# Patent

## Title
Methods of diagnosing susceptibility to myocardial infarction and screening for an LTA4H haplotype

## Abstract

Polymorphisms in the FLAP and LTA4H gene are shown by genetic association analysis to be susceptibility markers for myocardial infarction (MI) and ACS, as well as stroke and PAOD. Pathway targeting for treatment and diagnostic applications in identifying those who are at risk of developing MI, ACS, stroke or PAOD, in particular are described. The invention also provides methods of prophylaxis therapy for MI in human subjects having a race including black African ancestry by administering to the subject a composition comprising a therapeutically effective amount of MI therapeutic agent that inhibits leukotriene synthesis in vivo. The invention also provides for compositions comprising a leukotriene synthesis inhibitor and a statin and methods of using these compositions to reduce C-reactive protein in a human subject at risk of MI, ACS, stroke and/or PAOD.

## Background
This application is a continuation-in-part of U.S. patent application Ser. No. 11/096,191, filed Mar. 30, 2005 (now abandoned), which is incorporated herein by reference in its entirety.
The file copy of the sequence listing is submitted on a Compact-Disc Read Only Memory (CD-ROM). Three copies of the CD-ROM are submitted (denoted as Copy 1, Copy 2 and CRF) and the content of the 3 submitted CD-ROMs are identical . The sequence listing is saved on each CD-ROM as an ASCII text file named 40807.txt (571 kB), which was created on Nov. 8, 2005. The contents of each CD-ROM are incorporated herein by reference in its entirety.


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 >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.
Cardiovascular disease is one of the primary causes of death among African Americans and over 4 in every 10 non-Hispanic black adults have CVD, including myocardial infarction, stroke and congestive heart failure (American Heart Association, 2005 statistics). Race-based therapeutics are emerging to treat populations of human patients that are at a high risk based on their race. For example, BiDil (isosorbide dinitrate hydralazine hydrochloride) is currently approved for treatment of heart failure in self-identified black patients to improve survival, to prolong time to hospitalization for heart failure, and to improve patient-reported functional status. In addition, U.S. Pat. No. 6,465,463 purports to describe methods of reducing mortality associated with heart failure in black patients comprising administering to these patients hydralazine compounds in combination with isosorbide dinitrate or isosorbide mononitrate.
Although self-reporting is an effective way to identify race, ethnic ancestry and other family and medical history information that may be relevant to medical diagnosis and treatment, it is imperfect. A need exists for improved tools, especially genetic tools, to more reliably correlate patients with medical risks and susceptibilities. This may especially be true in the United States and other countries that are apt to have people of mixed ancestry.
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'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'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 Abl receptor tyrosine kinases indicates that Gleevec (STI571), a specific inhibitor of the Bcr-Abl kinase should be used for treatment of the cancer. For CML patients with such a genetic alteration, inhibition of the Bcr-Abl 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'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 Engld. 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 4th and 5th quintiles of CRP (relative risk of 1.7× and 1.9×, 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 (Dwyer et al. New England Journal of Medicine, 350: 29-37, 2004). 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.
One aspect of the invention includes materials and methods for identifying or diagnosing a person or screening for increased risk (susceptibility) to MI or other conditions based on FLAP and/or LTA4H markers that can be assayed from their DNA or RNA. For example, in some embodiments, the invention is a method of screening a human subject for susceptibility or elevated risk for MI or other adverse cardiovascular events by assaying nucleic acid from the subject for one or more polymorphisms, markers, haplotypes, alleles or genotypes, and assaying or predicting the presence or absence of the susceptibility or elevated risk from the presence or absence or identity of the polymorphisms, markers, haplotypes, etc. that are assayed. Numerous FLAP and LTA4H embodiments are described below in greater detail.
The invention also 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 (optionally) comprise monitoring myeloperoxidase level before and during the prophylaxis treatment, 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 or alternative inflammatory marker, such as C-reactive protein, in the human subject before and during the prophylaxis therapy.
In some 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 other preferred embodiments, the agent alters activity and/or nucleic acid expression of LTA4H. Preferred agents include those set forth in the Agent Table II 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 LTA4 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 B4 production by activated neutrophils, a 13% reduction of myeloperoxidase in whole blood, and a 27% increase in urinary leukotriene E4. 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 includes compositions comprising a leukotriene synthesis inhbitor and a statin. The invention also includes 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 comprise 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-((1,1dimethylethyl)thio)-alpha,alpha-dimethyl-5-(2quinolinylmethoxy)-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





