were used in the linkage analysis. For the microsatellite association study of the MI locus, 802 unrelated MI patients (n=233 females, n=624 males and n=302 early onset) and 837 population-based controls were used. For the SNP association study in and around the FLAP gene 779 unrelated MI patients were genotyped (n=293 females, n=486 males and n=358 early onset). The control group for the SNP association study was population based and comprised of 628 unrelated males and females in the age range of 30-85 years whose medical history was unknown.  
      The study was approved by the Data Protection Commission of Iceland and the National Bioethics Committee of Iceland. Informed consent was obtained from all study participants. Personal identifiers associated with medical information and blood samples were encrypted with a third party encryption system as previously described (Gulcher, J. R., Kristjansson, K., Gudbjartsson, H. &amp; Stefansson, K.,.  Eur J Hum Genet  8, 739-42 (2000)).  
     Statistical Analysis  
      A genome-wide scan was performed as previously described (Gretarsdottir, S. et al.  Am J Hum Genet  70, 593-603 (2002)), using a set of approximately 1000 microsatellite markers. Multipoint, affected-only allele-sharing methods (Kong, A. &amp; Cox, N. J.,  Am J Hum Genet  61, 1179-88 (1997)) were used to assess the evidence for linkage. All results were obtained using the program Allegro (Gudbjartsson, D. F., Jonasson, K., Frigge, M. L. &amp; Kong, A. Allegro,  Nat Genet  25, 12-3 (2000)) and the deCODE genetic map (Kong, A. et al.,  Nat Genet  31, 241-7 (2002)). The S pairs  scoring function (Whittemore, A. S. &amp; Halpern, J.,  Biometrics  50, 118-27 (1994); Kruglyak, L., Daly, M. J., Reeve-Daly, M. P. &amp; Lander, E. S.,  Am J Hum Genet  58, 1347-63 (1996)) was used, as was the exponential allele-sharing model (Kong, A. &amp; Cox, N. J.  Am J Hum Genet  61, 1179-88 (1997)) to generate the relevant 1-df (degree of freedom) statistics. When combining the family scores to obtain an overall score, a weighting scheme was used that is halfway on a log scale between weighting each affected pair equally and weighting each family equally. In the analysis, all genotyped individuals who are not affected are treated as “unknown”. Because of concern with small sample behaviour, corresponding P values were usually computed in two different ways for comparison, and the less significant one was reported. The first P value is computed based on large sample theory; Z lr =√(2 log e (10) LOD) and is distributed approximately as a standard normal distribution under the null hypothesis of no linkage (Kong, A. &amp; Cox, N. J.  Am J Hum Genet  61, 1179-88 (1997)). A second P value is computed by comparing the observed LOD score to its complete data sampling distribution under the null hypothesis (Gudbjartsson, D. F., Jonasson, K., Frigge, M. L. &amp; Kong, A. Allegro,.  Nat Genet  25, 12-3 (2000)). When a data set consists of more than a handful of families, these two P values tend to be very similar. The information measure that was used (Nicolae, D. University of Chicago (1999)), and is implemented in Allegro, is closely related to a classical measure of information (Dempster, A., Laird, N M, Rubin, D B.,  J R Stat Soc B  39, 1-38 (1977) and has a property that is between 0, if the marker genotypes are completely uninformative, and 1, if the genotypes determine the exact amount of allele sharing by descent among the affected relatives.  
      For single-marker association studies, Fisher&#39;s exact test was used to calculate two-sided P values for each allele. All P values were unadjusted for multiple comparisons unless specifically indicated. Allelic rather than carrier frequencies were presented for microsatellites, SNPs and haplotypes. To minimize any bias due to the relatedness of the patients that were recruited as families for the linkage analysis first and second-degree relatives were eliminated from the patient list. For the haplotype analysis, the program NEMO was used (Gretarsdottir, S. et al.,  Nat Genet  35, 131-8 (2003)), which handles missing genotypes and uncertainty with phase through a likelihood procedure, using the expectation-maximization algorithm as a computational tool to estimate haplotype frequencies. Under the null hypothesis, the affected individuals and controls are assumed to have identical haplotype frequencies. Under the alternative hypotheses, the candidate at-risk haplotype is allowed to have a higher frequency in the affected individuals than in controls, while the ratios of frequencies of all other haplotypes are assumed to be the same in both groups. Likelihoods are maximized separately under both hypotheses, and a corresponding 1-df likelihood ratio statistic used to evaluate statistical significance (id). Even though searches were only performed for haplotypes that increase the risk, all reported P values are two-sided unless otherwise stated. To assess the significance of the haplotype association corrected for multiple testing, a randomisation test was carried out using the same genotype data. The cohorts of affected individuals and controls were randomized, and the analysis was repeated. This procedure was repeated up to 1.000 times and the P value presented is the fraction of replications that produced a P value for a haplotype tested that is lower than or equal to the P value observed using the original patient and control cohorts.  
      For both single-marker and haplotype analysis, relative risk (RR) and population attributable risk was calculated assuming a multiplicative model (Terwilliger, J. D. &amp; Ott, J. A.,  Hum Hered  42, 337-46 (1992); Falk, C. T. &amp; Rubinstein, P.,  Ann Hum Genet  51 (Pt 3), 227-33 (1987)) in which the risk of the two alleles of haplotypes a person carries multiply. LD was calculated between pairs of SNPs using the standard definition of D′ (Lewontin, R. C.,  Genetics  50, 757-82 (1964)) and R 2  (Hill, W. G. &amp; Robertson, A.,  Genetics  60, 615-28 (1968)). Using NEMO, frequencies of the two marker allele combinations are estimated by maximum likelihood, and deviation from linkage equilibrium is evaluated by a likelihood ratio test. When plotting all SNP combinations to elucidate the LD structure in a particular region, D′ was plotted in the upper left corner and the P value in the lower right corner. In the LD plots presented, the markers are plotted equidistantly rather than according to their physical positions.  
     Identification of DNA Polymorphisms  
      New polymorphic repeats (e.g., dinucleotide or trinucleotide repeats) were identified with the Sputnik program. For microsatellite alleles: the CEPH sample 1347-02 (Centre d&#39;Etudes du Polymorphisme Humain, genomics repository) is used as a reference. The lower allele of each microsatellite in this sample is set at 0 and all other alleles in other samples are numbered according in relation to this reference. Thus allele1 is 1 bp longer than the lower allele in the CEPH sample, allele 2 is 2 bp longer than the lower allele in the CEPH sample, allele 3 is 3 bp longer than the lower allele in the CEPH sample, allele 4 is 4 bp longer than the lower allele in the CEPH sample, allele −1 is 1 bp shorter than the lower allele in the CEPH sample, allele −2 is 2 bp shorter than the lower allele in the CEPH sample, and so on. Single nucleotide polymorphisms in the gene were detected by PCR sequencing exonic and intronic regions from patients and controls. Public single nucleotide polymorphisms were obtained from the NCBI SNP database. SNPs were genotyped using a method for detecting SNPs with fluorescent polarization template-directed dye-terminator incorporation (SNP-FP-TDI assay) (Chen, X., Zehnbauer, B., Gnirke, A. &amp; Kwok, P. Y.,  Proc Natl Acad Sci USA  94, 10756-61. (1997)) and TaqMan assays (Applied Biosystems).  
     Results  
      Linkage Analysis  
      A genome wide scan was performed in search of MI susceptibility genes using a framework set of around 1000 microsatellite markers. The initial linkage analysis included 713 MI patients who fulfilled the WHO MONICA research criteria (The World Health Organization MONICA Project, WHO MONICA Project Principal Investigators,.  J Clin Epidemiol  41, 105-14 (1988)) and were clustered in 296 extended families. The linkage analysis was also repeated for early onset, male and female patients separately. Description of the number of patients and families in each analysis are provided in Table 1.  
               TABLE 1                          Number of patients that cluster into families and the       corresponding number of families used in the linkage analysis                                     Number of   Number of   Number of   Genotyped       Phenotype   patients   families   pairs   relatives a                                           All MI patients   713   296   863   1741       Males   575   248   724   1385       Females   140   56   108   366       Early onset   194   93   156   739                   a Genotyped relatives were used to increase the information on IBD sharing among the patients in the linkage analysis             
 
      None of these analyses yielded a locus of genome-wide significance. However, the most promising LOD score (LOD=2.86) was observed on chromosome 13q12-13 for female MI patients at the peak marker D13S289 (data not shown). This locus also had the most promising LOD score (LOD=2.03) for patients with early onset MI. After increasing the information on identity-by-descent sharing to over 90% by typing 14 additional microsatellite markers in a 30 centiMorgan (cM) region around D13S289, the LOD score from the female analysis dropped to 2.48 (P value=0.00036), while the highest LOD score remained at D13S289 ( FIG. 6 . 1 ).  
      Microsatellite Association Study  
      The 7.6 Mb region that corresponds to a drop of one in LOD score in the female MI linkage analysis, contains 40 known genes (Table 2).  
               TABLE 2                          Genes residing within the one LOD drop region of the chromosome       13q12-13 linkage peak.                     LL_Symbol   LL_gene_name               USP12L1   ubiquitin specific protease 12 like 1       RPL21   ribosomal protein L21       GTF3A   general transcription factor IIIA       MTIF3   mitochondrial translational initiation factor 3       PDZRN1   PDZ domain containing ring finger 1       MGC9850   hypothetical protein MGC9850       POLR1D   polymerase (RNA) I polypeptide D, 16 kDa       GSH1   GS homeobox 1       IPF1   insulin promoter factor 1, homeodomain transcription factor       CDX2   caudal type homeo box transcription factor 2       FLT3   fms-related tyrosine kinase 3       LOC255967   hypothetical protein LOC255967       FLT1   fms-related tyrosine kinase 1 (vascular endothelial growth           factor/vascular permeability factor receptor)       C13orf12   chromosome 13 open reading frame 12       LOC283537   hypothetical protein LOC283537       KIAA0774   KIAA0774 protein       SLC7A1   solute carrier family 7 (cationic amino acid transporter,           y+ system), member 1       UBL3   ubiquitin-like 3       MGC2599   hypothetical protein MGC2599 similar to katanin p60           subunit A 1 2599       HMGB1   high-mobility group box 1       D13S106E   highly charged protein       ALOX5AP   arachidonate 5-lipoxygenase-activating protein       FLJ14834   hypothetical protein FLJ14834       MGC40178   hypothetical protein MGC40178       HSPH1   heat shock 105 kDa/110 kDa protein 1       B3GTL   beta 3-glycosyltransferase-like       GREAT   similar to G protein coupled receptor affecting testicular           descent ( H. sapiens )       LOC196549   similar to hypothetical protein FLJ20897       13CDNA73   hypothetical protein CG003       BRCA2   breast cancer 2, early onset       CG018   hypothetical gene CG018       PRO0297   PRO0297 protein       LOC88523   CG016       CG012   hypothetical gene CG012       CG030   hypothetical gene CG030       CG005   hypothetical protein from BCRA2 region       APRIN   androgen-induced proliferation inhibitor       KL   Klotho       STARD13   START domain containing 13       RFC3   replication factor C (activator 1) 3, 38 kDa                  
 
      To determine which gene in this region most likely contributes to MI, 120 microsatellite markers positioned within this region were typed, and a case-control association study was performed using 802 unrelated MI patients and 837 population-based controls. The association study was also repeated for each of the three phenotypes that were used in the linkage study, i.e. early onset, male and female MI patients.  
      The initial association analysis was performed when the average spacing between microsatellite markers was approximately 100 kb. This analysis revealed several extended haplotypes composed of 4 and 5 microsatellite markers that were significantly associated with female MI (see  FIGS. 1 and 2 , and Tables 13 and 14). A region common to all these extended haplotypes, is defined by markers DG13S166 and D13S1238. This region included only one gene, the FLAP nucleic acid sequence. The two marker haplotype involving alleles 0 and −2 for markers DG13S166 and D13S1238, respectively, was found in excess in patients.  
      This was the first evidence that the FLAP gene might be involved in the pathogenesis of myocardial infarction.  
      Subsequent haplotype analysis that included more microsatellite markers (n=120) in the candidate region on chromosome 13q12-13, now with a marker density of 1 microsatellite marker per 60 kb, showed decreased significance of the original haplotype association. However, the haplotype association analysis using increased density of markers again pointed towards the FLAP gene. This analysis strongly suggested that a 300 kb region was involved in the susceptibility of myocardial infarction. As shown in  FIG. 5 . 2 , the haplotype that showed association to all MI with the lowest P value (0.00009) covered a region that contains 2 known genes, including the gene encoding arachidonate 5-lipoxygenase-activating protein (FLAP) and a gene with an unknown function called highly charged protein. However, the haplotype association to female MI in this region was less significant (P value=0.005) than for all MI patients and to our surprise, the most significant haplotype association was observed for male MI patients (P value=0.000002). This male MI haplotype was the only haplotype that remained significant after adjusting for all haplotypes tested.  
      In view of the association results described above, FLAP was an attractive candidate and therefore efforts were focused on this gene.  
      Screening for Polymorphisms in FLAP and Linkage Disequilibrium Mapping  
      To determine whether variations within the FLAP gene significantly associate with MI and to search for causal variations, the FLAP gene was sequenced in 93 patients and 93 controls. The sequenced region covers 60 kb containing the FLAP gene, including the 5 known exons and introns and the 26 kb region 5′ to the first exon and 7 kb region 3′ to the fifth exon. In all, 144 SNPs were identified, of those 96 were excluded from further analysis either because of low minor allele frequency or they were completely correlated with other SNPs and thus redundant.  FIG. 6  shows the distribution of the 48 SNPs, used for genotyping, relative to exons, introns and the 5′ and 3′flanking regions of the FLAP gene. Only one SNP was identified within a coding sequence (exon 2). This SNP did not lead to amino acid substitution. The locations of these SNPs in the NCBI human genome assembly, build 34, are listed in Table 3.  
               TABLE 3                          Locations of all genotyped SNPs in NCBI build 34 of the       human genome assembly                             SNP name   Build34 start                       SG13S381   29083350           SG13S366   29083518           SG13S1   29086224           SG13S2   29087473           SG13S367   29088090           SG13S10   29088473           SG13S3   29089044           SG13S368   29089886           SG13S4   29090997           SG13S5   29091307           SG13S90   29091780           SG13S6   29092536           SG13S371   29093964           SG13S372   29094259           SG13S373   29096688           SG13S375   29096874           SG13S376   29096962           SG13S25   29097553           SG13S377   29101965           SG13S100   29104271           SG13S95   29106329           SG13S191   29107830           SG13S106   29108579           SG13S114   29110096           SG13S121   29112174           SG13S122   29112264           SG13S43   29112455           SG13S192   29116308           SG13S88   29116401           SG13S137   29118118           SG13S86   29118815           SG13S87   29118873           SG13S39   29119740           SG13S26   29122253           SG13S27   29122283           SG13S29   29123643           SG13S89   29124441           SG13S96   29124906           SG13S30   29125840           SG13S97   29129139           SG13S32   29130547           SG13S41   29134045           SG13S42   29135877           SG13S34   29137100           SG13S35   29138117           SG13S181   29138633           SG13S184   29139435           SG13S188   29140805                      
 
      In addition to the SNPs, a polymorphism consisting of a monopolymer A repeat that has been described in the FLAP promoter region was typed (Koshino, T. et al.,.  Mol Cell Biol Res Commun  2, 32-5 (1999)).  
      The linkage disequilibrium (LD) block structure defined by the 48 SNPs that were selected for further genotyping is shown in  FIG. 8 . A strong LD was detected across the FLAP region, although it appears that at least one recombination may have occurred dividing the region into two strongly correlated LD blocks.  
      Haplotype Association to MI  
      To perform a case-control association study the 48 selected SNPs and the monopolymer A repeat marker were genotyped in a set of 779 unrelated MI patients and 628 population-based controls. Each of the 49 markers were tested individually for association to the disease. Three SNPs, one located 3 kb upstream of the first exon and the other two 1 and 3 kb downstream of the first exon, showed nominally significant association to MI (Table 4).  
               TABLE 4                          SNP allelic association in the MI cohort                                                 Pheno-       Al-   P       #   %   #   %       type   Marker   lele   value   RR   Pat.   Pat.   Ctrl   Ctrl                                                         All   SG13S106   G   0.0044   1.29   681   72.0   530   66.6       patients   SG13S100   A   0.020   1.29   388   69.6   377   63.9           SG13S114   T   0.021   1.21   764   70.0   602   65.8       Males   SG13S106   G   0.0037   1.35   422   72.9   530   66.6           SG13S100   A   0.0099   1.36   292   70.7   377   63.9           SG13S114   T   0.026   1.24   477   70.4   602   65.8       Early   SG13S100   A   0.0440   1.43   99   71.7   377   63.9       onset                  
 
      Nominally significant SNP association with corresponding number of patients (# Pat.) and controls (#Ctrl). RR refers to relative risk.  
      However, after adjusting for the number of markers tested, these results were not significant. A search was then conducted for haplotypes that show association to the disease using the same cohorts. For computational reasons, the search was limited to haplotype combinations constructed out of two, three or four SNPs and only haplotypes that were in excess in the patients were tested. The resulting P values were adjusted for all the haplotypes tested by randomizing the patients and controls (see Methods).  
      Several haplotypes were found that were significantly associated to the disease with an adjusted P value less that 0.05 (Table 5).  
               TABLE 5                       SNP haplotypes that significantly associate with Icelandic MI patients                                                                                    SG13S4   SG13S6   SG13S372   SG13S25   SG13S377   SG13S100   SG13S95   SG13S114   SG13S192   SG13S137   SG13S86   SG13S87   SG13S39                           G               T                   G               T               A                   G               T                   G       A                       A                   G           T   T                   G               T           G                   G       A                   G       A                   G               T                   G               T           G                   T   T                   G       A                   G                   G               T   A                   G               T           G                       T           G       G               T           G               A                   G       A           G               A                       A                   G       A           G                       T               A                   G       A           A                   G               T                   G           G               A           G                       T                   G       A           G                       T           G       G       A                   G       A           A                   G               T   A                   G       A           A                   G               T       C                   G               T                   G               T       C                   G   G   A           G                       T           G                       T           G           G               A                   G       A                           G       C   G               A           G                       T   A           G               A           G                       T                   G               T           G   G           A           G       G       A           A                                                                             SG13S27   SG13S89   SG13S96   SG13S32   SG13S41   SG13S42   SG13S34   SG13S188   P value a     P value b     Pat.frq   Ctrl.frq   RR   D′ c                     G       A                   0.0000023   0.005   0.158   0.095   1.80   1.00                   A                   0.0000030   0.006   0.158   0.095   1.78   1.00                   A           T       0.0000032   0.007   0.157   0.094   1.79   1.00                   A                   0.0000046   0.012   0.158   0.083   2.07   0.89                   A                   0.0000047   0.012   0.154   0.093   1.78   1.00                   A                   0.0000055   0.015   0.147   0.087   1.81   1.00                   A           T       0.0000061   0.017   0.157   0.083   2.07   0.89           G       A                   0.0000063   0.017   0.157   0.084   2.04   0.89                   A                   0.0000070   0.021   0.157   0.096   1.76   1.00               A   A                   0.0000075   0.022   0.149   0.089   1.78   1.00                   A                   0.0000083   0.024   0.208   0.139   1.62   0.99                   A                   0.0000084   0.026   0.145   0.074   2.14   0.88                   A                   0.0000084   0.026   0.139   0.082   1.82   1.00       G           A                   0.0000091   0.028   0.156   0.096   1.75   1.00                   A           T       0.0000094   0.028   0.210   0.141   1.61   0.99                   A                   0.0000100   0.028   0.156   0.096   1.74   1.00                   A               A   0.0000101   0.028   0.215   0.133   1.80   0.81                   A                   0.0000105   0.028   0.157   0.084   2.03   0.89                   A                   0.0000108   0.029   0.214   0.133   1.78   0.81               A   A                   0.0000110   0.030   0.146   0.075   2.10   0.88                   A                   0.0000112   0.030   0.212   0.144   1.60   1.00                               T       0.0000113   0.030   0.151   0.081   2.03   0.78                   A                   0.0000118   0.031   0.156   0.096   1.73   1.00                   A           T       0.0000126   0.034   0.212   0.131   1.79   0.79           G       A                   0.0000129   0.035   0.211   0.144   1.59   1.00       G           A                   0.0000134   0.035   0.156   0.084   2.01   0.89                   A                   0.0000136   0.036   0.211   0.143   1.60   1.00                   A                   0.0000137   0.036   0.156   0.085   2.00   0.89               A                       0.0000148   0.037   0.151   0.081   2.01   0.78                               T       0.0000150   0.037   0.160   0.099   1.73   0.87                   A                   0.0000150   0.037   0.130   0.066   2.13   0.90                               T       0.0000154   0.039   0.152   0.094   1.73   0.93                   A       A           0.0000154   0.040   0.155   0.097   1.70   1.00                   A                   0.0000157   0.040   0.141   0.085   1.76   1.00                   A                   0.0000158   0.040   0.152   0.084   1.94   0.90       G           A                   0.0000163   0.040   0.210   0.143   1.59   0.99                   A                   0.0000166   0.041   0.200   0.134   1.61   0.92           G       A                   0.0000168   0.042   0.213   0.133   1.76   0.81                   A                   0.0000168   0.042   0.156   0.084   2.00   0.89                   A                   0.0000171   0.042   0.211   0.136   1.70   0.81                   A                   0.0000183   0.043   0.192   0.128   1.62   0.85                   A                   0.0000184   0.043   0.212   0.132   1.77   0.81                       A       T       0.0000193   0.046   0.328   0.251   1.46   0.99       G                       T       0.0000194   0.046   0.175   0.115   1.64   0.98                   A                   0.0000202   0.048   0.210   0.136   1.70   0.81                                       0.0000209   0.049   0.151   0.082   2.00   0.76                   a Single test P values.              b P values adjusted for all the SNP haplotypes tested.              c Measure of correlation with Haplotype A4.             
 
      The most significant association was observed for a four SNP haplotype spanning 33 kb, including the first four exons of the gene ( FIG. 6 . 3 ), with a nominal P value of 0.0000023 and an adjusted P value of 0.005. This haplotype, labelled A4, has haplotype frequency of 15.8% (carrier frequency 30.3%) in patients versus 9.5% (carrier frequency 17.9%) in controls (Table 6).  
               TABLE 6                          Association of the A4 haplotype to MI, Stroke and PAOD                                     Phenotype (n)   Frq. Pat.   RR   PAR   P-value   P-value a                                               MI (779)   0.158   1.80   0.135   0.0000023   0.005       Males (486)   0.169   1.95   0.158   0.00000091   ND b         Females (293)   0.138   1.53   0.094   0.0098   ND       Early onset (358)   0.138   1.53   0.094   0.0058   ND       Stroke (702) c     0.149   1.67   0.116   0.000095   ND       Males (373)   0.156   1.76   0.131   0.00018   ND       Females (329)   0.141   1.55   0.098   0.0074   ND       PAOD (577) c     0.122   1.31   0.056   0.061   ND       Males (356)   0.126   1.36   0.065   0.057   ND       Females (221)   0.114   1.22   0.041   0.31   ND                   a P value adjusted for the number of haplotypes tested.              b Not done.              c Excluding known cases of MI. Shown is the FLAP A4 haplotype and corresponding number of patients (n), haplotype frequency in patients (Frq. pat.), relative risk (RR), population attributed risk (PAR) and P values.          # The A4 haplotype is defined by the following SNPs: SG13S25, SG13S114, SG13S89 and SG13S32 (Table 5). The same controls (n = 628) are used for the association analysis in MI, stroke and PAOD as well as for the male, female and early onset analysis.        # The A4 haplotype frequency in the control cohort is 0.095.           
 
      The relative risk conferred by The A4 haplotype compared to other haplotypes constructed out of the same SNPs, assuming a multiplicative model, was 1.8 and the corresponding population attributable risk (PAR) was 13.5%. As shown in Table 6, the A4 haplotype was observed in higher frequency in male patients (carrier frequency 30.9%) than in female patients (carrier frequency 25.7%). All the other haplotypes that were significantly associated with an adjusted P value less than 0.05, were highly correlated with the A4 haplotype and should be considered variants of that haplotype (Table 5). Table 6 shows the results of the haplotype A4 association study using 779 MI patients, 702 stroke patients, 577 PAOD patients and 628 controls. First and second degree relatives were excluded from the patient cohorts. All known cases of MI were removed from the stroke and PAOD cohorts before testing for association. A significant association of the A4 haplotype to stroke was observed, with a relative risk of 1.67 (P value=0.000095). In addition, it was determined whether the A4 haplotype was primarily associated with a particular sub-phenotype of stroke, and found that both ischemic and hemorrhagic stroke were significantly associated with the A4 haplotype (Table 22, below).  
      More Variants of Haplotype A4  
      Two correlated series of SNP haplotypes were observed in excess in patients, denoted as A and B in Table 7. The length of the haplotypes varies between 33 and 69 Kb, and the haplotypes cover one or two blocks of linkage disequilibrium. Both series of haplotypes contain the common allele G of the SNP SG13S25. All haplotypes in the A series contain the SNP SG13S114, while all haplotypes in the B series contain the SNP SG13S106. In the B series, the haplotypes B4, B5, and B6 have a relative risk (RR) greater than 2 and with allelic frequencies above 10%. The haplotypes in A series have slightly lower RR and lower p-values, but higher frequency (15-16%). The haplotypes in series B and A are strongly correlated, i.e. the haplotypes in B define a subset of the haplotypes in A. Hence, haplotypes B are more specific than A. Haplotypes A are however more sensitive, i.e. they capture more individuals with the putative mutation, as is observed in the population attributable risk which is less for B than for A. Furthermore, these haplotypes show similar risk ratios and allelic frequency for early-onset patients (defined as onset of first MI before the age of 55) and for both gender. In addition, analyzing various groups of patients with known risk factors, such as hypertension, high cholesterol, smoking and diabetes, did not reveal any significant correlation with these haplotypes, indicating that the haplotypes in the FLAP gene represent an independent genetic susceptibility factor for MI.  
               TABLE 7                       The selected SNP haplotypes and the corresponding p-values                                                                                    p-val   RR   #aff   aff.frq.   carr.frq.   #con   con.frq.   PAR   SG13S99   SG13S25   SG13S377               B4   4.80E−05   2.08   903   0.106   0.2   619   0.054   0.11       G       B5   2.40E−05   2.2   910   0.101   0.19   623   0.049   0.11   T   G       B6   1.80E−06   2.22   913   0.131   0.24   623   0.063   0.14   T   G   G       A4   5.10E−06   1.81   919   0.159   0.29   623   0.095   0.14       G       A5   2.60E−06   1.91   920   0.15   0.28   624   0.085   0.14   T   G                                                                 SG13S106   SG13S114   SG13S89   SG13S30   SG13S32   SG13S42   SG13S35                       B4   G           G       A           B5   G           G       A           B6   G               A       G           A4       T   G       A           A5       T   G       A                         Relative risk (RR),                number of patients (#aff),                allelic frequency in patients (aff.frq.),                carrier frequency in patients (carr.frq.),                number of controls (#con),                allelic frequency in controls (con.frq.),                population attributable risk (PAR).                The patients used for this analysis were all unrelated within 4 meioses.             
 
      Haplotype Association to Female MI  
      Before we had typed all the SNPs that eventually lead to the identification of A4 haplotype we performed a haplotype association analysis that included 437 female MI patients, 1049 male MI patients, and 811 controls that had been genotyped with several SNPs and 3 microsatellite markers. These markers were all located within 300 kb region encompassing the FLAP gene. In a case-control study of the MI patients using these data, several haplotypes were found, that were significantly over-represented in the female MI patients compared to controls (see Table 8). All these haplotypes were highly correlated with each other.  
               TABLE 8                       haplotypes in the FLAP region (FLAP and flanking nucleotide       sequences) that were associated with female MI.                                                                            SG13S421   SG13S418   SG13S419   SG13S420   DG13S166   SG13S106   SG13S114   SG13S121   SG13S122   SG13S88   SG13S181                   C       T   0           C       T   0       T       A   T           C       T   0       T           T           C       T   0       T   G       T           C       T   0       T           T           C   A   T   0       T           T           C       T   0       T       A   T           C       T   0       T           T   G           C       T   0           G       T           C       T   0           G       T           C       T   0   G               T           C       T   0               A   T           C       T   0               A   T           C       T   0           G   A   T       A   C       T   0           G       T       A   C       T   0                   T       A   C       T   0       T           T           C   A   T   0           G       T       A   C       T   0               A   T       A   C   A   T   0                   T                                                                         SG13S184   D13S1238   DG13S2605   p-val   N_aff   aff.frq   N_ctrl   ctrl.frq   rel_risk   PAR   info                       G   −2       1.30E−05   455   0.108   811   0.048   2.4   0.122   0.615               −2   0   7.61E−06   455   0.065   812   0.02   3.45   0.091   0.615               −2   0   8.82E−06   455   0.065   812   0.02   3.47   0.092   0.602               −2   0   9.31E−06   455   0.065   812   0.02   3.39   0.089   0.611           G   −2   0   6.91E−06   455   0.063   812   0.019   3.54   0.09   0.624               −2   0   9.76E−06   455   0.063   812   0.019   3.51   0.089   0.606           G   −2       1.09E−05   455   0.063   811   0.019   3.41   0.086   0.618               −2   0   1.10E−05   455   0.063   812   0.019   3.44   0.087   0.611           G   −2   0   1.11E−05   455   0.063   812   0.018   3.56   0.086   0.589           G   −2       1.22E−05   455   0.063   811   0.018   3.6   0.087   0.577           G   −2   0   1.26E−05   455   0.063   812   0.02   3.35   0.088   0.629           G   −2   0   8.59E−06   455   0.062   812   0.018   3.53   0.085   0.62           G   −2       1.20E−05   455   0.062   811   0.019   3.42   0.086   0.617           G   −2       1.21E−05   455   0.062   811   0.019   3.43   0.086   0.619           G   −2       7.93E−06   455   0.061   811   0.016   3.95   0.088   0.562           G   −2       1.09E−05   455   0.061   811   0.017   3.85   0.09   0.56           G   −2       5.00E−06   455   0.06   811   0.015   4.11   0.087   0.576           G   −2       1.31E−05   455   0.06   811   0.017   3.66   0.085   0.586           G   −2       8.53E−06   455   0.059   811   0.016   3.85   0.085   0.593           G   −2       9.63E−06   455   0.058   811   0.015   4.03   0.085   0.565                      
 
      Table 9 shows two haplotypes that are representative of these female MI risk haplotypes. The relative risk of these haplotypes were 2.4 and 4, and they were carried by 23% and 13% of female MI patients, respectively.  
               TABLE 9                       Two representative variants of the female MI “at risk” haplotypes                                                                    SG13S418   SG13S420   DG13S166   SG13S114   SG13S88   SG13S184   D13S1238               Female MI   C   T   0   T   T   G   −2           C   T   0           G   −2                                                                     p-val   N_aff   aff.frq   N_ctrl   ctrl.frq   rel_risk   PAR   info                       Female MI   6.38E−06   454   0.059   809   0.015   4.05   0.086   0.577               2.74E−05   447   0.106   809   0.048   2.33   0.116   0.623                         p-val: p-value for the association.                N_aff: Number of patients used in the analysis.                aff. frq: haplotype frequency in patients.                N_ctrl: number of controls used in the analysis.                ctrl.frq: Haplotype frequency in controls.                rel_risk: Relative risk of the haplotype.                PAR: population attributable risk.                info: information content.             
 
      Table 10 shows that these same haplotypes were also over-represented in male MI patients compared to controls, although with lower relative risk. It should be noted that after typing and analysing more SNPs in the FLAP region these female MI “at risk” haplotypes could no longer be considered significant after adjusting for multiple testing.  
               TABLE 10                       The frequencies of the female MI “at risk” haplotypes in male patients vs controls.                                                                    SG13S418   SG13S420   DG13S166   SG13S114   SG13S88   SG13S184   D13S1238               Male MI   C   T   0   T   T   G   −2           C   T   0           G   −2                                                                     p-val   N_aff   aff.frq   N_ctrl   ctrl.frq   rel_risk   PAR   Info                       Male MI   3.37E−01   1087   0.027   809   0.021   1.32   0.013   0.577               5.39E−01   1087   0.056   809   0.05   1.13   0.013   0.568                         p-val: p-value for the association.                N_aff: Number of patients used in the analysis.                aff.frq: haplotype frequency in patients.                N_ctrl: number of controls used in the analysis.                ctrl.frq: Haplotype frequency in controls.                rel_risk: Relative risk of the haplotype.                PAR: population attributable risk.                Info: information content.             
 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                   
               
               
                 The marker map for chromosome 13 used in the linkage analysis. 
               
            
           
           
               
               
            
               
                 Location (cM) 
                 Marker 
               
               
                   
               
            
           
           
               
               
            
               
                 6 
                 D13S175 
               
               
                 9.8 
                 D13S1243 
               
               
                 13.5 
                 D13S1304 
               
               
                 17.2 
                 D13S217 
               
               
                 21.5 
                 D13S289 
               
               
                 25.1 
                 D13S171 
               
               
                 28.9 
                 D13S219 
               
               
                 32.9 
                 D13S218 
               
               
                 38.3 
                 D13S263 
               
               
                 42.8 
                 D13S326 
               
               
                 45.6 
                 D13S153 
               
               
                 49.4 
                 D13S1320 
               
               
                 52.6 
                 D13S1296 
               
               
                 55.9 
                 D13S156 
               
               
                 59.8 
                 D13S1306 
               
               
                 63.9 
                 D13S170 
               
               
                 68.7 
                 D13S265 
               
               
                 73 
                 D13S167 
               
               
                 76.3 
                 D13S1241 
               
               
                 79.5 
                 D13S1298 
               
               
                 81.6 
                 D13S1267 
               
               
                 84.7 
                 D13S1256 
               
               
                 85.1 
                 D13S158 
               
               
                 87 
                 D13S274 
               
               
                 93.5 
                 D13S173 
               
               
                 96.7 
                 D13S778 
               
               
                 102.7 
                 D13S1315 
               
               
                 110.6 
                 D13S285 
               
               
                 115 
                 D13S293 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                   
               
               
                 Marker Map for the second step of Linkage Analysis 
               
            
           
           
               
               
            
               
                 Location (cM) 
                 Marker 
               
               
                   
               
            
           
           
               
               
            
               
                 1.758 
                 D13S175 
               
               
                 9.235 
                 D13S787 
               
               
                 11.565 
                 D13S1243 
               
               
                 16.898 
                 D13S221 
               
               
                 17.454 
                 D13S1304 
               
               
                 18.011 
                 D13S1254 
               
               
                 18.59 
                 D13S625 
               
               
                 19.308 
                 D13S1244 
               
               
                 19.768 
                 D13S243 
               
               
                 22.234 
                 D13S1250 
               
               
                 22.642 
                 D13S1242 
               
               
                 22.879 
                 D13S217 
               
               
                 25.013 
                 D13S1299 
               
               
                 28.136 
                 D13S289 
               
               
                 28.678 
                 D13S290 
               
               
                 29.134 
                 D13S1287 
               
               
                 30.073 
                 D13S260 
               
               
                 31.98 
                 D13S171 
               
               
                 32.859 
                 D13S267 
               
               
                 33.069 
                 D13S1293 
               
               
                 33.07 
                 D13S620 
               
               
                 34.131 
                 D13S220 
               
               
                 36.427 
                 D13S219 
               
               
                 39.458 
                 D13S1808 
               
               
                 40.441 
                 D13S218 
               
               
                 41.113 
                 D13S1288 
               
               
                 41.996 
                 D13S1253 
               
               
                 42.585 
                 D13S1248 
               
               
                 44.288 
                 D13S1233 
               
               
                 44.377 
                 D13S263 
               
               
                 45.535 
                 D13S325 
               
               
                 45.536 
                 D13S1270 
               
               
                 45.537 
                 D13S1276 
               
               
                 49.149 
                 D13S326 
               
               
                 49.532 
                 D13S1272 
               
               
                 52.421 
                 D13S168 
               
               
                 52.674 
                 D13S287 
               
               
                 60.536 
                 D13S1320 
               
               
                 64.272 
                 D13S1296 
               
               
                 71.287 
                 D13S156 
               
               
                 76.828 
                 D13S1306 
               
               
                 77.86 
                 D13S170 
               
               
                 82.828 
                 D13S265 
               
               
                 91.199 
                 D13S1241 
               
               
                 93.863 
                 D13S1298 
               
               
                 97.735 
                 D13S779 
               
               
                 100.547 
                 D13S1256 
               
               
                 102.277 
                 D13S274 
               
               
                 111.885 
                 D13S173 
               
               
                 112.198 
                 D13S796 
               
               
                 115.619 
                 D13S778 
               
               
                 119.036 
                 D13S1315 
               
               
                 126.898 
                 D13S285 
               
               
                 131.962 
                 D13S293 
               
               
                   
               
            
           
         
       
     