Target
Compound 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
Penning, T. D. et. al. Bioorg Med.




acid
Chem. Letters, (2002), 12, 3383-3386


LTA4H
SC-56938
Ethyl-1-[2-[4-
Penning, T. D. et. al. Bioorg Med.


Inhibitor

(phenylmethyl)phenoxy]ethyl]-
Chem. Letters (2003), 13, 1137-1139.




4-piperidine-
Penning, T. D. et. al. Bioorg Med.




carboxylate
Chem. Letters, (2002), 12, 3383-3386.





U.S. Pat. No. 6,506,87 6A1


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



LTA4H
SA-9499/
(R)-3-(4-



Inhibitor
SA-6541
Dimethylamino-





benzylsulfanyl)-2-((R)-





3-mercapto-2-methyl-





propionylamino)-





propionic acid



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-





oxopentyl]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., U.S. Pat. No. 6,040,147, 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. Reductions in CRP can also be measured relative to quartile or quintile distribution of CRP in the normal population (i.e., from 4th quartile to 3rd quartile or from 3rd quartile to 2nd quartile). For example, upper quartiles, having serum CRP greater than about 2.0 mg/L, defines a population to target for treatment according to the invention to reduce CRP an/or redule risk of MI or other adverse events.
The invention also includes 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 4th quartile to 3rd or from 3rd to 2nd) 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.
Soluble CD40 ligand (sCD40L) is a marker of inflammation as well as a marker for pro-thrombotic and pro-atherogenic processes. Elevated levels of sCD40L are associated with increased cardiovascular risk, including a heightened risk of death in patients with acute coronary syndromes (Varo et al. Circulation 108:1049-1052, 2003 and Schonbeck et al. Circ Res 89:1092-1103, 2001). Patients having an increased risk of ACS, such as MI, have levels of sCD40L that are greater than the 75th percentile, particularly patients in the 90-99th percentile are at a great risk. In addition, patients having an increased risk of ACS, including MI, have levels of sCD40L that are in the third or forth quartile.
The invention also contemplates methods of reducing serum sCD40L levels and method of monitoring serum sCD40L levels. Reductions in sCD40L can be measured on a concentration basis, where compositions and methods that reduce sCD40L levels relative to the quartile distribution of sCD40L in the normal population (i.e., from 4th quartile to 3rd or from 3rd to 2nd) are preferred. Reductions in sCD40L also can be measured on a percentage basis, where clinical effectiveness is evaluated as a percentage reduction in sCD40L in a patient compared to no drug therapy or compared to single drug therapy. Depending on the initial sCD40L measurement, compositions and methods that reduce sCD40L 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. Adminsitration of leukotriene synthesis inhibitors, such as the molecules described herein, is specificially contemplated in amounts effective to achieve the desired reduction in sCD40 lignad. Repeated measurements to monitor and maintain the sCD40L reduction and adjust dosing is specifically contemplated.
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 described 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 amount 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 21; 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 include 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, sCD40L 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 further 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.
In another embodiment, the invention provides methods of screening a human subject for susceptibility to MI comprising analyzing nucleic acid of a human subject for the presence or absence of the LTA4H haplotype comprised of markers: DG12S1664, SG12S16, SG12S17, SG12S18, SG12S21, SG12S22, SG12S23, SG12S24, SG12S25, SG12S26, DG12S1666, SG12S100, 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 DG12S 1664, 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.
Table 42 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 42 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.