      Table 13 shows the exons with positions that encode the FLAP protein, markers, polymorphisms and SNPs identified within the genomic sequence by the methods described herein.  
                                                                                                           start   end       NCBI   NCBI                           minor   position   position       build34;   build34;                           allele   in   in       start on chr.   stop on chr.   SNP/marker/           public   Vari-   minor   frequency   SEQ ID   SEQ ID       13 (bp)   13 (bp)   exon name   alias1   alias2   SNP   ation   allele   (%)   NO: 1   NO: 1                                                                            28932432   28932432   SG13S421       DG00AAFQR   rs1556428   A/G   G   10.32   432   432       28960356   28960356   SG13S417       SNP13B_R1028729   rs1028729   C/T   G   30.46   28356   28356       28965803   28965803   SG13S418       SNP13B_Y1323898   rs1323898   A/G   T   37.38   33803   33803       28974627   28974627   SG13S44               A/G   G   0.545   42627   42627       28975101   28975101   SG13S45               C/G   G   1.111   43101   43101       28975315   28975315   SG13S46               A/G   G   0.328   43315   43315       28975353   28975353   SG13S50               C/T   C   0.495   43353   43353       28975774   28975774   SG13S52               A/G   A   6.993   43774   43774       28985244   28985244   SG13S53           rs1408167   A/C   C   30.876   53244   53244       28985303   28985303   SG13S55           rs1408169   A/G   G   6.731   53303   53303       28985423   28985423   SG13S56               G/T   T   0.353   53423   53423       28985734   28985734   SG13S57           rs6490471   C/T   C   31.356   53734   53734       28985902   28985902   SG13S58           rs6490472   A/G   A   30.935   53902   53902       29003869   29003869   SG13S59               C/G   G   5.492   71869   71869       29004696   29004696   SG13S60               A/G   A   1.812   72696   72696       29007670   29007670   SG13S419       SNP13B_K912392   rs912392   C/T   G   35.00   75670   75670       29015410   29015410   SG13S61               C/T   C   1.314   83410   83410       29025792   29025792   SG13S62               C/T   T   3.521   93792   93792       29026202   29026202   SG13S63           rs7997114   A/G   A   30.031   94202   94202       29026668   29026668   SG13S64               A/G   A   1.724   94668   94668       29038707   29038707   SG13S65               A/G   A   0.369   106707   106707       29042180   29042180   SG13S420       DG00AAFIV   rs2248564   A/T   A   13.66   110180   110180       29049355   29049355   SG13S66               A/G   A   20.779   117355   117355       29049446   29049446   SG13S67               C/T   T   5.965   117446   117446       29050416   29050416   SG13S69               A/C   A   16.923   118416   118416       29059348   29059348   SG13S70               A/G   A   34.364   127348   127348       29059383   29059383   SG13S71               A/G   A   8.537   127383   127383       29059402   29059402   SG13S72               G/T   T   25.536   127402   127402       29063702   29063949   D13S289                           131702   131949       29064359   29064753   DG13S166                           132359   132753       29066272   29066272   SG13S73               A/G   A   37.302   134272   134272       29070551   29070551   SG13S99   SNP_13_Y1323892   DG00AAFIU   rs1323892   C/T   C   6.25   138551   138551       29081983   29081983   SG13S382   FLA267479           A/G   A   0.49   149983   149983       29082200   29082200   SG13S383   FLA267696           A/G   A   14.08   150200   150200       29082357   29082357   SG13S384   FLA267853           A/G   G   0.62   150357   150357       29083350   29083350   SG13S381   FLA268846   DG00AAJER       C/G   G   14.01   151350   151350       29083518   29083518   SG13S366   FLA269014   DG00AAJES   rs4312166   A/G   T   0.58   151518   151518       29085102   29085102   SG13S385   FLA270742           C/T   C   30.21   153102   153102       29085190   29085190   SG13S386   FLA270830           A/G   A   10.95   153190   153190       29086224   29086224   SG13S1   FLA271864           G/T   G   30.00   154224   154224       29087473   29087473   SG13S2   FLA273371           A/G   A   27.95   155473   155473       29088090   29088090   SG13S367   FLA273988   DG00AAJEU   rs4474551   A/G   G   2.41   156090   156090       29088186   29088186   SG13S388   FLA274084           A/G   A   0.39   156186   156186       29088473   29088473   SG13S10   FLA274371           A/T   T   10.23   156473   156473       29089044   29089044   SG13S3   FLA274942           C/T   T   15.17   157044   157044       29089886   29089886   SG13S368   FLA275784   DG00AAJEV       C/T   T   13.60   157886   157886       29090025   29090025   SG13S369   FLA275923   DG00AAJEW       G/T   G   12.44   158025   158025       29090054   29090054   SG13S370   FLA275952   DG00AAJEX       A/G   A   13.45   158054   158054       29090997   29090997   SG13S4   FLA276895           G/C   G   14.59   158997   158997       29091307   29091307   SG13S5   FLA277205       rs4238133   G/T   T   26.84   159307   159307       29091580   29091580   SG13S389   FLA277478           A/G   A   12.73   159580   159580       29091780   29091780   SG13S90   FLA277678           A/C   C   43.67   159780   159780       29092287   29092287   SG13S390   FLA278185       rs5004913   A/G   A   12.18   160287   160287       29092536   29092536   SG13S6   FLA278434           A/G   A   8.38   160536   160536       29092594   29092594   SG13S391   FLA278492           A/G   G   0.62   160594   160594       29092947   29092947   SG13S392   FLA278845           G/T   T   12.34   160947   160947       29093964   29093964   SG13S371   FLA279888   DG00AAJEY   rs4409939   A/G   G   25.34   161964   161964       29094259   29094259   SG13S372   FLA280183   DG00AAJEZ       A/G   C   0.24   162259   162259       29094999   29094999   SG13S393   FLA280923           A/T   T   25.66   162999   162999       29096688   29096688   SG13S373   FLA282612   DG00AAJFA       A/G   A   14.84   164688   164688       29096813   29096813   SG13S374   FLA282737   DG00AAJFB       A/G   G   12.37   164813   164813       29096874   29096874   SG13S375   FLA282798   DG00AAJFC       C/T   C   14.55   164874   164874       29096962   29096962   SG13S376   FLA282886   DG00AAJFD       A/G   G   11.99   164962   164962       29097476   29097476   SG13S394   FLA283400           C/G   C   14.66   165476   165476       29097553   29097553   SG13S25   FLA283477           A/G   A   12.21   165553   165553       29098486   29098486   SG13S395   FLA284410           A/G   A   0.79   166486   166486       29098891   29098891   SG13S396   FLA284815           A/C   C   10.15   166891   166891       29098979   29098979   SG13S397   FLA284903           C/T   C   3.53   166979   166979       29101965   29101965   SG13S377   FLA287889   DG00AAJFF       A/G   A   12.45   169965   169965       29103909   29103909   SG13S189   FLA289833           C/G   C   0.62   171909   171909       29104271   29104271   SG13S100   FLA290195   DG00AAHIK   rs4073259   A/G   G   31.55   172271   172271       29104629   29104629   SG13S398   FLA290553           C/G   G   4.94   172629   172629       29104646   29104646   SG13S94   FLA290570       rs4073261   C/T   C   15.51   172646   172646       29105099   29105099   SG13S101   FLA291023       rs4075474   C/T   T   27.91   173099   173099       29106329   29106329   SG13S95   FLA292253           G/T   G   14.74   174329   174329       29106652   29106652   SG13S102   FLA292576           A/T   T   1.17   174652   174652       29107138   29107138   SG13S103   FLA293062           C/T   T   1.28   175138   175138       29107404   29107404   SG13S104   FLA293328           A/G   A   2.17   175404   175404       29107668   29107812   EXON1                           175668   175812       29107830   29107830   SG13S191   FLA293754   DG00AAFJT   rs4769055   A/C   A   30.11   175830   175830       29108398   29108398   SG13S105   FLA294322           A/G   G   0.66   176398   176398       29108579   29108579   SG13S106   FLA294503   DG00AAHII       A/G   A   28.31   176579   176579       29108919   29108919   SG13S107   FLA294843       rs4075131   A/G   G   14.85   176919   176919       29108972   29108972   SG13S108   FLA294896       rs4075132   C/T   C   1.21   176972   176972       29109112   29109112   SG13S109   FLA295036           A/G   A   1.04   177112   177112       29109182   29109182   SG13S110   FLA295106           A/G   G   0.88   177182   177182       29109344   29109344   SG13S111   FLA295268       rs4597169   C/T   C   1.14   177344   177344       29109557   29109557   SG13S112   FLA295481           C/T   T   7.10   177557   177557       29109773   29109773   SG13S113   FLA295697       rs4293222   C/G   C   22.52   177773   177773       29110096   29110096   SG13S114   FLA296020   DG00AAHID       A/T   A   20.86   178096   178096       29110178   29110178   SG13S115   FLA296102           A/T   T   13.83   178178   178178       29110508   29110508   SG13S116   FLA296432       rs4769871   C/T   T   4.05   178508   178508       29110630   29110630   SG13S117   FLA296554       rs4769872   A/G   A   4.07   178630   178630       29110689   29110689   SG13S118   FLA296613       rs4769873   C/T   T   4.07   178689   178689       29110862   29110862   SG13S119   FLA296786           A/G   A   1.06   178862   178862       29111889   29111889   SG13S120   FLA297813           C/T   C   16.00   179889   179889       29112174   29112174   SG13S121   FLA298098   DG00AAHIJ   rs4503649   A/G   G   49.36   180174   180174       29112264   29112264   SG13S122   FLA298188   DG00AAHIH       A/G   A   29.75   180264   180264       29112306   29112306   SG13S123   FLA298230           C/T   T   5.06   180306   180306       29112455   29112455   SG13S43   FLA298379       rs3885907   A/C   C   46.23   180455   180455       29112583   29112583   SG13S399   FLA298507           A/C   C   1.59   180583   180583       29112680   29112680   SG13S124   FLA298604       rs3922435   C/T   T   1.45   180680   180680       29113139   29113139   SG13S125   FLA299063           A/G   G   11.32   181139   181139       29114056   29114056   SG13S400   FLA299980           A/G   A   3.25   182056   182056       29114738   29114738   SG13S126   FLA300662           A/G   A   34.12   182738   182738       29114940   29114940   SG13S127   FLA300864           A/G   G   29.63   182940   182940       29115878   29115878   SG13S128   FLA302094       rs4254165   A/G   A   45.68   183878   183878       29116020   29116020   SG13S129   FLA302236       rs4360791   A/G   G   36.65   184020   184020       29116068   29116068   SG13S130   FLA302284           G/T   G   8.07   184068   184068       29116196   29116296   EXON2                           184196   184296       29116249   29116249   SG13S190   FLA302465           C/T   T   1.02   184249   184249       29116308   29116308   SG13S192   FLA302524   B_SNP_302524   rs3803277   A/C   A   49.57   184308   184308       29116344   29116344   SG13S193   FLA302560           A/G   A   0.58   184344   184344       29116401   29116401   SG13S88   FLA302617   B_SNP_302617   rs3803278   C/T   C   24.71   184401   184401       29116688   29116688   SG13S131   FLA302904           C/T   T   7.19   184688   184688       29117133   29117133   SG13S132   FLA303349           A/C   A   1.10   185133   185133       29117546   29117546   SG13S133   FLA303762       rs4356336   C/T   T   37.65   185546   185546       29117553   29117553   SG13S38   FLA303769       rs4584668   A/T   A   45.50   185553   185553       29117580   29117580   SG13S134   FLA303796           C/T   T   1.22   185580   185580       29117741   29117741   SG13S135   FLA303957       rs4238137   C/T   T   0.89   185741   185741       29117954   29117954   SG13S136   FLA304170       rs4147063   C/T   T   36.69   185954   185954       29118118   29118118   SG13S137   FLA304334   DG00AAHIG   rs4147064   C/T   T   29.11   186118   186118       29118815   29118815   SG13S86   FLA305031           A/G   A   30.19   186815   186815       29118873   29118873   SG13S87   FLA305089   DG00AAHOJ       A/G   G   3.29   186873   186873       29119069   29119069   SG13S138   FLA305285           C/T   T   36.96   187069   187069       29119138   29119138   SG13S139   FLA305354           C/G   G   36.63   187138   187138       29119289   29119289   SG13S140   FLA305505           A/G/T   T   37.34   187289   187289       29119462   29119462   SG13S141   FLA305678           C/T   C   1.15   187462   187462       29119740   29119740   SG13S39   FLA305956           G/T   T   9.91   187740   187740       29120939   29120939   SG13S142   FLA307155       rs4387455   C/T   C   3.36   188939   188939       29120949   29120949   SG13S143   FLA307165       rs4254166   C/T   T   36.24   188949   188949       29121342   29121342   SG13S144   FLA307558       rs4075692   A/G   A   31.58   189342   189342       29121572   29121572   SG13S145   FLA307788           C/G   G   0.45   189572   189572       29121988   29121988   SG13S146   FLA308204           C/T   T   1.14   189988   189988       29122253   29122253   SG13S26   FLA308469           C/T   T   46.57   190253   190253       29122283   29122283   SG13S27   FLA308499           A/G   A   10.34   190283   190283       29122294   29122294   SG13S147   FLA308510           C/T   T   8.00   190294   190294       29122298   29122298   SG13S28   FLA308514           G/T   T   33.71   190298   190298       29122311   29122311   SG13S148   FLA308527           G/T   T   2.29   190311   190311       29123370   29123370   SG13S98   FLA309586           G/T   G   1.19   191370   191370       29123635   29123635   SG13S149   FLA309851           A/G   A   1.01   191635   191635       29123643   29123643   SG13S29   FLA309859           A/C   A   47.88   191643   191643       29124188   29124259   EXON3                           192188   192259       29124441   29124441   SG13S89   FLA310657   B_SNP_310657   rs4769874   A/G   A   4.68   192441   192441       29124906   29124906   SG13S96   FLA311122       rs4072653   A/G   G   29.72   192906   192906       29125032   29125032   SG13S150   FLA311248           C/G   C   8.22   193032   193032       29125521   29125521   SG13S401   FLA311737           C/T   C   21.10   193521   193521       29125822   29125822   SG13S151   FLA312038           C/T   T   8.57   193822   193822       29125840   29125840   SG13S30   FLA312056           G/T   T   23.23   193840   193840       29127301   29127301   SG13S31   FLA313550           C/T   T   24.20   195301   195301       29128080   29128162   EXON4                           196080   196162       29128284   29128284   SG13S152   FLA314500           C/G   C   23.89   196284   196284       29128316   29128316   SG13S402   FLA314532       rs4468448   C/T   T   19.33   196316   196316       29128798   29128798   SG13S403   FLA315014       rs4399410   A/G   G   11.50   196798   196798       29129016   29129016   SG13S153   FLA315232           A/T   T   3.08   197016   197016       29129139   29129139   SG13S97   FLA315355           A/G   A   9.72   197139   197139       29129154   29129154   SG13S154   FLA315370           C/T   T   0.98   197154   197154       29129395   29129395   SG13S40   FLA315611           G/T   T   2.24   197395   197395       29129915   29129915   SG13S155   FLA316131       rs4769875   A/G   A   1.43   197915   197915       29130192   29130192   SG13S156   FLA316408           A/C   A   1.80   198192   198192       29130256   29130256   SG13S157   FLA316472           A/G   G   2.38   198256   198256       29130299   29130299   SG13S158   FLA316515           A/C   A   0.61   198299   198299       29130353   29130353   SG13S159   FLA316569           G/T   G   2.55   198353   198353       29130391   29130391   SG13S160   FLA316607           C/T   T   0.83   198391   198391       29130547   29130547   SG13S32   FLA316763           A/C   C   48.50   198547   198547       29131280   29131280   SG13S161   FLA317496           A/G   G   2.44   199280   199280       29131403   29131403   SG13S162   FLA317619           A/G   G   2.45   199403   199403       29131404   29131404   SG13S163   FLA317620           C/T   C   2.45   199404   199404       29131431   29131431   SG13S164   FLA317647       rs4769058   C/T   C   2.55   199431   199431       29131517   29131517   SG13S165   FLA317733           A/T   T   20.00   199517   199517       29131528   29131528   SG13S166   FLA317744       rs4769059   C/T   T   2.46   199528   199528       29131599   29131599   SG13S167   FLA317815       rs4769876   A/G   A   3.50   199599   199599       29132003   29132003   SG13S168   FLA318219           A/C   C   8.39   200003   200003       29133753   29133753   SG13S33   FLA319969           G/T   T   8.99   201753   201753       29134045   29134045   SG13S41   FLA320261           A/G   G   5.41   202045   202045       29134177   29134177   SG13S169   FLA320393           A/G   G   4.12   202177   202177       29134379   29134379   SG13S404   FLA320595       rs4427651   G/T   G   38.33   202379   202379       29135558   29135558   SG13S170   FLA321774       rs3935645   C/T   C   32.77   203558   203558       29135640   29135640   SG13S171   FLA321856       rs3935644   A/G   G   48.03   203640   203640       29135750   29135750   SG13S172   FLA321966           A/G   G   1.67   203750   203750       29135809   29135809   SG13S173   FLA322025           A/T   A   0.68   203809   203809       29135877   29135877   SG13S42   FLA322093       rs4769060   A/G   G   42.44   203877   203877       29136080   29136556   EXON5                           204080   204556       29136290   29136290   SG13S194   FLA322506           C/T   T   0.30   204290   204290       29136462   29136462   SG13S195   FLA322678       rs1132340   A/G   G   2.46   204462   204462       29136797   29136797   SG13S174   FLA323013           A/G   G   0.56   204797   204797       29137100   29137100   SG13S34   FLA323316           G/T   G   30.23   205100   205100       29137150   29137150   SG13S175   FLA323366           A/G   A   2.40   205150   205150       29137607   29137607   SG13S176   FLA323823           A/G   A   2.24   205607   205607       29137651   29137651   SG13S177   FLA323867           C/T   T   1.64   205651   205651       29137905   29137905   SG13S178   FLA324121           C/G   C   1.40   205905   205905       29138117   29138117   SG13S35   FLA324333           A/G   A   9.52   206117   206117       29138375   29138375   SG13S179   FLA324591           A/G   A   48.14   206375   206375       29138385   29138385   SG13S180   FLA324601           C/T   T   2.50   206385   206385       29138633   29138633   SG13S181   FLA324849   DG00AAHIF   rs4420371   C/G   C   49.41   206633   206633       29139153   29139153   SG13S182   FLA325369           C/T   T   2.36   207153   207153       29139277   29139277   SG13S183   FLA325493       rs4466940   C/T   T   12.07   207277   207277       29139435   29139435   SG13S184   FLA325651   DG00AAHOI   rs4445746   A/G   A   16.67   207435   207435       29139971   29139971   SG13S185   FLA326187           A/G   G   7.66   207971   207971       29140441   29140441   SG13S405   FLA326657           A/G   A   9.66   208441   208441       29140649   29140649   SG13S91   FLA326865           A/G   A   7.78   208649   208649       29140695   29140695   SG13S186   FLA326911       rs4769877   A/T   A   25.71   208695   208695       29140703   29140703   SG13S187   FLA326919           A/G   A   1.43   208703   208703       29140805   29140805   SG13S188   FLA327021   DG00AAJFE       A/G   G   4.71   208805   208805       29141049   29141049   SG13S406   FLA327265           C/T   T   0.56   209049   209049       29142392   29142392   SG13S92   FLA328644       rs4429158   C/T   T   8.33   210392   210392       29142397   29142397   SG13S93   FLA328649           A/G   A   7.23   210397   210397       29142712   29142712   SG13S36   FLA328964           C/T   C   15.88   210712   210712       29144013   29144013   SG13S407   FLA330265           C/T   T   3.29   212013   212013       29144203   29144203   SG13S408   FLA330455           C/T   T   0.30   212203   212203       29144234   29144589   D13S1238                           212234   212589       29144255   29144255   SG13S7   FLA330507           C/T   T   16.28   212255   212255       29144877   29144877   SG13S37   FLA331129           A/G   G   16.70   212877   212877       29144982   29144982   SG13S409   FLA331234           A/G   A   1.93   212982   212982       29144983   29144983   SG13S8   FLA331235       rs4491352   A/C   C   30.64   212983   212983       29145122   29145122   SG13S410   FLA331374       rs4319601   C/T   T   20.57   213122   213122       29145143   29145143   SG13S411   FLA331395           A/G   A   1.54   213143   213143       29145171   29145171   SG13S9   FLA331423           C/T   C   16.37   213171   213171       29145221   29145221   SG13S412   FLA331473       rs4769062   A/G   A   7.42   213221   213221       29145265   29145265   SG13S413   FLA331517       rs4238138   C/T   T   1.91   213265   213265                  
 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                   
               
               
                 Extended 4 microsatellite marker haplotypes 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 4 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 markers 
                 : 
                 pos.rr-frqgt1perc 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Length 
                 p-val 
                 RR 
                 N_af 
                 P_al 
                 P_ca 
                 N_ct 
                 P_al 
                 P_ca 
                 Alleles 
                   
                   
                 Markers 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 0.88 
                 4.71E−06 
                 6.23 
                 428 
                 0.065 
                 0.125 
                 721 
                 0.011 
                 0.022 
                 0 
                 −12 
                 −6 
                 0 
                 DG13S80 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S83 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1110 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S163 
               
               
                 0.82 
                 8.60E−06 
                 INF 
                 438 
                 0.032 
                 0.062 
                 720 
                 0 
                 0 
                 0 
                 4 
                 2 
                 14 
                 DG13S1111 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1103 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S1287 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                 0.67 
                 6.98E−06 
                 19.91 
                 435 
                 0.03 
                 0.059 
                 721 
                 0.002 
                 0.003 
                 8 
                 6 
                 0 
                 8 
                 DG13S1103 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S163 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S290 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                 0.767 
                 4.85E−06 
                 26.72 
                 436 
                 0.048 
                 0.094 
                 721 
                 0.002 
                 0.004 
                 0 
                 0 
                 2 
                 12 
                 DG13S1101 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S166 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S1287 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                 0.515 
                 1.93E−06 
                 INF 
                 422 
                 0.048 
                 0.094 
                 721 
                 0 
                 0 
                 2 
                 0 
                 0 
                 6 
                 DG13S166 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S163 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S290 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                 0.864 
                 1.68E−06 
                 INF 
                 424 
                 0.024 
                 0.048 
                 717 
                 0 
                 0 
                 0 
                 2 
                 0 
                 −16 
                 DG13S166 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S163 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S293 
               
               
                 0.927 
                 5.38E−06 
                 INF 
                 435 
                 0.034 
                 0.067 
                 720 
                 0 
                 0 
                 4 
                 2 
                 14 
                 3 
                 DG13S1103 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S1287 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S301 
               
               
                   
               
               
                   Alleles #&#39;s: For SNP alleles A = 0, C = 1, G = 2, T = 3;    
               
               
                   for microsatellite alleles: the CEPH sample (Centre d&#39;Etudes du Polymorphisme Humain, genomics repository) is used as a reference, as described above.    
               
               
                   Length = length of haplotype in Mb.    
               
               
                   p-val = p-value.    
               
               
                   RR = Relative risk.    
               
               
                   N_af = Number of patients.    
               
               
                   P_al = allelic frequency of haplotype.    
               
               
                   P_ca = carrier frequency of haplotype.    
               
               
                   N_ct = number of controls.    
               
               
                   Alleles = alleles in the haplotype.    
               
               
                   Markers = markers in the haplotype.    
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                   
               
               
                 Extended 5 microsatellite marker haplotypes 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 5 markers 
                 : 
                 pos.rr-frqgt1perc 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Length 
                 p-val 
                 RR 
                 N_af 
                 P_al 
                 P_ca 
                 N_ct 
                 P_al 
                 P_ca 
                 Alleles 
                   
                   
                   
                 Markers 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 0.851 
                 7.45E−06 
                 15.43 
                 413 
                 0.034 
                 0.067 
                 715 
                 0.002 
                 0.005 
                 0 
                 18 
                 0 
                 0 
                 0 
                 DG13S79 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S1299 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S87 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S1246 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S166 
               
               
                 0.964 
                 8.07E−06 
                 INF 
                 437 
                 0.023 
                 0.045 
                 721 
                 0 
                 0 
                 0 
                 −12 
                 6 
                 8 
                 6 
                 DG13S79 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S83 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1104 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1103 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S163 
               
               
                 0.964 
                 2.38E−06 
                 INF 
                 437 
                 0.026 
                 0.052 
                 720 
                 0 
                 0 
                 0 
                 6 
                 0 
                 8 
                 6 
                 DG13S79 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1104 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S172 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1103 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S163 
               
               
                 0.931 
                 7.05E−06 
                 5.8 
                 429 
                 0.068 
                 0.131 
                 721 
                 0.012 
                 0.025 
                 0 
                 −6 
                 0 
                 0 
                 −2 
                 DG13S79 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1110 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S175 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S166 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S1238 
               
               
                 0.964 
                 8.13E−06 
                 INF 
                 434 
                 0.021 
                 0.041 
                 721 
                 0 
                 0 
                 0 
                 3 
                 8 
                 2 
                 6 
                 DG13S79 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1098 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1103 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S166 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S163 
               
               
                 0.597 
                 9.78E−06 
                  4.58 
                 428 
                 0.074 
                 0.143 
                 717 
                 0.017 
                 0.034 
                 −6 
                 0 
                 0 
                 0 
                 −2 
                 DG13S1110 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S89 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S175 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S166 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S1238 
               
               
                 0.896 
                 6.92E−06 
                 INF 
                 428 
                 0.026 
                 0.051 
                 721 
                 0 
                 0 
                 −12 
                 −6 
                 0 
                 −2 
                 2 
                 DG13S83 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1110 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S166 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S1238 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S290 
               
               
                 0.722 
                 2.18E−06 
                 INF 
                 453 
                 0.026 
                 0.051 
                 738 
                 0 
                 0 
                 −6 
                 0 
                 0 
                 −2 
                 2 
                 DG13S1110 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S289 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S166 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S1238 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S290 
               
               
                 0.982 
                 7.88E−06 
                 INF 
                 437 
                 0.028 
                 0.055 
                 721 
                 0 
                 0 
                 0 
                 0 
                 4 
                 2 
                 14 
                 DG13S87 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S175 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1103 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S1287 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                 0.841 
                 8.88E−06 
                 INF 
                 438 
                 0.032 
                 0.062 
                 720 
                 0 
                 0 
                 0 
                 0 
                 4 
                 2 
                 14 
                 DG13S89 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1111 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1103 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S1287 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                 0.841 
                 9.67E−07 
                 INF 
                 435 
                 0.029 
                 0.057 
                 721 
                 0 
                 0 
                 0 
                 8 
                 6 
                 0 
                 8 
                 DG13S89 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1103 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S163 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S290 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                 0.982 
                 7.90E−06 
                 18.63 
                 437 
                 0.026 
                 0.052 
                 721 
                 0.001 
                 0.003 
                 0 
                 4 
                 0 
                 2 
                 14 
                 DG13S87 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1103 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S166 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S1287 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                 0.841 
                 3.52E−06 
                 28.52 
                 436 
                 0.048 
                 0.094 
                 721 
                 0.002 
                 0.004 
                 0 
                 0 
                 0 
                 2 
                 12 
                 DG13S89 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1101 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S166 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S1287 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                 0.705 
                 5.28E−06 
                 INF 
                 435 
                 0.027 
                 0.053 
                 721 
                 0 
                 0 
                 0 
                 8 
                 6 
                 0 
                 8 
                 DG13S175 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1103 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S163 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S290 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                 0.841 
                 4.21E−06 
                 INF 
                 422 
                 0.048 
                 0.093 
                 721 
                 0 
                 0 
                 0 
                 2 
                 0 
                 0 
                 6 
                 DG13S89 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S166 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S163 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S290 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                 0.767 
                 4.02E−06 
                 28.11 
                 436 
                 0.049 
                 0.095 
                 721 
                 0.002 
                 0.004 
                 0 
                 0 
                 0 
                 2 
                 12 
                 DG13S1101 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S175 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S166 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S1287 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                 0.767 
                 1.29E−06 
                 31.07 
                 436 
                 0.047 
                 0.092 
                 721 
                 0.002 
                 0.003 
                 0 
                 0 
                 0 
                 2 
                 12 
                 DG13S1101 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S172 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S166 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S1287 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                 0.705 
                 4.25E−07 
                 INF 
                 422 
                 0.048 
                 0.093 
                 721 
                 0 
                 0 
                 0 
                 2 
                 0 
                 0 
                 6 
                 DG13S175 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S166 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S163 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S290 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                 0.683 
                 6.58E−06 
                 INF 
                 437 
                 0.029 
                 0.056 
                 721 
                 0 
                 0 
                 0 
                 4 
                 0 
                 2 
                 14 
                 DG13S172 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1103 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S166 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S1287 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                 0.767 
                 2.85E−06 
                 32.43 
                 436 
                 0.044 
                 0.087 
                 721 
                 0.001 
                 0.003 
                 0 
                 0 
                 0 
                 2 
                 12 
                 DG13S1101 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S166 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S290 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S1287 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                 0.865 
                 9.58E−06 
                 18.39 
                 451 
                 0.023 
                 0.045 
                 739 
                 0.001 
                 0.003 
                 0 
                 0 
                 2 
                 2 
                 −16 
                 D13S289 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S166 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S163 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S1287 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S293 
               
               
                 0.865 
                 5.08E−06 
                 INF 
                 453 
                 0.019 
                 0.038 
                 739 
                 0 
                 0 
                 0 
                 0 
                 2 
                 0 
                 −16 
                 D13S289 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S166 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S163 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S293 
               
               
                 0.927 
                 1.02E−07 
                 27.65 
                 437 
                 0.037 
                 0.073 
                 721 
                 0.001 
                 0.003 
                 4 
                 0 
                 2 
                 14 
                 3 
                 DG13S1103 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S166 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 D13S1287 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S1061 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 DG13S301 
               
               
                   
               
               
                   Length = length of haplotype in Mb.    
               
               
                   p-val = p-value.    
               
               
                   RR = Relative risk.    
               
               
                   N_af = Number of patients.    
               
               
                   P_al = allelic frequency of haplotype.    
               
               
                   P_ca = carrier frequency of haplotype.    
               
               
                   N_ct = number of controls.    
               
               
                   Alleles = alleles in the haplotype.    
               
               
                   Markers = markers in the haplotype    
               
            
           
         
       
     
     EXAMPLE 2  
     Relationship Between Polymorphism in the 5-Lipoxygenase Promoter and MI  
      A family of mutations in the G-C rich transcription factor binding region of the 5-LO gene has previously been identified. These mutations consist of deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. These naturally occurring mutations in the human 5-LO gene promoter have been shown to modify transcription factor binding and reporter gene transcription. The capacity of the mutant forms of DNA to promote transcription of CAT reporter constructs have been shown to be significantly less than that of the wild type DNA ( J. Clin. Invest.  Volume 99, Number 5, March 1997, 1130-1137).  
      To test whether 5-LO is associated with the atherosclerotic diseases, particularly myocardial infarction (MI) in the human population, this promoter polymorphism, consisting of variable number of tandem Sp1/Egr-1 binding sites, was genotyped in 1112 MI patients, 748 patients with PAOD, and 541 stroke patients.  
      The results, shown in Table 16, demonstrate that the wild type allele (which represents the allele with the most active promoter and thus with the highest expression of the 5-LO mRNA;  J. Clin. Invest.  Volume 99, Number 5, March 1997, 1130-1137) is significantly associated with MI (RR=1.2, p&lt;0.05). The results are consistent with a disease hypothesis that increased expression of the 5-LO plays a role in the pathogenesis of MI.  
                                           TABLE 16                                                   Risk   P-           N_aff   Frq_aff   N_ctrl   Frq_ctrl   Ratio   value                                                                MI patients   1112   0.8701   734   0.8501   1.1803   0.048       Independent   969   0.8720   734   0.8501   1.2013   0.037       Males   646   0.8740   734   0.8501   1.2232   0.039       Females   465   0.8645   734   0.8501   1.1249   0.180       Age of   522   0.8745   734   0.8501   1.2286   0.046       onset &lt;60       Males   353   0.8768   734   0.8501   1.2542   0.053       Females   169   0.8698   734   0.8501   1.1779   0.202       PAOD patients   748   0.8763   734   0.8501   1.2492   0.022       Independent   703   0.8755   734   0.8501   1.2400   0.027       Males   473   0.8774   734   0.8501   1.2613   0.033       Females   275   0.8745   734   0.8501   1.2289   0.092       Stroke   541   0.8743   734   0.8501   1.2262   0.046       patients       Males   326   0.8758   734   0.8501   1.2427   0.067       Females   215   0.8721   734   0.8501   1.2019   0.144       Cardio/Large V   202   0.8861   734   0.8501   1.3719   0.038       Cardioembolic   114   0.8772   734   0.8501   1.2592   0.165       Large Vessel   88   0.8977   734   0.8501   1.5474   0.053       Small Vessel   77   0.8831   734   0.8501   1.2791   0.163       Hemorrhagic   27   0.9259   734   0.8501   2.2035   0.081                 Single sided p-values: Fisher exact test.            N_aff = number of affected individuals;            Frq_aff = frequency of the wild type allele of the promoter polymorphism in the affected group;            N_ctrl = number of controls;            Frq_ctrl = frequency of the wild type allele of the promoter polymorphism in the control group;             
 
     EXAMPLE 3  
     Elevated LTE4 Biosynthesis in Individuals with the Flap MI-Risk Haplotype  
      Based on the known function of the end products of the leukotriene pathway and based on our 5-LO association data, the association of the FLAP haplotype with MI is best explained by increased expression and/or increased function of the FLAP gene. In other words, those individuals that have a “at risk” FLAP haplotype have either, or both, increased amount of FLAP, or more active FLAP. This would lead to increased production of leukotrienes in these individuals.  
      To demonstrate this theory, LTE4, a downstream leukotriene metabolite, was measured in patient serum samples. A quantitative determination of LTE4 in human serum was performed by liquid chromatography coupled with tandem mass spectrometry. The protocol was performed as follows:  
      Analytical Method  
               TABLE P1                       (Protocol 1): List of Abbreviations                                                CAN   Acetonitrile           IS   Internal standard           LC-MS/MS   Liquid chromatography tandem mass spectrometry           LOQ   Limit of quantification           QCs   Quality controls           R 2     Coefficient of determination           SS   Spiking solution                      
 
      Apparatus and Conditions  
               TABLE P2                          Analytical apparatus and conditions                     Instruments/Conditions   Details               Analytical column   Zorbax extend C 18 , 3.5 μm (50 × 2.1 mm)       Column temperature   Ambient       Pump and flow   Hewlett Packard Series 1100 Binary pump delivering 0.3 ml/min       Mobile phase   A: Buffer: Acetonitrile:H 2 O (5:95% v/v). (Containing 10 mM           Ammonium Acetate and 0.1% Acetic acid at pH 4.6).           B: Buffer: Acetonitrile:H 2 O (95:5% v/v). (Containing 10 mM           Ammonium Acetate and 0.1% Acetic acid at pH 4.6).                                     Gradient   Time   % A   % B   Flow rate               0.00   30   70   0.3 ml/min           1.00   30   70   0.3 ml/min           1.50   90   10   0.3 ml/min           6.00   90   10   0.3 ml/min           6.50   30   70   0.3 ml/min           10.00   30   70   0.3 ml/min                     Sample injection   HTC PAL autosampler 10 μl onto the HPLC column       Mass Spectrometric   Quattro Ultima ™ Tandem MS/MS, Micromass. England.       system       Recording and   Mass Lynx, version 3.5. All chromatograms and reports are       integration   printed out in hardcopy and stored in electronic form on the           workstation hard disk drive. Recording time was 10 min.       Retentions times   LTE 4 ˜3.05 min.           LTE 4 -d 3 ˜3.05 min.       Ionization mode   Electrospray atmospheric pressure in negative ion mode       Scan mode   Multiple reaction monitoring (MRM)                                 Compound   Parent ion   Daughter ion           LTE 4     438.2   333.2           LTE 4 -d 3     441.2   336.2                  
 
      Other Instruments  
               TABLE P3                          The apparatus used for sample treatment and measurements                                 Apparatus   Brand   Type                       Pipette   Eppendorf   Edos 5221           Pipette   Labsystems   Finnpipette 200 μl           Centrifuge   Eppendorf   5417C           Evaporation unit   Porvair   Ultravap           Vibrofix   Ika-Werk   VF-1               Thermolyne   Maxi-mix III ™, 65800           Balance   Sartorius   LA 120 S           Ultra sonic bath   Cole Parmer   8891                      
 
      Materials  
               TABLE P4                          Reagents for sample treatment and measurements                             Reagent   Manufacturer   Quality   Art no.               Acetonitrile (ACN)   Rathburn   HPLC grade   RH 1016       Methanol   Rathburn   HPLC grade   RH 1019       Ammonium acetate   Merck   Pro analysis   1116                  
 
     
       
         
           
               
             
               
                 TABLE P5 
               
             
            
               
                   
               
               
                   
               
               
                 Reference substances 
               
            
           
           
               
               
               
            
               
                   
                 Details 
                 Reference 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                 Reference standards 
                 Leukotrine E 4  from Cayman 
                 20410 
               
               
                   
                 Chemical, MI, USA 
               
               
                 Internal standards 
                 Leukotriene E 4 -20, 20, 20-d 3  from 
                 S10120 
               
               
                   
                 Biomol, PA, USA 
               
               
                   
               
            
           
         
       
     
      Stock Solutions  
      A stock solution of LTE 4  was prepared by the supplier at a concentration of 100 μg/ml in methanol. The stock solution was diluted to a concentration of 20 μg/ml in methanol and this working solution (WS-1) was kept refrigerated at 2-8° C.  
      An internal standard stock solution (LTE 4 -d 3 ) was prepared by the supplier at concentration of 49.5 μg/ml. The stock solution was diluted to a concentration of 1 μg/ml in methanol and this working solution was kept refrigerated at 2-8° C.  
      Preparation of spiking solutions, calibration standards and quality control samples  
      Spiking solutions (SS) in the concentration range of 1 ng/ml to 10000 ng/ml were prepared by dilution of the working Solution.  
      The following spiking solutions were prepared:  
               TABLE P6                          Spiking solutions for calibration standards                             Concentration           SS   (ng/ml)   Preparation                                 1   10000   500 μl of WS-1 (20 μg/ml) diluted to 1.0 ml with               70% MeOH/water       2   1000   100 μl of SS-1 was diluted to 1.0 ml with 70%               MeOH/water       3   100   100 μl of SS-2 was diluted to 1.0 ml with 70%               MeOH/water       4   30   300 μl of SS-3 was diluted to 1.0 ml with 70%               MeOH/water       5   20   200 μl of SS-3 was diluted to 1.0 ml with 70%               MeOH/water       6   16   160 μl of SS-3 was diluted to 1.0 ml with 70%               MeOH/water       7   12   120 μl of SS-3 was diluted to 1.0 ml with 70%               MeOH/water       8   8.0   400 μl of SS-5 was diluted to 1.0 ml with 70%               MeOH/water       9   4.0   200 μl of SS-5 was diluted to 1.0 ml with 70%               MeOH/water       10   2.0   100 μl of SS-5 was diluted to 1.0 ml with 70%               MeOH/water       11   1.4   175 μl of SS-8 was diluted to 1.0 ml with 70%               MeOH/water       12   1.0   125 μl of SS-8 was diluted to 1.0 ml with 70%               MeOH/water                  
 
     
       
         
           
               
             
               
                 TABLE P7 
               
             
            
               
                   
               
               
                   
               
               
                 Spiking solutions for quality controls 
               
            
           
           
               
               
               
            
               
                   
                 Concentration 
                   
               
               
                 SS 
                 (ng/ml) 
                 Preparation 
               
               
                   
               
               
                 13 
                 14 
                 140 μl of SS-3 was diluted to 1.0 ml with 70% 
               
               
                   
                   
                 MeOH/water 
               
               
                 14 
                 6.0 
                 300 μl of SS-5 was diluted to 1.0 ml with 70% 
               
               
                   
                   
                 MeOH/water 
               
               
                 15 
                 2.4 
                 120 μl of SS-5 was diluted to 1.0 ml with 70% 
               
               
                   
                   
                 MeOH/water 
               
               
                   
               
            
           
         
       
     
      After preparation, spiking solutions for calibration standards and quality controls were kept refrigerated at 2-8° C.  
     Preparation of Calibration Standards and Quality Controls  
      Fresh calibration standards and quality controls (QCs) were prepared each day by spiking blank plasma as described in Tables P8 and P9, respectively.  
               TABLE P8                          Preparation of calibration standards                         Concentration               (ng/ml)   SS (μl)   Blank Plasma                                 1500   20 μl of the SS-4 (30 ng/ml)   380 μl       1000   20 μl of the SS-5 (20 ng/ml)   380 μl       800   20 μl of the SS-6 (16 ng/ml)   380 μl       600   20 μl of the SS-7 (12 ng/ml)   380 μl       400   20 μl of the SS-8 (8 ng/ml)   380 μl       200   20 μl of the SS-9 (4.0 ng/ml)   380 μl       100   20 μl of the SS-10 (2.0 ng/ml)   380 μl       70   20 μl of the SS-11 (1.4 ng/ml)   380 μl       50   20 μl of the SS-12 (1.0 ng/ml)   380 μl                  
 
     
       
         
           
               
             
               
                 TABLE P9 
               
             
            
               
                   
               
               
                   
               
               
                 Preparation of quality controls 
               
            
           
           
               
               
               
            
               
                 Concentration 
                   
                   
               
               
                 (ng/ml) 
                 SS (μl) 
                 Blank Plasma 
               
               
                   
               
            
           
           
               
               
               
            
               
                 800 
                 20 μl of the SS-13 (14 ng/ml) 
                 380 μl 
               
               
                 40 
                 20 μl of the SS-14 (6.0 ng/ml) 
                 380 μl 
               
               
                 8.0 
                 20 μl of the SS-15 (2.4 ng/ml) 
                 380 μl 
               
               
                   
               
            
           
         
       
     
      Sample Preparation  
      Aliquots of 400 μl of each study sample, calibration standards, QC samples and control blank are pipetted into an eppendorf vial. All samples apart from blank are then spiked with 20 μl of internal standard working solution and the samples are then vortex-mixed for few seconds. The pH of the plasma samples is adjusted to pH 4.5 using 60 μl of 10% acetic acid and centrifuged for 10 min. at 4100 rpm immediately before the extraction. The solid phase extraction 96-well plate is conditioned with 1 ml methanol and 1 ml water. Then 400 μl of the plasma samples are loaded on the plate. Vacuum is applied, followed by drying the disk for 1 min. After being washed with 2 ml water and 1 ml 30% methanol in 2% acetic acid. Next the plate is eluted with 0.6 ml methanol. The plate is then dried for few minutes. The methanol eluate is evaporated almost to dryness under a stream of nitrogen at 45° C. The residue is reconstituted in 30 μl mobile phase and vortex-mixed for few min. Subsequently, the solutions are centrifuged for 10 min at 10.000 rpm. and 10 μl are injected by the autosampler into the LC-MS/MS system for quantification.  
     Performance Of Measurements  
      The samples will be prepared and measured in batches and a typical batch will consist of:  
      One set of calibration standards, one extra lowest calibration standard and one blank.  
      Samples collected from a 16 individuals and one set of control samples (C L , C M , C H )  
      Samples collected from a 17 individuals and one set of control samples (C L , C M , C H )  
     Quantitative Determination of Analyte in Plasma Samples  
      The standard curve is calculated from the peak area ratios ANALYTE/INTERNAL STANDARD of the calibration standards and their nominal ANALYTE concentrations. The unknown samples for LTE 4  were calculated from a quadratic regression equation where a weighted curve, 1/X 2 , is used. The measured peak area of the samples was converted into pictogram of ANALYTE per milliliter (pg/ml) of plasma according to the calculated equation for the standard curve.  
      The calculation of the regression for the standard curve and the calculations of the concentration of the unknown samples and the control samples are performed with MassLynx Software.  
     Acceptance Criteria  
      Calibration Standards  
      The coefficient of determination (R 2 ) for the calibration curve must exceed 0.98.  
      The calibration curve included the concentration range from 50 pg/ml-1500 pg/ml.  
      Concentration of the calibration standards must be within ±25% of their nominal value when recalculated from the regression equation. Calibration standards that fail these criteria (at most 3 in each run) are rejected and the calibration performed again with the remaining standards. If the standard curve is not accepted, the samples must be reanalyzed.  
      Control Samples  
      At least two thirds of the analysed quality controls must be within ±25% of their nominal value when calculated from regression equation. If more than a third of the controls fail, the samples must be reanalyzed.  
     Results  
      Table 17 (below) shows that the female MI “at risk” haplotype was more associated with female MI patients who have high LTE4 levels (top 50th percentile), than with female MI patients who have low levels of LTE4 (bottom 50th percentile).  
      In addition, haplotype analysis, comparing female MI patients with high levels of LTE4 with female patients with low levels, showed that those with high levels had increased prevalence of the “at risk” haplotype by 1.6 fold (see Table 18). Although the association did not rise to the level of statistical significance, the results show clearly that the “at risk” haplotypes are enriched in the MI patient group that has high levels of LTE4. The carrier frequency of the “at risk” haplotypes are 12% and 20%, respectively, in the whole female MI group, but go up to 15% and 24%, respectively, in the female MI group that has high levels of LTE4. Correspondingly, the carrier frequency of the “at risk” haplotypes decrease to 8% and 18%, respectively, in the group of female MI that has low levels of LTE4 (Note carrier frequencies are twice the disease allele frequency times 1 minus the disease allele frequency plus the square of the disease allele frequency).  
      Note that LTE4 may simply reflect the leukotriene synthesis rate of the leukotriene synthetic pathway upstream of the key leukotriene metabolite involved in MI risk. For example, leukotriene B4 is probably more likely than leukotriene E4 to be involved in the inflammatory aspects of MI plaques but since B4 has a short half life, it is difficult to measure reliably in serum samples, while E4 has long term stability.  
               TABLE 17                       Association of the at risk haplotypes for female MI, with       female MI who also have high levels of LTE4 (&gt;50 pg/ml (roughly the upper 50th       percentile).                                                                    SG13S418   SG13S420   DG13S166   SG13S114   SG13S88   SG13S184   D13S1238               High   C   T   0   T   T   G   −2       LTE4   C   T   0           G   −2       Low   C   T   0   T   T   G   −2       LTE4   C   T   0           G   −2                                                             p-val   N_aff   aff.frq   N_ctrl   ctrl.frq   rel_risk   PAR   info               High LTE4   3.72E−06   221   0.075   809   0.014   5.51   0.115   0.565           2.30E−05   220   0.122   809   0.046   2.89   0.154   0.608       Low LTE4   4.65E−02   185   0.040   809   0.015   2.67   0.048   0.511           2.88E−02   182   0.087   809   0.048   1.89   0.08    0.622                 P-val: p-value for the association.            N_aff: Number of patients used in the analysis.            Aff. frq: haplotype frequency in patients.            N_ctrl: number of controls used in the analysis.            Ctrl.frq: Haplotype frequency in controls.            Rel_risk: Relative risk of the haplotype.            PAR: population attributable risk.            Info: information content. Less association was found between the at risk haplotype for female MI, with female MI who also have low levels of LTE4 (&lt;50 pg/ml).             
 
     
       
         
           
               
             
               
                 TABLE 18 
               
               
                   
               
               
                   
               
               
                 Association between haplotypes that were most significantly 
               
               
                 associated with female MI, and serum LTE4 levels. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 SG13S418 
                 SG13S420 
                 DG13S166 
                 SG13S114 
                 SG13S88 
                 SG13S184 
               
               
                   
               
               
                 High vs low LTE4 
                 C 
                 T 
                 0 
                 T 
                 T 
                 G 
               
               
                   
                 C 
                 T 
                 0 
                   
                   
                 G 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 D13S1238 
                 p-val 
                 N_aff 
                 aff.frq 
                 N_ctrl 
                 ctrl.frq 
                 rel_risk 
                 PAR 
                 info 
               
               
                   
               
               
                 High 
                 −2 
                 1.61E−01 
                 221 
                 0.084 
                 185 
                 0.054 
                 1.61 
                 0.063 
                 0.689 
               
               
                 vs 
                 −2 
                 1.20E−01 
                 220 
                 0.13  
                 182 
                 0.088 
                 1.54 
                 0.089 
                 0.686 
               
               
                 low 
               
               
                 LTE4 
               
               
                   
               
               
                   P-val: p-value for the association.    
               
               
                   N_aff: Number of patients used in the analysis.    
               
               
                   Aff. frq: haplotype frequency in patients.    
               
               
                   N_ctrl: number of controls used in the analysis.    
               
               
                   Ctrl.frq: Haplotype frequency in controls.    
               
               
                   Rel_risk: Relative risk of the haplotype.    
               
               
                   PAR: population attributable risk.    
               
               
                   Info: information content. Here, the group of affected individuals were defined as female MI patients with high serum LTE4 (higher than 50 pg/ml) and the control group is defined as female MI patients with low serum LTE4 (below 50 pg/ml).    
               
            
           
         
       
     
     EXAMPLE 4  
     Elevated LTE4 Correlated with Elevated C-Reactive Protein (CRP)  
      The relationship between the increased production of leukotrienes and the inflammatory marker CRP, a well established risk factor for MI, was then explored. As shown in  FIG. 4 , a significant positive correlation was found between serum LTE4 levels and serum CRP levels.  
     EXAMPLE 5  
     Assessment of Level of CRP in Patients with at-Risk Haplotype  
      The level of CRP in female patients with female MI at-risk haplotypes was assessed, in order to assess whether there was a presence of a raised level of inflammatory marker in the presence of the female MI at-risk haplotype. Results are shown in Table 19. Although the association did not rise to the level of statistical significance, it was demonstrated that the average CRP was elevated in those patients with the at-risk haplotype versus those without it.  
               TABLE 19                          All female patients                                 no    Mean CRP   SE CRP                                              affecteds:   With haplotype.   155   4.91   8.7           Not with haplotype.   218   4.35   6.13                  
 
     EXAMPLE 6  
     Elevated Serum LTE4 Levels in MI Patients Versus Controls  
      The end products of the leukotriene pathway are potent inflammatory lipid mediators that can potentially contribute to development of atherosclerosis and destabilization of atherosclerotic plaques through lipid oxidation and/or proinflammatory effects. Examples one through five show that: 1) MI correlates with genetic variation at FLAP; 2) MI correlates with high expression promoter polymorphism at 5-LO; 3) C-reactive protein levels correlate with serum leukotriene E4; and 4) Patients with MI-risk FLAP haplotypes have higher levels of serum leukotriene E4 and CRP. Based on these data, it was hypothesized that serum leukotriene E4 levels correlate with MI risk.  
      To test this hypothesis, LTE4, a downstream leukotriene metabolite, was measured in 488 female MI patient and 164 control serum samples. The LTE4 levels for the patients were higher than that for the controls using a one-sided Wilcoxon rank-sum test. The p-value of the difference was 0.0092, thus confirming our hypothesis. Therefore, elevated leukotriene E4 represents a risk factor for MI. Serum or plasma LTE4 levels may be used to profile the MI risk for individuals to aid in deciding which treatment and lifestyle management plan is best for primary or secondary MI prevention. In the same way other leukotriene metabolites may be used to risk profile for MI.  
     EXAMPLE 7  
     Increased LTB4 Production in Activated Neutrophils from MI Patients  
      A principal bioactive product of one of the two branches of the 5-LO pathway is LTB4. To determine whether the patients with past history of MI have increased activity of the 5-LO pathway compared to controls, the LTB4 production in isolated blood neutrophils was measured before and after stimulation in vitro with the calcium ionophore, ionomycin. No difference was detected between the LTB4 production in resting neutrophils from MI patients or controls (results not shown). In contrast, the LTB4 generation by neutrophils from MI patients stimulated with the ionophore was significantly greater than by neutrophils from controls at 15 and 30 minutes, respectively ( FIG. 5 . 1 ). Moreover, as shown in  FIG. 5 . 2 , the observed increase in the LTB4 release was largely accounted for by male carriers of haplotype A4, whose cells produced significantly more LTB4 than cells from controls (P value=0.0042) (Table 20). As shown in Table 20, there was also a heightened LTB4 response in males who do not carry HapA but of borderline significance. This could be explained by additional variants in the FLAP gene that have not been uncovered, or alternatively in other genes belonging to the 5-LO pathway, that may account for upregulation in the LTB4 response in some of the patients without the FLAP at-risk haplotype. As shown in Table 20, differences in LTB4 response were not detected in females. However, due to a small sample size this cannot be considered conclusive. Taken together, the elevated levels of LTB4 production of stimulated neutrophils from male carriers of the at-risk haplotype suggest that the disease associated variants in the FLAP gene increase FLAP&#39;s response to factors that stimulate inflammatory cells, resulting in increased leukotriene production and increased risk for MI.  
     Methods  
      Isolation and Activation of Peripheral Blood Neutrophils  
      50 ml of blood were drawn into EDTA containing vacutainers from 43 MI patients and 35 age and sex matched controls. All blood was drawn at the same time in the early morning after 12 hours of fasting. The neutrophils were isolated using Ficoll-Paque PLUS (Amersham Biosciences).  
      Briefly, the red cell pellets from the Ficoll gradient were harvested and red blood cells subsequently lysed in 0.165 M NH 4 CL for 10 minutes on ice. After washing with PBS, neutrophils were counted and plated at 2×10 6  cells/ml in 4 ml cultures of 15% Fetal calf serum (FCS) (GIBCO BRL) in RPMI-1640 (GIBCO BRL). The cells were then stimulated with maximum effective concentration of ionomycin (1 μM). At 0, 15, 30, 60 minutes post ionomycin addition 600 μl of culture medium was aspirated and stored at −80C for the measurement of LTB4 release as described below. The cells were maintained at 37° C. in a humidified atmosphere of 5% CO 2 /95% air. All samples were treated with indomethasine (1 μM ) to block the cyclooxygenase enzyme.  
      Ionomycin-induced Release of LTB4 in Neutrophils  
      LTB4 Immunoassay (R&amp;D systems) was used to quantitate LTB4 concentration in supernatant from cultured ionomycin stimulated neutrophils. The assay used is based on the competitive binding technique in which LTB4 present in the testing samples (200 □l) competes with a fixed amount of alkaline phosphatase-labelled LTB4 for sites on a rabbit polyclonal antibody. During the incubation, the polyclonal Ab becomes bound to a goat anti-rabbit Ab coated onto the microplates. Following a wash to remove excess conjugate and unbound sample, a substrate solution is added to the wells to determine the bound enzyme activity. The color development is stopped and the absorbance is read at 405 nm. The intensity of the color is inversely proportional to the concentration of LTB4 in the sample. Each LTB4 measurement using the LTB4 Immunoassay, was done in duplicate.  
               TABLE 20                          LTB4 levels after ionomycin stimulation of isolated neutrophils a                               After 15 Minutes   After 30 Minutes                                 Phenotype (n)   Mean (SD)    P value   Mean (SD)   P value                                         Controls (35)   4.53 (1.00)       4.67 (0.88)           Males (18)   4.61 (1.10)       4.68 (1.07)       Females (17)   4.51 (0.88)       4.67 (0.62)       MI (41)   5.18 (1.09)   0.011   5.24 (1.06)   0.016       Carriers(16)   5.26 (1.09)   0.027   5.27 (1.09)   0.051       Non-carriers (24)   5.12 (1.08)   0.040   5.22 (1.03)   0.035       MI males (28)   5.37 (1.10)   0.0033   5.38 (1.09)   0.0076       Carriers(10)   5.66 (1.04)   0.0042   5.58 (1.12)   0.013       Non-carriers (18)   5.20 (1.09)   0.039   5.26 (1.05)   0.041       MI females (13)   4.78 (0.95)   0.46   4.95 (0.92)   0.36       Carriers(6)   4.59 (0.80)   0.90   4.75 (0.82)   0.85       Non-carriers (7)   4.94 (1.04)   0.34   5.12 (0.96)   0.25                   a Mean ± SD of log-transformed values of LTB4 levels of ionomycin-stimulated neutrophils from MI patients and controls. Results are shown for two time points: 15 and 30 minutes. The results for males and females and for MI male and female carriers and non-carriers of          #the at-risk haplotype HapA are shown separately. Two-sided p values corresponding to a standard two-sample test of the difference in the mean values between the MI patients, their various sub-cohorts and the controls are shown.           
 
     EXAMPLE 8  
     Haplotypes Associated With MI also Confer Risk of Stroke and PAOD  
      Because stroke and PAOD are diseases that are closely related to MI (all occur on the basis of atherosclerosis), it was examined whether the SNP haplotype in the FLAP gene that confers risk to MI also conferred risk of stroke and/or PAOD. The ‘at risk’ haplotype (A4 haplotype)can be defined by the following 4 SNPs: SG13S25 with allele G, SG13S114 with allele T, SG13S89 with allele G, and SG13S32 with allele A.  
      Table 21 shows that the haplotype A4 increases the risk of having a stroke to a similar extent as it increases the risk of having an MI. The ‘at risk’ haplotype is carried by 28% of stroke patients and 17% of controls, meaning that the relative risk of having stroke for the carriers of this haplotype is 1.7 (p-value=5.8 10 −06 ). Although not as significant, the ‘at risk’ haplotype also confers risk of having PAOD.  
                                               TABLE 21                              p-val   r   #aff   aff.frq.   #con   con.frq.   info   SG13S25               MI haplotypes       All MI patients       A4   5.3E−07   1.80   1407   0.16   614   0.09   0.82   G       B4   1.0E−04   1.87   1388   0.10   612   0.06   0.67   G       Males MI       A4   2.5E−08   2.00   864   0.17   614   0.09   0.82   G       B4   1.1E−05   2.12   852   0.11   612   0.06   0.67   G       Females MI       A4   1.9E−02   1.44   543   0.13   614   0.09   0.73   G       B4   7.9E−02   1.45   536   0.08   612   0.06   0.60   G       Replication in stroke       All stroke patients       A4   5.8E−06   1.73   1238   0.15   614   0.09   0.80   G       B4   2.3E−04   1.83   1000   0.10   612   0.06   0.71   G       Males stroke       A4   1.1E−06   1.91   710   0.17   614   0.09   0.79   G       B4   3.1E−05   2.11   574   0.11   612   0.06   0.72   G       Females stroke       A4   9.9E−03   1.49   528   0.13   614   0.10   0.74   G       B4   6.3E−02   1.47   426   0.08   612   0.06   0.70   G       All stroke excluding MI   8.4E−05   1.65   1054   0.15   614   0.09   0.78   G       Males stroke excluding MI   6.4E−05   1.78   573   0.16   614   0.09   0.75   G       Females stroke excluding MI   1.2E−02   1.49   481   0.14   614   0.10   0.72   G       Cardioembolic stroke   6.6E−04   1.87   248   0.16   614   0.10   0.74   G       Cardioembolic stroke excluding MI   3.8E−02   1.56   191   0.14   614   0.10   0.70   G       Large vessel stroke   8.0E−02   1.47   150   0.13   614   0.09   0.83   G       Large vessel stroke excluding MI   2.9E−01   1.31   114   0.12   614   0.09   0.80   G       Small vessel stroke   7.2E−04   2.05   166   0.18   614   0.09   0.71   G       Small vessel stroke excluding MI   1.0E−04   2.31   152   0.20   614   0.10   0.71   G       Hemorrhagic stroke   4.4E−02   1.73   97   0.15   614   0.09   0.72   G       Hemorrhagic stroke excluding MI   3.9E−02   1.78   92   0.16   614   0.09   0.71   G       Unknown cause stroke   1.3E−04   1.88   335   0.16   614   0.09   0.75   G       Unknown cause stroke excluding   6.5E−04   1.82   297   0.16   614   0.09   0.72   G       MI       MI and stroke together       All patients       Best haplo A4   4.1E−07   1.75   2659   0.15   614   0.09   0.82   G       B4   4.1E−05   1.85   2205   0.10   612   0.06   0.70   G       Males       A4   1.4E−08   1.93   1437   0.17   614   0.09   0.82   G       B4   2.0E−06   2.11   1290   0.11   612   0.06   0.70   G       Females       A4   3.6E−03   1.47   1024   0.13   614   0.09   0.77   G       B4   2.8E−02   1.48   915   0.08   612   0.06   0.66   G       Patients with both MI and stroke       A4   6.1E−05   2.10   184   0.18   614   0.09   0.86   G       Replication in PAOD       All PAOD patients   3.6E−02   1.31   920   0.12   614   0.10   0.84   G       Males PAOD   1.8E−02   1.40   580   0.13   614   0.10   0.84   G       Females PAOD   3.7E−01   1.17   340   0.11   614   0.10   0.83   G       All PAOD excluding MI   1.1E−01   1.24   750   0.12   614   0.10   0.83   G       Males PAOD excluding MI   8.3E−02   1.30   461   0.12   614   0.10   0.83   G       Males PAOD excluding MI and   8.7E−02   1.32   388   0.12   614   0.10   0.83   G       stroke                                                     SG13S106   SG13S114   SG13S89   SG13S30   SG13S32   SG13S42               MI haplotypes       All MI patients       A4       T   G       A       B4   G           G       A       Males MI       A4       T   G       A       B4   G           G       A       Females MI       A4       T   G       A       B4   G           G       A       Replication in stroke       All stroke patients       A4       T   G       A       B4   G           G       A       Males stroke       A4       T   G       A       B4   G           G       A       Females stroke       A4       T   G       A       B4   G           G       A       All stroke excluding MI       T   G       A       Males stroke excluding MI       T   G       A       Females stroke excluding MI       T   G       A       Cardioembolic stroke       T   G       A       Cardioembolic stroke excluding M   T   G       A   I       Large vessel stroke       T   G       A       Large vessel stroke excluding MI   T   G       A           Small vessel stroke       T   G       A       Small vessel stroke excluding MI   T   G       A           Hemorrhagic stroke       T   G       A       Hemorrhagic stroke excluding MI       T   G       A       Unknown cause stroke       T   G       A       Unknown cause stroke excluding       T   G       A       MI       MI and stroke together       All patients       Best haplo A4       T   G       A       B4   G           G       A       Males       A4       T   G       A       B4   G           G       A       Females       A4       T   G       A       B4   G           G       A       Patients with both MI and stroke       A4       T   G       A       Replication in PAOD       All PAOD patients       T   G       A       Males PAOD       T   G       A       Females PAOD       T   G       A       All PAOD excluding MI       T   G       A       Males PAOD excluding MI       T   G       A       Males PAOD excluding MI and       T   G       A       stroke                  
 
      The patient cohorts used in the association analysis shown in Table 21 may include first and second degree relatives.  
      Table 21, discussed above, shows the results of the haplotype A4 association study using 779 MI patients, 702 stroke patients, 577 PAOD patients and 628 controls. First and second degree relatives were excluded from the patient cohorts. All known cases of MI were removed from the stroke and PAOD cohorts before testing for association. A significant association of the A4 haplotype to stroke was observed, with a relative risk of 1.67 (P value=0.000095). In addition, it was determined whether the A4 haplotype was primarily associated with a particular sub-phenotype of stroke, and found that both ischemic and hemorrhagic stroke were significantly associated with the A4 haplotype (Table 22).  
               TABLE 22                          Association of the A4 haplotype to subgroups of stroke                                         Phenotype (n)   Pat. Frq.   RR   PAR   P-value                                                     Stroke a  (702)   0.149   1.67   0.116   0.000095           Ischemic (484)   0.148   1.65   0.113   0.00053           TIA (148)   0.137   1.51   0.090   0.058           Hemorrhagic (68)   0.167   1.91   0.153   0.024                           a Excluding known cases of MI.             
 
      Finally, the A4 haplotype was less significantly associated with PAOD (Table 21). It should be noted that similar to the stronger association of the A4 haplotype to male MI compared to female MI, it also shows stronger association to male stroke and PAOD (Table 21).  
     Study Population  
      The stroke and PAOD cohorts used in this study have previously been described (Gretarsdottir, S. et al.  Nat Genet  35, 131-8 (2003); Gretarsdottir, S. et al.,  Am J Hum Genet  70, 593-603 (2002); Gudmundsson, G. et al.,  Am J Hum Genet  70, 586-92-(2002)). For the stroke linkage analysis, genotypes from 342 male patients with ischemic stroke or TIA-that were linked to at least one other male patient within and including 6 meioses in 164 families were used. For the association studies 702 patients with all forms of stroke (n=329 females and n=373 males) and 577 PAOD patients (n=221 females and n=356 males) were analysed. Patients with stroke or PAOD that also had MI were excluded. Controls used for the stroke and PAOD association studies were the same as used in the MI SNP association study (n=628).  
      The study was approved by the Data Protection Commission of Iceland and the National Bioethics Committee of Iceland. Informed consent was obtained from all study participants. Personal identifiers associated with medical information and blood samples were encrypted with a third party encryption system as previously described (Gulcher, J. R., Kristjansson, K., Gudbjartsson, H. &amp; Stefansson, K.,.  Eur J Hum Genet  8, 739-42 (2000)).  
      In addition, in an independent linkage study of male patients with ischemic stroke or transient ischemic attack, linkage to the same locus was observed with a LOD score of 1.51 at the same peak marker ( FIG. 7 ), further suggested that a cardiovascular susceptibility factor might reside at this locus.  
     EXAMPLE 9  
     Haplotype Association to FLAP in a British Cohort  
      In an independent study, it was determined whether variants in the FLAP gene also have impact on risk of MI in a population outside Iceland. The four SNPs, defining the A4 haplotype, were typed in a cohort of 750 patients from the United Kingdom who had sporadic MI, and in 728 British population controls. The patients and controls come from 3 separate study cohorts recruited in Leicester and Sheffield. No significant differences were found in the frequency of the haplotype between patients and controls (16.9% versus 15.3%, respectively). However, when an. additional 9 SNPs, distributed across the FLAP gene, were typed in the British cohort and searched for other haplotypes that might be associated with MI, two SNPs showed association to MI with a nominally significant P value (data not shown). Moreover, three and four SNP haplotype combinations increased the risk of MI in the British cohort further and the most significant association was observed for a four SNP haplotype with a nominal P value=0.00037 (Table 23).  
               TABLE 23                          Association of the HapB haplotype to British MI patients                                     Phenotype (n)   Frq. Pat.   RR   PAR   P-value   P-value a                                               MI (750)   0.075   1.95   0.072   0.00037   0.046       Males (546)   0.075   1.97   0.072   0.00093   ND       Females (204)   0.073   1.90   0.068   0.021   ND                   a P value adjusted for the number of haplotypes tested using 1,000 randomization tests.            Shown are the results for HapB that shows the strongest association in British MI cohort. HapB is defined by the following SNPs: SG13S377, SG13S114, SG13S41 and SG13S35 (that have the following alleles A, A, A and G, respectively. In all three phenotypes shown the same set of n = 728 British controls is used and the frequency of HapB in the          # control cohort is 0.040. Number of patients (n), haplotype frequency in patients (Frq. pat.), relative risk (RR) and population attributed risk (PAR).           
 
      This was called haplotype HapB. The haplotype frequency of HapB is 7.5% in the MI patient cohort (carrier frequency 14.4%), compared to 4.0% (carrier frequency 7.8%) in controls, conferring a relative risk of 1.95 (Table 23). This haplotype remained significant after adjusting for all haplotypes tested, using 1000 randomisation steps, with an adjusted P value=0.046. No other SNP haplotype had an adjusted P value less than 0.05. The two at-risk haplotypes A4 and HapB appear to be mutually exclusive with no instance where the same chromosome carries both haplotypes.  
      British Study Population  
      The method of recruitment of 3 separate cohorts of British subjects has been described previously (Steeds, R., Adams, M., Smith, P., Channer, K. &amp; Samani, N. J.,  Thromb Haemost  79, 980-4 (1998); Brouilette, S., Singh, R. K., Thompson, J. R., Goodall, A. H. &amp; Samani, N. J.,  Arterioscler Thromb Vasc Biol  23, 842-6 (2003)). In brief, in the first two cohorts a total of 547 patients included those who were admitted to the coronary care units (CCU) of the Leicester Royal Infirmary, Leicester (July 1993-April 1994) and the Royal Hallamshire Hospital, Sheffield (November 1995-March 1997) and satisfied the World Health Organisation criteria for acute MI in terms of symptoms, elevations in cardiac enzymes or electrocardiographic changes (Nomenclature and criteria for diagnosis of ischemic heart disease. Report of the Joint International Society and Federation of Cardiology/World Health Organization task force on standardization of clinical nomenclature.  Circulation  59, 607-9 (1979)). A total of 530 control subjects were recruited in each hospital from adult visitors to patients with non-cardiovascular disease on general medical, surgical, orthopaedic and obstetric wards to provide subjects likely to be representative of the source population from which the subjects originated. Subjects who reported a history of coronary heart disease were excluded.  
      In the third cohort, 203 subjects were recruited retrospectively from the registries of 3 coronary care units in Leicester. All had suffered an MI according to WHO criteria before the age of 50 years. At the time of participation, patients were at least 3 months from the acute event. The control cohort comprised 180 subjects with no personal or family history of premature coronary heart disease, matched for age, sex, and current smoking status with the cases. Control subjects were recruited from 3 primary care practices located within the same geographical area. In all cohorts subjects were white of Northern European origin.  
     Discussion  
      These results show that variants of the gene encoding FLAP associate with increased risk of MI and stroke. In the Icelandic cohort, a haplotype that spans the FLAP gene is carried by 30% of all MI patients and almost doubles the risk of MI. These findings were subsequently replicated in an independent cohort of stroke patients. In addition, another haplotype that spans the FLAP gene is associated with MI in a British cohort. Suggestive linkage to chromosome 13q12-13 was observed with several different phenotypes, including female MI, early onset MI of both sexes, and ischemic stroke or TIA in males. However, surprisingly, the strongest haplotype association was observed to males with MI or stroke. Therefore, there may be other variants or haplotypes within the FLAP gene, or in other genes within the linkage region, that also may confer risk to these cardiovascular phenotypes.  
      These data also show that the at-risk haplotype of the FLAP gene has increased frequency in all subgroups of stroke, including ischemic, TIA, and hemorrhagic stroke. Of interest is that the A4 haplotype confers significantly higher risk of MI and stroke than it does of PAOD. This could be explained by differences in the pathogenesis of these diseases. Unlike PAOD patients who have ischemic legs because of atherosclerotic lesions that are responsible for gradually diminishing blood flow to the legs, the MI and stroke patients have suffered acute events, with disruption of the vessel wall suddenly decreasing blood flow to regions of the heart and the brain.  
      Association was not found between the A4 haplotype and MI in a British cohort. However, significant association to MI was found with a different variant spanning the FLAP gene. The fact that different haplotypes of the gene are found conferring risk to MI in a second population is not surprising. A common disease like MI associates with many different mutations or sequence variations, and the frequencies of these disease associated variants may differ between populations. Furthermore, the same mutations may be seen arising on different haplotypic backgrounds.  
      In summary, it has been found that: MI correlates with genetic variation at FLAP; MI correlates with high expression promoter polymorphism at 5-LO; patients with female MI at-risk FLAP haplotypes have higher levels of serum LTE4; LTE4 levels correlate with CRP levels in serum; and patients with MI at-risk FLAP haplotypes have elevated CRP. In addition, we have shown that isolated neutrophils from MI patients, produce more LTB4 when stimulated with ionomycin compared to controls. Taken together, these results show that increased leukotriene synthesis is a risk factor for MI, and that this risk is driven in part by variants in FLAP and 5-LO genes and are captured in part by measurement of levels of serum LTE4 and CRP. Furthermore, the SNP haplotype in the FLAP gene that confers risk to MI also confers risk of stroke and/or PAOD.  
     Markers Utilized Herein  
     
       
         
           
               
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                   
               
               
                 Basepair position of microsatellite markers (start and stop of the 
                   
               
               
                 amplimersin NCBI sequence assembly build 34 and primer sequences (forward 
               
               
                 and reverse). 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 basepair 
                 basepair 
                   
               
               
                 Marker 
                   
                   
                 start 
                 stop 
               
               
                 name 
                 forward primer 
                 reverse primer 
                 position 
                 position 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 DG13S2393 
                 CCTTTGCTTTGTTCCTATTTCTTT 
                 TCCCATTGCCCAGAGTTAAT 
                 22831401 
                 22831787 
                   
               
               
                   
                 (SEQ ID NO. 4) 
                 (SEQ ID NO. 5) 
               
               
                   
               
               
                 DG13S2070 
                 TCCTCATGTCTTCACCTAGAAGC 
                 CCACTCATGAGGGAGCTGTT 
                 23020439 
                 23020651 
               
               
                   
                 (SEQ ID NO. 6) 
                 (SEQ ID NO. 7) 
               
               
                   
               
               
                 DG13S2071 
                 TGTCACAGGCACACACTCTCT 
                 GAGTATGGCTGCTGCTCCTC 
                 23066973 
                 23067076 
               
               
                   
                 (SEQ ID NO. 8) 
                 (SEQ ID NO. 9) 
               
               
                   
               
               
                 DG13S2072 
                 ATGGCTCACACTGGCCTAAA 
                 TGAACAGACCAATAATAGTGCAG 
                 23136964 
                 23137114 
               
               
                   
                 (SEQ ID NO. 10) 
                 (SEQ ID NO. 11) 
               
               
                   
               
               
                 DG13S2078 
                 AAGCCACCCTTTAAACAGCA 
                 GCTGAGGAAGCAACTCCACT 
                 23591927 
                 23592081 
               
               
                   
                 (SEQ ID NO. 12) 
                 (SEQ ID NO. 13) 
               
               
                   
               
               
                 DG13S2079 
                 GCTCTGAATTCCCTGGCATA 
                 TTAGCCCTAGTCCCACTCTCC 
                 23646974 
                 23647183 
               
               
                   
                 (SEQ ID NO. 14) 
                 (SEQ ID NO. 15) 
               
               
                   
               
               
                 DG13S2082 
                 CAAGAGGCCTGCATAAGGAA 
                 AGATTGCCGGTGGCTTAAAT 
                 23807898 
                 23808174 
               
               
                   
                 (SEQ ID NO. 16) 
                 (SEQ ID NO. 17) 
               
               
                   
               
               
                 DG13S2083 
                 TGTCTGTTCCCGTCTGTCTG 
                 TTCATCCTCTGCCAAATTCC 
                 23882291 
                 23882532 
               
               
                   
                 (SEQ ID NO. 18) 
                 (SEQ ID NO. 19) 
               
               
                   
               
               
                 DG13S2086 
                 GGCATGTATTCACTGCCTGA 
                 AAACCCATTCTTCTTCCTCTTAC 
                 24069346 
                 24069771 
               
               
                   
                 (SEQ ID NO. 20) 
                 (SEQ ID NO. 21) 
               
               
                   
               
               
                 DG13S2089 
                 TATGTGTTCAGCCCAGACCTC 
                 CCCTGCCATGTGCATTTAC 
                 24274920 
                 24275129 
               
               
                   
                 (SEQ ID NO. 22) 
                 (SEQ ID NO. 23) 
               
               
                   
               
               
                 DG13S44 
                 CATTTCGGAAGGCAAAGAAA 
                 TTGCAATGAGGAATGAAGCA 
                 24413148 
                 24413382 
               
               
                   
                 (SEQ ID NO. 24) 
                 (SEQ ID NO. 25) 
               
               
                   
               
               
                 DG13S2095 
                 TCCATTATCCATCTGTTCATTCA 
                 GAAGAATTAATTGTAGGAGGCAA 
                 24621830 
                 24622121 
               
               
                   
                 (SEQ ID NO. 26) 
                 GA (SEQ ID NO. 27) 
               
               
                   
               
               
                 DG13S46 
                 CTGACATCACCACATTGATCG 
                 CATACACAGCCATGTGGAATTA 
                 24652046 
                 24652291 
               
               
                   
                 (SEQ ID NO. 28) 
                 (SEQ ID NO. 29) 
               
               
                   
               
               
                 DG13S2101 
                 ACGGTGATGACGCCTACATT 
                 TCACATGGACCAATTACCTAGAA 
                 24863557 
                 24863744 
               
               
                   
                 (SEQ ID NO. 30) 
                 (SEQ ID NO. 31) 
               
               
                   
               
               
                 D13S1254 
                 AAATTACTTCATCTTGACGATAA 
                 CTATTGGGGACTGCAGAGAG 
                 25316434 
                 25316657 
               
               
                   
                 CA (SEQ ID NO. 32) 
                 (SEQ ID NO. 33) 
               
               
                   
               
               
                 DG13S55 
                 AGCCAGTGTCCACAAGGAAGGAGG 
                 GTGAGACACATCTCTGG 
                 25337471 
                 25337753 
               
               
                   
                 (SEQ ID NO. 34) 
                 (SEQ ID NO. 35) 
               
               
                   
               
               
                 DG13S54 
                 AATCGTGCCTCAGTTCCATC 
                 CCACCAGGAACAACACACAC 
                 25377308 
                 25377463 
               
               
                   
                 (SEQ ID NO. 36) 
                 (SEQ ID NO. 37) 
               
               
                   
               
               
                 D13S625 
                 TTGCTCTCCAGCCTGGGC 
                 TTCCTCTGGCTGCCTGCG 
                 25391207 
                 25391395 
               
               
                   
                 (SEQ ID NO. 38) 
                 (SEQ ID NO. 39) 
               
               
                   
               
               
                 DG13S2695 
                 TCCTGCATGAGAAGGAACTG 
                 CGACATTCACTGTGGCTCTT 
                 25415551 
                 25415807 
               
               
                   
                 (SEQ ID NO. 40) 
                 (SEQ ID NO. 41) 
               
               
                   
               
               
                 DG13S1479 
                 TTTGATTCCGTGGTCCATTA 
                 TTATTTGGTCGGTGCACCTTT 
                 25459039 
                 25459368 
               
               
                   
                 (SEQ ID NO. 42) 
                 (SEQ ID NO. 43) 
               
               
                   
               
               
                 DG13S2696 
                 GGTGCACCGACCAAATAAGT 
                 CCAGCTTATTCTCTCTGCCTTC 
                 25459351 
                 25459478 
               
               
                   
                 (SEQ ID NO. 44) 
                 (SEQ ID NO. 45) 
               
               
                   
               
               
                 DG13S1440 
                 GGTAGGTTGAAATGGGCTAACA 
                 TCATGACAAGGTGTTGGATTT 
                 25520858 
                 25520987 
               
               
                   
                 (SEQ ID NO. 46) 
                 (SEQ ID NO. 47) 
               
               
                   
               
               
                 DG13S1890 
                 CCTCCTCTGCCATGAAGCTA 
                 CTATTTGGTCTGCGGGTTGT 
                 25672727 
                 25673140 
               
               
                   
                 (SEQ ID NO. 48) 
                 (SEQ ID NO. 49) 
               
               
                   
               
               
                 DG13S1540 
                 TACTGGGTTATCGCCTGACC 
                 CCAATGGACCTCTTGGACAT 
                 25704358 
                 25704504 
               
               
                   
                 (SEQ ID NO. 50) 
                 (SEQ ID NO. 51) 
               
               
                   
               
               
                 DG13S59 
                 TTTCGGCACAGTCCTCAATA 
                 CAGCTGGGTGTGGTGACAT 
                 25720194 
                 25720421 
               
               
                   
                 (SEQ ID NO. 52) 
                 (SEQ ID NO. 53) 
               
               
                   
               
               
                 DG13S1545 
                 CAGAGAGGAACAGGCAGAGG 
                 AGTGGCTGGGAAGCCTTATT 
                 25760018 
                 25760404 
               
               
                   
                 (SEQ ID NO. 54) 
                 (SEQ ID NO. 55) 
               
               
                   
               
               
                 DG13S1524 
                 AGGTGAGAGAACAAACCTGTCTT 
                 GCCTTCCTTCTAAGGCCAAC 
                 25843657 
                 25843768 
               
               
                   
                 (SEQ ID NO. 56) 
                 (SEQ ID NO. 57) 
               
               
                   
               
               
                 DG13S1529 
                 CTGTAGACTTTATCCCTGACTTAC 
                 CAATGAATGATGAAGATTCCACT 
                 26098943 
                 26099063 
               
               
                   
                 TG (SEQ ID NO. 58) 
                 C (SEQ ID NO. 59) 
               
               
                   
               
               
                 DG13S1908 
                 TGACACCATGTCTTACTGTTGC 
                 GAGGATACAATGAGAACCAAATC 
                 26110282 
                 26110493 
               
               
                   
                 (SEQ ID NO. 60) 
                 TC (SEQ ID NO. 61) 
               
               
                   
               
               
                 DG13S2525 
                 CAGGATCATCAGCCAGGTTT 
                 GCTGCATGTCACTAGGCATT 
                 26123233 
                 26123381 
               
               
                   
                 (SEQ ID NO. 62) 
                 (SEQ ID NO. 63) 
               
               
                   
               
               
                 DG13S1546 
                 CCACAGAATGCTCCAAAGGT 
                 GAGTTCAAGTGATGGATGACGA 
                 26159644 
                 26159995 
               
               
                   
                 (SEQ ID NO. 64) 
                 (SEQ ID NO. 65) 
               
               
                   
               
               
                 DG13S1444 
                 CAGATAGATGAATAGGTGGATGG 
                 CACTGTTCCAAGTGCTTTGC 
                 26207544 
                 26207727 
               
               
                   
                 A (SEQ ID NO. 66) 
                 (SEQ ID NO. 67) 
               
               
                   
               
               
                 DG13S66 
                 TATGCGTTGTGTGTGCTGTG 
                 GGGCCTTAGATTCTTGTAGTGG 
                 26279746 
                 26279962 
               
               
                   
                 (SEQ ID NO. 68) 
                 (SEQ ID NO. 69) 
               
               
                   
               
               
                 DG13S1907 
                 TGTCCAGACTGCCTCCTACA 
                 TGCAACACCTGGTTCACAAT 
                 26378401 
                 26378521 
               
               
                   
                 (SEQ ID NO. 70) 
                 (SEQ ID NO. 71) 
               
               
                   
               
               
                 DG13S68 
                 TTTGCGAGTCCTTGTGGAGT 
                 ACAGTCCGCTCCCTCCTAAT 
                 26511587 
                 26511825 
               
               
                   
                 (SEQ ID NO. 72) 
                 (SEQ ID NO. 73) 
               
               
                   
               
               
                 DG13S69 
                 ATGCTTGGCCCTCAGTTT 
                 TTGGCAACCCAAGCTAATATG 
                 26518188 
                 26518483 
               
               
                   
                 (SEQ ID NO. 74) 
                 (SEQ ID NO. 75) 
               
               
                   
               
               
                 D13S1250 
                 CTCCACAGTGACAGTGAGG 
                 GAGAGGTTCCCAATCCC 
                 26721525 
                 26721686 
               
               
                   
                 (SEQ ID NO. 76) 
                 (SEQ ID NO. 77) 
               
               
                   
               
               
                 DG13S574 
                 CAGCTCCTGGCCATATTTCT 
                 GAGCCATTTCTCTGGGTCTG 
                 26853541 
                 26853693 
               
               
                   
                 (SEQ ID NO. 78) 
                 (SEQ ID NO. 79) 
               
               
                   
               
               
                 DG13S73 
                 GGTCCGTGTCAACCCTTAGA 
                 CAGGTTGATGGGAGGGAAA 
                 26878938 
                 26879133 
               
               
                   
                 (SEQ ID NO. 80) 
                 (SEQ ID NO. 81) 
               
               
                   
               
               
                 DG13S1532 
                 CGGGAAATGACAGTGAGACC 
                 TGCCTAGATTCTCCCGTAAG 
                 26899505 
                 26899652 
               
               
                   
                 (SEQ ID NO. 82) 
                 (SEQ ID NO. 83) 
               
               
                   
               
               
                 D13S1242 
                 GTGCCCAGCCAGATTC 
                 GCCCCCAGTCAGGTTT 
                 26943073 
                 26943316 
               
               
                   
                 (SEQ ID NO. 84) 
                 (SEQ ID NO. 85) 
               
               
                   
               
               
                 DG13S576 
                 TTTCTCTCTCCACGGAATGAA 
                 AACCCATTCTCACAGGGTGTA 
                 27121599 
                 27121797 
               
               
                   
                 (SEQ ID NO. 86) 
                 (SEQ ID NO. 87) 
               
               
                   
               
               
                 DG13S1917 
                 AGGAGTGTGGCAGCTTGAG 
                 TGGATTCCCGTGAGTACCAG 
                 27135092 
                 27135232 
               
               
                   
                 (SEQ ID NO. 88) 
                 (SEQ ID NO. 89) 
               
               
                   
               
               
                 D13S217 
                 ATGCTGGGATCACAGGC 
                 AACCTGGTGGACTTTTGCT 
                 27169880 
                 27170051 
               
               
                   
                 (SEQ ID NO. 90) 
                 (SEQ ID NO. 91) 
               
               
                   
               
               
                 DG13S581 
                 AGCATTTCCAATGGTGCTTT 
                 CATGTTGATATGCCTGAAGGA 
                 27318359 
                 27318725 
               
               
                   
                 (SEQ ID NO. 92) 
                 (SEQ ID NO. 93) 
               
               
                   
               
               
                 DG13S1471 
                 CACTGTCTGCTGCCACTCAT 
                 AGAGATTATGTGATGTACCCTCTC 
                 27403303 
                 27403544 
               
               
                   
                 (SEQ ID NO. 94) 
                 TAT (SEQ ID NO. 95) 
               
               
                   
               
               
                 DG13S2505 
                 TGATGAAGATCTGGGCGTTA 
                 TGCCTGTGCTCACTCACTCT 
                 27493479 
                 27493626 
               
               
                   
                 (SEQ ID NO. 96) 
                 (SEQ ID NO. 97) 
               
               
                   
               
               
                 D13S120 
                 ATGACCTAGAAATGATACTGGC 
                 CAGACACCACAACACACATT 
                 27540983 
                 27541093 
               
               
                   
                 (SEQ ID NO. 98) 
                 (SEQ ID NO. 99) 
               
               
                   
               
               
                 D13S1486 
                 TGGTTTAAAAACCTCATGCC 
                 ATCCCAAACTCTGTACTTATGTAG 
                 27623349 
                 27623496 
               
               
                   
                 (SEQ ID NO. 100) 
                 G(SEQ ID NO. 101) 
               
               
                   
               
               
                 DG13S1495 
                 CCTTGGCTGTTGTGACTGGT 
                 CACTCAGGTGGGAGGATCAC 
                 27668199 
                 27668471 
               
               
                   
                 (SEQ ID NO. 102) 
                 (SEQ ID NO. 103) 
               
               
                   
               
               
                 DG13S1845 
                 CACTTTTGCCAGTAGCCTTGA 
                 TTGGGAAAGTTAACCCAGAGA 
                 27788787 
                 27789056 
               
               
                   
                 (SEQ ID NO. 104) 
                 (SEQ ID NO. 105) 
               
               
                   
               
               
                 DG13S1030 
                 TTTGGGAAGAGCCATGAGAC 
                 CTCTGGGCATTGGAGGATTA 
                 27872811 
                 27873164 
               
               
                   
                 (SEQ ID NO. 106) 
                 (SEQ ID NO. 107) 
               
               
                   
               
               
                 DG13S584 
                 GGGAGACAAGTCAGGTGAGG 
                 CTGAGTATGGAGTCTTCATCATTA 
                 27924334 
                 27924484 
               
               
                   
                 (SEQ ID NO. 108) 
                 TC (SEQ ID NO. 109) 
               
               
                   
               
               
                 DG13S79 
                 TGCTACTAGATTTGACCAACCA 
                 GACTTGTAAAGGATTTAGTGATTT 
                 28213368 
                 28213495 
               
               
                   
                 (SEQ ID NO. 110) 
                 CG(SEQ ID NO. 111) 
               
               
                   
               
               
                 DG13S80 
                 GTGGAAGGCCTCTCTCTGTG 
                 TGCTTCTTGAGGGAAAGCAT 
                 28297121 
                 28297353 
               
               
                   
                 (SEQ ID NO. 112) 
                 (SEQ ID NO. 113) 
               
               
                   
               
               
                 DG13S1934 
                 CCTTCAGAGGATTTCCCTTTC 
                 CTGGTTTGACTCCAGCTTCA 
                 28461787 
                 28462194 
               
               
                   
                 (SEQ ID NO. 114) 
                 (SEQ ID NO. 115) 
               
               
                   
               
               
                 DG13S1104 
                 CCTGGCACGGAATAGACACT 
                 GGCCTCCTTTGCTCTGAAG 
                 28497694 
                 28498071 
               
               
                   
                 (SEQ ID NO. 116) 
                 (SEQ ID NO. 117) 
               
               
                   
               
               
                 DG13S1097 
                 CATCCCTGTGGCTGATTAAGA 
                 AACAGTTCCAGCCCGTTCTA 
                 28532382 
                 28532543 
               
               
                   
                 (SEQ ID NO. 118) 
                 (SEQ ID NO. 119) 
               
               
                   
               
               
                 DG13S1110 
                 TTTCAAAGGAATATCCAAGTGC 
                 TGGCGTACCATATAAACAGTTCTC 
                 28547636 
                 28547900 
               
               
                   
                 (SEQ ID NO. 120) 
                 (SEQ ID NO. 121) 
               
               
                   
               
               
                 DG13S87 
                 TTCAATGAAGGTGCCGAAGT 
                 TGTCTATCCCAAAGCTGCAA 
                 28597688 
                 28597905 
               
               
                   
                 (SEQ ID NO. 122) 
                 (SEQ ID NO. 123) 
               
               
                   
               
               
                 DG13S2400 
                 GCTCAGTCCAAGTTCATGCTC 
                 TGGGATTGGGTTCTGGATAC 
                 28671947 
                 28672231 
               
               
                   
                 (SEQ ID NO. 124) 
                 (SEQ ID NO. 125) 
               
               
                   
               
               
                 DG13S3114 
                 CCTACTTTTCCATCTCCTCCTTG 
                 TGGAGTAAGTTGGAGAATTGTTG 
                 28678081 
                 28678248 
               
               
                   
                 (SEQ ID NO. 126) 
                 A(SEQ ID NO. 127) 
               
               
                   
               
               
                 DG13S1111 
                 GCAAGACTCTGTTGAAGAAGAAG 
                 TCCCTCTGTTTGAGTTTCTCG 
                 28760422 
                 28760531 
               
               
                   
                 A (SEQ ID NO. 128) 
                 (SEQ ID NO. 129) 
               
               
                   
               
               
                 DG13S3122 
                 CCTTGGGCAGTCAGAGAAAC 
                 CCCGTGAAGTCTGAGAGGTG 
                 28778662 
                 28778906 
               
               
                   
                 (SEQ ID NO. 130) 
                 (SEQ ID NO. 131) 
               
               
                   
               
               
                 DG13S1101 
                 AGGCACAGTCGCTCATGTC 
                 AAACTTTAGCTAATGGTGGTCAA 
                 28812542 
                 28812874 
               
               
                   
                 (SEQ ID NO. 132) 
                 A (SEQ ID NO. 133) 
               
               
                   
               
               
                 D13S1246 
                 GAGCATGTGTGACTTTCATATTC 
                 AGTGGCTATTCATTGCTACAGG 
                 28903534 
                 28903738 
               
               
                   
                 AG (SEQ ID NO. 134) 
                 (SEQ ID NO. 135) 
               
               
                   
               
               
                 DG1351103 
                 TTGCTGGATGCTGGTTTCTA 
                 AAAGAGAGAGAGAAAGAGAAAG 
                 28910502 
                 28910765 
               
               
                   
                 (SEQ ID NO. 136) 
                 AAAGA (SEQ ID NO. 137) 
               
               
                   
               
               
                 DG1353147 
                 AAAGTGGATGCAGTTTGAGGTTT 
                 GCTAGCCATTACAGACAACCAA 
                 29018341 
                 29018591 
               
               
                   
                 (SEQ ID NO. 138) 
                 (SEQ ID NO. 139) 
               
               
                   
               
               
                 DG13S3150 
                 CAGGGCTCCATGTATCCATAA 
                 CAATCTTTGGCTTTGGGTTT 
                 29042766 
                 29042948 
               
               
                   
                 (SEQ ID NO. 140) 
                 (SEQ ID NO. 141) 
               
               
                   
               
               
                 D13S289 
                 CTGGTTGAGCGGCATT 
                 TGCAGCCTGGATGACA 
                 29063702 
                 29063949 
               
               
                   
                 (SEQ ID NO. 142) 
                 (SEQ ID NO. 143) 
               
               
                   
               
               
                 DG13S166 
                 CCTATGGAAGCATAGGGAAGAA 
                 CCCACTTCTGAGTCTCCTGAT 
                 29064359 
                 29064753 
               
               
                   
                 (SEQ ID NO. 144) 
                 (SEQ ID NO. 145) 
               
               
                   
               
               
                 DG13S3156 
                 GGGAAATGGAGCTGCTGTTAT 
                 GAGTGGGTGAGTGCAAGGAT 
                 29111037 
                 29111416 
               
               
                   
                 (SEQ ID NO. 146) 
                 (SEQ ID NO. 147) 
               
               
                   
               
               
                 D13S1238 
                 CTCTCAGCAGGCATCCA 
                 GCCAACGTAATTGACACCA 
                 29144427 
                 29144579 
               
               
                   
                 (SEQ ID NO. 148) (SEQ ID NO. 149) 
               
               
                   
               
               
                 DG13S2605 
                 TGAAAGGAAGGTCCCTGAGTT 
                 CCCTGCTTTGCACAAGTTATC 
                 29145896 
                 29146055 
               
               
                   
                 (SEQ ID NO. 150) 
                 (SEQ ID NO. 151) 
               
               
                   
               
               
                 DG13S163 
                 CACATGAGGCTGTATGTGGA 
                 TGTGCAGGAATGAGAAGTCG 
                 29177152 
                 29177313 
               
               
                   
                 (SEQ ID NO. 152) 
                 (SEQ ID NO. 153) 
               
               
                   
               
               
                 D13S290 
                 CCTTAGGCCCCATAATCT 
                 CAAATTCCTCAATTTGCAAAAT 
                 29227323 
                 29227512 
               
               
                   
                 (SEQ ID NO. 154) 
                 (SEQ ID NO. 155) 
               
               
                   
               
               
                 D13S1229 
                 GGTCATTCAGGGAGCCATTC 
                 CCATTATATTTCACCAAGAGGCTG 
                 29282262 
                 29282396 
               
               
                   
                 SEQ ID NO. 156) 
                 C SEQ ID NO. 157) 
               
               
                   
               
               
                 DG13S2358 
                 AGTCAAGGCTGACAGGGAAG 
                 GCTCTCAGCCCTCAATGTGT 
                 29342275 
                 29342399 
               
               
                   
                 (SEQ ID NO. 158) 
                 (SEQ ID NO. 159) 
               
               
                   
               
               
                 DG13S2658 
                 ATTTGGGTTCCTCTCCCAAT 
                 ACAAACTCTTGCTGCTGGTG 
                 29348162 
                 29348426 
               
               
                   
                 (SEQ ID NO. 160) 
                 (SEQ ID NO. 161) 
               
               
                   
               
               
                 DG13S1460 
                 TGCCTGGTCATCTACCCATT 
                 TCTACTGCAGCGCTGATCTT 
                 29389048 
                 29389297 
               
               
                   
                 (SEQ ID NO. 162) 
                 (SEQ ID NO. 163) 
               
               
                   
               
               
                 DG13S2434 
                 TCCTTCCAGAAGGTTTGCAT 
                 TGCAAAGTTGTTCAAGAGAGACA 
                 29485254 
                 29485392 
               
               
                   
                 (SEQ ID NO. 164) 
                 (SEQ ID NO. 165) 
               
               
                   
               
               
                 DG13S1448 
                 CAGCAGGAAGATGGACAGGT 
                 CACACTGCATCACACATACCC 
                 29499404 
                 29499531 
               
               
                   
                 (SEQ ID NO. 166) 
                 (SEQ ID NO. 167) 
               
               
                   
               
               
                 D13S1287 
                 TATGCCAGTATGCCTGCT 
                 GTCACATCAGTCCATTTGC 
                 29513830 
                 29514063 
               
               
                   
                 (SEQ ID NO. 168) 
                 (SEQ ID NO. 169) 
               
               
                   
               
               
                 DG13S2665 
                 GGTTTATGTCTGTGTGTGTGTGC 
                 TGAGGGATGTCAGAGAAATATGC 
                 29747845 
                 29747984 
               
               
                   
                 (SEQ ID NO. 170) 
                 (SEQ ID NO. 171) 
               
               
                   
               
               
                 DG13S1904 
                 TGATGAAATTGCCTAGTGATGC 
                 GGATCCAATCGTAGGCTACC 
                 29767797 
                 29767922 
               
               
                   
                 (SEQ ID NO. 172) 
                 (SEQ ID NO. 173) 
               
               
                   
               
               
                 DG13S1490 
                 ACCTAAACACCACGGACTGG 
                 CAGGTATCGACATTCTTCCAAA 
                 29908555 
                 29908958 
               
               
                   
                 (SEQ ID NO. 174) 
                 (SEQ ID NO. 175) 
               
               
                   
               
               
                 DG13S2637 
                 GGTGATCTAGGGAATTATTTGTC 
                 TTGGCCACTAAGGTCCAGAT 
                 29941956 
                 29942120 
               
               
                   
                 TTC (SEQ ID NO. 176) 
                 (SEQ ID NO. 177) 
               
               
                   
               
               
                 DG13S96 
                 CCTTTGAGGCTGGATCTGTT 
                 TTTCCTTATCATTCATTCCCTCA 
                 30166433 
                 30166650 
               