Particular LTA4H haplotypes, HapK, HapL and HapQ, are set out in Table 47. The invention includes methods of screening a human subject for susceptibility to MI comprising analyzing nucleic acid of a human subject for the presence or absence of the HapK haplotype comprised of the following markers: SG12S16, SG12S21, SG12S23, SG12S25, SG12S26, SG12S100, SG12S28, SG12S143, SG12S144, SG12S221 with alleles C, G, T, A, T, T, T, C, G and G, respectively. The invention also provides methods of screening a human subject for susceptibility to MI comprising analyzing nucleic acid of a human subject for the presence or absence of the HapL haplotype comprised of the following markers: SG12S16, SG12S21, SG12S23, SG12S25, SG12S26, SG12S100, SG12S28, SG12S143, SG12S144, SG12S221 with alleles C, G, T, A, T, T, T, T, A and G, respectively. The invention also provides methods of screening a human subject for susceptibility to MI comprising analyzing nucleic acid of a human subject for the presence or absence of the HapQ haplotype comprised of the following markers SG12S16, SG12S21, SG12S23, SG12S25, SG12S26, SG12S100, SG12S28, SG12S143, SG12S144, SG12S221 with alleles C, G, T, G, T, C, T, T, A and G, respectively.
As described herein, smaller subsets of markers, or even single individual markers, may be used as a surrogate for screening for a predictive haplotype because the subsets/markers with predictive values include marker SG12S551 having allele T or SG12S540 having allele A or marker rs250477 having allele C and marker rs2660896 having allele T or marker SG12S540 (re2540500) having allele A and marker rs107353540 having allele A.
For example, the invention provides for methods of assessing a susceptibility to myocardial infarction (MI) or acute coronary syndrome (ACS) in a human individual, comprising screening nucleic acid of the individual for at least one polymorphism in a LTA4H nucleic acid that correlates with increased occurrence of myocardial infarction or ACS in a human population, wherein the presence of the at least one polymorphism identifies the individual as having elevated susceptibility to MI or ACS, and wherein the absence of the at least one polymorphism in the nucleic acid identifies the individual as not having the elevated susceptibility. Such polymorphisms include allele T at marker SG12S551 or allele A at marker SG12S540 or allele C at marker rs250477 and allele T at marker rs2660896 or allele A at marker SG12S540 (re2540500) and allele A at marker rs107353540
The study described in Example 21 demonstrates that the frequency of the LTA4H haplotype, HapK, is rare in black African American subjects but confers a surprisingly great relative risk for MI. The invention also provides for methods of prophylaxis therapy for myocardial infarction (MI) in a human comprising selecting a human subject having a race that includes black African ancestry, and administering to the subject a composition comprising a therapeutically effective amount of an MI therapeutic agent that inhibits leukotriene synthesis in vivo.
Human subjects having a race that includes black African ancestry may be persons of African descent or lineage. Black African ancestry may be determined by self reporting as African-Americans, Afro-Americans, Black Americans, being a member of the black race or being a member of the negro race. For example, African Americans or Black Americans are those persons living in North America and having origins in any of the black racial groups of Africa. For example, self-reported persons of black African ancestry may have at least one parent of black African ancestry or at least one grandparent of black African ancestry.
Human subjects having a race that includes black African ancestry may also be determined by genetic analysis. Genetic analysis of ancestry may be carried out using unlinked microsatellite markers such as those set out in Smith et al. Am J Hum Genet 74, 1001-13 (2004). Particularly, African ancestry may be determined using the unlinked microsatellite markers set out in Table 54 herein and described in Example 21. A person's alleles for each tested marker are identified and using standard computational techniques, compared to the allelic frequencies of each marker in different races. The use of increasing numbers of these markers generally provides an increasing arsenal of information to assess probable racial origin/mixture. For example, the selecting of human subjects of black African ancestry comprises analyzing nucleic acid from the human subject to determine a racial genotype comprised of at least 8 microsatellite markers set forth in Table 54, and selecting for therapy a subject with a genotype that correlates with the race. Preferably, the racial genotype is comprised of alleles of the markers D1S466, D3S1583, D3S4011, D4S3014, D6S1037, D9S1777, D20S113 and D22S1172, that correlate with African and European ancestry.