               
                   
                 (SEQ ID NO. 178) 
                 (SEQ ID NO. 179) 
               
               
                   
               
               
                 D13S260 
                 AGATATTGTCTCCGTTCCATGA 
                 CCCAGATATAAGGACCTGGCTA 
                 30234833 
                 30234997 
               
               
                   
                 (SEQ ID NO. 180) 
                 (SEQ ID NO. 181) 
               
               
                   
               
               
                 DG13S17 
                 TTTTAAGCCCTGTGGAATGTATTT 
                 GACATTGCAGGTCAAGTAGGG 
                 30288392 
                 30288544 
               
               
                   
                 (SEQ ID NO. 182) 
                 (SEQ ID NO. 183) 
               
               
                   
               
               
                 DG13S306 
                 TGCATAAGGCTGGAGACAGA 
                 CACAGCAGATGGGAGCAAA 
                 30404049 
                 30404203 
               
               
                   
                 (SEQ ID NO. 184) 
                 (SEQ ID NO. 185) 
               
               
                   
               
               
                 DG13S2486 
                 AGCCAGTTGTCTTTCATCCTG 
                 TGCCTGTGCTTGTATATTCTGTG 
                 30411508 
                 304TT55 
               
               
                   
                 (SEQ ID NO. 186) 
                 (SEQ ID NO. 187) 
               
               
                   
               
               
                 DG13S18 
                 GTGCATGTGCATACCAGACC 
                 GGCAAGATGACCTCTGGAAA 
                 30456875 
                 30457193 
               
               
                   
                 (SEQ ID NO. 188) 
                 (SEQ ID NO. 189) 
               
               
                   
               
               
                 DG13S1062 
                 TTTGTGTTCCAGGTGAGAATTG 
                 GAACCATATCCCAAGGCACT 
                 30551596 
                 30551715 
               
               
                   
                 (SEQ ID NO. 190) 
                 (SEQ ID NO. 191) 
               
               
                   
               
               
                 DG13S1093 
                 TTGTTCCCACATTCATTCTACA 
                 TTAAACTCGTGGCAAAGACG 
                 30625918 
                 30626190 
               
               
                   
                 (SEQ ID NO. 192) 
                 (SEQ ID NO. 193) 
               
               
                   
               
               
                 DG13S1059 
                 CACCATGCCTGGCTCTTT 
                 AACTTCTCCAGTTGTGTGGTTG 
                 30822917 
                 30823246 
               
               
                   
                 (SEQ ID NO. 194) 
                 (SEQ ID NO. 195) 
               
               
                   
               
               
                 D13S171 
                 CCTACCATTGACACTCTCAG 
                 TAGGGCCATCCATTCT 
                 31051937 
                 31052167 
               
               
                   
                 (SEQ ID NO. 196) 
                 (SEQ ID NO. 197) 
               
               
                   
               
               
                 DG13S2359 
                 TCTGTGTGTATTGTGTACTCCTCT 
                 TCACACAATTTGAACCAATCCT 
                 31073673 
                 31073849 
               
               
                   
                 G (SEQ ID NO. 198) 
                 (SEQ ID NO. 199) 
               
               
                   
               
               
                 DG13S1092 
                 ACCAAGATATGAAGGCCAAA 
                 CCTCCAGCTAGAACAATGTGAA 
                 31113759 
                 31113934 
               
               
                   
                 (SEQ ID NO. 200) 
                 (SEQ ID NO. 201) 
               
               
                   
               
               
                 DG13S2629 
                 TGATCATGTCAGCAGCAGAAG 
                 AGTAACAGGTGAGGGCATGG 
                 3TT9791 
                 3TT9953 
               
               
                   
                 (SEQ ID NO. 202) 
                 (SEQ ID NO. 203) 
               
               
                   
               
               
                 DG13S1449 
                 TGTCCATAGCTGTAGCCCTGT 
                 CTCAATGGGCATCTTTAGGC 
                 31199228 
                 31199498 
               
               
                   
                 (SEQ ID NO. 204) 
                 (SEQ ID NO. 205) 
               
               
                   
               
               
                 DG13S312 
                 CAAACAAACAAACAAGCAAACC 
                 TGGACGTTTCTTTCAGTGAGG 
                 31280202 
                 31280550 
               
               
                   
                 (SEQ ID NO. 206) 
                 (SEQ ID NO. 207) 
               
               
                   
               
               
                 DG13S1511 
                 TGATAACTTACCAGCATGTGAGC 
                 TCACCTCACCTAAGGATCTGC 
                 31321562 
                 31321854 
               
               
                   
                 (SEQ ID NO. 208) 
                 (SEQ ID NO. 209) 
               
               
                   
               
               
                 DG13S2454 
                 GCTAGCAAATCTCTCAACTTCCA 
                 TCTTCTCCATGCTGCTTCCT 
                 31352662 
                 31352803 
               
               
                   
                 (SEQ ID NO. 210) 
                 (SEQ ID NO. 211) 
               
               
                   
               
               
                 DG13S314 
                 CATGCAATTGCCCAATAGAG 
                 TTGGGCTTGTCTACCTAGTTCA 
                 31379760 
                 31380086 
               
               
                   
                 (SEQ ID NO. 212) 
                 (SEQ ID NO. 213) 
               
               
                   
               
               
                 DG13S1071 
                 GCTGCACGTATTTGTTGGTG 
                 AAACAGCAGAAATGGGAACC 
                 31447431 
                 31447669 
               
               
                   
                 (SEQ ID NO. 214) 
                 (SEQ ID NO. 215) 
               
               
                   
               
               
                 DG13S1068 
                 CCGTGGGCTATCAATTTCTG 
                 AAGATGCAATCTGGTTCCAA 
                 31553333 
                 31553570 
               
               
                   
                 (SEQ ID NO. 216) 
                 (SEQ ID NO. 217) 
               
               
                   
               
               
                 DG13S1077 
                 CCCAAGACTGAGGAGGTCAA 
                 GCTGACGGAGAGGAAAGAGA 
                 31569360 
                 31569733 
               
               
                   
                 (SEQ ID NO. 218) 
                 (SEQ ID NO. 219) 
               
               
                   
               
               
                 DG13S2343 
                 TCACAAAGCAAGCAATCACA 
                 TGATGGATGCACCATGTTA 
                 31653489 
                 31653608 
               
               
                   
                 (SEQ ID NO. 220) 
                 (SEQ ID NO. 221) 
               
               
                   
               
               
                 DG13S316 
                 TGAGAAGCCTGGGCATTAAG 
                 ACAAGCTCATCCAGGGAAAG 
                 31708002 
                 31708244 
               
               
                   
                 (SEQ ID NO. 222) 
                 (SEQ ID NO. 223) 
               
               
                   
               
               
                 DG13S1558 
                 AGAGCTGATCTGGCCGAAG 
                 GGTGGACACAGAATCCACACT 
                 31986248 
                 31986627 
               
               
                   
                 (SEQ ID NO. 224) 
                 (SEQ ID NO. 225) 
               
               
                   
               
               
                 D13S267 
                 GGCCTGAAAGGTATCCTC 
                 TCCCACCATAAGCACAAG 
                 32062233 
                 32062380 
               
               
                   
                 (SEQ ID NO. 226) 
                 (SEQ ID NO. 227) 
               
               
                   
               
               
                 DG13S1478 
                 TCAACCTAGGATTGGCATTACA 
                 TCTAGGATTTGTGCCTTTCCA 
                 32157761 
                 32158137 
               
               
                   
                 (SEQ ID NO. 228) 
                 (SEQ ID NO. 229) 
               
               
                   
               
               
                 DG13S1551 
                 ATTCGTGCAGCTGTTTCTGC 
                 GCATGACATTGTAAATGGAGGA 
                 32364898 
                 32365153 
               
               
                   
                 (SEQ ID NO. 230) 
                 (SEQ ID NO. 231) 
               
               
                   
               
               
                 DG13S1884 
                 GGTGGGAATGTGTGACTGAA 
                 CCAGGTACAACATTCTCCTGAT 
                 32451203 
                 32451315 
               
               
                   
                 (SEQ ID NO. 232) 
                 (SEQ ID NO. 233) 
               
               
                   
               
               
                 D13S1293 
                 TGCAGGTGGGAGTCAA 
                 AAATAACAAGAAGTGACCTTCCT 
                 32536337 
                 32536467 
               
               
                   
                 (SEQ ID NO. 234) 
                 A (SEQ ID NO. 235) 
               
               
                   
               
               
                 DG13S1518 
                 AAAGGATGCATTCGGTTAGAG 
                 ACTGTCCTGTGCCTGTGCTT 
                 32588965 
                 32589321 
               
               
                   
                 (SEQ ID NO. 236) 
                 (SEQ ID NO. 237) 
               
               
                   
               
               
                 D13S620 
                 GTCCACCTAATGGCTCATTC 
                 CAAGAAGCACTCATGTTTGTG 
                 32627749 
                 32627947 
               
               
                   
                 (SEQ ID NO. 238) 
                 (SEQ ID NO. 239) 
               
               
                   
               
               
                 DG13S1866 
                 AGCCTGTGATTGGCTGAGA 
                 GGCTTACAGCTGCCTCCTTT 
                 32633306 
                 32633709 
               
               
                   
                 (SEQ ID NO. 240) 
                 (SEQ ID NO. 241) 
               
               
                   
               
               
                 DG13S1927 
                 CCCACAGAGCACTTTGTTAGA 
                 GCCTCCCTTAAGCTGTTATGC 
                 32691932 
                 32692304 
               
               
                   
                 (SEQ ID NO. 242) 
                 (SEQ ID NO. 243) 
               
               
                   
               
               
                 DG13S1503 
                 CACTCTTTACTGCCAATCACTCC 
                 GCCGTGTGGGTGTATGAAT 
                 32699827 
                 32700058 
               
               
                   
                 (SEQ ID NO. 244) 
                 (SEQ ID NO. 245) 
               
               
                   
               
               
                 DG13S332 
                 TTGTACCAGGAACCAAAGACAA 
                 CACAGACAGAGGCACATTGA 
                 32764576 
                 32764751 
               
               
                   
                 (SEQ ID NO. 246) 
                 (SEQ ID NO. 247) 
               
               
                   
               
               
                 DG13S333 
                 GCTCTGGTCACTCCTGCTGT 
                 CATGCCTGGCTGATTGTTT 
                 32872275 
                 32872720 
               
               
                   
                 (SEQ ID NO. 248) 
                 (SEQ ID NO. 249) 
               
               
                   
               
               
                 D13S220 
                 CCAACATCGGGAACTG 
                 TGCATTCTTTTAAGTCCATGTC 
                 32967602 
                 32967793 
               
               
                   
                 (SEQ ID NO. 250) 
                 (SEQ ID NO. 251) 
               
               
                   
               
               
                 DG13S1919 
                 CAGCAACTGACAACTCATCCA 
                 CCTCAATCCTCAGCTCCAAC 
                 33014255 
                 33014477 
               
               
                   
                 (SEQ ID NO. 252) 
                 (SEQ ID NO. 253) 
               
               
                   
               
               
                 DG13S2383 
                 TGATTGGTTCTGTTGTTGCTG 
                 AGCCCAAGGCTCTTTGTGAG 
                 33053369 
                 33053553 
               
               
                   
                 (SEQ ID NO. 254) 
                 (SEQ ID NO. 255) 
               
               
                   
               
               
                 DG13S1439 
                 TCCTTCACAGCTTCAAACTCA 
                 AGTGAGAAGCTTCCATACTGGT 
                 33070030 
                 33070264 
               
               
                   
                 (SEQ ID NO. 256) 
                 (SEQ ID NO. 257) 
               
               
                   
               
               
                 DG13S335 
                 GCCAACCGTTAGACAAATGA 
                 CTACATGTGCACCACAACACC 
                 33102278 
                 33102478 
               
               
                   
                 (SEQ ID NO. 258) 
                 (SEQ ID NO. 259) 
               
               
                   
               
               
                 DG13S340 
                 AGTTTATTGCCGCCGAGAG 
                 ACCCACCACATTCACAAGC 
                 33124866 
                 33125238 
               
               
                   
                 (SEQ ID NO. 260) 
                 (SEQ ID NO. 261) 
               
               
                   
               
               
                 DG13S1496 
                 CGATTGCCATGTCTCTTGA 
                 GAGATCTGGCCTGGATTTGT 
                 33215915 
                 33216066 
               
               
                   
                 (SEQ ID NO. 262) 
                 (SEQ ID NO. 263) 
               
               
                   
               
               
                 DG13S347 
                 TCATTGTCAGCACAGAATGAACT 
                 GGAGGGAGGGAAGAAAGAGA 
                 33280351 
                 33280688 
               
               
                   
                 (SEQ ID NO. 264) 
                 (SEQ ID NO. 265) 
               
               
                   
               
               
                 DG13S339 
                 GGGAAGAGGAGATTGACTTGTT 
                 GGAACACCATCATTCCAACC 
                 33352425 
                 33352656 
               
               
                   
                 (SEQ ID NO. 266) 
                 (SEQ ID NO. 267) 
               
               
                   
               
               
                 DG13S1926 
                 TACAAGCTCCACCGTCCTTC 
                 TGAGTTGCTGCCTCTTCAAA 
                 33388692 
                 33388919 
               
               
                   
                 (SEQ ID NO. 268) 
                 (SEQ ID NO. 269) 
               
               
                   
               
               
                 DG13S1469 
                 TGCTAATGGGCCAAGGAATA 
                 GCTAAATGTCCTCATGAATAGCC 
                 33416571 
                 33416940 
               
               
                   
                 (SEQ ID NO. 270) 
                 (SEQ ID NO. 271) 
               
               
                   
               
               
                 DG13S351 
                 TGTCCTGCAGACAGATGGTC 
                 CCTCCGGAGTAGCTGGATTA 
                 33497762 
                 33498055 
               
               
                   
                 (SEQ ID NO. 272) 
                 (SEQ ID NO. 273) 
               
               
                   
               
               
                 DG13S26 
                 GAGACTGGCCCTCATTCTTG 
                 AAGAAGCCAGAGACAAAGAAATA 
                 33584096 
                 33584425 
               
               
                   
                 (SEQ ID NO. 274) 
                 CA (SEQ ID NO. 275) 
               
               
                   
               
               
                 DG13S30 
                 CATCTATCTTGGATTCAGTGGTG 
                 TGCTCCCAACATCTTACCAG 
                 33731684 
                 33732071 
               
               
                   
                 (SEQ ID NO. 276) 
                 (SEQ ID NO. 277) 
               
               
                   
               
               
                 DG13S1435 
                 TGTCCTCTGGTCATTTCTATGGT 
                 CATGAATGAGAAGTGATGAATGG 
                 33762069 
                 33762285 
               
               
                   
                 (SEQ ID NO. 278) 
                 (SEQ ID NO. 279) 
               
               
                   
               
               
                 DG13S356 
                 CAGACACTGTAAACTGGCTTCG 
                 CCACATTGCTATCAGCGTA 
                 33908746 
                 33908957 
               
               
                   
                 (SEQ ID NO. 280) 
                 (SEQ ID NO. 281) 
               
               
                   
               
               
                 DG13S2316 
                 ATGTGCTGTGGTCCAGATTT 
                 CCTACTACTGCAATTACTCCCTAC 
                 33913787 
                 33913954 
               
               
                   
                 (SEQ ID NO. 282) 
                 C (SEQ ID NO. 283) 
               
               
                   
               
               
                 DG13S357 
                 TGTCATAGGCTGCGGTATT 
                 TTGGTAGGGTCCTTTCCTTTT 
                 33935177 
                 33935378 
               
               
                   
                 (SEQ ID NO. 284) 
                 (SEQ ID NO. 285) 
               
               
                   
               
               
                 DG13S1032 
                 GCCTGCTCACTGTTGTTTGA 
                 CGGTTATCAGAGACTGGTGGT 
                 33967059 
                 33967269 
               
               
                   
                 (SEQ ID NO. 286) 
                 (SEQ ID NO. 287) 
               
               
                   
               
               
                 DG13S1557 
                 GGCTTATTTCATGTACGGCTA 
                 GGTTAAACTCTACTTAGTCCTGAT 
                 33996100 
                 33996249 
               
               
                   
                 (SEQ ID NO. 288) 
                 GC (SEQ ID NO. 289) 
               
               
                   
               
               
                 DG13S1925 
                 GAACTCTGCAGGCACCTCTT 
                 CCTGAAGCGCTTGTACTGAA 
                 34079148 
                 34079570 
               
               
                   
                 (SEQ ID NO. 290) 
                 (SEQ ID NO. 291) 
               
               
                   
               
               
                 DG13S360 
                 TTGGCTTCTCGCTCTTTCTT 
                 AGCCATCAGTCACATGCAAA 
                 34138872 
                 34139221 
               
               
                   
                 (SEQ ID NO. 292) 
                 (SEQ ID NO. 293) 
               
               
                   
               
               
                 DG13S1522 
                 AGATCTCCAGGGCAGAGGAC 
                 CCTTCCTCCCTCCTTCTCTC 
                 34195314 
                 34195659 
               
               
                   
                 (SEQ ID NO. 294) 
                 (SEQ ID NO. 295) 
               
               
                   
               
               
                 DG13S2324 
                 CAGTCAAATGTCTCAACCTTCC 
                 CTAGCAACATGGCCAAGAAA 
                 34224040 
                 34224206 
               
               
                   
                 (SEQ ID NO. 296) 
                 (SEQ ID NO. 297) 
               
               
                   
               
               
                 DG13S1517 
                 CGTCATTGATCCCAATCATCT 
                 GGCTGATAGCCTCCCTTGTA 
                 34271358 
                 34271587 
               
               
                   
                 (SEQ ID NO. 298) 
                 (SEQ ID NO. 299) 
               
               
                   
               
               
                 DG13S364 
                 ACCTTCAAGCTTCCGGTTT 
                 TTCCATCCGTCCATCTATCC 
                 34323307 
                 34323478 
               
               
                   
                 (SEQ ID NO. 300) 
                 (SEQ ID NO. 301) 
               
               
                   
               
               
                 DG13S1036 
                 TTAAAGTCACTTGTCTGTGGTCA 
                 TTTGTAGGAATCAAGTCAAATAAT 
                 34525065 
                 34525280 
               
               
                   
                 (SEQ ID NO. 302) 
                 GTA (SEQ ID NO. 303) 
               
               
                   
               
               
                 DG13S1037 
                 CTTTCGGAAGCTTGAGCCTA 
                 CCCAAGACCACTGCCATATT 
                 34616658 
                 34616926 
               
               
                   
                 (SEQ ID NO. 304) 
                 (SEQ ID NO. 305) 
               
               
                   
               
               
                 DG13S1854 
                 TGACAGGTTTGGGTATATTGGA 
                 TGCTTAATGTAGTGGCAGCA 
                 34622055 
                 34622151 
               
               
                   
                 (SEQ ID NO. 306) 
                 (SEQ ID NO. 307) 
               
               
                   
               
               
                 DG13S1038 
                 TCCTGCCTTTGTGAATTCCT 
                 GTTGAATGAGGTGGGCATTA 
                 34702405 
                 34702738 
               
               
                   
                 (SEQ ID NO. 308) 
                 (SEQ ID NO. 309) 
               
               
                   
               
               
                 DG13S2366 
                 TTGGGAATAAATCAGGTGTTGA 
                 GCAGCAGCTCAGCATTTCTC 
                 34735455 
                 34735583 
               
               
                   
                 (SEQ ID NO. 310) 
                 (SEQ ID NO. 311) 
               
               
                   
               
               
                 DG13S1039 
                 CCATTTAATCCTCCAGCCATT 
                 GCTCCACCTTGTTACCCTGA 
                 34743651 
                 34743817 
               
               
                   
                 (SEQ ID NO. 312) 
                 (SEQ ID NO. 313) 
               
               
                   
               
               
                 DG13S1840 
                 ACAACCCTGGAATCTGGACT 
                 GAAGGAAAGGAAAGGAAAGAAA 
                 34805466 
                 34805682 
               
               
                   
                 (SEQ ID NO. 314) 
                 (SEQ ID NO. 315) 
               
               
                   
               
               
                 DG13S369 
                 TGACAAGACTGAAACTTCATCAG 
                 GATGCTTGCTTGGGAGGTA 
                 34815499 
                 34815755 
               
               
                   
                 (SEQ ID NO. 316) 
                 (SEQ ID NO. 317) 
               
               
                   
               
               
                 DG13S2481 
                 CAGGTTAGAGCCCATCCAAG 
                 AGGCTCAGCTTCATCCACAT 
                 34867728 
                 34867872 
               
               
                   
                 (SEQ ID NO. 318) 
                 (SEQ ID NO. 319) 
               
               
                   
               
               
                 D13S219 
                 AAGCAAATATGCAAAATTGC 
                 TCCTTCTGTTTCTTGACTTAACA 
                 34956581 
                 34956707 
               
               
                   
                 (SEQ ID NO. 320) 
                 (SEQ ID NO. 321) 
               
               
                   
               
               
                 DG13S2351 
                 GGGAACAGGTCACAGGTCAT 
                 GGAAGACTGGGTGGTCACAG 
                 35099146 
                 35099320 
               
               
                   
                 (SEQ ID NO. 322) 
                 (SEQ ID NO. 323) 
               
               
                   
               
               
                 DG13S384 
                 TTCCTTCTGCTTGTGAGCTG 
                 TACCCTCACCTCCTCATGC 
                 35499548 
                 35499763 
               
               
                   
                 (SEQ ID NO. 324) 
                 (SEQ ID NO. 325) 
               
               
                   
               
               
                 DG13S1507 
                 GAAGACATTGGCAGGTCTGG 
                 GAGCCCTCATGTTGGGATAA 
                 35557977 
                 35558206 
               
               
                   
                 (SEQ ID NO. 326) 
                 (SEQ ID NO. 327) 
               
               
                   
               
               
                 DG13S1512 
                 TTGTTGATTCTCCCATTCTGTG 
                 TCACCTACCTCATCTCATACTCAA 
                 35668964 
                 35669201 
               
               
                   
                 (SEQ ID NO. 328) 
                 A (SEQ ID NO. 329) 
               
               
                   
               
               
                 DG13S1556 
                 TCTTCCGGACAAGTTTCCAA 
                 TGGGTCATCTGGACATTCA 
                 35791215 
                 35791467 
               
               
                   
                 (SEQ ID NO. 330) 
                 (SEQ ID NO. 331) 
               
               
                   
               
               
                 DG13S388 
                 GCAAATGAGGCTGGTAAGGT 
                 TGCACTGTGGTAGAGGGAAA 
                 35817061 
                 35817320 
               
               
                   
                 (SEQ ID NO. 332) 
                 (SEQ ID NO. 333) 
               
               
                   
               
               
                 DG13S1442 
                 CAACATACTCCTATGCCTAGAAA 
                 CTCACCAGGCAGAAACAGGT 
                 35842967 
                 35843335 
               
               
                   
                 GAAA (SEQ ID NO. 334) 
                 (SEQ ID NO. 335) 
               
               
                   
               
               
                 DG13S1045 
                 CCCAATGGCATGCTTCACT 
                 GGTTCTCCCAGCATTGGTT 
                 35928180 
                 35928324 
               
               
                   
                 (SEQ ID NO. 336) 
                 (SEQ ID NO. 337) 
               
               
                   
               
               
                 DG13S2452 
                 AAGGCCTCTGGGTAGGTAGG 
                 AAGCAATCCTTATGGGCTCT 
                 35948528 
                 35948826 
               
               
                   
                 (SEQ ID NO. 338) 
                 (SEQ ID NO. 339) 
               
               
                   
               
               
                 DG13S2350 
                 CCAGGTAATCAGAAGCCTCA 
                 TTCCGTTAAATCCAGCCATC 
                 36011840 
                 36011961 
               
               
                   
                 (SEQ ID NO. 340) 
                 (SEQ ID NO. 341) 
               
               
                   
               
               
                 DG13S2483 
                 CAGGGACTGCAGTGTCTCAA 
                 ATGCCACATTTGCCTCTCTC 
                 36027396 
                 36027703 
               
               
                   
                 (SEQ ID NO. 342) 
                 (SEQ ID NO. 343) 
               
               
                   
               
               
                 DG13S1100 
                 CCACCTTCCACTTAATACAAACT 
                 GAAGCAATCCATTCCAAGAAA 
                 36056838 
                 36057115 
               
               
                   
                 TC (SEQ ID NO. 344) 
                 (SEQ ID NO. 345) 
               
               
                   
               
               
                 DG13S1501 
                 GTCCTGAGGGTGTCCAGGTA 
                 GCTGGAGAACTCCTATTCTGCT 
                 36215761 
                 36215909 
               
               
                   
                 (SEQ ID NO. 346) 
                 (SEQ ID NO. 347) 
               
               
                   
               
               
                 DG13S1868 
                 TGGAGCTATTGCGGTTCTCT 
                 TCAAATCTCTCTTTCCTCCTCCT 
                 36313203 
                 36313417 
               
               
                   
                 (SEQ ID NO. 348) 
                 (SEQ ID NO. 349) 
               
               
                   
               
               
                 DG13S395 
                 CAGTTCCAGCTACGGGAGAA 
                 CCGCATTTAGGCAAGTCTCA 
                 36317151 
                 36317507 
               
               
                   
                 (SEQ ID NO. 350) 
                 (SEQ ID NO. 351) 
               
               
                   
               
               
                 D13S1491 
                 AAGCACACACAGATGCTAGG 
                 CCTCAGCCTCCATAATCTCA 
                 36361442 
                 36361571 
               
               
                   
                 (SEQ ID NO. 352) 
                 (SEQ ID NO. 353) 
               
               
                   
               
               
                 DG13S400 
                 GTACAGAGCCCACCTTCTGG 
                 TCACTATGCTGCAAGGCAAG 
                 36369862 
                 36370134 
               
               
                   
                 (SEQ ID NO. 354) 
                 (SEQ ID NO. 355) 
               
               
                   
               
               
                 D13S894 
                 GGTGCTTGCTGTAAATATAATTG 
                 CACTACAGCAGATTGCACCA 
                 36536509 
                 36536706 
               
               
                   
                 (SEQ ID NO. 356) 
                 (SEQ ID NO. 357) 
               
               
                   
               
               
                 D13S218 
                 GATTTGAAAATGAGCAGTCC 
                 GTCGGGCACTACGTTTATCT 
                 36830331 
                 36830519 
               
               
                   
                 (SEQ ID NO. 358) 
                 (SEQ ID NO. 359) 
               
               
                   
               
               
                 DG13S1553 
                 TGGGTGAAGATGCTACCTGA 
                 CCCTTCTTCCTTTCCCTCTC 
                 36898814 
                 36899040 
               
               
                   
                 (SEQ ID NO. 360) 
                 (SEQ ID NO. 361) 
               
               
                   
               
               
                 DG13S411 
                 TGCCAGGTCTGAGTTGTAAGC 
                 CAGCATGAGACCCTGTCAAA 
                 36908058 
                 36908265 
               
               
                   
                 (SEQ ID NO. 362) 
                 (SEQ ID NO. 363) 
               
               
                   
               
               
                 DG13S1870 
                 GAAAGAAAGAAAGAAAGAAGAA 
                 AATCACCAAACCTGGAAGCA 
                 36927423 
                 36927632 
               
               
                   
                 AGAAA (SEQ ID NO. 364) 
                 (SEQ ID NO. 365) 
               
               
                   
               
               
                 DG13S1870 
                 GAAAGAAAGAAAGAAAGAAGAA 
                 AATCACCAAACCTGGAAGCA 
                 36927485 
                 36927632 
               
               
                   
                 AGAAA (SEQ ID NO. 366) 
                 (SEQ ID NO. 367) 
               
               
                   
               
               
                 DG13S39 
                 TCTGAGTTAAACACTTGAGTTGC 
                 CCAGTAAATGGCAGTGTGGTT 
                 36957292 
                 36957640 
               
               
                   
                 TG (SEQ ID NO. 368) 
                 (SEQ ID NO. 369 
               
               
                   
               
               
                 DG13S2415 
                 TGTCATGGATATTTCTACATAAA 
                 TGAAGATGGTTATTGCTTCCTTC 
                 36984719 
                 36984955 
               
               
                   
                 CCAA (SEQ ID NO. 370) 
                 (SEQ ID NO. 371) 
               
               
                   
               
               
                 DG13S412 
                 CGCTTTGTTTGGTTTGGTTT 
                 ATGCAGTTGTCCCACATGCT 
                 37036929 
                 37037137 
               
               
                   
                 (SEQ ID NO. 372) 
                 (SEQ ID NO. 373) 
               
               
                   
               
               
                 DG13S414 
                 TCCTGCACTCCAAAGGAAAC 
                 AACTCTGGTTTAATTCAGCTTTGT 
                 37047489 
                 37047713 
               
               
                   
                 (SEQ ID NO. 374) 
                 C (SEQ ID NO. 375) 
               
               
                   
               
               
                 DG13S1872 
                 TTCTTGAGGGCATAAAGCTGA 
                 CACACTCACCAGGCACTCTG 
                 37119505 
                 37119608 
               
               
                   
                 (SEQ ID NO. 376) 
                 (SEQ ID NO. 377) 
               
               
                   
               
               
                 DG13S416 
                 CAGGTTTGATGAAGGAAATATGC 
                 GGGATCCTCTGCATTTCTCTAA 
                 37125983 
                 37126184 
               
               
                   
                 (SEQ ID NO. 378) 
                 (SEQ ID NO. 379) 
               
               
                   
               
               
                 DG13S2607 
                 TTTGCCAAATCAACCTTCAG 
                 CCTGCTTCACACCTCTGACC 
                 37317455 
                 37317831 
               
               
                   
                 (SEQ ID NO. 380) 
                 (SEQ ID NO. 381) 
               
               
                   
               
               
                 DG13S1898 
                 ACTCACACACAACCACCACA 
                 GCTACTGGTGGGTCGTAAGC 
                 37318932 
                 37319055 
               
               
                   
                 (SEQ ID NO. 382) 
                 (SEQ ID NO. 383) 
               
               
                   
               
               
                 D13S1288 
                 TTCAGAGACCATCACGGC 
                 CTGGAAAAATCAGTTGAATCCTA 
                 37321295 
                 37321486 
               
               
                   
                 (SEQ ID NO. 384) 
                 GC (SEQ ID NO. 385) 
               
               
                   
               
               
                 DG13S2567 
                 AGGAAAGCCGAGAAAGCATA 
                 CATGTATCCACATGCCCAGA 
                 37416093 
                 37416462 
               
               
                   
                 (SEQ ID NO. 386) 
                 (SEQ ID NO. 387) 
               
               
                   
               
               
                 DG13S418 
                 CCTTCAGCGCAGCTACATCT 
                 AGAACTGCGAGGTCCAAGTG 
                 37473016 
                 37473380 
               
               
                   
                 (SEQ ID NO. 388) 
                 (SEQ ID NO. 389) 
               
               
                   
               
               
                 DG13S419 
                 GGGAGAAAGAGAGGTAGGAAGG 
                 TTCCCAAGTTAGCAGCATCC 
                 37532947 
                 37533123 
               
               
                   
                 (SEQ ID NO. 390) 
                 (SEQ ID NO. 391) 
               
               
                   
               
               
                 DG13S1051 
                 TTCTAGAGGAGTCTATTTCTTTAC 
                 GGAGCTGTCACTTGAGCTTTG 
                 37694432 
                 37694579 
               
               
                   
                 TGG (SEQ ID NO. 392) 
                 (SEQ ID NO. 393) 
               
               
                   
               
               
                 DG13S1841 
                 CCGTGACCTACAGGGAACAT 
                 GGCATCGGGTGTTTCTATTC 
                 37715601 
                 37715829 
               
               
                   
                 (SEQ ID NO. 394) 
                 (SEQ ID NO. 395) 
               
               
                   
               
               
                 DG13S1052 
                 AGACCTGCCTGTGTTCTGGT 
                 GGAGTGAAATAAGTGGAACTGGA 
                 37831275 
                 37831438 
               
               
                   
                 (SEQ ID NO. 396) 
                 (SEQ ID NO. 397) 
               
               
                   
               
               
                 DG13S1053 
                 CATTAAATGAGTCATAAAGGTCA 
                 AACATTGTTGCTTTGCTGGA 
                 37935190 
                 37935311 
               
               
                   
                 TGG (SEQ ID NO. 398) 
                 (SEQ ID NO. 399) 
               
               
                   
               
               
                 DG13S423 
                 GGCCTTAGCTCAGTTTCTGG 
                 TGCAAAGACATTTGCGGATA 
                 37941221 
                 37941411 
               
               
                   
                 (SEQ ID NO. 400) 
                 (SEQ ID NO. 401) 
               
               
                   
               
               
                 D13S1253 
                 CCTGCATTTGTGTACGTGT 
                 CAGAGCCGTGGTAGTATATTTTT 
                 37944396 
                 37944533 
               
               
                   
                 (SEQ ID NO. 402) 
                 (SEQ ID NO. 403) 
               
               
                   
               
               
                 DG13S2539 
                 GGAACCAGTCATTGGGTGT 
                 TTATTGCTCCCTCGTCCAAG 
                 38050898 
                 38051253 
               
               
                   
                 (SEQ ID NO. 404) 
                 (SEQ ID NO. 405) 
               
               
                   
               
               
                 DG13S2509 
                 TGCCTTAAGGTCTATTATTTCCTT 
                 ACCAATGCAGGAAGACTCAA 
                 38067039 
                 38067186 
               
               
                   
                 TC (SEQ ID NO. 406) 
                 (SEQ ID NO. 407) 
               
               
                   
               
               
                 DG13S1863 
                 CTGATGAAAGGACACACATGC 
                 TGCATTAACTATGCAGCTTGAAA 
                 38092085 
                 38092353 
               
               
                   
                 (SEQ ID NO. 408) 
                 (SEQ ID NO. 409) 
               
               
                   
               
               
                 DG13S2510 
                 GTCGTGCAATCCCGAGAG 
                 GGATTCCTGCTGGCTCTTCT 
                 38197807 
                 38198059 
               
               
                   
                 (SEQ ID NO. 410) 
                 (SEQ ID NO. 411) 
               
               
                   
               
               
                 DG13S1909 
                 CTGGTGTGGTCAGGAAATGA 
                 GTGCTAAACACATGTGAGTGAGA 
                 38309328 
                 38309442 
               
               
                   
                 (SEQ ID NO. 412) 
                 G (SEQ ID NO. 413) 
               
               
                   
               
               
                 DG13S428 
                 TTTGACCATGCTTTCTCTTTGA 
                 GCTTGATGACTCCCTGCTGT 
                 38346716 
                 38347069 
               
               
                   
                 (SEQ ID NO. 414) 
                 (SEQ ID NO. 415) 
               
               
                   
               
               
                 DG13S1858 
                 AAGCCATTGAAAGGCAGGTA 
                 GGGACTTTCCGGCTTCTATT 
                 38371574 
                 38371742 
               
               
                   
                 (SEQ ID NO. 416) 
                 (SEQ ID NO. 417) 
               
               
                   
               
               
                 DG13S1911 
                 TGGTTGGGAACCATTCTCCT 
                 GCAGAGAAGGGATTTACTCCAG 
                 38475656 
                 38475877 
               
               
                   
                 (SEQ ID NO. 418) 
                 (SEQ ID NO. 419) 
               
               
                   
               
               
                 DG13S433 
                 ACTTGACATGGAGCAAGCTG 
                 AGCTCATCATGCTGTAAGGAG 
                 38516056 
                 38516191 
               
               
                   
                 (SEQ ID NO. 420) 
                 (SEQ ID NO. 421) 
               
               
                   
               
               
                 DG13S2421 
                 CACAGGCTCTCACATTCTCG 
                 TGACACTCATCCCTCTGCTG 
                 38534972 
                 38535357 
               
               
                   
                 (SEQ ID NO. 422) 
                 (SEQ ID NO. 423) 
               
               
                   
               
               
                 DG13S2375 
                 TGAGTTTCATAAGTTTACTACCTG 
                 GGCAGGGAGAAAGGACAAAT 
                 38548257 
                 38548440 
               
               
                   
                 CTG (SEQ ID NO. 424) 
                 (SEQ ID NO. 425) 
               
               
                   
               
               
                 D13S1248 
                 TCCCTTATGTGGGATTAGTTGA 
                 CAGACATGGAACTGAGATTTTTT 
                 38558005 
                 38558267 
               
               
                   
                 (SEQ ID NO. 426) 
                 (SEQ ID NO. 427) 
               
               
                   
               
               
                 DG13S1856 
                 TGTTTCCATCTCTCTACCCATGT 
                 TCAATGTTCTTATTGAGTGGGAAA 
                 38577323 
                 38577506 
               
               
                   
                 (SEQ ID NO. 428) 
                 (SEQ ID NO. 429) 
               
               
                   
               
               
                 DG13S435 
                 ATATCCACCCACCCACACAT 
                 TAGCTCTGAGGGCAGAGACC 
                 38591043 
                 38591261 
               
               
                   
                 (SEQ ID NO. 430) 
                 (SEQ ID NO. 431) 
               
               
                   
               
               
                 DG13S2459 
                 CCGTCCTTCCTCCACTGAT 
                 AGAGCACTGAGGGAGCAAAT 
                 38596056 
                 38596299 
               
               
                   
                 (SEQ ID NO. 432) 
                 (SEQ ID NO. 433) 
               
               
                   
               
               
                 DG13S438 
                 AGCTACAGCACGAGGCAGTT 
                 TTTGAATTGAGTTGCTGTTCG 
                 38676957 
                 38677248 
               
               
                   
                 (SEQ ID NO. 434) 
                 (SEQ ID NO. 435) 
               
               
                   
               
               
                 DG13S1865 
                 TGTACACCACCAACCATTCTG 
                 GGGAAGAAAGGCAAATAGCA 
                 38684800 
                 38684904 
               
               
                   
                 (SEQ ID NO. 436) 
                 (SEQ ID NO. 437) 
               
               
                   
               
               
                 DG13S2354 
                 GGATTGGCAATTAGCAGGTC 
                 GCCTGGTCAAAGATAACAGACG 
                 38773862 
                 38774026 
               
               
                   
                 (SEQ ID NO. 438) 
                 (SEQ ID NO. 439) 
               
               
                   
               
               
                 DG13S2534 
                 CCTGATTAAGCTGGCCTTTG 
                 ATCCTTCTGGGACCCTCATC 
                 38801698 
                 38801951 
               
               
                   
                 (SEQ ID NO. 440) 
                 (SEQ ID NO. 441) 
               
               
                   
               
               
                 DG13S1903 
                 GCTTTGCTTCCTTCTTGGTG 
                 CAACATTACGGCCAGTCTCA 
                 38802843 
                 38803052 
               
               
                   
                 (SEQ ID NO. 442) 
                 (SEQ ID NO. 443) 
               
               
                   
               
               
                 DG13S1896 
                 GGTGCATCTGATAAGCCAAA 
                 GCTGTCTTGGACACAGTGGA 
                 38815291 
                 38815405 
               
               
                   
                 (SEQ ID NO. 444) 
                 (SEQ ID NO. 445) 
               
               
                   
               
               
                 DG13S443 
                 CACCATCATCATCTGGTTGG 
                 GAGCTCATTGAAAGGCAGGA 
                 38838839 
                 38839093 
               
               
                   
                 (SEQ ID NO. 446) 
                 (SEQ ID NO. 447) 
               
               
                   
               
               
                 DG13S445 
                 CCATCCATCTATCCATTTATCTCT 
                 GGATTTATCCTTGCCCTGCT 
                 38840399 
                 38840584 
               
               
                   
                 (SEQ ID NO. 448) 
                 (SEQ ID NO. 449) 
               
               
                   
               
               
                 DG13S447 
                 CTATCATCCATCCATCCTATTTG 
                 TTAGGGCAGCTACCTGGAAA 
                 38840751 
                 38840928 
               
               
                   
                 (SEQ ID NO. 450) 
                 (SEQ ID NO. 451) 
               
               
                   
               
               
                 D13S1233 
                 AGGACTANAGATGAATGCTC 
                 GACATGACTCCATGTTTGGT 
                 38875108 
                 38875292 
               
               
                   
                 (SEQ ID NO. 452) 
                 (SEQ ID NO. 453) 
               
               
                   
               
               
                 DG13S2320 
                 CCTCACCTTGCAATTTCCTG 
                 CTGACTTGCCTGTTGGCATA 
                 38957405 
                 38957570 
               
               
                   
                 (SEQ ID NO. 454) 
                 (SEQ ID NO. 455) 
               
               
                   
               
               
                 DG13S451 
                 TTTGGGATCTTGAAGACCTTT 
                 TTGTGGCATGTCCTTGGTT 
                 39032835 
                 39033191 
               
               
                   
                 (SEQ ID NO. 456) 
                 (SEQ ID NO. 457) 
               
               
                   
               
               
                 DG13S180 
                 TGTACACTGCAAACATTGCTAAA 
                 TTGTCCTTTCATTATGACGTGTCT 
                 39233968 
                 39234350 
               
               
                   
                 (SEQ ID NO. 458) 
                 (SEQ ID NO. 459) 
               
               
                   
               
               
                 DG13S458 
                 AAGCCTGAAAGGATACACACAA 
                 CAGGATCCCAGACTTTCCAG 
                 39475899 
                 39476187 
               
               
                   
                 A (SEQ ID NO. 460) 
                 (SEQ ID NO. 461) 
               
               
                   
               
               
                 DG13S2547 
                 GGTGAATCCCACCCTCATAC 
                 TTGGTATGTTTCCTATTGTTGCAT 
                 39612492 
                 39612849 
               
               
                   
                 (SEQ ID NO. 462) 
                 (SEQ ID NO. 463) 
               
               
                   
               
               
                 D13S244 
                 GAACCAGTGAGTTTTATTAC 
                 AGACACAGCATATAATACATG 
                 39665226 
                 39665353 
               
               
                   
                 (SEQ ID NO. 464) 
                 (SEQ ID NO. 465) 
               
               
                   
               
               
                 DG13S2435 
                 TGAAGCTTTGTGGCTTGTTG 
                 GACTGAGTCCACAGCCCATT 
                 39863067 
                 39863301 
               
               
                   
                 (SEQ ID NO. 466) 
                 (SEQ ID NO. 467) 
               
               
                   
               
               
                 D13S263 
                 CCTGGCCTGTTAGTTTTTATTGTT 
                 CCCAGTCTTGGGTATGTTTTTA 
                 39878976 
                 39879126 
               
               
                   
                 A (SEQ ID NO. 468) 
                 (SEQ ID NO. 469) 
               
               
                   
               
               
                 DG13S188 
                 CCACCATGCAAGAACAGATG 
                 GCTTTGCACTTGGCTGTCTT 
                 39935769 
                 39936103 
               
               
                   
                 (SEQ ID NO. 470) 
                 (SEQ ID NO. 471) 
               
               
                   
               
               
                 DG13S189 
                 TTTGCATGAAGTAAAGTATCCCTG 
                 CACAAACCACAAGATGATTGG 
                 39968676 
                 39969030 
               
               
                   
                 T (SEQ ID NO. 472) 
                 (SEQ ID NO. 473) 
               
               
                   
               
               
                 DG13S190 
                 GGGCATCATGTCTACAACTCA 
                 ACCAAGGGCACTTGCTGATA 
                 40027542 
                 40027801 
               
               
                   
                 (SEQ ID NO. 474) 
                 (SEQ ID NO. 475) 
               
               
                   
               
               
                 DG13S2370 
                 AGGATGAAGAGGGAGGAAGG 
                 CCAGACTGATCTTCCTTAATTAGT 
                 40159684 
                 40159812 
               
               
                   
                 (SEQ ID NO. 476) 
                 TG (SEQ ID NO. 477) 
               
               
                   
               
               
                 DG13S196 
                 CCTCCTCTTTCTGCTGCTGT 
                 AGCCAAAGAACCCAAAGAAAC 
                 40251445 
                 40251793 
               
               
                   
                 (SEQ ID NO. 478) 
                 (SEQ ID NO. 479) 
               
               
                   
               
               
                 DG13S2457 
                 GCCCTACTTTGCCTCAGAAA 
                 GCAACTCATGCCAGCCTCTA 
                 40376042 
                 40376447 
               
               
                   
                 (SEQ ID NO. 480) 
                 (SEQ ID NO. 481) 
               
               
                   
               
               
                 DG13S2445 
                 AACTGTGTTAATGATGGGCAAA 
                 AACGAGCGCATGAAACCTAT 
                 40422793 
                 40423200 
               
               
                   
                 (SEQ ID NO. 482) 
                 (SEQ ID NO. 483) 
               
               
                   
               
               
                 DG13S211 
                 CCTGGTCAATTGAACCCAAA 
                 TGAAGGAAGATAAAGCAGGGTAA 
                 40434073 
                 40434172 
               
               
                   
                 (SEQ ID NO. 484) 
                 (SEQ ID NO. 485) 
               
               
                   
               
               
                 DG13S472 
                 CTCTCTCTGGCCCTCTCTTG 
                 GGTAACTGCCATTCTTCTACCA 
                 40476985 
                 40477395 
               
               
                   
                 (SEQ ID NO. 486) (SEQ ID NO. 487) 
               
               
                   
               
               
                 DG13S207 
                 ACTCCACCTGAAGGGAGAAA 
                 TGGAAGCCACTAATTGGAGAA 
                 40545942 
                 40546202 
               
               
                   
                 (SEQ ID NO. 488) 
                 (SEQ ID NO. 489) 
               
               
                   
               
               
                 DG13S200 
                 AATGGATGGATACCTCCTTATCA 
                 CTCATTGTGGCTTTCTGTGC 
                 40737337 
                 40737570 
               
               
                   
                 (SEQ ID NO. 490) 
                 (SEQ ID NO. 491) 
               
               
                   
               
               
                 DG13S198 
                 GTACCCACACCTCACCAAGC 
                 CGTAGCTCACATTCCCAACA 
                 40811813 
                 40812059 
               
               
                   
                 (SEQ ID NO. 492) 
                 (SEQ ID NO. 493) 
               
               
                   
               
               
                 DG13S215 
                 GGCGAGTGAAAGAGAGGACA 
                 GGGTGGTAATTCCCAGATGA 
                 40871695 
                 40871992 
               
               
                   
                 (SEQ ID NO. 494) 
                 (SEQ ID NO. 495) 
               
               
                   
               
               
                 DG13S221 
                 TCTGCAACAGCCAGAATCAA 
                 TGTCTGTTTGGCAACTTTCTGTC 
                 41107773 
                 41108TT 
               
               
                   
                 (SEQ ID NO. 496) 
                 (SEQ ID NO. 497) 
               
               
                   
               
               
                 DG13S219 
                 AGGTGAACCCAGTCCAGCTA 
                 TCTTAGGCAAAGGAGCCAGT 
                 41127591 
                 41127734 
               
               
                   
                 (SEQ ID NO. 498) 
                 (SEQ ID NO. 499) 
               
               
                   
               
               
                 D13S1270 
                 ACATGAGCACTGGTGACTG 
                 GGCCTCAAATGTTTTAAGCA 
                 41161654 
                 41161831 
               
               
                   
                 (SEQ ID NO. 500) 
                 (SEQ ID NO. 501) 
               
               
                   
               
               
                 DG13S225 
                 TTCTGGGTGTTCGCTATTCC 
                 TTTCCTGTCCAGTCCTGACC 
                 41212951 
                 41213310 
               
               
                   
                 (SEQ ID NO. 502) 
                 (SEQ ID NO. 503) 
               
               
                   
               
               
                 D13S1276 
                 GTTTTGCAGGTCTAGGTCACAC 
                 AGGATAGCTTGAGCCCG 
                 41213917 
                 41214090 
               
               
                   
                 (SEQ ID NO. 504) 
                 (SEQ ID NO. 505) 
               
               
                   
               
            
           
         
       
     
      All references cited herein are incorporated by reference in their entirety. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.  
     EXAMPLE 10  
     Randomized, Placebo-Controlled, Crossover Clinical Trial Demonstrates Inhibition of FLAP Reduced Biomarkers of Risk of Myocardial Infarction  
      The 5-lipoxygenase pathway, through FLAP, leads to the production of leukotriene B 4 , one of the most potent chemokine mediators of arterial inflammation. The experiments described in Example 7 showed that MI patients make more LTB 4  than do controls. Hence, it appears that the at-risk variant upregulates the leukotriene pathway. A clinical trial was carried out to demonstrate that patients with the genetic variation in FLAP that predisposes to MI could benefit from inhibiting FLAP, with the FLAP inhibitor DG-031. In the short term study, changes in levels of biomarkers that are associated with risk of MI were scored as evidence of changes in the risk of MI.  
      Patient Population  
      All patients in the study had a history of MI and were carriers of specific MI-associated haplotypes in the FLAP and/or the LTA4 hydrolase genes (See U.S. patent application Ser. No. 10/944,272 and PCT Application No. PCT/2004/030582, incorporated by reference in its entirety. The recruitment process included individuals who had previously participated in a study of the genetics of MI (Helgadottir et al., Nat. Genet. 2004;36(3):233-9. 2004). Apart from FLAP, the LTA4 hydrolase gene. also shows significant association to MI in Iceland and baseline mRNA expression of the LTA4 hydrolase gene is greater in MI patients than in control subjects; that is subjects with at-risk variants in either the FLAP or LTA4 hydrolase genes are at increased risk of sustaining MI. Thus, carriers of either the FLAP or LTA4 hydrolase at-risk haplotypes were recruited and their haplotypes were confirmed by analysis of DNA from blood sample collected in the study.  
      Nine Single Nucleotide Polymorphism (SNP) markers were genotyped to define the at-risk haplotypes. These SP markers are set out in Table 25 below and are described in detail in Example 1. SNPs genotyping within the FLAP and LTA4 hydrolase genes was performed using SNP-based Taqman platform (ABI) as described in Helgadottir et al., 2004 March;36(3):233-9. The haplotypes carried by each individual were estimated using the program NEMO (version 1.01) and 902 in-house population controls, as described in Gretarsdottir et al., Nat Genet 35:131-8, 2003.  
               TABLE 25                          Genotypes used to derive FLAP and LTA 4  hydrolase at-risk       haplotypes.                                                 Haplotype   Allele   SNP   Allele   SNP   Allele   SNP               1   A3 (FLAP gene)   G   SG13S25   T   SG13S114   A   SG13S32       2   AF (FLAP gene)   G   SG13S25   T   SG13S114       3   NA3 (FLAP gene)   A   SG13S122   C   SG13S32   C   SG13S8       4   HF (LTA 4 -OH gene)   A   SG12S25   C   SG12S223       5   GF (LTA 4 -OH gene)   A   SG12S225   T   SG12S233                  
 
      The recruits were asked for permission for the use of their medical and genetic information already collected at deCODE genetics (Reykjavik, Iceland) for the clinical trial. Of over 900 patients identified as eligible by clinical and genotypic criteria, 640 returned their signed consent providing permission to use their genetic and medical data. The genotypes for the FLAP and LTA4 hydrolase genes were subsequently reconfirmed, and those who were carriers of variants in the FLAP and/or LTA4 hydrolase genes were judged eligible for the study if they also met the other inclusion criteria and none of the exclusion criteria set out in Table 26. The baseline characteristics of the patients participating in the study are set out in Table 27. All patients who participated gave informed consent and the protocol was approved by the National Bioethics Committee in Iceland.  
               TABLE 26                       Study eligibility criteria.                  Inclusion criteria                 Age 40 to 75.       Carrier of the FLAP and/or the LTA 4  hydrolase haplotype       Documented CAD with previous history of MI       Women of childbearing potential must have a negative urine pregnancy       test at visit 1 and are required to use 2 adequate barrier methods       of contraception throughout the study.       Understanding of the study procedures and agreement to participate in       the study by giving written informed consent.                 Exclusion criteria                 Confirmed diagnosis of congestive heart failure (CHF).       Any experimental treatment within 2 months of screening or planned       for the following 3 months.       Acute CV event (such as ACS, MI or stroke) within 1 month prior to       enrolment.       Elevated CPK above 3 fold upper normal limit (UNL). Other liver       function tests and kidney function tests above 1.5 fold upper       normal limit.       Immunocompromised subjects, including subjects known to be HIV       positive or with malignant disease and/or on chronic       immunosuppressive therapy.       Subjects known to have positive serology results for HBsAg, HCV Ab.       Treatment with immunosuppressive cytotoxic drugs or corticosteroids       within 6 weeks or during conduct of study.       Major surgery within 6 weeks prior to enrolment.       Any other major intercurrent illness and other condition, which, in the       investigator&#39;s judgement, will interfere with the subject&#39;s       participation in this study.       Subjects not willing to return for follow-up or with known history of non-       compliance.       Patients who consume more than 2 alcoholic drinks/day or ≧10       drinks/week, or history of alcohol abuse within the past 2 years.       Patients must agree to comply with the restrictions on alcohol       (≦2 drinks/day and &lt;10 drinks/week and no alcohol intake within       48 hours of study visits).       Pregnant or lactating women.       Poor mental function or any other reason to expect patient difficulty in       complying with the requirements of the study.                  
 
     
       
         
           
               
             
               
                 TABLE 27 
               
             
            
               
                   
               
               
                   
               
               
                 Baseline characteristics of the study cohort. 
               
            
           
           
               
               
               
               
            
               
                   
                 250 mg/day 
                 500 mg/day 
                 750 mg/day 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Active- 
                 Placebo- 
                 Active- 
                 Placebo- 
                 Active- 
                 Placebo- 
               
               
                   
                 placebo 
                 active 
                 placebo 
                 active 
                 placebo 
                 active 
               
               
                 Characteristic 
                 (n = 32) 
                 (n = 32) 
                 (n = 32) 
                 (n = 32) 
                 (n = 32) 
                 (n = 31) 
               
               
                   
               
               
                 Demography 
                   
                   
                   
                   
                   
                   
               
               
                 Male/Female 
                 24/8 
                 24/8 
                 24/8 
                 24/8 
                 24/8 
                 24/7 
               
               
                 Age (SD), years 
                   66 (8) 
                   66 (8) 
                   65 (7) 
                   67 (7) 
                   64 (8) 
                   67 (7) 
               
               
                 Age range, years 
                 47-75 
                 47-75 
                 51-75 
                 52-75 
                 47-75 
                 56-75 
               
               
                 Age &gt; 60 years, % 
                 78% 
                 75% 
                 78% 
                 78% 
                 69% 
                 74% 
               
               
                 Weight (SD), kg 
                   86 (11) 
                   87 (12) 
                   86 (14) 
                   92 (18) 
                   91 (13) 
                   93 (19) 
               
               
                 Height (SD), cm 
                  173 (8) 
                  174 (7) 
                  173 (8) 
                  174 (9) 
                  174 (7) 
                  173 (10) 
               
               
                 BMI (SD), kg/m 2   
                   29 (3) 
                   29 (3) 
                   29 (4) 
                   30 (6) 
                   30 (4) 
                   31 (5) 
               
               
                 Cardiovascular history 
               
               
                 Two or more prev. infarcts 
                   3 (9%) 
                   6 (19%) 
                   3 (9%) 
                   7 (22%) 
                   6 (19%) 
                   8 (26%) 
               
               
                 Time since last MI (mo&#39;s) 
                  146 (63) 
                  137 (73) 
                  143 (65) 
                  121 (68) 
                  129 (71) 
                  131 (59) 
               
               
                 Hypertension (current) 
                   5 (16%) 
                   10 (31%) 
                   4 (12%) 
                   4 (12%) 
                   8 (25%) 
                   7 (23%) 
               
               
                 Diabetes 
                   10 (31%) 
                   8 (25%) 
                   6 (19%) 
                   12 (38%) 
                   8 (25%) 
                   10 (32%) 
               
               
                 Haplotype frequency 
               
               
                 A3 carrier (FLAP) 
                   8 (25%) 
                   7 (22%) 
                   8 (25%) 
                   5 (16%) 
                   11 (34%) 
                   13 (42%) 
               
               
                 AF carrier (FLAP)* 
                   29 (91%) 
                   27 (84%) 
                   28 (88%) 
                   28 (88%) 
                   28 (88%) 
                   26 (84%) 
               
               
                 NA3 carrier (FLAP) 
                   5 (16%) 
                   5 (16%) 
                   4 (12%) 
                   3 (9%) 
                   3 (9%) 
                   0 (0%) 
               
               
                 HF carrier (LTA 4 -OH) 
                   13 (41%) 
                   18 (56%) 
                   19 (59%) 
                   22 (69%) 
                   13 (41%) 
                   20 (65%) 
               
               
                 GF carrier (LTA 4 -OH) 
                   3 (9%) 
                   9 (28%) 
                   5 (16%) 
                   6 (19%) 
                   7 (22%) 
                   3 (10%) 
               
               
                 NA3/A3 &amp; HF/GF carrier 
                   11 (34%) 
                   12 (38%) 
                   9 (28%) 
                   15 (47%) 
                   9 (28%) 
                   14 (45%) 
               
               
                 Relevant medication 
               
               
                 Statins (%) 
                   27 (84%) 
                   28 (88%) 
                   26 (81%) 
                   28 (88%) 
                   25 (78%) 
                   27 (87%) 
               
               
                 Other chol&#39;l low&#39;ng drug (%) 
                   0 (0%) 
                   0 (0%) 
                   3 (9%) 
                   1 (3%) 
                   1 (3%) 
                   1 (3%) 
               
               
                 Aspirin (%) 
                   28 (88%) 
                   28 (88%) 
                   28 (88%) 
                   25 (78%) 
                   27 (84%) 
                   26 (84%) 
               
               
                 Nitrates (%) 
                   13 (41%) 
                   12 (38%) 
                   10 (31%) 
                   8 (25%) 
                   8 (25%) 
                   12 (39%) 
               
               
                 Ca-channel blockers (%) 
                   9 (28%) 
                   6 (19%) 
                   9 (28%) 
                   7 (22%) 
                   7 (22%) 
                   8 (26%) 
               
               
                 ACE-inhibitors (%) 
                   7 (22%) 
                   10 (31%) 
                   12 (38%) 
                   10 (31%) 
                   10 (31%) 
                   13 (42%) 
               
               
                 Beta-blockers (%) 
                   22 (69%) 
                   23 (72%) 
                   23 (72%) 
                   18 (56%) 
                   24 (75%) 
                   22 (71%) 
               
               
                 Diuretics (%) 
                   9 (28%) 
                   13 (41%) 
                   7 (22%) 
                   7 (22%) 
                   11 (34%) 
                   9 (29%) 
               
               
                 Plasma lipids 
               
               
                 Cholesterol (SD), mmol/L 
                  5.0 (1.0) 
                  5.0 (0.8) 
                  5.2 (1.0) 
                  4.8 (1.1) 
                  5.2 (1.2) 
                  5.0 (1.0) 
               
               
                 HDL (SD), mmol/L 
                  1.4 (0.3) 
                  1.4 (0.3) 
                  1.5 (0.3) 
                  1.4 (0.5) 
                  1.4 (0.4) 
                  1.4 (0.3) 
               
               
                 LDL (SD), mmol/L 
                  3.0 (1.0) 
                  3.0 (0.7) 
                  3.1 (1.0) 
                  2.9 (1.0) 
                  3.2 (1.0) 
                  3.0 (0.9) 
               
               
                 Triglycerides (SD), mmol/L 
                  1.4 (0.8) 
                  1.5 (0.7) 
                  1.7 (1.6) 
                  1.3 (0.7) 
                  1.4 (0.7) 
                  1.4 (0.6) 
               
               
                 Blood pressure 
               
               
                 Diastolic (SD), mmHg 
                   79 (8) 
                   78 (7) 
                   81 (11) 
                   79 (9) 
                   78 (12) 
                   78 (7) 
               
               
                 Systolic (SD), mmHg 
                  137 (13) 
                  133 (19) 
                  139 (22) 
                  136 (17) 
                  141 (22) 
                  140 (17) 
               
               
                 Smoking habits 
               
               
                 Never smoked 
                   9 (28%) 
                   4 (13%) 
                   5 (16%) 
                   3 (9%) 
                   8 (25%) 
                   7 (23%) 
               
               
                 Prior history of smoking 
                   18 (56%) 
                   20 (63%) 
                   19 (59%) 
                   24 (75%) 
                   19 (59%) 
                   16 (52%) 
               
               
                 Current smoker 
                   5 (16%) 
                   8 (25%) 
                   8 (25%) 
                   5 (16%) 
                   5 (16%) 
                   8 (26%) 
               
               
                 Alcohol use 
               
               
                 Never used alcohol 
                   4 (13%) 
                   4 (13%) 
                   2 (6%) 
                   5 (16%) 
                   5 (16%) 
                   5 (16%) 
               
               
                 Prior use of alcohol 
                   1 (3%) 
                   5 (16%) 
                   4 (13%) 
                   3 (9%) 
                   4 (13%) 
                   2 (6%) 
               
               
                 Current use of alcohol 
                   27 (84%) 
                   23 (72%) 
                   26 (81%) 
                   24 (75%) 
                   23 (72%) 
                   24 (77%) 
               
               
                   
               
               
                   *a common low-risk haplotype (RR 1.3) carried by 85-90% of study subjects    
               
            
           
         
       
     
     Study Conduct  
      All study participants lived in the Reykjavik metropolitan area or its neighboring townships. All study participants were followed by the designated cardiologists at the University Hospital of Iceland, at their outpatient or private clinics, and all subjects had participated in a study on the genetics of MI. After the subject had given informed consent, a medical and medication history was completed, including co-morbidities, concomitant medications and specific details about the subject&#39;s cardiovascular history, including current status. All study participants were fasting and had not taken their medications prior to the study visit. Cardiologists examined the patients at all 8 visits and completed the case report forms. All blood was collected and processed immediately after sampling. All blood specimens used for the biomarker studies were processed within 2 hours of blood sampling.  
     Study Drug  
      Patients (191 subjects) who met the study eligebility criteria were enrolled and randomized into 3 different dose-level groups: (1) 64 patients on 250 mg/day therapy with DG-031 (250 mg q.d.), vs placebo; (2) 64 patients on 500 mg/day therapy with DG-031 (250 mg b.i.d.) vs placebo; and (3) 63 patients on 750 mg/day therapy with DG-031 (250 mg t.i.d.) vs placebo. The 750 mg/day dose was well tolerated in previous phase I-III human studies (Dahlen et al., Thorax 1997; 52:342-7; Hamilton et al., Thorax 1997; 52:348-54), conducted on healthy volunteers and patients with asthma as a part of the drug development program for Bayer x 1005 (now DG-031). All patients received 3 tablets per day. Treatment periods, 4 weeks in duration each, were separated by a 2-week washout period. The placebo tablets were identical in shape, color, form and taste to the active tablets except that they contained no active drug ingredients. Treatment with DG-031 or placebo was in addition to the subject&#39;s standard care, including all medications and treatment plan as prescribed by the subject&#39;s cardiologist prior to enrollment. The cross-over study design is summarized in  FIG. 9 . Due to early termination of 19 subjects (primarily related to unavailability due to travel), 11 were replaced prior to enrollment closure. Thus, a total of 191 subjects were enrolled, with 172 completing all 8 visits or 8 patients (4.4%) short of target. Three subjects did not return for early termination visit.  
     Data Analysis, Randomization and Statistical Considerations  
      All data were analyzed according to a pre-established analysis plan and by intention-to-treat. Hypotheses were tested at a two-sided nominal significance level of 0.05. Each arm of the study, as well as pooled sets (combining dose levels), was considered for the primary analysis. Each such set is a standard AB/BA cross-over design and in the primary analysis of efficacy, the levels of biomarkers of MI risk at the end of the treatment periods (visits 4 and 7) were used as primary response variables. The difference between DG-031 and placebo treatment was the primary outcome, assessed separately for each of the biomarkers. Treatment effect was tested using a two-sample t-test on the period differences for suitably transformed response variables, under an assumption of normality of the transformed data. We report treatment effect as one half of the observed mean differences in the two-sample t-test, with a 95% CI. No pre-tests for carry-over effect were performed as a part of the primary analysis. Tests for carry-over were done and are reported separately from results of primary analysis. As was prespecified for the primary analysis, a simple Bonferroni-adjustment based on 10 biomarkers for the primary objective for the pooled set of the two highest doses, was used to report the outcome of the primary objective. All p-values reported are nominal.  
      To cancel out potential seasonal effects, carry-over effects were also studied with two-sample t-tests that compare maesurements of the AB (drug/placebo) group with the measurenents of the BA (placebo/drug) group. To estimate the effect of the drug at visit 3 for the AB group, (v3-v2), with v3 and v2 denoting, respectively, measurements at visit 3 and visit 2, was used. Similarly (v4-v2) and (v5-v2) were used to estimate effects at visits 4 and 5. For estimating the effect at visit 6, [(v6-v2)+(v3-v2)] was used. Note that v6 from the BA group includes the drug effect after two weeks which cancels the drug effect at visit 3 from the AB group. Similarly, [(v7-v2)+(v4-v2)] was used to estimate the effect at visit 7. The two higher dose AB groups were used for all visits. All 3 BA groups are used for visits 3, 4 and 5 since they had all received the same treatment until visit 5, but only the two higher dose BA groups are used for visits 6 and 7.  
      The sample size for this study was chosen so that each of the three arms provided, after up to 5% dropout, at least 80% power (with α=0.05, two-sided) to detect a relative lowering of 15% for a log-normal response variable, given that an assay for that variable has a coefficient of variation of 20% and the intra-person coefficient of variation is as high as 25%. Based on these assumptions, the recruitment target included 180 subjects with randomization into 3 different dose-level groups as described above.  
      At the enrollment visit, an independent study nurse who was blinded to the drug content, dispensed medication kits according to a computer generated randomization list. Randomization of study patients was stratified according to sex. For both strata, a permuted block design with block size 12 was used to assign patients into each of the six sequences of the study. All biomarkers were transformed using a shifted log transform (transformed value is natural log of original value plus a shifting constant for each assay). Missing data were filled in using a simple last observation carried forward (LOCF) scheme, in cases where no previous measurement existed, next observation was carried back. Statistical outliers for data sets were brought in based on IQR distance from median.  
     Biomarker Measurements  
      The ELISA and mass spectrometry assays were used to measure the levels of the MI at risk biomarkers and are summarized in Table 28. Apart from measurements in plasma, LTB 4  and MPO were also measured in whole blood preparations ex vivo following ionomycin-activation of leukocytes, using ELISA and mass spectrometry. Both dose- and time-dependent stimulations were performed to determine the maximum LTB 4  and MPO output of the cells. Correction was made for white blood cell count, as the amount of these mediators produced relates to the number of cells in a fixed volume. On the log scale the adjustment was based on a linear model, with coefficients determined empirically at time of blind review. Several tertiary markers were also measured including: IL-6, IL-12p40, TNFα, MMP-9, sICAM, sVCAM, P-selectin, E-selectin, MCP-1 and oxidised LDL.  
               TABLE 28                          Methods and assays used to quantify study biomarkers.                                 Assay   Supplier   Name of kit   Catalog nr.   Principle of the method                         ELISA method                                 Myeloperoxidase (MPO)   Assay Design,   Titerzyme EIA   # 900-115   A quantitative solid phase           Inc.           sandwich ELISA       LTB 4     R&amp;D   LTB 4     # DE0275   A competitive binding                       immunoassay       Amyloid A   Biosource   Human SAA kit   # KHA0012   A quantitative solid phase                       sandwich ELISA       Cysteinyl Leukotriene   R&amp;D   Cysteinyl   # DE3200   A competitive binding               Leukotriene       immunoassay       Nitrotyrosine   OxisResearch   Bioxytech Nitro   # 21055   A quantitative solid phase               tyrosine-EIA       sandwich ELISA       TNF-α   R&amp;D   Quantikine HS   # HSTA00C   A quantitative solid phase               Human TNF-α       sandwich ELISA       IL6   R&amp;D   Quantikine HS   # HS600B   A quantitative solid phase               Human IL-6       sandwich ELISA       IL-12p40   R&amp;D   Quantikine Human   # DP400   A quantitative solid phase               IL-12p40       sandwich ELISA       MCP-1   R&amp;D   Quantikine Human   # DCP00   A quantitative solid phase               MCP-1       sandwich ELISA       ICAM   R&amp;D   Parameter human   # BBE 1B   A quantitative solid phase               sICAM-1       sandwich ELISA       sE-Selectin   R&amp;D   Parameter human   # BBE 2B   A quantitative solid phase               sEselectin       sandwich ELISA       sP-Selectin   R&amp;D   Parameter human   # BBE 6   A quantitative solid phase               sPselectin       sandwich ELISA       VCAM   R&amp;D   Parameter human   # BBE 3   A quantitative solid phase               sVCAM-1       sandwich ELISA       MMP 9   R&amp;D   Quantikine Human   # DMP900   A quantitative solid phase               MMP-9(total)       sandwich ELISA       Oxidised LDL   Mercodia   Oxidised LDL Elisa   # 10-1143-01   A quantitative solid phase                       sandwich ELISA       Lp-PLA 2     Diadexus San   PLAC test       A quantitative solid phase           Fransisco, CA           sandwich ELISA                 Other methods                                 Hs-CRP   Roche Hitachi   Hs-CRP   11972855   Immunoturbidimetric assay           912 analyser       LTB 4  (MS)   LC/MS/MS   LTB 4  assay       Mass spectrometer with                       internal standard                  
 
     Clinical Outcome  
      Baseline values for the biomarker variables prior to treatment are shown in Table 29. For the primary efficacy endpoint, as specified in the statistical analysis plan, 10 variables were considered in the pooled set of subjects on 500 mg and 750 mg arms and the data is set out in Table 30. The primary efficacy endpoint of the study was confirmed by showing that DG-031 reduces levels of LTB 4  produced by ionomycin-activated neutrophils ex vivo for the pooled set of 500 mg and 750 mg arms (nominal p=0.0042), and this is statistically significant after correction for multiple testing. As shown in Table 30, the maximum reduction in LTB 4  and MPO production amounted to 26% for LTB 4  (nominal p=0.0026) and 13% for MPO (nominal p=0.023) at the 750 mg/day dose of DG-031. DG-031 also reduced significantly serum sICAM-1 (nominal p=0.02), but no effects were observed on other tertiary markers. Lp-PLA 2  increased by 9% (nominal p=0.0056) in response to the highest dose of DG-031 and there was comparable increase observed in LDL cholesterol (8%) that correlated with Lp-PLA 2 . In contrast, the effects of the 2 lower doses (250 mg/day and 500 mg/day) on Lp-PLA 2  were not significant. Urine levels of LTE 4  increased by 27% in response to the highest dosage of DG-031 (nominal p=0.00002)). Significant correlation was observed between the inhibition of LTB 4  and MPO production in response to DG-031 (r=0.65, p&lt;0.00001).  
               TABLE 29                          Summary statistics of baseline biomarker values.                                 250 mg/day   500 mg/day   750 mg/day                                             Active-   Placebo-   Active-   Placebo-   Active-   Placebo-           placebo   active   placebo   active   placebo   active       Assay   (n = 32)   (n = 32)   (n = 32)   (n = 32)   (n = 32)   (n = 31)                         Primary objectives                                         Amyloid A   9.92 (0.94)   9.84 (0.75)   9.78 (0.91)   9.44 (0.43)   9.74 (0.43)   9.63 (0.50)           n = 32   n = 32   n = 32   n = 32   n = 32   n = 31       Hs-CRP   0.78 (0.88)   0.95 (1.12)   0.75 (1.19)   0.46 (0.73)   0.89 (0.81)   0.77 (0.86)           n = 32   n = 32   n = 32   n = 32   n = 32   n = 31       Lp-PLA 2     5.47 (0.33)   5.50 (0.29)   5.49 (0.27)   5.32 (0.39)   5.51 (0.41)   5.42 (0.22)           n = 32   n = 32   n = 32   n = 32   n = 32   n = 31       LTB 4  in whole   10.78 (0.85)    11.02 (0.85)    10.41 (0.65)    10.54 (0.75)    10.74 (0.57)    10.85 (0.87)        blood †     n = 32   n = 32   n = 32   n = 31   n = 32   n = 30       LTB 4  in w.b.*,   8.14 (0.71)   8.24 (0.76)   7.79 (0.68)   7.95 (0.69)   8.11 (0.59)   8.11 (0.72)       corr for wbc †,‡     n = 32   n = 32   n = 32   n = 31   n = 32   n = 30       LTE 4  in urine   6.57 (0.33)   6.69 (0.32)   6.53 (0.40)   6.55 (0.38)   6.67 (0.48)   6.69 (0.40)           n = 31   n = 31   n = 32   n = 32   n = 32   n = 31       MPO in plasma   3.72 (0.51)   3.71 (0.55)   3.47 (0.41)   3.51 (0.37)   3.71 (0.45)   3.72 (0.52)           n = 32   n = 32   n = 32   n = 32   n = 31   n = 31       MPO in whole   6.54 (0.49)   6.67 (0.37)   6.47 (0.44)   6.38 (0.49)   6.56 (0.34)   6.64 (0.47)       blood   n = 31   n = 32   n = 32   n = 31   n = 32   n = 31       MPO in w. b.*,   4.69 (0.39)   4.74 (0.37)   4.65 (0.37)   4.58 (0.44)   4.73 (0.33)   4.75 (0.34)       corr. for wbc ‡     n = 31   n = 32   n = 32   n = 31   n = 32   n = 31       N-tyrosine   3.18 (0.73)   3.40 (1.02)   3.25 (0.95)   3.81 (1.48)   3.50 (0.99)   3.66 (1.42)           n = 31   n = 31   n = 28   n = 29   n = 31   n = 30                 Tertiary objectives                                         ICAM   5.67 (0.27)   5.67 (0.21)   5.65 (0.29)   5.66 (0.20)   5.69 (0.25)   5.68 (0.23)           n = 32   n = 32   n = 32   n = 32   n = 32   n = 31       IL12p40   4.98 (0.41)   4.87 (0.48)   4.86 (0.36)   5.04 (0.42)   5.02 (0.50)   4.96 (0.43)           n = 32   n = 32   n = 32   n = 32   n = 32   n = 31       IL6   0.87 (0.40)   1.15 (0.63)   1.07 (0.89)   0.90 (0.32)   0.95 (0.71)   1.11 (0.42)           n = 32   n = 32   n = 32   n = 32   n = 32   n = 31       MCP-1   5.92 (0.39)   5.90 (0.23)   5.86 (0.21)   5.94 (0.24)   5.92 (0.23)   5.91 (0.23)           n = 32   n = 32   n = 32   n = 31   n = 32   n = 31       MMP 9   6.34 (0.39)   6.40 (0.44)   6.10 (0.47)   6.20 (0.42)   6.15 (0.43)   6.15 (0.52)           n = 32   n = 32   n = 32   n = 32   n = 32   n = 31       Oxidized - LDL   11.09 (0.33)    11.06 (0.37)    11.03 (0.29)    11.12 (0.30)    11.07 (0.33)    11.08 (0.30)            n = 32   n = 32   n = 32   n = 32   n = 32   n = 31       sE-Selectin   4.20 (0.19)   4.21 (0.28)   4.12 (0.25)   4.19 (0.33)   4.22 (0.26)   4.24 (0.36)           n = 32   n = 32   n = 32   n = 32   n = 32   n = 31       sP-Selectin   4.85 (0.30)   5.00 (0.47)   4.72 (0.28)   4.72 (0.30)   4.90 (0.48)   4.77 (0.35)           n = 32   n = 31   n = 31   n = 31   n = 32   n = 30       sVCAM   6.09 (0.18)   6.06 (0.15)   6.08 (0.17)   6.07 (0.19)   6.09 (0.24)   6.10 (0.17)           n = 32   n = 32   n = 32   n = 32   n = 30   n = 31       TNF-α   0.64 (0.48)   0.56 (0.54)   0.51 (0.49)   0.54 (0.39)   0.53 (0.46)   0.47 (0.43)           n = 26   n = 27   n = 27   n = 30   n = 29   n = 27                 *w.b. = whole blood              † baseline is not available for LTB 4  measured using mass spectrometry              ‡ corr. for wbc = corrected for white blood cell count             
 
      Table 30. Treatment effect based on two sample t-test for the treatment groups, the pooled sets for the two highest doses and all doses (natural log scale).  
               TABLE 30                          Treatment effect based on two sample t-test for the treatment groups, the pooled sets for the two       highest doses and all doses (natural log scale).                                         250 mg/day   500 mg/day   750 mg/day   500 &amp; 750 mg/day   250, 500 &amp; 750       Assay   (n = 64)   (n = 64)   (n = 63)   (n = 127)   mg/day (n = 191)                         Primary objectives                                     Amyloid A     0.03 [−0.09, 0.15]   −0.05 [−0.17, 0.06]   −0.01 [−0.11, 0.09]   −0.03 [−0.11, 0.05]   −0.01 [−0.07, 0.05]           (p = 0.61)   (p = 0.36)   (p = 0.90)   (p = 0.43)   (p = 0.77)       Hs-CRP     0.05 [−0.14, 0.24]     0.09 [−0.09, 0.26]     0.04 [−0.13, 0.21]     0.06 [−0.06, 0.18]     0.06 [−0.04, 0.16]           (p = 0.59)   (p = 0.34)   (p = 0.66)   (p = 0.32)   (p = 0.26)       Lp-PLA 2       0.05 [−0.03, 0.12]     0.03 [−0.04, 0.10]     0.09 [0.03, 0.15]     0.06 [0.01, 0.10]     0.05 [0.01, 0.09]           (p = 0.24)   (p = 0.37)   (p = 0.0056)   (p = 0.012)   (p = 0.0073)       LTB 4  in w.b.*, mass   −0.11 [−0.29, 0.06]   −0.09 [−0.28, 0.11]   −0.26 [−0.46, −0.06]   −0.17 [−0.31, −0.04]   −0.15 [−0.26, −0.05]       spec. †     (p = 0.19)   (p = 0.38)   (p = 0.010)   (p = 0.013)   (p = 0.0051)       LTB 4  in w.b.*, corr.   −0.11 [−0.28, 0.05]   −0.08 [−0.26, 0.09]   −0.30 [−0.49, −0.11]   −0.19 [−0.32, −0.06]   −0.16 [−0.27, −0.06]       for wbc ‡ , m.s. §     (p = 0.18)   (p = 0.35)   (p = 0.0026)   (p = 0.0042)   (p = 0.0018)       LTB 4  in whole blood †     −0.13 [−0.35, 0.09]   −0.19 [−0.44, 0.06]   −0.30 [−0.56, −0.04]   −0.24 [−0.42, −0.07]   −0.21 [−0.34, −0.07]           (p = 0.24)   (p = 0.13)   (p = 0.025)   (p = 0.0073)   (p = 0.0036)       LTB 4  in w.b.*, corr.   −0.13 [−0.35, 0.08]   −0.18 [−0.42, 0.05]   −0.34 [−0.59, −0.09]   −0.26 [−0.43, −0.09]   −0.22 [−0.35, −0.08]       for wbc †,‡     (p = 0.22)   (p = 0.12)   (p = 0.0089)   (p = 0.0027)   (p = 0.0014)       LTE 4  in urine     0.14 [0.03, 0.24]     0.15 [0.05, 0.24]     0.24 [0.14, 0.34]     0.19 [0.12, 0.26]     0.17 [0.12, 0.23]           (p = 0.011)   (p = 0.0030)   (p = 0.00002)   (p &lt; 0.00001)   (p &lt; 0.00001)       MPO in plasma   −0.07 [−0.22, 0.07]     0.08 [−0.04, 0.21]   −0.04 [−0.17, 0.09]   0.02 [−0.07, 0.11]   −0.01 [−0.09, 0.06]           (p = 0.32)   (p = 0.20)   (p = 0.49)   (p = 0.68)   (p = 0.76)       MPO in whole blood †       0.01 [−0.08, 0.11]   −0.01 [−0.13, 0.11]   −0.11 [−0.22, 0.00]   −0.06 [−0.14, 0.02]   −0.04 [−0.10, 0.03]           (p = 0.78)   (p = 0.85)   (p = 0.056)   (p = 0.14)   (p = 0.27)       MPO in w. b.*, corr.     0.01 [−0.08, 0.11]   0.00 [−0.11, 0.12]   −0.13 [−0.24, −0.02]   −0.06 [−0.14, 0.02]   −0.04 [−0.10, 0.02]       for wbc ‡     (p = 0.76)   (p = 0.94)   (p = 0.023)   (p = 0.12)   (p = 0.24)       N-tyrosine   −0.03 [−0.15, 0.09]   −0.03 [−0.13, 0.08]     0.03 [−0.08, 0.14]     0.00 [−0.07, 0.08]   −0.01 [−0.07, 0.05]           (p = 0.60)   (p = 0.60)   (p = 0.56)   (p = 0.96)   (p = 0.78)                 Tertiary objectives                                     ICAM     0.00 [−0.04, 0.03]     0.00 [−0.04, 0.03]   −0.03 [−0.06, 0.00]   −0.02 [−0.04, 0.00]   −0.01 [−0.03, 0.01]           (p = 0.83)   (p = 0.81)   (p = 0.025)   (p = 0.10)   (p = 0.16)       IL12p40     0.01 [−0.04, 0.06]     0.02 [−0.04, 0.08]     0.01 [−0.04, 0.06]     0.01 [−0.02, 0.05]     0.01 [−0.02, 0.04]           (p = 0.69)   (p = 0.53)   (p = 0.70)   (p = 0.46)   (p = 0.40)       IL6   −0.02 [−0.13, 0.09]     0.06 [−0.03, 0.16]   −0.01 [−0.10, 0.09]     0.03 [−0.04, 0.09]     0.01 [−0.05, 0.07]           (p = 0.68)   (p = 0.19)   (p = 0.87)   (p = 0.40)   (p = 0.69)       MCP-1   −0.02 [−0.07, 0.03]     0.02 [−0.03, 0.08]   −0.03 [−0.08, 0.03]     0.00 [−0.04, 0.04]   −0.01 [−0.04, 0.02]           (p = 0.51)   (p = 0.35)   (p = 0.32)   (p = 0.98)   (p = 0.69)       MMP 9   −0.03 [−0.12, 0.05]     0.02 [−0.06, 0.11]   −0.02 [−0.11, 0.06]     0.00 [−0.06, 0.06]   −0.01 [−0.06, 0.04]           (p = 0.47)   (p = 0.58)   (p = 0.60)   (p = 0.97)   (p = 0.69)       Oxidized - LDL     0.00 [−0.08, 0.07]     0.02 [−0.07, 0.11]     0.06 [−0.03, 0.16]     0.04 [−0.02, 0.11]     0.03 [−0.02, 0.08]           (p = 0.91)   (p = 0.65)   (p = 0.16)   (p = 0.18)   (p = 0.28)       sE-Selectin     0.03 [−0.03, 0.09]   −0.01 [−0.06, 0.04]   −0.04 [−0.09, 0.01]   −0.02 [−0.06, 0.01]     0.00 [−0.04, 0.03]           (p = 0.30)   (p = 0.82)   (p = 0.11)   (p = 0.20)   (p = 0.75)       sP-Selectin   −0.02 [−0.11, 0.06]     0.00 [−0.08, 0.08]     0.09 [0.01, 0.16]     0.04 [−0.02, 0.10]     0.02 [−0.03, 0.07]           (p = 0.58)   (p = 0.97)   (p = 0.034)   (p = 0.15)   (p = 0.40)       sVCAM     0.00 [−0.05, 0.04]   −0.01 [−0.06, 0.04]   −0.03 [−0.07, 0.02]   −0.02 [−0.05, 0.01]   −0.01 [−0.04, 0.01]           (p = 0.85)   (p = 0.60)   (p = 0.24)   (p = 0.24)   (p = 0.28)       TNF-α     0.00 [−0.08, 0.09]   −0.02 [−0.10, 0.07]     0.01 [−0.07, 0.08]     0.00 [−0.06, 0.06]     0.00 [−0.05, 0.05]           (p = 0.93)   (p = 0.70)   (p = 0.85)   (p = 0.90)   (p = 0.95)                 *w.b. = whole blood              † measurement is not part of the primary analysis wrt adjustment for multiple testing              ‡ corr. for wbc = corrected for white blood cell count              § m.s. = mass spec. = mass spectrometry             
 
     Tests for Carry-over Effects  
      A test for carry-over effects from the treatment phase to the placebo phase was performed as a two-sample t-test on the differences between visit 2 and 5 for patients on drug and placebo, respectively. The cohort taking drug consists of patients on 500 mg/day and 750 mg/day treatment and the placebo cohort includes patients on placebo from all 3 tracks. The resulting p-values and confidence intervals for the effect are given in Table 31 (data were not available for Lp-PLA 2  and N-tyrosine). No carry over effects were observed with LTB 4  and MPO. In contrast, marked carry over effects were observed for CRP and SAA, with reduction in CRP that was significant at the 5% level (p=0.017). SAA showed similar carry over effects that was slightly below this significance level (p==0.051).  
               TABLE 31                          Test for carry-over effect for each study period.                             Assay   p-value   Effect   95% Cl                                     CRP   0.017   −0.28   [−0.52, −0.05]       Amyloid A   0.051   −0.14   [−0.29, 0.00]       LTE 4  in urine   0.48   −0.06   [−0.22, 0.10]       MCP-1   0.084   0.07   [−0.01, 0.15]       MMP 9   0.56   −0.04   [−0.16, 0.09]       MPO in plasma   0.28   −0.11   [−0.31, 0.09]       White blood cell count   0.57   −0.01   [−0.06, 0.03]       LTB 4  in whole blood, corr.   0.45   −0.10   [−0.36, 0.16]       for wbc ‡         MPO in whole blood, corr.   0.93   0.01   [−0.13, 0.15]       for wbc ‡         LTB 4  in whole blood,   0.45   0.19   [−0.33, 0.71]       mass spec. §         LTB 4  in whole blood, corr.   0.64   0.12   [−0.40, 0.64]       for wbc, m.s. §                     ‡ corr. for wbc = corrected for white blood cell count              § m.s. = mass spec. = mass spectrometry             
 
       FIG. 10  shows the estimated mean effects on CRP and SAA for the subjects receiving the two higher drug doses in the first period. Note that measurements from subjects receiving the placebo first also contribute to these estimates to cancel out potential seasonal effects. For visits 3 (after 2 weeks on therapy) and 4 (after 4 weeks on therapy), this constitutes the treatment effect, whereas the carry-over effects appear between visits 5 to 7.  
      The level of CRP dropped at visits 3 and 4, but not significantly. The reduction became more pronounced, about 25%, and significant at visit 5 (p=0.017), and seems to persists until visit 7, during the time the subjects were on placebo. This prolonged effect is part of the reason that the drug effect was not detected in the primary analysis which did not take this scenario into account. The design of this trial does not have maximal power for studying such effects which is reflected by the large standard errors in the estimates, particularly for visits 6 and 7. Even though measurements at visits 3 and 6 are not available for SAA, the observed changes of CRP and Amyloid A between visits 2 and 5 are highly correlated (r=0.68, p&lt;0.00001). Hence it appears that the drug has similar effects on both biomarkers.  
      No difference was detected in the effects of DG-031 on biomarkers of MI risk between patients with FLAP or LTA4 hydrolase haplotypes when the data were analysed separately.  
      There was no difference in serious adverse events between the treatment groups or dose armns in the study cohort. In particular, no difference was detected in liver transaminases between the groups on active drug or placebo. The only symptom that was significantly more often reported for active drug was dizziness, experienced by 6 patients on active drug (any dose) and none on placebo (p=0.032). This did not interfere with the daily activities of the subjects.  
      When taken together, the data generated through the MI gene-isolation (Example 1) and the clinical trial reported herein, show that DG031 is a safe and well tolerated drug that can, at least in part, correct a biochemical defect that confers a relative risk of acute cardiovascular events that is similar to or greater than the risk conferred by the top quintile of LDL cholesterol. Indeed, the data suggest that DG-031 reduces serum levels of CRP and SAA by approximately 25%, suggesting that this will cause reduction in the risk of acute cardiovascular events.  
     EXAMPLE 11  
     Clinical Trial Investigating the Effect of Compositions Comprising a Leukotriene Synthesis Inhibitor and a Statin on Biomarkers of Risk of Myocardial Infarction  
      A randomized, placebo-controlled crossover-clinical trial, as described in Example 10, is carried out to investigate the effect of compositions comprising a leukotriene synthesis inhibitor and a statin on the levels of biomarkers of risk of MI. The participants for the study optionally are carriers of variants in the FLAP and/or LTA4 hydrolase genes set out in Table 25. One group of participants receives a leukotriene synthesis inhibitor alone, such as DG031. Another group of participants receives a statin alone. A third group of participants receives a composition comprising both a leukotriene synthesis inhibitor and a statin. The forth group of participants receives a placebo.  
      Each participant receives the treatment for at least two months and the levels of biomarkers set out in Table 28 are monitored in each participant for at least three months. It is expected that the group receiving a leukotriene synthesis inhibitor alone will have a 25% decrease in CRP levels and the group receiving a statin alone will also have a 25% decrease in CRP levels. More substantial decrease in CRP from combination therapy is evidence that the combination therapy is beneficial. In view of the data from the clinical trial described in Example 10, wherein almost all (about 85%) of the participants were on statin therapy, it is expected that the group receiving the combination therapy will exhibit a 50% decrease in CRP levels.  
     EXAMPLE 12  
     Association of variants in the gene encoding ALOX5AP/FLAP to MI in a North American Population  
      As described in Example 1, a variant in the gene encoding 5-Lipoxygenase activating protein (ALOX5AP/FLAP) confers risk to both MI and stroke in Iceland. Another SNP-based haplotype within ALOXSAP, HapB, showed significant association to MI in British cohorts as described in Example 9. using similar techniques, the association between HapA and HapB and MI in a North American (“Cleveland”) cohort was analyzed.  
      The ALOX5AP haplotype HapA is also associated to MI in a North-American population. The SNPs defining HapA (SG13S25, SG13S114, SG13S89, and SG13S32) and HapB (SG13S377, SG13S114, SG13S41 and SG13S35) were genotyped in 696 MI patients (553 males and 143 females) and 698 controls (314 males and 384 females). The majority of the study subjects were Caucasians and approximately 10% were African American. Information on the ethnicity at an individual level was not available.  
      The SNP haplotype analysis was done using the program NEMO (Gretarsdottir et al.,  Nat Genet  35:131-8, 2003). NEMO handles missing genotypes and uncertainty with phase through a likelihood procedure, using the expectation-maximization algorithm as a computational tool to estimate haplotype frequencies. For the at-risk haplotypes we calculated the relative risk (RR) assuming a multiplicative model (Falk&amp; Rubinstein P Ann Hum Genet 51 (Pt 3):227-33, 1987: Terwilliger &amp; Ott Hum Hered. 42:337-46, 1992) in which the risk of the two alleles of haplotypes a person carries multiply.  
      The results of the haplotype association analysis for HapA and HapB are shown in Table 32. As demonstrated in the Icelandic population (see Example 1), the estimated frequency of HapA was significantly greater in the patient group than in the control group. In the total cohort, the allelic/haplotype frequency of HapA was 16.9% and 13.6% in patients and controls respectively (P=0.014), which corresponds to a 29% increase in risk of MI for each copy of HapA carried. The relative risk of MI in the total group was 1.29 and a P-value for the association was 0.014. In addition, HapA was overrepresented in patients who had experienced an MI relatively early in life (males before the age of 55 and females before the age of 65). As shown in Table 32, the relative risk of early onset MI (males before the age of 55 and females before the age of 65) was 1.61 and the P-value for the association was 0.0034.  
               TABLE 32                          Association of HapA and HapB with MI                             HapA   HapB                                         Phenotype (n)   Frequency   RR   P   Frequency   RR   P                                                 Total cohort                               Controls (698)   0.136           0.074       MI (696)   0.169   1.29   0.014   0.081   1.1   NS       Early onset   0.205   1.61   0.0034       (170)                  
 