In some variations of the invention, the methods comprise selecting a human subject having both a race that includes black African ancestry and at least one additional genetic, family or medical history risk factor of cardiovascular disease. In some variations, the additional family or medical history risk factor may be a race that includes European ancestry, whereby the subject has mixed African and European ancestry. An exemplary subject having a race including a mixed African and European ancestry may have at least one parent or grandparent of African ancestry and at least one parent or grandparent of European ancestry. European ancestry may be determined by self reporting as European ancestor(s) (from a country in Europe), European American, white or Caucasian. European ancestry may also be determined by genetic analysis using unlinked microsatellite markers as described in Example 21 and set out in Table 54.
In some variations, the additional risk factor comprises a genetic variation that correlates with susceptibility to MI, in at least one gene selected from 5-lipoxygenase activating protein (FLAP) and leukotriene A4 hydrolase (LTA4H). In some variations of the method, the selecting step comprises determining a FLAP genotype or haplotype of a human subject, and selecting for therapy a human subject with the race and with a FLAP genotype that correlates with susceptibility to MI. In particular, the FLAP genotype or haplotype that correlates with susceptibity of MI is HapA or HapB or the haplotypes provided in Table 5, Table 7 and Table 34.
In other variations of the method, the selecting step comprises determining a LTA4H genotype or haplotype of a human subject, and selecting for therapy a human subject with the race and with a LTA4H genotype that correlates with susceptibility to MI. In particular, the LTA4H genotype or haplotype that correlates with susceptibility to MI is HapK, HapL or HapQ or the haplotypes provided in Table 47 or 51. The selected human subject may be determined to have a LTA4H haplotype comprising markers SG12S25 having allele A, SG12S26 having allele T, SG12S143 having allele C, SG12S144 having allele G and SG12S221 having allele G. The selected human subject may also be determined to have a LTA4H haplotype comprising markers SG12S25 having allele A, SG12S26 having allele T, SG12S100 having allele T, SG12S28 having allele T, SG12S143 having allele C, SG12S144 having allele G and SG12S221 having allele G. In addition, selected human subject may also be determined to have a LTA4H haplotype comprising markers SG12S16 having allele C, SG12S21 having allele G, SG12S23 having allele T, SG12S25 having allele A, SG12S26 having allele T, SG12S100 having allele T, SG12S28 having allele T, SG12S143 having allele C, SG12S144 having allele G and SG12S221 having allele G.
A surrogate haplotype is a haplotype that correlates with the original haplotype, but may be defined by different markers. A perfect surrogate haplotype is one that will identify exactly the same individuals as the original haplotype. For example, all subjects that carry the original haplotype will also carry the the surrogate haplotype and all that carry the surrogate also carry the original haplotype. A perfect surrogate will have a correlation coefficient (R2) of 1. Surrogate haplotypes may not be perfect, for example if the frequency of the surrogate is different than that of original haplotype then it is likely that all subjects that carry the original haplotype will also carry the surrogate haplotype but not all that carry the surrogate will carry the original (if the surrogate is more frequent than the original haplotype). In this case the R2 is less than 1.
In some variations of the invention, the selected human subject may be determined to have a HapK surrogate haplotype. For example, the selected human subject may be determined to have a LTA4H genotype comprising marker SG12S551 having allele T, or SG12S540 having allele A. The SG12S551 may serve as a single SNP surrogate for HapK, since its T allele correlates to HapK in HapMap CEU samples (The International HapMap Project. Nature 426, 789-96 (2003)) with R2=0.74, D′=1. The SG12S540 may also serve as a single SNP surrogate for HapK, since its A allele correleates to HapK in HapMap CEU samples with R2=0.81 and D′=1.
In another variation of the invention, the selected human subject may be determined to have the HapK surrogate haplotype comprising marker rs250477 having allele C and marker rs2660896 having allele T. The correlation of this 2 SNP surrogate haplotype to HapK is R2=0.95 for both African American patients and Caucasian patients. In addition, the selected human subject may be determined to have the HapK surrogate haplotype comprising marker SG12S540 (re2540500) having allele A and marker rs107353540 having allele A. The correlation of this 2 SNP haplotype to HapK is R2=0.95 for both African American patients and Caucasian patients.