      The association of HapB to MI in the study cohort was also studied. HapB has previously been shown to confer risk of MI in an English cohort (see Example 9). A slight excess of HapB was observed in the total patient group (8.1%) compared to all controls (7.4%), but it was not significant (Table 32).  
      This analysis demonstrated that an ALOX5AP haplotype, HapA, previously reported to confer risk of MI and stroke in an Icelandic cohort (Example 1), and to stroke in a Scottish cohort (described below in Example 14), associates with MI in an North-American population. HapB that confers risk of MI in an British cohort (Example 9) was not associated with MI in this North-American cohort.  
     EXAMPLE 13  
     Additional ALOX5AP/FLAP Haplotype Associated with MI in a North American Population  
      From the analysis of the Cleveland cohort described in Example 12, another haplotype was identified which significantly associated with MI. This haplotype is denoted as HapC, and 5 variations of this haplotype were identified (HapC1, HapC2, HapC3, HapC4-A, HapC4-B). These haplotypes show the most significant association to MI in the Cleveland cohort.  
      These haplotypes are defined in Table 33. HapC1 is the T allele of marker SG13S375. HapC2 has T allele of marker SG13S375 and the G allele of SG13S25. HapC3 adds allele A of SG13S32 plus T allele of marker SG13S375 and the G allele of SG13S25. The addition of the fourth SNP or SG13S106 splits HapC3 into two parts, or HapC4-A and HapC4-B. Allele G of SNP SG13S25, which is in HapC2, HapC3, HapC4-A and HapC4-B is also a characteristic of HapA.  
      The frequency of HapC1, HapC2, HapC3 and HapC4-A and B in different populations are shown in the Table 33. HapC1, HapC2, and HapC3 are over-represented in the patient groups in all populations tested. In the Iceland and UK cohors studied, the HapC4-A part of HapC3 seems to be the one that captures all of the risk conferred by HapC3.  
      All HapC variants except HapC4-B are correlated with HapA, meaning the chromosomes that carry HapC also tend to carry HapA. The correlation between HapC4-A and HapA is defined by a correlation coefficient (R2) of 0.52; the linkage disequilibrium (D′) of 0.77 and the P-value (measure of significance) of 6.4×10-312. HapC is also correlated with HapB, although HapA and HapB are negatively correlated. The correlation between HapC4-A and HapB haplotypes is defined by a correlation coefficient (R2) of 0.08; the linkage disequilibrium (D′) of 0.05 and the P-value of 2.2×10-39.  
                                                           TABLE 33                                   # of   Aff.   # of   Control                           Haplotype   p-value   RR   Affected   Frequency   Controls   Frequency   Info   SG13S375   SG13S25   SG13S106   SG13S32                                    Total Cleveland Cohort                                                             HapC1   0.002089   1.385   666   0.86036   662   0.816465   1   T                   HapC2   0.000416   1.3722   683   0.774968   671   0.715071   1   T   G       HapC3   0.000148   1.4012   695   0.346955   695   0.274923   0.9   T   G       A       HapC4-A   0.038937   1.2662   696   0.184999   698   0.152022   0.8   T   G   G   A       HapC4-B   0.003222   1.4174   696   0.166372   698   0.123424   0.8   T   G   A   A                 United Kingdom Cohort                                                             HapC1   3.00E−01   1.15   559   0.894   591   0.881   1   T                   HapC2   6.40E−02   1.2   741   0.808   708   0.778   0.9   T   G       HapC3   3.90E−02   1.2   747   0.341   719   0.302   0.8   T   G       A       HapC4-A   1.10E−02   1.31   749   0.21   721   0.169   0.8   T   G   G   A       HapC4-B   8.90E−01   1.02   749   0.134   721   0.132   0.7   T   G   A   A                 Iceland MI Cohort                                                             HapC1   2.90E−01   1.14   645   0.886   575   0.872   1   T                   HapC2   1.10E−01   1.16   774   0.756   612   0.728   0.9   T   G       HapC3   4.50E−02   1.19   775   0.33   618   0.292   0.9   T   G       A       HapC4-A   8.00E−04   1.5   777   0.169   622   0.119   0.8   T   G   G   A       HapC4-B   5.40E−01   0.93   777   0.162   622   0.172   0.9   T   G   A   A                 Iceland Stroke Cohort                                                             HapC1   4.80E−01   1.09   683   0.881   575   0.872   1   T                   HapC2   2.90E−01   1.1   697   0.749   612   0.73   0.9   T   G       HapC3   1.90E−02   1.23   700   0.343   618   0.297   0.9   T   G       A       HapC4-A   1.90E−04   1.58   702   0.181   622   0.122   0.8   T   G   G   A       HapC4-B   4.70E−01   0.92   702   0.16   622   0.171   0.9   T   G   A   A                  
 
     EXAMPLE 14  
     Association of Variants in the Gene Encoding ALOX5AP/FLAP to Stroke in a Scottish Population  
      Analysis of HapA and HapB haplotypes was carried out in a Scottish cohort as described in Example 1 and 9. The SNPs defining HapA (SG13S25, SG13S114, SG13S89, and SG13S32) and HapB (SG13S377, SG13S114, SG13S41 and SG13S35) were genotyped in 450 Scottish stroke patients and 710 controls. The patient and control cohorts have been described previously (MacLeod et al., Neurology 53:418-20, 1999; Meiklejohn et al., Stroke 32:57-62, 2001; Duthie et al., Am J Clin. Nutr. 75:908-13, 2002; Whalley et al., Am J Clin. Nutr., 2004). In brief, 450 patients from North East Scotland with CT confirmation of ischemic stroke. (including 26 patients with transient ischemic attack (TIA)) were recruited between 1997 and 1999, within one week of admission to the Acute stroke unit at Aberdeen Royal Infirmary. Patients were further subclassified according to the TOAST research criteria (Adams et al., Stroke 24:35-41, 1993). One hundred and fifty five patients (34%) had large vessel stroke, 96 (21.3%) had cardiogenic stroke and 109 (24.2%) had small vessel stroke. In 5 cases (1.1%) stroke with other determined etiology was diagnosed, 7 (1.5%) had more than one etiology, and 78 (17.3%) had unknown cause of stroke despite extensive evaluation. Seven hundred and ten controls with no history of stroke or TIA were recruited as a part of the 1921 (n=227) and 1936 (n=371) Aberdeen Birth Cohort Studies (Duthie et al., Am. J. Clin. Nutr. 75:908-13, 2002; Whalley et al., Am J Clin. Nutr., 2004), and from primary care (n=l 12) (Meiklejohn et al., Stroke 32:57-62, 2001).  
      The SNP haplotype analysis was done using the program NEMO (Gretarsdottir et al.,  Nat Genet  35:131-8, 2003). NEMO handles missing genotypes and uncertainty with phase through a likelihood procedure, using the expectation-maximization algorithm as a computational tool to estimate haplotype frequencies. As the two haplotypes tested had previously been shown to confer risk of MI and stroke in an Icelandic cohort, and MI in an English cohort, the reported P-values are one-sided. For the at-risk haplotypes we calculated the relative risk (RR) assuming a multiplicative model (Falk &amp; Rubinstein P Ann Hum Genet 51 (Pt 3):227-33, 1987: Terwilliger &amp; Ott Hum Hered. 42:337-46, 1992) in which the risk of the two alleles of haplotypes a person carries multiply.  
      The results of the haplotype association analysis for HapA and HapB are shown in Table 34. The haplotype frequencies of HapA in the Scottish stroke and control populations were higher than in the corresponding Icelandic populations. As demonstrated in the Icelandic population, the estimated frequency of HapA was significantly greater in Scottish stroke patients than in Scottish controls. The carrier frequency of HapA in Scottish patients and controls were 33.4% and 26.4%, respectively, resulting in a relative risk of 1.36 (P=0.007) and a corresponding PAR 9.6%. In the Icelandic population, a higher frequency of HapA was observed in male patients when compared to female patients with either stroke or MI. This gender difference in the frequency of HapA was not observed in the Scottish population.  
               TABLE 34                          Association of HapA and HapB with ischemic stroke                             HapA                                     Fre-       HapB                                         Phenotype (n)   quency   RR   P-value   Frequency   RR   P-value                                                 Scotland                               Controls (710)   0.142           0.058       Ischemic stroke   0.184   1.36   0.007   0.068   1.20   NS       (450) a         Males (253)   0.183   1.35   0.023   0.092   1.65   0.016       Females (181)   0.179   1.34   0.044   0.035   0.58   NS       Iceland       Controls (624)   0.095           0.07       Ischemic stroke   0.147   1.63   0.00013   0.073   1.09   NS       (632)       Males (335)   0.155   1.75   0.0002   0.086   1.31   NS       Females (297)   0.138   1.51   0.0079   0.058   0.86   NS                  
 
      The association of HapB to stroke in the Scottish cohort was also investigated. HapB has previously been shown to confer risk of MI in an English cohort (Example 9). A slight excess of HapB was observed in the patient group (6.8%) compared to controls (5.8%), but it was not significant (Table 34). However, gender specific analysis showed that the frequency of HapB was higher in males with ischemic stroke (9.2%) than in controls, resulting in a RR of 1.65 (P=0.016). The frequency of HapB in females with ischemic stroke was 3.5% which was lower but not significantly different from controls. The frequencies of HapB in males and females with ischemic stroke differed significantly (P=0.0021). As shown in Table 34, similar trends were observed in our Icelandic cohort; the frequency of HapB being greater in males with ischemic stroke (8.6%) than in females with ischemic stroke (5.8%), although this was not significant (P=0.055).  
      Thus, HapA, the risk haplotype of ALOX5AP, associates with ischemic stroke in a Scottish cohort. HapB was not associated with ischemic stroke in the Scottish cohort. However, HapB was overrepresented in male patients.  
     EXAMPLE 15  
     Identification of LTA4H Haplotypes Associated with MI Study Population  
      Patients entering the study were defined from a myocardial infarction (MI) registry that includes all MIs (over 8,000 patients) in Iceland from 1981 to 2002. This registry is a part of the World Health Organization MONICA Project (The World Health Organization MONICA Project (monitoring trends and determinants in cardiovascular disease): a major international collaboration. WHO MONICA Project Principal Investigators.  J. Clin. Epidemiol.  1988; 41:105-14). Diagnosis of all patients in the registry follow strict diagnostic rules based on symptoms, electrocardiograms, cardiac enzymes, and necropsy findings.  
      Blood samples from over 1500 MI patients, both cases with a family history and sporadic cases were collected. For each patient that participated, blood was collected from 2 relatives (unaffected or affected). Their genotypes were used to help with construction of haplotypes. Blood samples from over 950 controls were also collected. The control cohort was population based.  
     Linkage Analysis  
      In an effort to enrich for those patients who had stronger genetic factors contributing to their risk for MI, we fractionated the MI cohort to those patients with earlier onset MI. We chose different age cutoffs for male and females since the average age of MI in females is 10 years older than for males. Using MI onset at age less than 50 in males and less than 60 in females, 196 patients were clustered within 67 Pedigrees. These pedigrees included related earlier onset MI patients such that each patient is related to at least one other patient up to and including six meiotic events. The information regarding the relatedness of patients was obtained from an encrypted genealogy database that covers the entire Icelandic nation (Gulcher et al,  Eur. J. Hum. Genet.  8: 739-742 (2000)). A genome-wide scan was performed using a framework map of 1000 microsatellite markers, using protocols described elsewhere (Gretarsdottir S., et al.  Am. J Hum. Genet., 70: 593-603, 2002)). The marker order and positions were obtained from deCODE genetic&#39;s high resolution genetic map (Kong A, et al.,  Nat. genet.,  31: 241-247 (2002)). All markers used in the linkage analysis are publicly available microsatellite markers. The population-based allele frequencies were constructed from a cohort of more than 30,000 Icelanders who have participated in genetic studies of various disease projects.  
      For statistical analysis, multipoint, affected only allele-sharing methods were used to assess evidence for linkage. All results, both the LOD and the non-parametric linkage (NPL) score, were obtained using the program ALLEGRO (Gudbjartsson D. F., et al.,  Nat Genet.,  25: 12-13(2000)). The baseline linkage analysis (Gretarsdottir S., et al.,  Am. J. Hum. Genet.  70: 593-603, (2002)) uses the Spairs scoring function (Whittermore A S, and Haplem J A.,  Biometrics  50: 118-127 (1994)) and Kruglyak et al.,  Am. J. Hum. Genet.,  58:1347-1363 (1996)) the exponential allele-sharing model (Kong A., and Cox N. J.,  Am. J. Hum. Genet.  61:1179-1188 (1997)), and a family weighting scheme which is halfway, on the log-scale, between weighing each affected pairs equally and weighing each family equally.  
     Fine Mapping  
      A candidate susceptibility locus was defined as the region under the LOD score curve where the score was one lower than the highest lod score ((peak lod score−1)one lod drop). This region (approx. 12 Mb) was finemapped with microsatellite markers with an average spacing between markers of approximately 1.5 cM.  
     Case-control Haplotype Association Analysis  
      A large case-control analysis was initially carried out using over 560 male MI patients and 338 female MI patients and 480 population-based controls in an effort to find the MI gene within the linkage peak on chromosome 12 found in genome-wide linkage analysis. Given that a member of the leukotriene biosynthetic pathway, LTA4H, was near the peak microsatellite marker, an effort was made to identify microsatellite markers positioned close to, or within, the LTA4H gene. Three microsatellite markers were identified within the deCODE genetics modified assembly of the public UCSC human genome sequence assembly and they were subsequently genotyped. In addition, SNPs were identified within the LTA4H gene by sequencing 93 patients. Out of the 90 SNPs that were identified 12 were selected to genotype 894 patients and 462 controls. These three microsatellite markers and 12 SNPs, were subsequently used for haplotype analysis. Results from the initial haplotype analysis are shown in Table 38 and Table 39.  
      We then typed a subset of the markers on more MI patients and controls. This subset included 8 SNPs and 3 microsatellite markers. In addition, we typed 9 new SNPs on the total cohort which now included 1560 MI patients and 953 controls. Results from the haplotype association analysis, using the extended cohort and a total of 17 SNPs and 3 microsatellite markers, are shown in Table 39.  
      The frequencies of haplotypes in the patient and the control groups using an expectation-maximization algorithm were estimated (Dempster A. P. et al.,  J. R. Stat. Soc. B.  39: 1-389 (1977)). An implementation of this algorithm that can handle missing genotypes and uncertainty with the phase was used. Under the null hypothesis, the patients and the controls are assumed to have identical frequencies. Using a likelihood approach, an alternative hypothesis where a candidate at-risk-haplotype is allowed to have a higher frequency in patients than controls, while the ratios of the frequencies of other haplotypes are assumed to be the same in both groups was tested. Likelihoods are maximized separately under both hypothesis and a corresponding 1-df likelihood ratio statistics is used to evaluate the statistic significance.  
      To assess the significance of the haplotype association corrected for multiple testing, we carried out a randomisation test using the same genotype data. We randomised the cohorts of patients and controls and repeated the analysis. This procedure was repeated up to 500 times and the adjusted P value is the fraction of replications that produced a P value for some haplotype tested that is lower than or equal to the P value we observed using the original patient and control cohorts.  
     Results  
      Table 35 shows the results of the first step of the linkage analysis; multipoint non-parametric LOD scores for a framework marker map on chromosome 12. A LOD score suggestive of linkage of 1.95 was found at marker D12S2081. This linkage peak was one of the highest peaks found for the earlier onset MI phenotype. Table 36 shows the results of the second step of the linkage analysis; multipoint non-parametric LOD scores for the families after adding 20 fine mapping markers to the candidate region. The inclusion of additional microsatellite markers increased the information on sharing by decent from 0.8 to 0.9, around the markers that gave the highest LOD scores. The lodscore in this locus increased to 2.01 and the peak marker was D12S348 at centimorgin distance 110.6. Thus the locus remained suggestive for linkage suggesting that a gene conferring risk for MI was within the 10 million bases defined by the width of the linkage peak.  
      One of the genes close to the peak marker at this linkage peak (that is, the marker with the highest sharing or lodscore) was LTA4H. Our previous genetic work with FLAP showed that the leukotriene biosynthetic pathway plays a major role in MI risk. Since LTA4H encodes a major member of the leukotriene biosynthetic pathway converting Leukotriene A to Leukotriene B, we chose to test it for association to MI in a case-control study using 894 MI patients and 462 population-based controls.  
      The genomic sequence of the LTA4H gene is set out as SEQ ID NO:. 718. The sequence of the LTA4H mRNA is set out as SEQ ID NO: 719. The sequence of the LTA4H polypeptide is set out as SEQ ID NO: 720.  
      Table 37 shows SNPs that were found by sequencing the LTA4H gene. One of the SNPs, LTA4H — 31334, is in the coding region. The polymorphism, AG, does not change the amino acid sequence in the protein. The rest of the SNPs were outside the coding exons of LTA4H and were within introns or flanking regions of LTA4H.  
      Table 38 shows results from the initial haplotype association analysis using 894 MI patients and 462 controls that were typed with 3 microsatellite markers and 12 SNPs. The following markers show a significant association with MI in males: DG12S1664, SG12S16, SG12S17, SG12S18, SG12S21, SG12S22, SG12S23, SG12S24, SG12S25, SG12S26, DG12S1666, SG12S1O0, SG12S28, and SG12S144, with alleles 0, C, A, T, G, G, T, T, A, T, 0, and T, T, and A, respectively. The allelic frequency of a shorter version of this haplotype including markers DG12S1664, SG12S26, DG12S1666, and SG12S144, with alleles 0, T, 0, and A, respectively, is 51% in male MI patients and 43% in controls (carried by 76% of male patients and 67% of controls). Allelic frequency of this haplotype is higher, or 56%, in a subgroup of patients that have had more than one MI (see Table 38).  
      Table 39 shows the results of the haplotype association analysis using 1560 unrelated MI patients and 953 unrelated population controls. A haplotype comprised of the consecutive markers was highly significant in MI patients that had also had either stroke or peripheral arterial occlusive disease (PAOD) (P-value adjusted for multiple comparisons=0.007). The fact that the haplotypes shown in Table 39 are more significant in MI patients that have more than one clinically evident cardiovascular complication might indicate that the gene played a role in clinical activity or severity of the atherosclerotic disease. The significantly associated haplotype is comprised of the following consecutive markers; SG12S438, DG12S1664, SG12S16, SG12S21, SG12S23, SG12S25, SG12S26, DG12S1666, SG12S100, SG12S28, SG12S143,SG12S144, SG12S221, SG12S222, SG12S223, SG12S225, SG12S226, SG12S233, SG12S237, and DG12S1668 with alleles C, 0, C, G, T, A, T, 0, T, T, T, A, G, C, C, G, G, C,T,and 0. Also shown in Table 39 is a shorter version of the consecutive haplotype and a haplotype that shows a significant protection against MI involving more than one clinically evident cardiovascular complication.  
      In summary, it has been shown for the first time that genetic variants of LTA4H show significant association to MI. The results complement previous work showing that variants in FLAP are significantly associated with MI. In both cases the risk ratio is similar to or higher than the conventional and well-known risk factors for MI including smoking, hypercholesterolemia, hypertension and diabetes anong others.  
      All embodiments of the invention in relation to FLAP genetic discoveries, including but not limited to genetic and protein materials, genetic testing materials and methods, and testing and therapy are also applicable in the context of LTA4H genetic discoveries, and are repeated here by reference. Agents that target any point in the leukotriene biosynthesis pathway are contemplated and expected to be useful in therapy or prophylaxis for subjects with any of the genetic predispositions identified herein. For example, FLAP inhibitors are specifically contemplated for therapy of subjects at risk due to a LTA4H genotype.  
               TABLE 35                          The marker map for chromosome 12 and LOD scores in the first step       of the linkage analysis.                                         location   LOD   dhat   NPL   Zlr   Info   marker                                                 0   1.2574   −0.4865   −1.6783   −2.4063   0.5456   D12S352       3.083   1.7993   −0.5525   −2.1441   −2.8786   0.6374   D12S1608       3.554   1.8107   −0.5494   −2.1696   −2.8877   0.6472   D12S1656       6.566   1.8434   −0.5493   −2.2066   −2.9136   0.6591   D12S1626       7.956   1.8748   −0.5527   −2.2239   −2.9383   0.6638   D12S372       12.93   1.5997   −0.4719   −2.166   −2.7142   0.7291   D12S1725       13.761   1.6842   −0.4859   −2.2249   −2.785   0.732    D12S314       16.166   1.6989   −0.5279   −2.0948   −2.7971   0.6467   D12S374       24.078   1.0258   −0.4043   −1.5861   −2.1734   0.6036   D12S336       26.254   1.0166   −0.3907   −1.6163   −2.1637   0.6338   D12S1697       31.288   0.9373   −0.3846   −1.5323   −2.0775   0.6     D12S364       34.202   0.8469   −0.3806   −1.4006   −1.9748   0.5518   D12S1728       39.399   0.8692   −0.4163   −1.3441   −2.0007   0.4871   D12S1682       44.135   0.7789   −0.3786   −1.306   −1.894   0.5121   D12S1591       49.974   0.7977   −0.3819   −1.3162   −1.9166   0.5166   D12S1640       52.254   0.8638   −0.3759   −1.4437   −1.9945   0.5749   D12S1704       53.951   0.8005   −0.3442   −1.4441   −1.92   0.6191   D12S1681       55.792   0.4155   −0.2301   −1.0815   −1.3833   0.6554   D12S345       57.468   0.2695   −0.1842   −0.8653   −1.114   0.6382   D12S1668       61.09   0.6674   −0.3134   −1.2999   −1.7531   0.6074   D12S85       67.239   0.9722   −0.3854   −1.5762   −2.116   0.6203   D12S368       74.802   0.8922   −0.3971   −1.4186   −2.027   0.5412   D12S83       76.789   0.9969   −0.4272   −1.4897   −2.1426   0.5351   D12S329       84.363   0.0618   −0.103   −0.3514   −0.5333   0.4367   D12S313       92.292   0.0266   0.052   0.2826   0.3497   0.6444   D12S326       96.995   0.2219   0.1438   0.8312   1.0108   0.6496   D12S1708       102.426   1.0345   0.2707   2.0001   2.1827   0.7615   D12S351       103.746   1.4296   0.3119   2.3732   2.5659   0.7625   D12S95       109.914   1.9537   0.3537   2.8183   2.9995   0.7796   D12S2081       112.689   1.4231   0.2984   2.4796   2.56   0.84    D12S346       114.367   1.1079   0.2685   2.1563   2.2588   0.8307   D12S1727       117.962   1.2498   0.2916   2.2133   2.3991   0.7773   D12S78       123.398   0.2995   0.1592   1.012   1.1744   0.7055   D12S1613       126.542   0.1457   0.1139   0.6968   0.819   0.6986   D12S1583       132.981   0.0058   0.0232   0.1392   0.1631   0.7222   D12S354       133.655   0.0011   0.0106   0.0607   0.0725   0.6962   D12S369       133.964   0.0012   0.0107   0.0608   0.0728   0.6913   D12S79       139.646   0.0742   0.0823   0.4953   0.5844   0.701    D12S366       142.505   0.1383   0.1088   0.694   0.7979   0.7292   D12S395       143.459   0.0732   0.0795   0.5072   0.5805   0.7417   D12S2073       143.698   0.0886   0.0875   0.5572   0.6387   0.7369   D12S1349       144.394   0.0604   0.0727   0.4591   0.5275   0.7376   D12S378       148.306   0   0.0013   0.0084   0.0096   0.7673   D12S1614       151.275   0.0125   0.0351   0.1985   0.2397   0.6764   D12S324       155.308   0.3155   0.1758   0.9568   1.2054   0.6008   D12S2075       156.144   0.2797   0.1706   0.8734   1.1348   0.5679   D12S1675       158.207   0.3194   0.1834   0.9265   1.2128   0.5549   D12S1679       162.448   0.3706   0.1872   1.0567   1.3063   0.6156   D12S1659       164.59   0.368   0.1876   1.0474   1.3019   0.6084   D12S367       172.615   0.3231   0.1872   0.9214   1.2199   0.5371   D12S1723       174.333   0.2827   0.1781   0.847   1.1411   0.5229   D12S1638                  
 
     
       
         
           
               
             
               
                 TABLE 36 
               
             
            
               
                   
               
               
                   
               
               
                 The marker map for chromosome 12 and LOD scores, in the second 
               
               
                 step of the linkage analysis. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 location 
                 LOD 
                 dhat 
                 NPL 
                 Zlr 
                 Info 
                 marker 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 0 
                 1.6956 
                 −0.6253 
                 −1.8379 
                 −2.7944 
                 0.4963 
                 D12S352 
               
               
                 3.758 
                 2.024 
                 −0.6098 
                 −2.2287 
                 −3.053 
                 0.6154 
                 D12S1608 
               
               
                 4.239 
                 2.0532 
                 −0.6089 
                 −2.262 
                 −3.0749 
                 0.6257 
                 D12S1656 
               
               
                 4.899 
                 2.0351 
                 −0.6062 
                 −2.2476 
                 −3.0614 
                 0.6244 
                 D12S100 
               
               
                 4.949 
                 2.0335 
                 −0.6059 
                 −2.2466 
                 −3.0601 
                 0.6243 
                 D12S1694 
               
               
                 5.825 
                 1.9982 
                 −0.5969 
                 −2.2337 
                 −3.0335 
                 0.6278 
                 D12S1615 
               
               
                 7.41 
                 1.895 
                 −0.5609 
                 −2.2259 
                 −2.9541 
                 0.6556 
                 D12S1626 
               
               
                 8.241 
                 1.9046 
                 −0.5627 
                 −2.2255 
                 −2.9616 
                 0.6556 
                 D12S372 
               
               
                 9.071 
                 1.8945 
                 −0.5659 
                 −2.197 
                 −2.9537 
                 0.6463 
                 D12S835 
               
               
                 9.239 
                 1.8908 
                 −0.5659 
                 −2.1919 
                 −2.9509 
                 0.6452 
                 D12S1050 
               
               
                 9.628 
                 1.8804 
                 −0.5648 
                 −2.1812 
                 −2.9427 
                 0.6435 
                 D12S1652 
               
               
                 13.786 
                 1.6009 
                 −0.4751 
                 −2.1492 
                 −2.7152 
                 0.7218 
                 D12S1725 
               
               
                 14.624 
                 1.596 
                 −0.4767 
                 −2.1379 
                 −2.7111 
                 0.7157 
                 D12S314 
               
               
                 15.679 
                 1.7102 
                 −0.5249 
                 −2.1113 
                 −2.8064 
                 0.6569 
                 D12S328 
               
               
                 15.729 
                 1.7111 
                 −0.5255 
                 −2.1102 
                 −2.8071 
                 0.656  
                 D12S93 
               
               
                 15.917 
                 1.7113 
                 −0.5272 
                 −2.1062 
                 −2.8073 
                 0.6527 
                 D12S99 
               
               
                 16.495 
                 1.6721 
                 −0.5331 
                 −2.0411 
                 −2.7749 
                 0.6266 
                 D12S1673 
               
               
                 16.684 
                 1.6562 
                 −0.5339 
                 −2.0199 
                 −2.7617 
                 0.6192 
                 D12S356 
               
               
                 17.131 
                 1.6124 
                 −0.5336 
                 −1.9702 
                 −2.725 
                 0.6035 
                 D12S374 
               
               
                 20.18 
                 1.4787 
                 −0.5541 
                 −1.7482 
                 −2.6095 
                 0.5214 
                 D12S1625 
               
               
                 23.545 
                 1.1182 
                 −0.4645 
                 −1.5402 
                 −2.2693 
                 0.5229 
                 D12S397 
               
               
                 24.869 
                 0.9441 
                 −0.4038 
                 −1.4682 
                 −2.0852 
                 0.5568 
                 D12S1695 
               
               
                 24.979 
                 0.9297 
                 −0.3985 
                 −1.4625 
                 −2.0692 
                 0.5606 
                 D12S336 
               
               
                 25.269 
                 0.9337 
                 −0.399 
                 −1.4663 
                 −2.0736 
                 0.5617 
                 D12S1674 
               
               
                 25.559 
                 0.9367 
                 −0.3992 
                 −1.4704 
                 −2.077 
                 0.5632 
                 D12S1690 
               
               
                 25.772 
                 0.9384 
                 −0.3989 
                 −1.4735 
                 −2.0788 
                 0.5648 
                 D12S1696 
               
               
                 25.793 
                 0.9385 
                 −0.3989 
                 −1.4738 
                 −2.0789 
                 0.5649 
                 D12S77 
               
               
                 26.767 
                 0.9395 
                 −0.3946 
                 −1.4893 
                 −2.08 
                 0.5758 
                 D12S827 
               
               
                 27.155 
                 0.937 
                 −0.3915 
                 −1.4961 
                 −2.0773 
                 0.5821 
                 D12S1697 
               
               
                 27.325 
                 0.938 
                 −0.3939 
                 −1.4894 
                 −2.0784 
                 0.5766 
                 D12S89 
               
               
                 28.883 
                 0.9248 
                 −0.4057 
                 −1.4313 
                 −2.0636 
                 0.5411 
                 D12S391 
               
               
                 30.851 
                 0.8473 
                 −0.39 
                 −1.3665 
                 −1.9754 
                 0.5299 
                 D12S1581 
               
               
                 31.936 
                 0.7765 
                 −0.3651 
                 −1.3345 
                 −1.891 
                 0.5429 
                 D12S1580 
               
               
                 32.188 
                 0.7575 
                 −0.3576 
                 −1.3274 
                 −1.8677 
                 0.5489 
                 D12S320 
               
               
                 32.238 
                 0.7536 
                 −0.356 
                 −1.326 
                 −1.863 
                 0.5503 
                 D12S364 
               
               
                 32.735 
                 0.7445 
                 −0.3581 
                 −1.3038 
                 −1.8516 
                 0.538  
                 D12S308 
               
               
                 34.013 
                 0.7073 
                 −0.3557 
                 −1.2478 
                 −1.8048 
                 0.5172 
                 D12S2210 
               
               
                 34.335 
                 0.6949 
                 −0.3532 
                 −1.2338 
                 −1.7889 
                 0.5143 
                 D12S1303 
               
               
                 35.153 
                 0.6582 
                 −0.3436 
                 −1.1984 
                 −1.741 
                 0.5108 
                 D12S1728 
               
               
                 36.074 
                 0.693 
                 −0.3705 
                 −1.1841 
                 −1.7864 
                 0.4727 
                 D12S1715 
               
               
                 37.358 
                 0.7161 
                 −0.3917 
                 −1.1671 
                 −1.816 
                 0.4445 
                 D12S310 
               
               
                 37.716 
                 0.723 
                 −0.3955 
                 −1.1681 
                 −1.8247 
                 0.4414 
                 D12S1669 
               
               
                 39.199 
                 0.7267 
                 −0.3952 
                 −1.1753 
                 −1.8294 
                 0.4443 
                 D12S1650 
               
               
                 40.35 
                 0.7034 
                 −0.3777 
                 −1.1844 
                 −1.7998 
                 0.4644 
                 D12S1682 
               
               
                 45.086 
                 0.6102 
                 −0.3149 
                 −1.1956 
                 −1.6764 
                 0.5509 
                 D12S1591 
               
               
                 46.757 
                 0.645 
                 −0.3251 
                 −1.2237 
                 −1.7234 
                 0.5509 
                 D12S1057 
               
               
                 47.216 
                 0.6504 
                 −0.3287 
                 −1.2219 
                 −1.7307 
                 0.5449 
                 D12S1617 
               
               
                 49.098 
                 0.6565 
                 −0.332 
                 −1.2227 
                 −1.7387 
                 0.5404 
                 D12S1596 
               
               
                 50.007 
                 0.6508 
                 −0.3269 
                 −1.2292 
                 −1.7312 
                 0.5503 
                 D12S1034 
               
               
                 50.925 
                 0.6382 
                 −0.3169 
                 −1.2391 
                 −1.7144 
                 0.5696 
                 D12S1640 
               
               
                 53.204 
                 0.7066 
                 −0.3153 
                 −1.3729 
                 −1.8039 
                 0.6362 
                 D12S1704 
               
               
                 53.205 
                 0.7066 
                 −0.3153 
                 −1.373 
                 −1.8039 
                 0.6362 
                 D12S1643 
               
               
                 54.901 
                 0.6809 
                 −0.2936 
                 −1.4087 
                 −1.7708 
                 0.695  
                 D12S1681 
               
               
                 55.526 
                 0.5731 
                 −0.2654 
                 −1.301 
                 −1.6245 
                 0.6994 
                 D12S1648 
               
               
                 55.827 
                 0.5217 
                 −0.2504 
                 −1.25 
                 −1.55 
                 0.7065 
                 D12S61 
               
               
                 56.499 
                 0.4119 
                 −0.2146 
                 −1.1385 
                 −1.3772 
                 0.737  
                 ATA73C05 
               
               
                 56.549 
                 0.4041 
                 −0.2119 
                 −1.1303 
                 −1.3641 
                 0.7401 
                 D12S1621 
               
               
                 56.793 
                 0.3671 
                 −0.1986 
                 −1.0906 
                 −1.3002 
                 0.7572 
                 D12S345 
               
               
                 57.118 
                 0.3602 
                 −0.1959 
                 −1.0835 
                 −1.288 
                 0.7615 
                 D12S2080 
               
               
                 58.072 
                 0.3416 
                 −0.1881 
                 −1.0664 
                 −1.2542 
                 0.7782 
                 D12S1048 
               
               
                 58.469 
                 0.3345 
                 −0.1849 
                 −1.0609 
                 −1.2411 
                 0.7867 
                 D12S1668 
               
               
                 59.057 
                 0.3671 
                 −0.1944 
                 −1.1109 
                 −1.3002 
                 0.7874 
                 D12S1589 
               
               
                 59.716 
                 0.4056 
                 −0.2045 
                 −1.1706 
                 −1.3667 
                 0.7932 
                 D12S291 
               
               
                 60.054 
                 0.4612 
                 −0.221 
                 −1.2374 
                 −1.4573 
                 0.7826 
                 D12S1301 
               
               
                 61.826 
                 0.7555 
                 −0.2833 
                 −1.6011 
                 −1.8652 
                 0.8213 
                 D12S1713 
               
               
                 62.09 
                 0.7752 
                 −0.2879 
                 −1.6189 
                 −1.8894 
                 0.819  
                 D12S85 
               
               
                 63.701 
                 0.8433 
                 −0.309 
                 −1.6549 
                 −1.9707 
                 0.7867 
                 D12S1701 
               
               
                 64.377 
                 0.8374 
                 −0.3088 
                 −1.6463 
                 −1.9637 
                 0.7819 
                 D12S2199 
               
               
                 64.888 
                 0.821 
                 −0.3047 
                 −1.6355 
                 −1.9445 
                 0.785  
                 D12S1590 
               
               
                 65.096 
                 0.8096 
                 −0.3025 
                 −1.6239 
                 −1.9309 
                 0.784  
                 D12S1627 
               
               
                 65.665 
                 0.8586 
                 −0.3194 
                 −1.6441 
                 −1.9884 
                 0.756  
                 D12S1620 
               
               
                 65.666 
                 0.8587 
                 −0.3194 
                 −1.6441 
                 −1.9885 
                 0.7561 
                 D12S1635 
               
               
                 66.235 
                 0.8957 
                 −0.3295 
                 −1.6678 
                 −2.031 
                 0.7474 
                 D12S1633 
               
               
                 66.236 
                 0.8958 
                 −0.3295 
                 −1.6678 
                 −2.0311 
                 0.7473 
                 D12S1629 
               
               
                 66.838 
                 0.9205 
                 −0.3325 
                 −1.6967 
                 −2.0589 
                 0.7558 
                 D12S347 
               
               
                 67.205 
                 0.9208 
                 −0.3307 
                 −1.7028 
                 −2.0592 
                 0.7633 
                 D12S1677 
               
               
                 68.24 
                 1.1611 
                 −0.3656 
                 −1.9527 
                 −2.3124 
                 0.8101 
                 D12S368 
               
               
                 68.854 
                 1.1354 
                 −0.3678 
                 −1.9021 
                 −2.2867 
                 0.7842 
                 D12S96 
               
               
                 69.118 
                 1.1237 
                 −0.3682 
                 −1.8815 
                 −2.2749 
                 0.7746 
                 D12S398 
               
               
                 70.315 
                 1.0649 
                 −0.3662 
                 −1.7961 
                 −2.2145 
                 0.7407 
                 D12S1604 
               
               
                 70.523 
                 1.0539 
                 −0.3653 
                 −1.7827 
                 −2.2031 
                 0.7365 
                 D12S359 
               
               
                 70.637 
                 1.0579 
                 −0.3678 
                 −1.7787 
                 −2.2072 
                 0.7304 
                 D12S1651 
               
               
                 71.597 
                 1.0794 
                 −0.3844 
                 −1.7459 
                 −2.2296 
                 0.6917 
                 D12S1724 
               
               
                 71.8 
                 1.0813 
                 −0.3867 
                 −1.7392 
                 −2.2315 
                 0.6859 
                 D12S1707 
               
               
                 72.252 
                 1.0822 
                 −0.3904 
                 −1.7247 
                 −2.2324 
                 0.6753 
                 D12S2191 
               
               
                 73.451 
                 1.0636 
                 −0.3917 
                 −1.6882 
                 −2.2132 
                 0.6601 
                 D12S1632 
               
               
                 74.528 
                 1.0229 
                 −0.3828 
                 −1.6582 
                 −2.1704 
                 0.6601 
                 D12S90 
               
               
                 74.775 
                 1.0106 
                 −0.3795 
                 −1.6517 
                 −2.1573 
                 0.6617 
                 D12S305 
               
               
                 74.919 
                 1.0029 
                 −0.3773 
                 −1.648 
                 −2.1491 
                 0.6631 
                 D12S1298 
               
               
                 75.69 
                 0.9563 
                 −0.363 
                 −1.6289 
                 −2.0985 
                 0.6753 
                 D12S1700 
               
               
                 75.691 
                 0.9562 
                 −0.3629 
                 −1.6288 
                 −2.0984 
                 0.6756 
                 D12S1056 
               
               
                 75.744 
                 0.9527 
                 −0.3618 
                 −1.6276 
                 −2.0946 
                 0.6767 
                 D12S1662 
               
               
                 75.802 
                 0.9487 
                 −0.3605 
                 −1.6262 
                 −2.0902 
                 0.6779 
                 D12S83 
               
               
                 75.803 
                 0.9487 
                 −0.3605 
                 −1.6262 
                 −2.0902 
                 0.6779 
                 D12S1655 
               
               
                 76.339 
                 0.9582 
                 −0.3657 
                 −1.6221 
                 −2.1006 
                 0.6682 
                 D12S298 
               
               
                 76.916 
                 0.9668 
                 −0.3701 
                 −1.62 
                 −2.1101 
                 0.6606 
                 D12S1726 
               
               
                 77.789 
                 0.9767 
                 −0.3743 
                 −1.621 
                 −2.1209 
                 0.6546 
                 D12S329 
               
               
                 80.622 
                 0.7896 
                 −0.3801 
                 −1.2958 
                 −1.9068 
                 0.5155 
                 D12S1649 
               
               
                 83.513 
                 0.4582 
                 −0.2911 
                 −0.9752 
                 −1.4527 
                 0.4746 
                 D12S1601 
               
               
                 84.007 
                 0.3957 
                 −0.2648 
                 −0.9209 
                 −1.35 
                 0.4851 
                 D12S1294 
               
               
                 84.428 
                 0.3441 
                 −0.2407 
                 −0.8746 
                 −1.2588 
                 0.5003 
                 D12S335 
               
               
                 85.558 
                 0.2207 
                 −0.1753 
                 −0.75 
                 −1.0081 
                 0.573  
                 D12S313 
               
               
                 86.414 
                 0.2075 
                 −0.1672 
                 −0.7361 
                 −0.9775 
                 0.5883 
                 D12S375 
               
               
                 86.588 
                 0.2051 
                 −0.1658 
                 −0.7331 
                 −0.9718 
                 0.5905 
                 D12S1680 
               
               
                 87.042 
                 0.198 
                 −0.1615 
                 −0.7253 
                 −0.9549 
                 0.5991 
                 D12S1693 
               
               
                 88.586 
                 0.1683 
                 −0.1407 
                 −0.7008 
                 −0.8803 
                 0.6584 
                 D12S1040 
               
               
                 89.237 
                 0.1545 
                 −0.1303 
                 −0.6917 
                 −0.8436 
                 0.6988 
                 D12S299 
               
               
                 89.238 
                 0.1545 
                 −0.1303 
                 −0.6917 
                 −0.8435 
                 0.6987 
                 D12S92 
               
               
                 89.781 
                 0.143 
                 −0.1214 
                 −0.6848 
                 −0.8116 
                 0.7399 
                 D12S1052 
               
               
                 90.368 
                 0.131 
                 −0.1118 
                 −0.6779 
                 −0.7767 
                 0.7921 
                 D12S337 
               
               
                 91.289 
                 0.155 
                 −0.1175 
                 −0.7641 
                 −0.8449 
                 0.8534 
                 D12S1660 
               
               
                 91.913 
                 0.087 
                 −0.0886 
                 −0.5648 
                 −0.6331 
                 0.8225 
                 D12S1684 
               
               
                 92.02 
                 0.0761 
                 −0.0831 
                 −0.5262 
                 −0.5921 
                 0.8142 
                 D12S350 
               
               
                 93.288 
                 0.0009 
                 −0.0089 
                 −0.0583 
                 −0.0652 
                 0.8082 
                 D12S326 
               
               
                 97.989 
                 0.2109 
                 0.123 
                 0.9332 
                 0.9855 
                 0.8597 
                 D12S1297 
               
               
                 97.99 
                 0.2119 
                 0.1234 
                 0.9351 
                 0.9879 
                 0.8588 
                 D12S106 
               
               
                 97.991 
                 0.213 
                 0.1237 
                 0.9371 
                 0.9903 
                 0.8578 
                 D12S1708 
               
               
                 99.524 
                 0.6535 
                 0.201 
                 1.7426 
                 1.7347 
                 0.9295 
                 D12S1667 
               
               
                 99.525 
                 0.6535 
                 0.201 
                 1.7427 
                 1.7348 
                 0.9296 
                 D12S319 
               
               
                 100.397 
                 0.7234 
                 0.208 
                 1.8684 
                 1.8252 
                 0.9553 
                 D12S323 
               
               
                 100.398 
                 0.7235 
                 0.208 
                 1.8686 
                 1.8253 
                 0.955  
                 D12S88 
               
               
                 100.399 
                 0.7301 
                 0.2091 
                 1.8758 
                 1.8336 
                 0.9533 
                 D12S1719 
               
               
                 100.519 
                 0.7536 
                 0.2127 
                 1.9016 
                 1.8629 
                 0.947  
                 D12S1593 
               