In some variations of the method, the selecting of human subjects comprises analyzing nucleic acid of a human subject for the presence or absence of at least one FLAP polymorphism or LTA4H polymorphism that correlates with a susceptibility to myocardial infarction, and selecting for therapy a human subject with the race and with the at least one polymorphism that correlates with susceptibility to MI. The FLAP polymorphic markers that correlate with MI include those SNPs set out in Table 5, Table 7 and Table 34 and those SNPs within the HapA or HapB FLAP haplotype. The LTA4H polymorphic markers that correlate with MI include those SNPs set out in Table 47 or 51 and those SNPs within the HapK, HapL and HapQ LTA4H haplotype.
The individuals or human subjects selected for treatment in any of the methods of the invention 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 >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 in any of the methods of the invention may have an elevated inflammatory marker, e.g., a marker such as an end product of the leukotriene pathway, such as LTB4, C-reactive protein (CRP), serum sCD40L, 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, individuals or human subjects selected for treatment in any of the methods of the invention have an elevated CRP level in excess of 3.0 mg/L, In addition, the invention contemplates selecting human subjects for treatment that have greater than 2.75 mg/L, greater than 2.5 mg/L or greater than 2.25 mg/L, greater than 2.0 mg/L. The invention also contemplates selecting human subjects for treatment that have a median CRP level, e.g. CRP level greater than 1.75mg/L or greater than 1.5 mg/L.
In some variations of the invention, individuals or human subjects selected for treatment in any of the methods of the invention have an elevated serum sCD40L level in excess of 1.4 ng/ml. In addition, the invention contemplates selecting human subjects for treatment that have greater than 1.5 ng/ml, greater than 2.0 ng/ml, greater than 2.5 ng/ml, greater than 3.0 ng/ml, greater than 3.5 ng/ml, greater than 4.0 ng/ml, greater than 4.5 ng/ml or greater than 5.0 ng/ml.
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.
In another 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 soluble CD40 ligand (CD40L) level in the serum of the human subject before and during the prophylaxis treatment, wherein the MI therapeutic agent is administered in an amount effective to reduce the sCD40L 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 using any of the preceding methods of the invention 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 sCD40, 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.
In jurisdictions that forbid the patenting of methods that are practiced on the human body, the following restrictions are intended: (1) the selecting of a human subject shall be construed to be restricted to selecting based on testing of a biological sample that has previously been removed from a human body and/or based on information obtained from a medical history, patient interview, or other activity that is not practiced on the human body; and (2) the administering of a composition to a human subject shall be restricted to prescribing a controlled substance that a human subject will self-administer by any technique (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation that is consistent with laws or regulations defining patentable subject matter is intended. In jurisdictions that do not forbid the patenting of methods that are practiced on the human body, the selecting of subjects and the administering of compositions includes both methods practiced on the human body and also the foregoing activities.
The invention further provides for methods of diagnosising susceptibility to myocardial infarction in a human subject comprising: screening for black African ancestry in the human subject, and screening for an LTA4H genotype or haplotype in a nucleic acid of the human subject, wherein the presence of the a black African ancestry and the genotype or haplotype in the nucleic acid is indicative of a susceptibility to cardiovascular disease. The screening for black African ancestry may be determined by any of the techniques described herein, such as patient self-reporting African ancestory or identification of African ancestory by genetic testing. Likewise, the at-risk haplotype is determined by any technique described herein, and is any at-risk haplotype described herein such as a HapK genotype or haplotype.