               
                 101.064 
                 0.8567 
                 0.2269 
                 2.0196 
                 1.9863 
                 0.9341 
                 D12S853 
               
               
                 101.841 
                 0.9732 
                 0.2384 
                 2.1747 
                 2.117 
                 0.951  
                 D12S1710 
               
               
                 102.131 
                 1.1754 
                 0.2589 
                 2.4086 
                 2.3266 
                 0.9561 
                 D12S1717 
               
               
                 103.423 
                 1.1442 
                 0.2555 
                 2.379 
                 2.2955 
                 0.9588 
                 D12S351 
               
               
                 104.343 
                 1.341 
                 0.2756 
                 2.5694 
                 2.485 
                 0.9479 
                 D12S311 
               
               
                 104.743 
                 1.6769 
                 0.3035 
                 2.8993 
                 2.7789 
                 0.952  
                 D12S95 
               
               
                 105.266 
                 1.7384 
                 0.3095 
                 2.9441 
                 2.8294 
                 0.9441 
                 D12S1345 
               
               
                 106.345 
                 1.8647 
                 0.326 
                 2.9793 
                 2.9304 
                 0.8988 
                 D12S1346 
               
               
                 110.627 
                 2.0063 
                 0.3408 
                 3.0437 
                 3.0397 
                 0.8726 
                 D12S348 
               
               
                 110.908 
                 1.9856 
                 0.337 
                 3.0533 
                 3.0239 
                 0.8861 
                 D12S1716 
               
               
                 110.909 
                 1.9854 
                 0.337 
                 3.053 
                 3.0238 
                 0.886  
                 D12S1657 
               
               
                 112.477 
                 1.3244 
                 0.2754 
                 2.5394 
                 2.4696 
                 0.9375 
                 D12S393 
               
               
                 112.658 
                 1.5716 
                 0.2988 
                 2.7576 
                 2.6903 
                 0.9246 
                 D12S1706 
               
               
                 113.456 
                 1.482 
                 0.2868 
                 2.7191 
                 2.6125 
                 0.9569 
                 D12S1600 
               
               
                 113.686 
                 1.4654 
                 0.2856 
                 2.7011 
                 2.5978 
                 0.9556 
                 D12S346 
               
               
                 114.583 
                 1.2538 
                 0.2643 
                 2.5203 
                 2.4029 
                 0.9739 
                 D12S1641 
               
               
                 114.628 
                 1.2491 
                 0.2637 
                 2.5166 
                 2.3984 
                 0.9748 
                 D12S306 
               
               
                 114.674 
                 1.2445 
                 0.2632 
                 2.5127 
                 2.3939 
                 0.9759 
                 D12S332 
               
               
                 115.043 
                 1.3131 
                 0.271 
                 2.5676 
                 2.4591 
                 0.9635 
                 D12S1041 
               
               
                 115.364 
                 1.1318 
                 0.2546 
                 2.3621 
                 2.283 
                 0.956  
                 D12S1727 
               
               
                 116.299 
                 1.1829 
                 0.2606 
                 2.4032 
                 2.334 
                 0.9477 
                 D12S1607 
               
               
                 116.948 
                 1.2361 
                 0.2691 
                 2.4273 
                 2.3859 
                 0.9221 
                 IGF1 
               
               
                 116.949 
                 1.2361 
                 0.2691 
                 2.4273 
                 2.3859 
                 0.9219 
                 D12S1030 
               
               
                 117.75 
                 1.5059 
                 0.2956 
                 2.6701 
                 2.6334 
                 0.9082 
                 PAH 
               
               
                 118.61 
                 1.2001 
                 0.2629 
                 2.4192 
                 2.3509 
                 0.9435 
                 D12S360 
               
               
                 118.899 
                 1.4558 
                 0.2869 
                 2.6729 
                 2.5893 
                 0.9393 
                 D12S78 
               
               
                 119.188 
                 1.399 
                 0.2838 
                 2.5969 
                 2.5382 
                 0.9253 
                 D12S338 
               
               
                 120.067 
                 1.3032 
                 0.2727 
                 2.5213 
                 2.4498 
                 0.943  
                 D12S1647 
               
               
                 120.068 
                 1.2993 
                 0.2723 
                 2.5179 
                 2.4461 
                 0.9436 
                 D12S317 
               
               
                 120.348 
                 1.4722 
                 0.2886 
                 2.6798 
                 2.6038 
                 0.9378 
                 D12S1597 
               
               
                 121.195 
                 1.3839 
                 0.2842 
                 2.5548 
                 2.5245 
                 0.9127 
                 D12S1683 
               
               
                 124.023 
                 0.6306 
                 0.2003 
                 1.693 
                 1.7041 
                 0.9045 
                 D12S1342 
               
               
                 124.297 
                 0.6069 
                 0.198 
                 1.6474 
                 1.6718 
                 0.8927 
                 D12S1613 
               
               
                 125.597 
                 0.483 
                 0.183 
                 1.4221 
                 1.4915 
                 0.8432 
                 D12S1605 
               
               
                 126.055 
                 0.451 
                 0.1786 
                 1.3612 
                 1.4411 
                 0.8293 
                 D12S84 
               
               
                 126.796 
                 0.3855 
                 0.1683 
                 1.2383 
                 1.3324 
                 0.8059 
                 D12S105 
               
               
                 127.545 
                 0.3132 
                 0.1527 
                 1.1129 
                 1.2009 
                 0.8072 
                 D12S1583 
               
               
                 129.188 
                 0.2211 
                 0.1354 
                 0.8864 
                 1.009 
                 0.7362 
                 D12S1344 
               
               
                 130.64 
                 0.141 
                 0.1122 
                 0.6858 
                 0.8058 
                 0.6977 
                 D12S1616 
               
               
                 133.986 
                 0.0109 
                 0.0313 
                 0.1941 
                 0.2238 
                 0.742  
                 D12S354 
               
               
                 134.268 
                 0.0114 
                 0.0321 
                 0.1973 
                 0.2287 
                 0.7353 
                 D12S1023 
               
               
                 134.818 
                 0.0122 
                 0.0336 
                 0.2027 
                 0.237 
                 0.7233 
                 D12S369 
               
               
                 134.959 
                 0.0122 
                 0.0336 
                 0.2019 
                 0.2365 
                 0.7205 
                 D12S1602 
               
               
                 135.149 
                 0.0121 
                 0.0335 
                 0.2006 
                 0.2356 
                 0.7164 
                 D12S79 
               
               
                 135.367 
                 0.0102 
                 0.0312 
                 0.1829 
                 0.217 
                 0.7035 
                 D12S1665 
               
               
                 137.617 
                 0.0008 
                 0.0093 
                 0.0498 
                 0.0617 
                 0.6492 
                 D12S1718 
               
               
                 140.815 
                 0.0287 
                 0.0511 
                 0.3109 
                 0.3633 
                 0.7212 
                 D12S366 
               
               
                 141.527 
                 0.0431 
                 0.0638 
                 0.374 
                 0.4458 
                 0.6902 
                 D12S349 
               
               
                 141.528 
                 0.0879 
                 0.0897 
                 0.5377 
                 0.6361 
                 0.6935 
                 D12S1619 
               
               
                 141.755 
                 0.0867 
                 0.0892 
                 0.5334 
                 0.6317 
                 0.6917 
                 D12S385 
               
               
                 143.676 
                 0.0629 
                 0.073 
                 0.476 
                 0.5383 
                 0.7618 
                 D12S395 
               
               
                 143.677 
                 0.0629 
                 0.073 
                 0.4759 
                 0.5382 
                 0.7615 
                 D12S321 
               
               
                 143.678 
                 0.0629 
                 0.073 
                 0.4759 
                 0.5381 
                 0.7613 
                 D12S1721 
               
               
                 143.824 
                 0.0588 
                 0.0707 
                 0.4601 
                 0.5205 
                 0.7614 
                 D12S1666 
               
               
                 144.632 
                 0.0428 
                 0.0604 
                 0.3929 
                 0.444 
                 0.7652 
                 D12S2073 
               
               
                 144.962 
                 0.0437 
                 0.0611 
                 0.3961 
                 0.4485 
                 0.7621 
                 D12S1349 
               
               
                 145.291 
                 0.037 
                 0.0563 
                 0.3644 
                 0.4128 
                 0.7628 
                 D12S1603 
               
               
                 145.426 
                 0.0331 
                 0.0534 
                 0.3446 
                 0.3907 
                 0.7623 
                 D12S378 
               
               
                 149.447 
                 0.0134 
                 −0.0352 
                 −0.2159 
                 −0.2483 
                 0.7658 
                 D12S1614 
               
               
                 149.448 
                 0.0134 
                 −0.0352 
                 −0.2158 
                 −0.2483 
                 0.7656 
                 D12S342 
               
               
                 152.517 
                 0.0049 
                 −0.0224 
                 −0.124 
                 −0.1505 
                 0.6847 
                 D12S324 
               
               
                 153.404 
                 0.0009 
                 −0.0099 
                 −0.0509 
                 −0.064 
                 0.6328 
                 D12S1634 
               
               
                 153.405 
                 0.0009 
                 −0.0098 
                 −0.0507 
                 −0.0638 
                 0.6382 
                 D12S307 
               
               
                 154.88 
                 0.0244 
                 0.0534 
                 0.2534 
                 0.3353 
                 0.561  
                 D12S1658 
               
               
                 155.819 
                 0.0768 
                 0.0941 
                 0.447 
                 0.5948 
                 0.549  
                 GATA41E12 
               
               
                 155.94 
                 0.0855 
                 0.0991 
                 0.472 
                 0.6275 
                 0.5489 
                 D12S2078 
               
               
                 157.397 
                 0.0566 
                 0.0832 
                 0.3729 
                 0.5104 
                 0.5228 
                 D12S1675 
               
               
                 159.342 
                 0.0829 
                 0.0973 
                 0.4654 
                 0.6179 
                 0.5526 
                 D12S1679 
               
               
                 161.157 
                 0.1143 
                 0.1111 
                 0.5609 
                 0.7255 
                 0.5776 
                 D12S1609 
               
               
                 163.425 
                 0.1165 
                 0.1067 
                 0.5964 
                 0.7324 
                 0.6407 
                 D12S834 
               
               
                 163.559 
                 0.1167 
                 0.1063 
                 0.5993 
                 0.733 
                 0.6461 
                 D12S1659 
               
               
                 165.72 
                 0.175 
                 0.1287 
                 0.7383 
                 0.8977 
                 0.6479 
                 D12S1714 
               
               
                 165.721 
                 0.175 
                 0.1287 
                 0.7383 
                 0.8978 
                 0.648  
                 D12S367 
               
               
                 168.245 
                 0.1739 
                 0.132 
                 0.7137 
                 0.8949 
                 0.6107 
                 D12S2069 
               
               
                 168.246 
                 0.1739 
                 0.132 
                 0.7138 
                 0.8949 
                 0.6105 
                 D12S97 
               
               
                 170.298 
                 0.2145 
                 0.1514 
                 0.7627 
                 0.9938 
                 0.5626 
                 D12S343 
               
               
                 170.824 
                 0.2262 
                 0.156 
                 0.78 
                 1.0207 
                 0.5566 
                 D12S1599 
               
               
                 171.817 
                 0.2496 
                 0.1638 
                 0.8178 
                 1.0722 
                 0.5531 
                 D12S392 
               
               
                 173.734 
                 0.2978 
                 0.1751 
                 0.9099 
                 1.171 
                 0.5715 
                 D12S1723 
               
               
                 175.333 
                 0.2667 
                 0.1709 
                 0.8351 
                 1.1083 
                 0.5393 
                 D12S357 
               
               
                 175.456 
                 0.2648 
                 0.1707 
                 0.8307 
                 1.1043 
                 0.5372 
                 D12S1638 
               
               
                 176.211 
                 0.2665 
                 0.1772 
                 0.8027 
                 1.1079 
                 0.4984 
                 D12S2343 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 37 
               
               
                   
               
               
                   
               
               
                 Build34 
                 Build34 
                 Marker 
                 Marker 
                   
                 Public 
                   
                 Minor 
                 Minor 
                 position in 
               
               
                 start 
                 stop 
                 name 
                 alias 
                 IUPAC 
                 SNP 
                 Variation 
                 allele 
                 allele % 
                 Sequence 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 94877218 
                 94877218 
                 SG12S432 
                   
                 R 
                 rs2270318 
                 A/G 
                 A 
                 12.75 
                 7218 
               
               
                 94885285 
                 94885285 
                 SG12S438 
                   
                 S 
                 rs2268517 
                 C/G 
                 G 
                 9.36 
                 15285 
               
               
                 94896055 
                 94896055 
                 SG12S16 
                 LTA4H_3645 
                 Y 
                   
                 C/T 
                 T 
                 22.64 
                 26055 
               
               
                 94896115 
                 94896115 
                 SG12S56 
                 LTA4H_3705 
                 K 
                   
                 G/T 
                 G 
                 4.14 
                 26115 
               
               
                 94896339 
                 94896339 
                 SG12S57 
                 LTA4H_3929 
                 Y 
                   
                 C/T 
                 C 
                 2.5 
                 26339 
               
               
                 94896351 
                 94896351 
                 SG12S58 
                 LTA4H_3941 
                 S 
                   
                 C/G 
                 C 
                 0.85 
                 26351 
               
               
                 94896393 
                 94896393 
                 SG12S37 
                 LTA4H_3983 
                 W 
                   
                 A/T 
                 T 
                 9.3 
                 26393 
               
               
                 94896705 
                 94896705 
                 SG12S59 
                 LTA4H_4295 
                 R 
                   
                 A/G 
                 A 
                 4.5 
                 26705 
               
               
                 94896786 
                 94896786 
                 SG12S60 
                 LTA4H_4376 
                 R 
                   
                 A/G 
                 A 
                 2.87 
                 26786 
               
               
                 94896832 
                 94896832 
                 SG12S61 
                 LTA4H_4422 
                 R 
                   
                 A/G 
                 G 
                 1.56 
                 26832 
               
               
                 94896897 
                 94896897 
                 SG12S29 
                 LTA4H_4487 
                 W 
                   
                 A/T 
                 T 
                 4.26 
                 26897 
               
               
                 94896985 
                 94896985 
                 SG12S17 
                 LTA4H_4575 
                 R 
                 rs11108372 
                 A/G 
                 A 
                 41.41 
                 26985 
               
               
                 94897845 
                 94897845 
                 SG12S62 
                 LTA4H_5435 
                 Y 
                   
                 C/T 
                 C 
                 1.17 
                 27845 
               
               
                 94898878 
                 94898878 
                 SG12S63 
                 LTA4H_6468 
                 Y 
                   
                 C/T 
                 T 
                 4.46 
                 28878 
               
               
                 94899057 
                 94899057 
                 SG12S64 
                 LTA4H_6647 
                 Y 
                   
                 C/T 
                 C 
                 2.99 
                 29057 
               
               
                 94899549 
                 94899549 
                 SG12S18 
                 LTA4H_7139 
                 W 
                   
                 A/T 
                 A 
                 21.72 
                 29549 
               
               
                 94900318 
                 94900318 
                 SG12S19 
                 LTA4H_7908 
                 W 
                   
                 A/T 
                 A 
                 10.9 
                 30318 
               
               
                 94900639 
                 94900639 
                 SG12S65 
                 LTA4H_8229 
                 K 
                   
                 G/T 
                 G 
                 5.09 
                 30639 
               
               
                 94900892 
                 94900892 
                 SG12S66 
                 LTA4H_8482 
                 R 
                   
                 A/G 
                 G 
                 0.59 
                 30892 
               
               
                 94901997 
                 94901997 
                 SG12S68 
                 LTA4H_9587 
                 W 
                   
                 A/T 
                 T 
                 3.63 
                 31997 
               
               
                 94902169 
                 94902169 
                 SG12S69 
                 LTA4H_9759 
                 W 
                   
                 A/T 
                 A 
                 0.88 
                 32169 
               
               
                 94902337 
                 94902337 
                 SG12S70 
                 LTA4H_9927 
                 M 
                   
                 A/C 
                 A 
                 24.09 
                 32337 
               
               
                 94902454 
                 94902454 
                 SG12S71 
                 LTA4H_10044 
                 Y 
                   
                 C/T 
                 C 
                 20.93 
                 32454 
               
               
                 94902928 
                 94902928 
                 SG12S72 
                 LTA4H_10518 
                 Y 
                   
                 C/T 
                 T 
                 1.35 
                 32928 
               
               
                 94903037 
                 94903037 
                 SG12S30 
                 LTA4H_10627 
                 W 
                 rs2540498 
                 A/T 
                 A 
                 22.36 
                 33037 
               
               
                 94903300 
                 94903300 
                 SG12S73 
                 LTA4H_10890 
                 Y 
                 rs2300559 
                 C/T 
                 C 
                 2.33 
                 33300 
               
               
                 94903618 
                 94903618 
                 SG12S20 
                 LTA4H_11208 
                 M 
                   
                 A/C 
                 C 
                 39.08 
                 33618 
               
               
                 94903720 
                 94903720 
                 SG12S21 
                 LTA4H_11310 
                 R 
                 rs2660880 
                 A/G 
                 A 
                 5.95 
                 33720 
               
               
                 94905002 
                 94905002 
                 SG12S38 
                 LTA4H_12592 
                 Y 
                 rs2110762 
                 C/T 
                 C 
                 34.92 
                 35002 
               
               
                 94905216 
                 94905216 
                 SG12S74 
                 LTA4H_12806 
                 Y 
                   
                 C/T 
                 T 
                 0.8 
                 35216 
               
               
                 94905667 
                 94905667 
                 SG12S22 
                 LTA4H_13257 
                 R 
                 rs2072510 
                 A/G 
                 A 
                 36.88 
                 35667 
               
               
                 94905821 
                 94905821 
                 SG12S75 
                 LTA4H_13411 
                 Y 
                   
                 C/T 
                 T 
                 1.39 
                 35821 
               
               
                 94906078 
                 94906078 
                 SG12S23 
                 LTA4H_13668 
                 Y 
                   
                 C/T 
                 C 
                 7.06 
                 36078 
               
               
                 94906362 
                 94906362 
                 SG12S31 
                 LTA4H_13952 
                 Y 
                   
                 C/T 
                 T 
                 5.67 
                 36362 
               
               
                 94906457 
                 94906457 
                 SG12S76 
                 LTA4H_14047 
                 W 
                 rs10492226 
                 A/T 
                 A 
                 1.18 
                 36457 
               
               
                 94906743 
                 94906743 
                 SG12S77 
                 LTA4H_14333 
                 W 
                   
                 A/T 
                 A 
                 24.77 
                 36743 
               
               
                 94907375 
                 94907375 
                 SG12S78 
                 LTA4H_14965 
                 Y 
                   
                 C/T 
                 T 
                 2.48 
                 37375 
               
               
                 94907545 
                 94907545 
                 SG12S24 
                 LTA4H_15135 
                 Y 
                 rs2660900 
                 C/T 
                 C 
                 23.76 
                 37545 
               
               
                 94907935 
                 94907935 
                 SG12S79 
                 LTA4H_15525 
                 S 
                   
                 C/G 
                 C 
                 0.83 
                 37935 
               
               
                 94908971 
                 94908971 
                 SG12S32 
                 LTA4H_16561 
                 R 
                 rs2540496 
                 A/G 
                 A 
                 31.11 
                 38971 
               
               
                 94909012 
                 94909012 
                 SG12S80 
                 LTA4H_16602 
                 W 
                   
                 A/T 
                 A 
                 0.74 
                 39012 
               
               
                 94909191 
                 94909191 
                 SG12S39 
                 LTA4H_16781 
                 K 
                 rs2540495 
                 G/T 
                 T 
                 30.74 
                 39191 
               
               
                 94909554 
                 94909554 
                 SG12S81 
                 LTA4H_17144 
                 R 
                 rs12319438 
                 A/G 
                 G 
                 4.12 
                 39554 
               
               
                 94910164 
                 94910164 
                 SG12S82 
                 LTA4H_17754 
                 R 
                   
                 A/G 
                 A 
                 0.4 
                 40164 
               
               
                 94910246 
                 94910246 
                 SG12S83 
                 LTA4H_17836 
                 W 
                   
                 A/T 
                 T 
                 1.21 
                 40246 
               
               
                 94910273 
                 94910273 
                 SG12S84 
                 LTA4H_17863 
                 R 
                   
                 A/G 
                 A 
                 2.82 
                 40273 
               
               
                 94911669 
                 94911669 
                 SG12S25 
                 LTA4H_19259 
                 R 
                 rs1978331 
                 A/G 
                 G 
                 31.68 
                 41669 
               
               
                 94911781 
                 94911781 
                 SG12S85 
                 LTA4H_19371 
                 Y 
                   
                 C/T 
                 T 
                 1.25 
                 41781 
               
               
                 94914296 
                 94914296 
                 SG12S40 
                 LTA4H_21886 
                 W 
                 rs7959337 
                 A/T 
                 A 
                 5.29 
                 44296 
               
               
                 94916236 
                 94916236 
                 SG12S86 
                 LTA4H_23826 
                 R 
                   
                 A/G 
                 G 
                 4.71 
                 46236 
               
               
                 94916445 
                 94916445 
                 SG12S87 
                 LTA4H_24035 
                 Y 
                   
                 C/T 
                 T 
                 1.27 
                 46445 
               
               
                 94916452 
                 94916452 
                 SG12S88 
                 LTA4H_24042 
                 R 
                 rs1990611 
                 A/G 
                 A 
                 33.76 
                 46452 
               
               
                 94916805 
                 94916805 
                 SG12S89 
                 LTA4H_24395 
                 R 
                 rs7981011 
                 A/G 
                 G 
                 4.91 
                 46805 
               
               
                 94916919 
                 94916919 
                 SG12S26 
                 LTA4H_24509 
                 Y 
                   
                 C/T 
                 C 
                 17.16 
                 46919 
               
               
                 94917444 
                 94917444 
                 SG12S90 
                 LTA4H_25034 
                 R 
                   
                 A/G 
                 A 
                 0.84 
                 47444 
               
               
                 94918851 
                 94918851 
                 SG12S91 
                 LTA4H_26441 
                 Y 
                 rs2660838 
                 C/T 
                 C 
                 25 
                 48851 
               
               
                 94919176 
                 94919176 
                 SG12S92 
                 LTA4H_26766 
                 Y 
                   
                 C/T 
                 C 
                 20.44 
                 49176 
               
               
                 94919667 
                 94919667 
                 SG12S93 
                 LTA4H_27257 
                 R 
                 rs2268516 
                 A/G 
                 A 
                 2.44 
                 49667 
               
               
                 94920368 
                 94920368 
                 SG12S94 
                 LTA4H_27958 
                 Y 
                 rs2660839 
                 C/T 
                 C 
                 31.82 
                 50368 
               
               
                 94921763 
                 94921763 
                 SG12S41 
                 LTA4H_29353 
                 Y 
                   
                 C/T 
                 C 
                 20.35 
                 51763 
               
               
                 94921923 
                 94921923 
                 SG12S95 
                 LTA4H_29513 
                 R 
                 rs4441106 
                 A/G 
                 G 
                 7.07 
                 51923 
               
               
                 94922409 
                 94922409 
                 SG12S96 
                 LTA4H_29999 
                 R 
                 rs763875 
                 A/G 
                 A 
                 5.92 
                 52409 
               
               
                 94922502 
                 94922502 
                 SG12S97 
                 LTA4H_30092 
                 Y 
                 rs763876 
                 C/T 
                 T 
                 2.1 
                 52502 
               
               
                 94922681 
                 94922681 
                 SG12S98 
                 LTA4H_30271 
                 Y 
                 rs763874 
                 C/T 
                 C 
                 32.42 
                 52681 
               
               
                 94923446 
                 94923446 
                 SG12S42 
                 LTA4H_31036 
                 Y 
                 rs2660892 
                 C/T 
                 C 
                 27.41 
                 53446 
               
               
                 94923744 
                 94923744 
                 SG12S55 
                 LTA4H_31334 
                 R 
                   
                 A/G 
                 A 
                 0.27 
                 53744 
               
               
                 94924037 
                 94924037 
                 SG12S99 
                 LTA4H_31627 
                 R 
                   
                 A/G 
                 A 
                 4.37 
                 54037 
               
               
                 94924845 
                 94924845 
                 SG12S100 
                 LTA4H_32435 
                 Y 
                 rs2247570 
                 C/T 
                 C 
                 27.79 
                 54845 
               
               
                 94924938 
                 94924938 
                 SG12S101 
                 LTA4H_32528 
                 R 
                   
                 A/G 
                 A 
                 1.5 
                 54938 
               
               
                 94925915 
                 94925915 
                 SG12S33 
                 LTA4H_33505 
                 Y 
                 rs2660895 
                 C/T 
                 C 
                 30.71 
                 55915 
               
               
                 94926590 
                 94926590 
                 SG12S34 
                 LTA4H_34180 
                 Y 
                 rs2247330 
                 C/T 
                 C 
                 30.9 
                 56590 
               
               
                 94926724 
                 94926724 
                 SG12S102 
                 LTA4H_34314 
                 R 
                 rs2247323 
                 A/G 
                 G 
                 31.85 
                 56724 
               
               
                 94926915 
                 94926915 
                 SG12S103 
                 LTA4H_34505 
                 Y 
                 rs2247313 
                 C/T 
                 T 
                 32.74 
                 56915 
               
               
                 94927010 
                 94927010 
                 SG12S104 
                 LTA4H_34600 
                 Y 
                 rs2247309 
                 C/T 
                 C 
                 32.74 
                 57010 
               
               
                 94927133 
                 94927133 
                 SG12S27 
                 LTA4H_34723 
                 Y 
                 rs2247304 
                 C/T 
                 C 
                 25.57 
                 57133 
               
               
                 94927900 
                 94927900 
                 SG12S35 
                 LTA4H_35490 
                 R 
                 rs2660897 
                 A/G 
                 A 
                 35.93 
                 57900 
               
               
                 94927959 
                 94927959 
                 SG12S105 
                 LTA4H_35549 
                 Y 
                 rs11108381 
                 C/T 
                 T 
                 2.4 
                 57959 
               
               
                 94928465 
                 94928465 
                 SG12S28 
                 LTA4H_36055 
                 K 
                 rs2660898 
                 G/T 
                 G 
                 29.36 
                 58465 
               
               
                 94928740 
                 94928740 
                 SG12S36 
                 LTA4H_36330 
                 Y 
                 rs2540490 
                 C/T 
                 T 
                 31 
                 58740 
               
               
                 94928970 
                 94928970 
                 SG12S106 
                 LTA4H_36560 
                 Y 
                 rs2540489 
                 C/T 
                 C 
                 30.89 
                 58970 
               
               
                 94929183 
                 94929183 
                 SG12S107 
                 LTA4H_36773 
                 Y 
                 rs11108382 
                 C/T 
                 T 
                 2.58 
                 59183 
               
               
                 94929213 
                 94929213 
                 SG12S108 
                 LTA4H_36803 
                 R 
                 rs2540488 
                 A/G 
                 A 
                 26.28 
                 59213 
               
               
                 94929761 
                 94929761 
                 SG12S109 
                 LTA4H_37351 
                 Y 
                 rs2300557 
                 C/T 
                 T 
                 4.76 
                 59761 
               
               
                 94929770 
                 94929770 
                 SG12S110 
                 LTA4H_37360 
                 W 
                 rs2246990 
                 A/T 
                 A 
                 28.57 
                 59770 
               
               
                 94929936 
                 94929936 
                 SG12S111 
                 LTA4H_37526 
                 W 
                   
                 A/T 
                 A 
                 2.81 
                 59936 
               
               
                 94930044 
                 94930044 
                 SG12S112 
                 LTA4H_37634 
                 M 
                   
                 A/C 
                 C 
                 46.15 
                 60044 
               
               
                 94930343 
                 94930343 
                 SG12S43 
                 LTA4H_37933 
                 K 
                 rs2246973 
                 G/T 
                 G 
                 32.93 
                 60343 
               
               
                 94930357 
                 94930357 
                 SG12S113 
                 LTA4H_37947 
                 Y 
                 rs2246972 
                 C/T 
                 T 
                 33.54 
                 60357 
               
               
                 94931246 
                 94931246 
                 SG12S114 
                 LTA4H_38836 
                 K 
                   
                 G/T 
                 T 
                 7.55 
                 61246 
               
               
                 94934775 
                 94934775 
                 SG12S141 
                   
                 R 
                 rs10777768 
                 A/G 
                   
                   
                 64775 
               
               
                 94934975 
                 94934975 
                 SG12S140 
                   
                 M 
                 rs2660840 
                 A/C 
                 C 
                 29.77 
                 64975 
               
               
                 94937348 
                 94937348 
                 SG12S143 
                   
                 Y 
                 rs2540482 
                 C/T 
                 C 
                 17.02 
                 67348 
               
               
                 94941021 
                 94941021 
                 SG12S144 
                   
                 R 
                 rs2660845 
                 A/G 
                 G 
                 19.43 
                 71021 
               
               
                 94943761 
                 94943761 
                 SG12S221 
                   
                 R 
                 rs2540475 
                 A/G 
                 A 
                 16.92 
                 73761 
               
               
                 94946089 
                 94946089 
                 SG12S222 
                   
                 Y 
                 rs2660850 
                 C/T 
                 C 
                 15.47 
                 76089 
               
               
                 94948016 
                 94948016 
                 SG12S460 
                   
                 M 
                 RS2660852 
                 A/C 
                 A 
                 37.22 
                 78016 
               
               
                 94949965 
                 94949965 
                 SG12S223 
                   
                 Y 
                 rs2660875 
                 C/T 
                 C 
                 43.79 
                 79965 
               
               
                 94950568 
                 94950568 
                 SG12S224 
                   
                 R 
                 rs2540473 
                 A/G 
                 G 
                 6.12 
                 80568 
               
               
                 94952847 
                 94952847 
                 SG12S225 
                   
                 R 
                 rs2540472 
                 A/G 
                 A 
                 5.63 
                 82847 
               
               
                 94953483 
                 94953483 
                 SG12S226 
                   
                 S 
                 rs2540471 
                 C/G 
                 C 
                 37.7 
                 83483 
               
               
                 94953798 
                 94953798 
                 SG12S227 
                   
                 R 
                   
                 A/G 
                   
                   
                 83798 
               
               
                 94953801 
                 94953801 
                 SG12S228 
                   
                 Y 
                 rs2660890 
                 C/T 
                 T 
                 46.96 
                 83801 
               
               
                 94953831 
                 94953831 
                 SG12S229 
                   
                 M 
                 rs2660889 
                 A/C 
                   
                   
                 83831 
               
               
                 94954155 
                 94954155 
                 SG12S230 
                   
                 R 
                 rs2660888 
                 A/G 
                 A 
                 35.68 
                 84155 
               
               
                 94954449 
                 94954449 
                 SG12S231 
                   
                 Y 
                 rs4762661 
                 C/T 
                   
                   
                 84449 
               
               
                 94958156 
                 94958156 
                 SG12S232 
                   
                 Y 
                   
                 C/T 
                   
                   
                 88156 
               
               
                 94958339 
                 94958339 
                 SG12S233 
                   
                 Y 
                 rs2660885 
                 C/T 
                 T 
                 15.18 
                 88339 
               
               
                 94962388 
                 94962388 
                 SG12S234 
                   
                 R 
                 rs5800242 
                 A/G 
                   
                   
                 92388 
               
               
                 94962435 
                 94962435 
                 SG12S235 
                   
                 Y 
                 rs759391 
                 C/T 
                   
                   
                 92435 
               
               
                 94963320 
                 94963320 
                 SG12S236 
                   
                 S 
                 rs2540467 
                 C/G 
                   
                   
                 93320 
               
               
                 94963655 
                 94963655 
                 SG12S237 
                   
                 Y 
                 rs2540466 
                 C/T 
                 T 
                 37.05 
                 93655 
               
               
                 94963774 
                 94963774 
                 SG12S238 
                   
                 Y 
                 rs10492225 
                 C/T 
                   
                   
                 93774 
               
               
                 94964298 
                 94964298 
                 SG12S239 
                   
                 W 
                 rs2660874 
                 A/T 
                   
                   
                 94298 
               
               
                 94966584 
                 94966584 
                 SG12S240 
                   
                 W 
                 rs2540461 
                 A/T 
                   
                   
                 96584 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 38A 
               
               
                   
               
               
                   
               
               
                 Haplotype association analysis including SNPs and microsatellite markers across the LTA4H gene. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 DG12S1664 
                 SG12S16 
                 SG12S17 
                 SG12S18 
                 SG12S21 
                 SG12S22 
                 SG12S23 
                 SG12S24 
                 SG12S25 
                 SG12S26 
               
               
                   
                   
               
            
           
           
               
            
               
                 All MI vs 
               
               
                 controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 short 
                 0 
                 C 
                 A 
                 T 
                 G 
                 G 
                 T 
                 T 
                 A 
                 T 
               
               
                 form 
                 0 
                   
                   
                   
                   
                   
                   
                   
                   
                 T 
               
            
           
           
               
            
               
                 MI males vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 short 
                 0 
                 C 
                 A 
                 T 
                 G 
                 G 
                 T 
                 T 
                 A 
                 T 
               
               
                 form 
                 0 
                   
                   
                   
                   
                   
                   
                   
                   
                 T 
               
            
           
           
               
            
               
                 MI females vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 short 
                 0 
                 C 
                 A 
                 T 
                 G 
                 G 
                 T 
                 T 
                 A 
                 T 
               
               
                 form 
                 0 
                   
                   
                   
                   
                   
                   
                   
                   
                 T 
               
            
           
           
               
            
               
                 Recurrent MI vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 short 
                 0 
                 C 
                 A 
                 T 
                 G 
                 G 
                 T 
                 T 
                 A 
                 T 
               
               
                 form 
                 0 
                   
                   
                   
                   
                   
                   
                   
                   
                 T 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 DG12S1666 
                 SG12S100 
                 SG12S28 
                 SG12S144 
                 p-val 
                 r 
                 #aff 
                 aff.frq. 
                 #con 
                 con.frq. 
               
               
                   
                   
               
            
           
           
               
            
               
                 All MI vs 
               
               
                 controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 short 
                 0 
                 T 
                 T 
                 A 
                 1.67E−02 
                 1.24 
                 590 
                 0.49 
                 481 
                 0.44 
               
               
                   
                 form 
                 0 
                   
                   
                 A 
                 3.20E−03 
                 1.32 
                 590 
                 0.5 
                 480 
                 0.43 
               
            
           
           
               
            
               
                 MI males vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 short 
                 0 
                 T 
                 T 
                 A 
                 5.10E−03 
                 1.34 
                 361 
                 0.51 
                 481 
                 0.44 
               
               
                   
                 form 
                 0 
                   
                   
                 A 
                 1.50E−03 
                 1.4 
                 361 
                 0.51 
                 480 
                 0.43 
               
            
           
           
               
            
               
                 MI females vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 short 
                 0 
                 T 
                 T 
                 A 
                 3.80E−01 
                 1.11 
                 229 
                 0.46 
                 481 
                 0.44 
               
               
                   
                 form 
                 0 
                   
                   
                 A 
                 1.35E−01 
                 1.2 
                 229 
                 0.47 
                 480 
                 0.43 
               
            
           
           
               
            
               
                 Recurrent MI vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 short 
                 0 
                 T 
                 T 
                 A 
                 1.50E−02 
                 1.51 
                 88 
                 0.54 
                 481 
                 0.44 
               
               
                   
                 form 
                 0 
                   
                   
                 A 
                 2.40E−03 
                 1.69 
                 88 
                 0.56 
                 480 
                 0.43 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
             
               
                 TABLE 38B 
               
               
                   
               
               
                   
               
               
                 Information on miCrosatellite markers that were 
                   
               
               
                 used in the haplotype assoCiation analysis shown 
               
               
                 in TABLE 38A. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Marker Name 
                 DG12S1664 
               
               
                   
               
               
                 Chr 
                 12 
               
               
                 Cytoband 
                 q23.1 
               
               
                 Start in SEQ_ID_NO_718 
                 7855 
               
               
                 (bp) 
               
               
                 NCBI_build33Start (Mb) 
                 96.317853 
               
               
                 Size 
                 238 
               
               
                 CEPH standard 
                 245 
               
               
                 (referenCe allele) 
               
               
                 Polymorphism type 
                 SNP 
               
               
                 Polymorphism Class 
                 in-del 
               
               
                 Heterozygosity ratio 
                 0.23 
               
               
                 Forward primer 
                 GGAAGGAGGACACTTCTGGA 
               
               
                   
                 (SEQ ID NO:721) 
               
               
                 Reverse primer 
                 GCTGTGAATGGCTAAACTTGG 
               
               
                   
                 (SEQ ID NO:722) 
               
               
                   
               
               
                   
               
               
                 Marker Name 
                 DG12S1666 
               
               
                   
               
               
                 Chr 
                 12 
               
               
                 Cytoband 
                 q23.1 
               
               
                 Start in SEQ_ID_NO_718 
                 38342 
               
               
                 (bp) 
               
               
                 NCBI_build33Start (Mb) 
                 96.34834 
               
               
                 Size 
                 188 
               
               
                 CEPH standard 
                 193 
               
               
                 (referenCe allele) 
               
               
                 Polymorphism type 
                 MiCrosatellite 
               
               
                 Polymorphism Class 
                 Di 
               
               
                 Heterozygosity ratio 
                 0.52 
               
               
                 Forward primer 
                 CACAGAAGCTGCAGTGGAAG 
               
               
                   
                 (SEQ ID NO:723) 
               
               
                 Reverse primer 
                 CAAATGGAGGAGTCAAGACCA 
               
               
                   
                 (SEQ ID NO:724) 
               
               
                   
               
               
                   
               
               
                 Marker Name 
                 DG12S1668 
               
               
                   
               
               
                 Chr 
                 12 
               
               
                 Cytoband 
                 q23.1 
               
               
                 Start in SEQ_ID_NO_718 
                 86595 
               
               
                 (bp) 
               
               
                 NCBI_build33Start (Mb) 
                 96.396593 
               
               
                 Size 
                 398 
               
               
                 CEPH standard 
                 398 
               
               
                 (referenCe allele) 
               
               
                 Polymorphism type 
                 MiCrosatellite 
               
               
                 Polymorphism Class 
                 Di 
               
               
                 Heterozygosity ratio 
                 0.72 
               
               
                 Forward primer 
                 GCAGTTTAAGCTGTATGTATATGAGG 
               
               
                   
                 SEQ ID NO:725) 
               
               
                 Reverse primer 
                 TGAAAGCCATCACTGTAAGGA 
               
               
                   
                 (SEQ ID NQ:726) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 39 
               
               
                   
               
               
                   
               
               
                 Haplotype association analysis including SNPs and microsatellite markers in the LTA4H 
               
               
                 gene region. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 SG12S438 
                 DG12S1664 
                 SG12S16 
                 SG12S21 
                 SG12S23 
                 SG12S25 
                 SG12S26 
                 DG12S1666 
                 SG12S100 
               
               
                   
               
            
           
           
               
            
               
                 All MI vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Consecutive 
                 C 
                 0 
                 C 
                 G 
                 T 
                 A 
                 T 
                 0 
                 T 
               
               
                 Short version 
                 C 
                 0 
                   
                   
                   
                   
                   
                   
                   
               
               
                 Protective variant 
                 C 
                   
                 C 
                   
                   
                   
                 T 
                   
                   
               
            
           
           
               
            
               
                 MI males vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Consecutive 
                 C 
                 0 
                 C 
                 G 
                 T 
                 A 
                 T 
                 0 
                 T 
               
               
                 Short version 
                 C 
                 0 
                   
                   
                   
                   
                   
                   
                   
               
               
                 Protective variant 
                 C 
                   
                 C 
                   
                   
                   
                 T 
                   
                   
               
            
           
           
               
            
               
                 MI females vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Consecutive 
                 C 
                 0 
                 C 
                 G 
                 T 
                 A 
                 T 
                 0 
                 T 
               
               
                 Short version 
                 C 
                 0 
                   
                   
                   
                   
                   
                   
                   
               
               
                 Protective variant 
                 C 
                   
                 C 
                   
                   
                   
                 T 
                   
                   
               
            
           
           
               
            
               
                 Recurrent MI vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Consecutive 
                 C 
                 0 
                 C 
                 G 
                 T 
                 A 
                 T 
                 0 
                 T 
               
               
                 Short version 
                 C 
                 0 
                   
                   
                   
                   
                   
                   
                   
               
               
                 Protective variant 
                 C 
                   
                 C 
                   
                   
                   
                 T 
                   
                   
               
            
           
           
               
            
               
                 MI plus stroke or PAOD vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Consecutive 
                 C 
                 0 
                 C 
                 G 
                 T 
                 A 
                 T 
                 0 
                 T 
               
               
                 Short version 
                 C 
                 0 
                   
                   
                   
                   
                   
                   
                   
               
               
                 Protective variant 
                 C 
                   
                 C 
                   
                   
                   
                 T 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 SG12S28 
                 SG12S143 
                 SG12S144 
                 SG12S221 
                 SG12S222 
                 SG12S223 
                 SG12S225 
                 SG12S226 
               
               
                   
                   
               
            
           
           
               
            
               
                 All MI vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Consecutive 
                 T 
                 T 
                 A 
                 G 
                 C 
                 C 
                 G 
                 G 
               
               
                 Short version 
                   
                   
                   
                   
                 C 
                   
                   
                 G 
               
               
                 Protective variant 
                   
                 T 
                   
                   
                   
                   
                   
                   
               
            
           
           
               
            
               
                 MI males vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Consecutive 
                 T 
                 T 
                 A 
                 G 
                 C 
                 C 
                 G 
                 G 
               
               
                 Short version 
                   
                   
                   
                   
                 C 
                   
                   
                 G 
               
               
                 Protective variant 
                   
                 T 
                   
                   
                   
                   
                   
                   
               
            
           
           
               
            
               
                 MI females vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Consecutive 
                 T 
                 T 
                 A 
                 G 
                 C 
                 C 
                 G 
                 G 
               
               
                 Short version 
                   
                   
                   
                   
                 C 
                   
                   
                 G 
               
               
                 Protective variant 
                   
                 T 
                   
                   
                   
                   
                   
                   
               
            
           
           
               
            
               
                 Recurrent MI vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Consecutive 
                 T 
                 T 
                 A 
                 G 
                 C 
                 C 
                 G 
                 G 
               
               
                 Short version 
                   
                   
                   
                   
                 C 
                   
                   
                 G 
               
               
                 Protective variant 
                   
                 T 
                   
                   
                   
                   
                   
                   
               
            
           
           
               
            
               
                 MI plus stroke or PAOD vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Consecutive 
                 T 
                 T 
                 A 
                 G 
                 C 
                 C 
                 G 
                 G 
               
               
                 Short version 
                   
                   
                   
                   
                 C 
                   
                   
                 G 
               
               
                 Protective variant 
                   
                 T 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 SG12S233 
                 SG12S237 
                 DG12S1668 
                 p-val 
                 P-val adj. 
                 r 
                 #aff 
                 aff.frq. 
                 #con 
                 con.frq. 
               
               
                   
                   
               
            
           
           
               
            
               
                 All MI vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Consecutive 
                 C 
                 T 
                 0 
                 6.2E−02 
                   
                 1.34 
                 1560 
                 0.051 
                 953 
                 0.039 
               
               
                 Short version 
                   
                   
                 0 
                 1.5E−03 
                   
                 1.63 
                 1556 
                 0.071 
                 951 
                 0.045 
               
               
                 Protective variant 
                   
                 C 
                   
                 7.5E−02 
                   
                 0.88 
                 1557 
                 0.290 
                 951 
                 0.317 
               
            
           
           
               
            
               
                 MI males vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Consecutive 
                 C 
                 T 
                 0 
                 2.2E−02 
                   
                 1.49 
                 1096 
                 0.051 
                 953 
                 0.035 
               
               
                 Short version 
                   
                   
                 0 
                 3.1E−03 
                   
                 1.66 
                 1093 
                 0.069 
                 951 
                 0.043 
               
               
                 Protective variant 
                   
                 C 
                   
                 6.3E−02 
                   
                 0.86 
                 1094 
                 0.283 
                 951 
                 0.314 
               
            
           
           
               
            
               
                 MI females vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Consecutive 
                 C 
                 T 
                 0 
                 4.3E−01 
                   
                 1.19 
                 464 
                 0.046 
                 953 
                 0.039 
               
               
                 Short version 
                   
                   
                 0 
                 1.6E−02 
                   
                 1.60 
                 463 
                 0.073 
                 951 
                 0.047 
               
               
                 Protective variant 
                   
                 C 
                   
                 3.1E−01 
                   
                 0.91 
                 463 
                 0.301 
                 951 
                 0.322 
               
            
           
           
               
            
               
                 Recurrent MI vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Consecutive 
                 C 
                 T 
                 0 
                 7.7E−02 
                   
                 1.52 
                 273 
                 0.060 
                 953 
                 0.040 
               
               
                 Short version 
                   
                   
                 0 
                 7.5E−02 
                   
                 1.54 
                 272 
                 0.067 
                 951 
                 0.045 
               
               
                 Protective variant 
                   
                 C 
                   
                 9.8E−02 
                   
                 0.82 
                 273 
                 0.274 
                 951 
                 0.316 
               
            
           
           
               
            
               
                 MI plus stroke or PAOD vs controls 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Consecutive 
                 C 
                 T 
                 0 
                 1.5E−03 
                 0.007 
                 1.97 
                 325 
                 0.073 
                 953 
                 0.038 
               
               
                 Short version 
                   
                   
                 0 
                 2.4E−05 
                 0.038 
                 2.39 
                 325 
                 0.099 
                 951 
                 0.044 
               
               
                 Protective variant 
                   
                 C 
                   
                 4.1E−05 
                   
                 0.61 
                 325 
                 0.220 
                 951 
                 0.315 
               
               
                   
               
               
                   P-val = p-value.    
               
               
                   P-val adj: P-value adjusted for multiple comparisons.    
               
               
                   r = Relative risk.    
               
               
                   #aff = Number of patients.    
               
               
                   #con = number of controls.    
               
               
                   Aff.frq = haplotype/allelic frequency in patients.    
               
               
                   Con.frq = haplotype/allelic frequency in controls.    
               
            
           
         
       
     
     Discussion  
      In a genome wide search for susceptibility genes for MI, a gene was mapped to 12q23. This locus was fine mapped with microsatellite markers. Haplotype analysis in a large case-control association study using markers spanning a 79 kb region across the LTA4H gene, shows that LTA4H is a significant susceptibility gene for MI.  
      The LTA4H gene encodes a protein that is required for leukotriene B4 synthesis. The leukotrienes are potent inflammatory lipid mediators derived from arachidonic acid. Given that our data shows that LTA4H shows significant association to MI, it may contribute to development of atherosclerosis in coronary arteries and/or to the destabilization of existing coronary atherosclerotic plaques through lipid oxidation and/or proinflammatory effects.  
      Dashwood and coworkers have studied expression of the enzymes that control the formation of leukotrienes in coronary arteries. They showed that cells showing positive antibody binding to 5-LO, FLAP (5-lipoxygenase activating protein), and leukotriene A4 hydrolase were present in the coronary arteries and had a similar distribution to macrophages. (Dashwood, et al.,  Circulation  1998 Jun. 23;97(24):2406-13). Thus, LTA4H and other members of the leukotriene pathway are expressed within cell types found in atherosclerotic lesions that form the basis for the final event of myocardial infarction. Their potential role in plaque instability may explain why many patients have stable angina for years without suffering a myocardial infarction (and therefore presumably have atherosclerotic lesions without the instability that leads to overriding thrombosis and MI) while others suffer MI with little or no period of stable angina. Those patients with elevated LTA4H enzymatic activity in atherosclerotic lesions may have more unstable plaques and higher MI rates. In addition, increased LTA4H activity may accelerate atherosclerosis lesion formation and progression.  
      This LTA4H data complements data presented above on the gene that encodes FLAP, which works with 5-LO to produce Leukotriene A4; that is, it is upstream of LTA4H. We found that variants in the FLAP gene more than double the risk of MI. LTA4H represents the second member of the leukotriene biosynthetic pathway that we have been the first to show confers substantially higher risk for MI.  
      Further work in animals which supports our discovery that LTA4H is a disease gene for MI comes from Aiello and coworkers. They have shown that leukotriene B4 receptor antagonism reduces monocytic foam cells in mice, suggesting that LTB4 has a role in the pathogenesis of atherosclerosis in mice. (Aiello, et al.,  Arteriosclerosis, Thrombosis and Vascular Biology.  2002;22:443.)  
      Finally, additional support of our human validation of the leukotriene pathways role in MI in general, and for LTA4H, in particular, comes from Mehrabian et al. who described the identification of 5-Lipoxygenase (5-LO) as a major gene contributing to atherosclerosis susceptibility in mice. Mehrabian et al. described that heterozygous deficiency for the enzyme in a knockout model decreased the atherosclerotic lesion size in LDL−/− mice by about 95%. Mehrabian et al show that the enzyme is expressed abundantly in macrophage-rich regions of atherosclerotic lesions, and suggested that 5-LO and/or its products might act locally to promote lesion development (Mehrabian et al.,  Circulation Research.  91:120 (2002)).  
      These results suggest that the Leukotriene B4 branch of the leukotriene pathway (as opposed to the other main end products of the leukotriene biosynthetic pathway: leukotriene C4, leukotriene D4, and leukotriene E4) may be more specifically involved in MI risk. If so, then medicants acting on this branch or blocking the effects of LTB4 may be more effective in preventing/treating MI than : those acting on the other branches of the pathway or that block the effects of LTC4, LTD4, or LTE4. However, our current data do not exclude these other branches of the leukotriene pathway; the data do suggest that at least the LTB4 side of the leukotriene pathway is important for MI. All medicaments that act on any branch of the pathway are contemplated herein for MI prophylaxis or therapy.  
      Mutations and /or polymorphisms within or near the LTA4H nucleic acid, and other members of the same pathway (i.e., leukotriene B4 receptor 1 and 2, leukotriene B4 omega-hydroxylase, leukotriene B4 12-hydroxydehydrogenase), that show association with the disease, may be used as a diagnostic test to predict those at risk for MI and ACS as well as those who might benefit from medicants directed against members of the leukotriene pathway. Therefore, there may be other members of the leukotriene pathway that may be valuable therapeutic targets for myocardial infarction in addition to LTA4H and FLAP.  
     EXAMPLE 16  
     mRNA Expression of the LTA4 Hydrolase Gene in White Blood Cells of MI Patients vs Control  
      mRNA expression was compared in white blood cells from patients with history of myocardial infarction (MI) and in age and sex matched controls without MI. The leucocyte population was separated into: 1) neutrophils and 2) peripheral blood mononuclear cells prior to RNA extraction using standardized methods as previously described (Helgadottir et al, Nature Genetics, 2004; Hakonarson et al, J Immunol, 2001).  
      RNA was isolated from PBM cells obtainted from 43 MI patients and 35 controls. RNA was separately analyzed from granulocytes from the same subjects. Sufficient amount for RNA was obtained from all PBM cell preparations, and granulocyte preparations from 35 MI patients and 29 controls. RNA was converted into cDNA using the protocol below. PCR was then run on the cDNA with the LTA4H Assay-on-Demand and Beta Actin Pre-Developed Assay Reagent from Applied Biosystems using the PCR parameters below.  
               TABLE 40                       PCR Parameters                                                    RT Reaction                       TaqMan RT Buffer   1×       MgCl2   5.5   mM       dNTP   0.5   mM per dNTP   25° C.   10′       Random Hexamers   2.5   uM   48° C.   30′       Rnase Inhibitor   0.4   U/uL   95° C.   5′       MultiScribe   1.25   U/uL       Reverse       Transcriptase       RNA   2   ng/uL           50   uL Reaction Volume       PCR Reaction       TaqMan Universal   1×       95° C.   10′       Master Mix                             TaqManAssay (20×)   1×       40 cycles:                                 cDNA   2   ng/ul (original RNA)   95° C.   15″           10   uL Reaction Volume   60° C.   60″                 All PCR reactions run in duplicates.             
 
      ABI7900 instrument was used to calculate CT (Threshold Cycle) values. Samples displaying a greater than I deltaCT between duplicates were not used in our analysis. Quantity was obtained using the formula 2ˆ-deltaCT where deltaCT represents the difference of CT values between target and housekeeping assay. mRNA expression was subsequently compared between patients and controls. To determine if there were differences between the groups, we used standardized Mann-Whitney analysis as well as Standard t tests, with p&lt;0.05 considered significant. Moreover, given our hypothesis of enhanced expression of the LTA4 hydrolase gene in patients compared to controls, we report both unpaired two-sided and unpaired one-sided t tests with Welch correction.  
               TABLE 41                          Results       Analysis                                 #   # 5% extr.   Ave Q −5% extr.                                                 PBMC                       Patients   43   2.15   1.954317191           Controls   35   1.75   1.72766267           Granulocytes           Patients   35   1.75   0.401265947           Controls   29   1.45   0.331226464                         Statistics Granulocytes MI patients vs controls           P = 0.0868           Mann-Whitney           two-sided test           P = 0.0635 Unpaired two-sided t test           P = 0.0318 Unpaired one-sided t test           P = 0.0556 Unpaired two-sided t test with Welch correction           P = 0.0278 Unpaired one-sided t test with Welch correction           Statistics PBMC Patients vs Control           P = 0.0456 Mann-Whitney two-sided test           P = 0.0591 Unpaired two-sided t test           P = 0.0296 Unpaired one-sided t test           P = 0.0656 Unpaired two-sided t test with Welch correction           P = 0.0328 Unpaired one-sided t test with Welch correction                      
 
      Relative to cells isolated from control subjects, mRNA expression of LTA4 hydrolase gene is significantly enhanced in both PBM cells and granulocytes isolated from patients with MI. These data further confirmed the role of this gene in MI.  
     EXAMPLE 17  
     SNPs in the LTA4H Gene and Haplotype Association  
      Ten SNPs in the LTA4H gene, 8 from the sequenced region and 2 public SNPs in the 5′ region of the gene, were selected for genotyping 1500 MI patients and 900 controls. The linkage disequilibrium (LD) pattern defined by the 10 genotyped SNPs is shown in  FIG. 13   a.  All haplotypes with greater than 1% allelic frequency that were found in the Icelandic cohort are shown in  FIG. 13   b.    
     Subjects from Icelans  
      The study cohort included 1496 unrelated Icelandic subjects with MI (1096 males, 400 females, and 432 with early onset MI), and 867 unrelated population controls (xx males, xx females). Early onset was defined for subjects having their MI before the age of 55 for males and 65 for females. Of the 1425 patients 345 were diagnosed with more than one atherosclerotic complication (including 157 with MI and stroke but without PAOD, 148 with MI and PAOD but without stroke, and 40 with all three diseases). The recruitment of the cohort has previously been described 2 In brief, MI patients were recruited from a registry that includes all MIs diagnosed before the age of 75 in Iceland from 1981-2002. Diagnosis of patients followed WHO-MONICA diagnostic criteria for acute MI in terms of elevations of cardiac enzymes and electrocardiographic changes 30. The stroke diagnosis was confirmed by neurologists after examining the patients and/or reviewing their medical and radiological records. The PAOD diagnosis was confirmed by angiography showing severe atherosclerosis, and the majority (85%) has undergone angioplasty and/or vascular surgery.  
      The Data Protection Commission of Iceland and the National Bioethics Committee of Iceland approved the study. Informed consent was obtained from all study participants. Personal identifiers associated with medical information and blood samples were encrypted with a third party encryption system as previously described  31 .  
     Subjects from the United States  
      Philadelphia  
      The patients and controls came from the same study cohort that was ascertained among individuals who underwent a coronary angiography in the cardiac catheterization laboratory of the University of Pennsylvania. Apart from history of MI, the diagnosis was confirmed by angiography. (Input from collaborators)  
      The patient group included 731 Caucasians, 103 African Americans, and 73 subjects of other or unknown ethnicity (including 4 Native Americans, 7 Hispanics, 9 of other (undefined) ethnicity and 51 of unknown ethnicity). The control group included 434 Caucasians, 128 African Americans, and 86 individuals of other or unknown ethnicity (including 5 Asians, 14 Hispanics, 11 of other (undefined) ethnicity, and 62 of unknown ethnicity).  
      Cleveland  
      The study group from Cleveland included 696 MI patients (553 males and 143 females) and 698 controls (314 males and 384 females). The majority of the study subjects were Caucasians and approximately 10% were African American. Information on the ethnicity of individuals was not available. To separate the study subjects into ethnic groups 8 microsatellite markers with different allele distributions in Caucasians and African Americans were genotyped on the study cohort. This procedure is described in detail elsewhere (submitted manuscript). In brief, the 8 markers were identified by genotyping approximately 2000 microsatellite markers in a group of 35 Caucasian Americans and 88 African Americans, with self-reported ethnicity, from many regions of the United States. The allelic frequencies of the microsatellite markers in this cohort were used as a reference when we evaluated the population stratification in the study cohort from Cleveland. Of the Caucasian MI patients 147 did also have a history of stroke and/or peripheral vascular disease.  
      The statistical analysis for the association study of the LTA4H gene haplotypes with MI is described above in Example 1.  
     Association Study  
      In a case-control association study, including 1500 MI patients and 900 population-based controls, 10 genotyped SNPs were tested for association to the disease. The frequency of all SNPs in the patient and control groups is shown in Table 42. One SNP or SG12S100 (rs2247570) showed nominally significant association to MI (Table 43). The frequency of allele T of this SNP was 72.4% and 69.6% in the patient and control groups respectively (P=0.043), which corresponds to a 15% increase in risk of MI for each copy of allele T carried (Table 43).  
               TABLE 42                          SNP allelic association to MI       Shown are the genotyped SNPs including public names if available (variation shown in brackets), and       the frequency of the underlined allele, in Caucasian, and African American cohorts, and in a cohort of       unknown or other ethnicity. Each column represents an individual cohort. The ethnicity of the cohort       is indicated, followed by a letter for the place from which the cohort came. The letters I identifies the       cohort from Iceland, the letter P identifies the cohort from Philadelphia and the letter C identifies the       cohorts from Cleveland. Also shown is the number (n) of subjects genotyped, the relative risk (RR)       and the P-value for the association with MI. The P-values are two sided and are obtained with a       likelihood ratio test. Public names for SNPs SG12S16 and SG12S26 are not available. The basepair       positions for these two SNPs in the human genome assembly build 34 (National Center for       Biotechnology Information (NCBI)) are 94896055 and 94916919, respectively                                                         Unknown   Afr.   Afr.       SNP name (variation)   Caucasian I   Caucasian P   Caucasian C   or other P   American P   American C               SG12S16 ( T /C)                               Frq. in patients (n)   0.232 (1521)   0.239 (547)   0.255 (606)   0.167 (60)   0.044 (80)   0.035 (57)       Frq. in controls (n)   0.233 (807)   0.239 (333)   0.228 (576)   0.143 (63)    0.06 (100)   0.045 (89)       RR/P-value       SG12S21/rs2660880 ( A /G)       Frq. in patients (n)   0.062 (1510)   0.073 (704)   0.066 (586)   0.028 (72)   0.021 (97)   0.009 (55)       Frq. in controls (n)   0.071 (829)   0.062 (422)   0.067 (568)   0.055 (82)   0.012 (124)   0.006 (87)       RR/P-value       SG12S23/rs6538697 ( T /C)       Frq. in patients (n)   0.926 (1430)   0.934 (715)   0.915 (597)   0.894 (71)   0.842 (98)   0.791 (55)       Frq. in controls (n)   0.928 (822)   0.911 (426)   0.917 (569)   0.907 (86)   0.849 (126)   0.858 (88)       RR/P-value       SG12S25/rs1978331 ( A /G)       Frq. in patients (n)   0.636 (1348)   0.603 (718)   0.582 (604)   0.575 (73)    0.38 (100)   0.373 (55)       Frq. in controls (n)   0.613 (815)   0.599 (423)   0.593 (577)   0.559 (85)   0.242 (126)   0.273 (88)       RR/P-value                   1.92/0.0016   1.58/0.077       SG12S26 ( T /C)       Frq. in patients (n)   0.831 (1442)   0.831 (699)   0.809 (606)   0.854 (72)   0.969 (96)   0.973 (55)       Frq. in controls (n)   0.827 (812)   0.813 (417)   0.828 (573)   0.894 (85)   0.955 (122)   0.961 (89)       RR/P-value       SG12S100/rs2247570       ( T /C)       Frq. in patients (n)   0.724 (1496)   0.699 (690)   0.688 (582)   0.671 (70)   0.626 (95)   0.644 (52)       Frq. in controls (n)   0.696 (805)   0.708 (409)   0.699 (568)    0.69 (84)   0.533 (123)   0.518 (85)       RR/P-value   1.15/0.043               1.47/0.049   1.69/0.039       SG12S28/rs2660898 ( T /G)       Frq. in patients (n)   0.695 (1375)   0.686 (711)   0.658 (603)   0.711 (71)   0.798 (99)   0.741 (56)       Frq. in controls (n)   0.678 (800)   0.665 (426)   0.674 (579)   0.738 (84)   0.798 (124)   0.817 (90)       RR/P-value       SG12S143/rs2540482       ( C /T)       Frq. in patients (n)   0.174 (1473)   0.263 (716)   0.247 (592)   0.368 (72)   0.285 (100)   0.286 (56)       Frq. in controls (n)   0.173 (833)   0.207 (423)   0.239 (562)   0.271 (85)   0.232 (125)   0.256 (88)       RR/P-value       1.37/0.0025       1.57/0.064       SG12S144/rs2660845       ( G /A)       Frq. in patients (n)   0.211 (1415)    0.30 (697)   0.293 (564)   0.426 (68)   0.337 (95)   0.349 (53)       Frq. in controls (n)   0.214 (807)   0.251 (417)   0.298 (552)   0.298 (84)    0.29 (124)   0.355 (83)       RR/P-value       1.28/0.012       1.75/0.02       SG12S221/rs2540475       ( G /A)       Frq. in patients (n)   0.821 (1438)   0.778 (718)    0.79 (603)   0.856 (73)   0.887 (97)   0.902 (56)       Frq. in controls (n)   0.821 (773)   0.772 (428)    0.8 (578)   0.853 (85)   0.861 (126)   0.864 (88)       RR/P-value                  
 
     
       
         
           
               
             
               
                 TABLE 43 
               
             
            
               
                   
               
               
                   
               
               
                 SNP allelic association to MI 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Frequency 
                   
                 # genotyped 
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Cohort 
                 SNPs 
                 Allele 
                 Patients 
                 Controls 
                   
                 Patients 
                 Controls 
                 RR 
                 P 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Iceland 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Caucasians 
                 SG12S100 
                 T 
                 0.724 
                 0.696 
                 # 
                 1496 
                 805 
                 1.15 
                 0.043 
               
               
                 Philadelphia 
               
               
                 Caucasians 
                 SG12S100 
                 T 
                 0.699 
                 0.708 
                 # 
                 690 
                 409 
                 0.96 
                 NS 
               
               
                   
                 SG12S143 
                 C 
                 0.263 
                 0.206 
                 # 
                 716 
                 424 
                 1.37 
                 0.002 
               
               
                   
                 SG12S144 
                 G 
                 0.30 
                 0.25 
                 # 
                 697 
                 418 
                 1.28 
                 0.011 
               
               
                   
                 SG12S23 
                 T 
                 0.934 
                 0.91 
                 # 
                 715 
                 427 
                 1.39 
                 0.04  
               
               
                 African 
                 SG12S100 
                 T 
                 0.626 
                 0.533 
                 # 
                 95 
                 123 
                 1.47 
                 0.049 
               
               
                 Americans 
                 SG12S143 
                 C 
                 0.285 
                 0.232 
                 # 
                 100 
                 125 
                 1.32 
                 NS 
               
               
                   
                 SG12S144 
                 G 
                 0.337 
                 0.29 
                 # 
                 95 
                 124 
                 1.24 
                 NS 
               
               
                   
                 SG12S23 
                 T 
                 0.842 
                 0.849 
                 # 
                 98 
                 126 
                 0.95 
                 NS 
               
               
                   
                 SG12S25 
                 A 
                 0.38 
                 0.242 
                 # 
                 100 
                 126 
                 1.92 
                 0.002 
               
               
                 Undefined 
                 SG12S100 
                 T 
                 0.671 
                 0.69 
                 # 
                 70 
                 84 
                 0.92 
                 NS 
               
               
                 ethnicity 
                 SG12S143 
                 C 
                 0.368 
                 0.262 
                 # 
                 72 
                 103 
                 1.64 
                 0.035 
               
               
                   
                 SG12S144 
                 G 
                 0.426 
                 0.291 
                 # 
                 68 
                 103 
                 1.8 
                 0.01  
               
               
                   
                 SG12S23 
                 T 
                 0.894 
                 0.907 
                 # 
                 71 
                 86 
                 0.87 
                 NS 
               
               
                   
                 SG12S25 
                 A 
                 0.575 
                 0.559 
                 # 
                 73 
                 85 
                 1.07 
                 NS 
               
               
                 Cleveland 
               
               
                 Caucasians 
                 SG12S100 
                 T 
                 0.688 
                 0.699 
                 # 
                 582 
                 568 
                 0.95 
                 NS 
               
               
                   
                 SG12S143 
                 C 
                 0.247 
                 0.239 
                 # 
                 592 
                 562 
                 1.05 
                 NS 
               
               
                   
                 SG12S144 
                 G 
                 0.293 
                 0.298 
                 # 
                 564 
                 552 
                 0.98 
                 NS 
               
               
                   
                 SG12S23 
                 T 
                 0.915 
                 0.917 
                 # 
                 597 
                 569 
                 0.99 
                 NS 
               
               
                   
                 SG12S25 
                 A 
                 0.582 
                 0.593 
                 # 
                 604 
                 577 
                 0.96 
                 NS 
               
               
                 African 
                 SG12S100 
                 T 
                 0.644 
                 0.518 
                 # 
                 52 
                 85 
                 1.69 
                 0.039 
               
               
                 Americans 
                 SG12S143 
                 C 
                 0.292 
                 0.246 
                 # 
                 53 
                 81 
                 1.26 
                 NS 
               
               
                   
                 SG12S144 
                 G 
                 0.349 
                 0.355 
                 # 
                 53 
                 83 
                 0.97 
                 NS 
               
               
                   
                 SG12S23 
                 T 
                 0.791 
                 0.858 
                 # 
                 55 
                 88 
                 0.63 
                 NS 
               
               
                   
                 SG12S25 
                 A 
                 0.373 
                 0.273 
                 # 
                 55 
                 88 
                 1.58 
                 0.077 
               
               
                   
               
            
           
         
       
     
      All haplotypes defined by the genotyped SNPs were tested for association to MI. As shown in Table 44 no haplotype is significantly over-represented in all MI patients. Since males with MI showed suggestive linkage to the chromosome 12q22-23 region, this phenotype was also tested for association. In addition we tested haplotype association to more severe MI phenotypes, or early onset MI, and MI with another clinical manifestation of atherosclerosis (PAOD and/or stroke). The phenotypes MI in males or early onset disease did not show significant association to any of the tested haplotypes. However, as shown in Table 45, MI with additional atherosclerotic manifestation showed an association to a haplotype that we call HapK. The allelic frequency of HapK in patients with additional atherosclerotic manifestation and in controls is 14.5% and 10.4%, respectively, which corresponds to a relative risk (RR) of 1.45 (P=0.0091), for each copy of HapK carried.  
      Since proper interpretation of the significance of the association of the SNP SG12S100 and haplotype HapK to MI has to take multiple comparisons into account, the P-values were adjusted for the number of tests made, corresponding to the number of haplotypes tested, by randomizing the patients and controls. The significance of the association of SNP SG12S100 to MI did not survive this adjustment at 0.05 level. However, the association of HapK to MI with additional atherosclerotic complication could still be considered significant with adjusted P-value 0.035. However, since the adjusted P-value still does not take the multiple phenotypes tested into account we consider this haplotype only marginally significant with the Icelandic data only. Hence, this candidate at-risk variant called for confirmation in an independent cohort.  
               TABLES 44                       Shown are all observed haplotypes with frequency greater than 1% in the population       controls for each indicated cohort, and the allelic frequency       in patients and controls. Also shown is the relative risk (RR)       and the P-value for the association to MI.                  44a. Iceland-Caucasians (1555patients/863controls)Patients = All MI                                                 SG12S16   SG12S21   SG12S23   SG12S25   SG12S26   SG12S100   SG12S28                 C       G       T       A       T       T       T           C       G       T       A       T       T       T         T   G   T   G   C   C   G       C   G   C   G   T   T   G       T   G   T   G   C   C   G       T   A   T   G   T   C   G       C   G   T   G   T   C   T       T   G   T   G   C   C   G       C   G   T   A   T   T   T       C   G   T   G   T   C   T       C   A   T   G   T   C   G       C   G   C   G   T   T   G                                     Frequency                                                 SG12S143   SG12S144   SG12S221   P   RR   Patients   Controls   Name                 T       A       G     2.7E−01   1.07   0.497   0.480   HapL         C       G       G     3.8E−01   1.09   0.112   0.104   HapK       T   A   A   2.3E−01   1.15   0.090   0.079       T   A   G   5.8E−01   1.07   0.064   0.060       C   G   G   9.2E−01   0.99   0.058   0.059       T   A   A   4.7E−01   0.90   0.048   0.053       T   G   G   2.9E−02   0.70   0.031   0.044       T   A   G   1.0E−01   0.71   0.022   0.031       T   A   A   7.4E−01   0.92   0.018   0.019       T   A   G   1.1E−01   0.61   0.008   0.013   HapQ       T   A   A   9.1E−02   0.57   0.006   0.011       T   A   A   7.0E−01   1.13   0.012   0.011                         44b. Iceland-Caucasians (325patients/863controls) Patients = MI with additional CVD                                                 SG12S16   SG12S21   SG12S23   SG12S25   SG12S26   SG12S100   SG12S28                 C       G       T       A       T       T       T           C       G       T       A       T       T       T         T   G   T   G   C   C   G       C   G   C   G   T   T   G       T   G   T   G   C   C   G       T   A   T   G   T   C   G       C   G   T   G   T   C   T       T   G   T   G   C   C   G       C   G   T   A   T   T   T       C   G   T   G   T   C   T       C   A   T   G   T   C   G       C   G   C   G   T   T   G                                     Frequency                                                 SG12S143   SG12S144   SG12S221   P   RR   Patients   Controls   Nameo                 T       A       G     8.7E−01   0.98   0.477   0.480   HapL         C       G       G     9.1E−03   1.45   0.145   0.104   HapK       T   A   A   5.5E−01   1.11   0.086   0.079       T   A   G   8.6E−01   0.97   0.058   0.060       C   G   G   5.4E−01   0.88   0.051   0.059       T   A   A   8.9E−01   0.97   0.051   0.053       T   G   G   8.8E−03   0.48   0.021   0.044       T   A   G   9.6E−01   0.99   0.032   0.031       T   A   A   1.4E−01   0.51   0.010   0.019       T   A   G   4.1E−01   0.67   0.009   0.013   HapQ       T   A   A   2.6E−01   0.54   0.006   0.011       T   A   A   9.5E−01   0.97   0.011   0.011                         44c. Philadelphia-Caucasians (731 patients/434controls)                                                 SG12S16   SG12S21   SG12S23   SG12S25   SG12S26   SG12S100   SG12S28                 C       G       T       A       T       T       T           C       G       T       A       T       T       T         T   G   T   G   C   C   G       C   G   C   G   T   T   G       T   G   T   G   C   C   G       T   A   T   G   T   C   G       C   G   T   A   T   T   T       C   G   C   G   T   T   G       C   G   T   G   T   C   T       T   G   T   G   C   C   G       C   G   T   G   T   C   T       C   G   T   G   T   T   T       C   G   T   G   T   T   T                                     Frequency                                                 SG12S143   SG12S144   SG12S221   P   RR   Patients   Controls   Name                 T       A       G     4.5E−02   0.83   0.363   0.406   HapL         C       G       G     1.5E−02   1.34   0.185   0.145   HapK       T   A   A   3.7E−01   0.88   0.098   0.110       T   A   G   8.9E−02   0.72   0.048   0.065       C   G   G   7.5E−01   0.94   0.048   0.051       T   A   A   3.3E−01   1.21   0.060   0.050       T   A   A   5.9E−01   1.17   0.030   0.026       T   A   A   1.3E−01   0.59   0.013   0.022       T   G   G   7.6E−01   1.10   0.023   0.021       T   A   G   8.8E−01   0.94   0.018   0.019       T   A   G   8.3E−02   1.75   0.027   0.016   HapQ       T   A   G   1.2E−01   0.47   0.007   0.014       T   G   G   3.7E−01   1.41   0.017   0.012                         44d. Cleveland-Caucasians (614patients/583controls)                                                 SG12S16   SG12S21   SG12S23   SG12S25   SG12S26   SG12S100   SG12S28                 C       G       T       A       T       T       T           C       G       T       A       T       T       T         T   G   T   G   C   C   G       C   G   C   G   T   T   G       T   G   T   G   C   C   G       T   A   T   G   T   C   G       C   G   T   G   T   C   T       C   G   T   G   T   C   T       C   G   T   A   T   T   T       C   G   C   G   T   T   G       T   G   T   G   C   C   G       C   G   T   G   T   T   T       C   G   T   A   T   T   T                                     Frequency                                                 SG12S143   SG12S144   SG12S221   P   RR   Patients   Controls   Name                 T       A       G     7.4E−01   0.97   0.380   0.386   HapL         C       G       G     8.8E−01   1.02   0.163   0.160   HapK       T   A   A   4.2E−01   1.12   0.100   0.091       T   A   G   7.9E−01   1.05   0.064   0.061       C   G   G   8.7E−01   0.97   0.057   0.058       T   A   A   7.8E−01   1.05   0.053   0.050       T   G   G   6.7E−01   0.90   0.030   0.033       T   A   G   9.8E−01   1.01   0.023   0.023   HapQ       T   A   A   9.1E−01   0.97   0.021   0.022       T   A   A   6.7E−01   0.86   0.016   0.019       T   A   G   6.5E−01   1.17   0.021   0.018       T   G   G   8.2E−01   0.92   0.013   0.014       C   A   G   3.9E−01   0.64   0.007   0.010                         44e. Philadelphia-Other or unknown ethnicity (73patients/86controls)                                                 SG12S16   SG12S21   SG12S23   SG12S25   SG12S26   SG12S100   SG12S28                 C       G       T       A       T       T       T           C       G       T       A       T       T       T         C   G   T   G   T   C   T       T   A   T   G   T   C   G       T   G   T   G   C   C   G       C   G   C   G   T   T   G       C   G   T   G   T   T   T       C   G   T   G   T   C   T       T   G   T   G   C   C   G       C   G   C   G   T   C   G       T   G   T   G   C   C   G       C   G   C   G   T   T   G       C   G   T   G   T   T   T       T   G   T   G   T   T   T       T   G   T   G   C   C   G                                     Frequency                                                 SG12S143   SG12S144   SG12S221   P   RR   Patients   Controls   Name                 T       A       G     7.7E−02   0.64   0.274   0.370   HapL         C       G       G     1.1E−02   2.04   0.283   0.162   HapK       T   A   G   3.2E−01   0.59   0.045   0.074   HapQ       T   A   A   2.5E−01   0.51   0.027   0.052       T   A   A   6.7E−01   1.24   0.059   0.048       T   A   G   9.0E−01   0.93   0.041   0.044       T   A   G   5.7E−01   0.69   0.027   0.039       T   G   G   1.8E−01   2.22   0.058   0.027       T   A   G   8.7E−01   0.84   0.021   0.025       C   G   G   9.4E−01   0.94   0.020   0.021       C   G   G   5.7E−02   3.88   0.063   0.017       T   A   A   1.8E−01   2.65   0.041   0.016       T   G   G   9.1E−02   0.00   0.000   0.014       C   G   G   1.2E−01   0.00   0.000   0.012       C   G   A   2.6E−01   0.00   0.000   0.010                         42f. Philadelphia-African Americans (103patients/128controls)                                                 SG12S16   SG12S21   SG12S23   SG12S25   SG12S26   SG12S100   SG12S28               C   G   T   G   T   C   T       C   G   T   G   T   T   T         C       G       T       A       T       T       T         C   G   C   G   T   T   G       C   G   T   G   T   C   T       C   G   T   G   T   C   T       C   G   C   G   T   C   G       C   G   T   A   T   C   T       C   G   T   G   T   C   T       C   G   C   G   T   C   G       C   G   C   G   T   T   G       C   G   T   A   T   T   T         C       G       T       A       T       T       T         C   G   T   G   T   T   T       T   G   T   G   C   C   G       C   G   T   A   T   T   T       T   G   T   G   C   C   G       C   G   T   G   T   T   T       T   G   T   G   C   C   G                                     Frequency                                                 SG12S143   SG12S144   SG12S221   P   RR   Patients   Controls   Name               T   A   G   3.4E−02   0.53   0.119   0.202   HapQ       T   A   G   4.8E−02   0.55   0.110   0.183         T       A       G     2.5E−02   1.77   0.235   0.148   HapL       T   A   G   4.7E−01   1.38   0.073   0.054       T   G   G   4.8E−01   1.34   0.070   0.054       C   G   G   1.9E−01   0.29   0.013   0.042       C   G   G   5.3E−01   0.65   0.026   0.039       C   G   A   5.4E−01   0.70   0.026   0.036       T   A   A   6.1E−02   0.18   0.006   0.033       T   A   G   3.6E−01   0.48   0.014   0.029       C   G   G   3.9E−01   0.48   0.014   0.029       T   A   A   9.0E−01   0.90   0.020   0.022         C       G       G     4.9E−04   5.48   0.105   0.021   HapK       C   G   G   5.9E−01   1.46   0.028   0.019       T   A   G   7.0E−01   0.70   0.012   0.012       C   G   A   7.6E−01   1.42   0.018   0.018       C   G   G   7.7E−01   0.74   0.009   0.009       T   G   G   3.0E−01   0.00   0.000   0.000       T   A   A   5.1E−01   0.17   0.002   0.002                         42g. Cleveland-African Americans (57patients/90controls)                                                 SG12S16   SG12S21   SG12S23   SG12S25   SG12S26   SG12S100   SG12S28               C   G   T   G   T   C   T         C       G       T       A       T       T       T         C   G   T   G   T   T   T       C   G   T   G   T   C   T       C   G   T   G   T   T   T         C       G       T       A       T       T       T         C   G   C   G   T   T   G       C   G   C   G   T   C   G       C   G   T   G   T   C   T       C   G   T   A   T   T   T       T   G   T   G   C   C   G       C   G   T   G   T   T   T       C   G   C   G   T   C   G       C   G   T   A   T   C   T       C   G   T   A   T   T   T       C   G   T   G   T   C   T                                     Frequency                                                 SG12S143   SG12S144   SG12S221   P   RR   Patients   Controls   Name               T   A   G   7.3E−02   0.51   0.116   0.206   HapQ         T       A       G     2.5E−01   1.51   0.196   0.140   HapL       T   A   G   8.3E−01   0.91   0.096   0.105       T   G   G   5.6E−02   0.33   0.031   0.087       C   G   G   4.4E−01   0.58   0.038   0.064         C       G       G     2.1E−02   2.89   0.153   0.059   HapK       T   A   G   1.9E−01   1.97   0.101   0.054       T   A   G   2.6E−01   1.92   0.083   0.045       C   G   G   8.8E−01   0.88   0.037   0.041       C   G   A   2.1E−02   0.00   0.000   0.041       T   A   A   4.0E−01   0.52   0.018   0.033       T   A   A   1.5E−01   0.00   0.000   0.014       C   G   G   4.9E−01   2.02   0.029   0.014       C   G   A   1.9E−01   0.00   0.000   0.013       T   A   A   5.1E−01   2.35   0.023   0.010       T   A   A   3.9E−01   3.26   0.031   0.010                  
 
     
       
         
           
               
             
               
                 TABLE 45 
               
             
            
               
                   
               
               
                   
               
               
                 The association of HapK to MI 
               
            
           
           
               
               
               
            
               
                   
                 Frequency 
                   
               
            
           
           
               
               
               
               
               
            
               
                 Cohorts (n) 
                 Patients 
                 Controls 
                 RR 
                 P-value 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Iceland 
                   
                   
                   
                   
               
               
                 Caucasians, all MI 
                 0.112 
                 0.104 
                 1.09 
                 NS 
               
               
                 (1555/863) 
               
               
                 Early onset (725/863) 
                 0.114 
                 0.104 
                 1.10 
                 NS 
               
               
                 MI and additional CVD 
                 0.145 
                 0.104 
                 1.45 
                 0.0091 
               
               
                 (325/863) 
               
               
                 Philadelphia 
               
               
                 Caucasians, all MI 
                 0.185 
                 0.145 
                 1.34 
                 0.015 
               
               
                 (731/434) 
               
               
                 African Americans, all 
                 0.105 
                 0.021 
                 5.28 
                 0.00049 
               
               
                 MI (103/128) 
               
               
                 Other or undefined 
                 0.283 
                 0.162 
                 2.04 
                 0.011 
               
               
                 ethnicity, all MI (73/86) 
               
               
                 Cleveland 
               
               
                 Caucasians, all MI 
                 0.163 
                 0.160 
                 1.02 
                 NS 
               
               
                 (614/583) 
               
               
                 MI and additional CVD 
                 0.183 
                 0.160 
                 1.20 
                 NS 
               
               
                 (147/601) 
               
               
                 African Americans, all 
                 0.153 
                 0.059 
                 2.89 
                 0.021 
               
               
                 MI (57/90) 
               
               
                   
               
            
           
         
       
     
     EXAMPLE 18  
     A Replication Study of SNPs in LTA4H Gene in US Cohorts  
      In a replication study, the putative association of HapK to MI was assessed in independent study cohorts from Philadelphia and Cleveland. The cohort from Philadelphia included 901 subjects who had suffered a MI and 738 controls, who had undergone coronary angiography and had no evidence of coronary artery disease. The ethnicity of the individuals from Philadelphia was self-reported. The cohort from Cleveland included 696 MI patients and 698 controls.  
      The 10 SNPs, genotyped in the Icelandic cohort, were typed in the US cohorts and in addition to testing haplotype association, association to single SNPs in each ethnic group was tested separately. Table 42 (above) shows the frequency of all genotyped SNPs in the US and Icelandic cohorts. The frequency of the SNPs is quite similar in the three Caucasian groups on one hand and in the two African American cohorts on the other hand. However, as expected the frequency of many of the SNPs differs between the two ethnic groups. As shown in Table 43 (above)the nominally significant association of SNP SG12S100 to MI in the Icelandic cohort does not hold up in the US Caucasians. However, the association of this SNP to MI is replicated in both African American groups. The frequency of allele T is 62.6% and 53.3% in African American patients and controls from Philadelphia respectively (P=0.049), which corresponds to a RR of 1.47 for each T allele. The corresponding frequency in African Americans from Cleveland is 64.4% and 51.8% (P=0.039) and the RR of MI for carriers of each T allele is 1.69.  
      Furthermore, another SNP, or SG12S25 (rs1978331), associates with MI in African Americans from Philadelphia; the frequency of allele A is 38.0% and 24.2% in patients and controls respectively (P=0.0016). Similar frequencies of this SNP were observed in the African American cohort from Cleveland, or 37.3% and 27.3% in patients and controls respectively (P-value 0.077). As shown in Table 42 (above), three SNPs, SG12S143 (rs2540482), SG12S144 (rs2660845), and SG12S23 (rs6538697) show single marker association to MI in Caucasians from Philadelphia, with respective P-values 0.0023, 0.011 and 0.04. The association of SG12S144 to MI was replicated in the cohort from Philadelphia that included subjects of other or unknown ethnicity, with P-value 0.02. Furthermore, we observed that the at-risk allele (C) of SG12S 143 is over-represented in the patients of unknown or other ethnicity and in both African American cohorts, and the at-risk allele (G) of SG12S144 in African Americans from Philadelphia, although the association was not significant in these cohorts. No significant association between SNPs and MI was observed in Caucasians from Cleveland (Table 43). However, we observed that allele C of SG12S143 is over-represented in the subgroup of MI patients that also had history of other atherosclerotic manifestations, including stroke or peripheral vascular disease; the frequency in this patient group is 29.2% compared to a 23.9% frequency in the control group (P=0.06). In addition, the frequency of allele G of SG12S144 in MI patients with additional atherosclerotic manifestations is 34.8% compared to a 29.8% frequency in the control group (P=0.09).  
      Table 44 (above) shows the frequency of all observed haplotypes in the US and Icelandic cohorts. In addition, LD maps for US Caucasians and African Americans are shown in a Table 42.  
      Table 45 shows haplotype association results for the Icelandic at-risk haplotype HapK in the US cohorts. As shown in Table 45, although the haplotype frequency of HapK differs substantially between the ethnic groups, HapK associates with MI in all groups except in Caucasians from Cleveland. The haplotype frequency of HapK in the Caucasian patient and control groups from Philadelphia is 18.5% and 14.5%, respectively (P=0.015), which corresponds to a 1.34 RR of MI for each copy of HapK carried. While the haplotype frequency of HapK was less in patients and controls of African American origin from Philadelphia, or 10.5% versus 2.1% respectively, the RR of MI for carriers of HapK in this cohort is more than 5 (P=0.00049) (Table 45). The haplotype frequency of HapK in the subjects from Philadelphia with other or unknown ethnicity, was 28.3% and 16.2% in the patients and controls respectively (P=0.011), which corresponds to a 2-fold increase in risk of MI for each copy of HapK carried. Furthermore, HapK significantly associated with MI in the small African American cohort from Cleveland, conferring almost 3-fold increase in risk of MI for each copy of HapK carried (P=0.021). Association of HapK to MI in the Caucasian cohort from Cleveland was not observed (Table 45). However, we observed that the frequency of HapK in the subgroup of MI patients that also had history of other atherosclerotic manifestations is greater than in the total MI group (Table 45).  
     Haplotype Diversity and Association in African Americans  
      The haplotype diversity in the LTA4H region in the African American cohorts is quite different from that in the Caucasian cohorts (Table 44). For example, the allelic frequency of the most common haplotype (HapL) in Caucasians is ˜40% and 48% in the US and Icelandic cohorts respectively. In contrast, the frequency of HapL in the African American controls is only 14-15%. In addition, the allelic frequency of the most common haplotype in African Americans, that we call HapQ, is 20%, but the frequency of HapQ in the Caucasian cohorts is ten times less. The haplotype diversity in the cohort including individuals of other or unknown ethnicity appears to be much closer to the diversity in Caucasians (Table 44).  
      Given the distinct haplotype diversity of African Americans, additional haplotypes for association to MI in this ethnic group were tested. Table 46 shows two haplotypes that in addition to HapK show association to MI in the African American cohorts. As shown in Table 46, the allelic frequency of HapL in African Americans from Philadelphia is 23.5% and 14.8% in patients and controls respectively (P=0.025) which corresponds to a RR of 1.77. Similar frequency of HapL was observed in the African American cohort from Cleveland or 19.6% and 14.0% in patients and controls respectively, with RR 1.51, but the sample size is small and the P-value was not significant in this cohort.  
      Furthermore we found a nominally significant association of HapQ, the most common haplotype in our African American cohorts, to MI. As shown in Table 46, the frequency of HapQ in the African Americans from Philadelphia was less in the patients than in controls, or 11.9% and 20.2% respectively (P=0.034). We further observed that the frequency of HapQ in the African American cohort from Cleveland was again similar to that in Philadelphia, or 11.6% and 20.6% in patients and controls respectively.  
      We repeated the association tests for HapL and HapQ in the African American cohorts from Philadelphia and Cleveland combined. In this combined group the RR of MI for HapL was 1.62 with P=0.019 and for HapQ the RR was 0.51 and P=0.0047 (Table 46).  
      In apparent contrast, the frequency of HapL in Caucasians from Philadelphia, is less in patients than in controls, or 36.3% and 40.6% respectively (P=0.045) (Table 44). Significant under-representation of HapL in patients is not observed in any other cohorts tested. HapQ, which is relatively rare (˜1-2% allelic frequency) in the Caucasian cohorts, did not show significant association to MI in these ethnic groups (Table 44).  
               TABLE 46                          Haplotypes associated with MI in African Americans                             Frequency                                         Cohorts (n)   Haplotype   Patients   Controls   RR   P                                             Philadelphia                           African Americans   HapL   0.235   0.148   1.77   0.025       (103/128)   HapQ   0.119   0.202   0.53   0.034       Cleveland       African Americans   HapL   0.196   0.14   1.51   NS       (57/90)   HapQ   0.116   0.206   0.51   NS       African American   HapL           1.62   0.019       cohorts combined   HapQ           0.51   0.0047       (160/213)                  
 
     EXAMPLE 19  
     Correlation Between LTB4 Production and HapK  
      To determine whether the MI-associated haplotype HapK correlates with LTB4 production we measured LTB4 from peripheral blood neutrophils isolated from 41 patients with a previous history of MI and 35 controls, whose medical history was unknown. A study including the same sample set, which showed that stimulated neutrophils from patients with a past history of MI produced more LTB4 than those of controls, is described above. To obtain the HapK-carrier status of these individuals, the SNPs defining HapK on DNA samples from the same subjects were genotyped. Seven patients and seven controls carried HapK. The ionomycin stimulated cells from individuals (both patients and controls) carrying the MI-associated haplotype, HapK, produced significantly more LTB4 than those from non-carriers. The P-values for the correlation with HapK are 0.01 and 0.008 for the LTB4 production after stimulation for 15 and 30 minutes respectively. Age and gender of the individuals does not correlate with the LTB4 production.