In an embodiment of the invention, the method of diagnosing comprises screening for a LTA4H haplotype comprising markers SG12S25 having allele A, SG12S26 having allele T, SG12S143 having allele C, SG12S144 having allele G and SG12S221 having allele G. The LTA4H haplotype screened for may also comprise markers SG12S25 having allele A, SG12S26 having allele T, SG12S100 having allele T, SG12S28 having allele T, SG12S143 having allele C, SG12S144 having allele G and SG12S221 having allele G. Further, the LTA4H haplotype screened for may comprise markers SG12S16 having allele C, SG12S21 having allele G, SG12S23 having allele T, SG12S25 having allele A, SG12S26 having allele T, SG12S100 having allele T, SG12S28 having allele T, SG12S143 having allele C, SG12S144 having allele G and SG12S221 having allele G.
A method of assessing a susceptibility to myocardial infarction (MI) or acute coronary syndrome (ACS) in a human individual, comprising screening nucleic acid of the individual for at least one polymorphism in a LTA4H nucleic acid that correlates with increased occurrence of myocardial infarction or ACS in a human population,
wherein the presence of the at least one polymorphism identifies the individual as having elevated susceptibility to MI or ACS, and
wherein the absence of the at least one polymorphism in the nucleic acid identifies the individual as not having the elevated susceptibility.
It is well established that different racial and ethnic groups show greater or lesser susceptibility to certain diseases and conditions, and there is at least one drug (BiDilTM) that is FDA-approved for race-specific administration. A need exists for improved materials and methods for diagnosing racial or ethnic ancestry using genetic techniques. Genetic techniques offer a potential improvement over self-reporting, especially for individuals having unknown parents, such as adoptees, or for providing information about prior generations that are beyond an individual's known family tree. Another aspect of the invention is novel materials and methods for genetic assessment of race. For example, the invention includes a method of determining racial ancestry of a human individual, comprising assaying or analyzing nucleic acid of a human individual to determine a racial genotype comprised of one or more microsatellite marker(s) selected from the group consisting of D1S493, D1S2866, D1S2630, D1S466, D1S2847, D2S166, D6S405 D6S446, DG8S156, D8S1719, D8S1831, D8S1746, D9S1839, D9S1777, D10S1698, D11S4130, D11S1321 D11S4206, D11S1320, D12S1723, D13S152, D14S741, D16S404, D17S745, D17S1799, D18S464, D19S534, D19S113, D20S878, and D21S1884; wherein racial ancestry is determined from alleles of the one or more microsatellite markers, and determining racial ancestry based on the racial genotype. In some variations, in addition to these one or more markers, one assays one or more of the additional Table 54 markers to generate the racial genotype.
The invention also includes methods of assessing risk for a disease with race-dependent risks in a human individual comprising analyzing nucleic acid of a human individual to determine a racial genotype comprised of one or more microsatellite marker(s) selected from the group consisting of D1S493, D1S2866, D1S2630, D1S466, D1S2847, D2S166, D6S405 D6S446, DG8S156, D8S1719, D8S1831, D8S1746, D9S1839, D9S1777, D10S1698, D11S4130, D11S1321 D11S4206, D11S1320, D12S1723, D13S152, D14S741, D16S404, D17S745, D17S1799, D18S464, D19S534, D19S113, D20S878, and D21S1884; wherein racial ancestry is determined from alleles of the one or more microsatellite markers, determining racial ancestry based on the racial genotype and evaluating risk for the disease based on the racial genotype.
Studies with the ethnicity panel of markers described herein have shown that ethnicity is a quantifiable trait. The applications of such an ethnicity panel extends beyond its utility for determining degree of ancestry in the context of HapK and MI risk. Although a strong correspondence between self-reported ethnicity and genetically estimated ancestry has been found, self-report is not entirely reliable. Therefore, there are several scenarios where the ethnicity panel of markers described herein (using some or all of our markers beyond those previously reported) can be useful.
In one embodiment, the determination of racial or ethnic ancestry using the panel of genetic markers may assess susceptibility or risk for certain diseases. Many diseases have race-dependent incidences or risks. For example, hypertension is more common in African-Americans than in Caucasians. Traditionally, ethnicity or race is taken into account when defining a given individual's risk for a disease. However, that depends on self-reporting by the individual of his/her ethnicity. Using an ethnicity panel of markers may improve reliability of risk assessments for any given disease that shows race-dependent risks.
In another embodiment, the determination of racial or ethnic ancestry using the panel of genetic markers will allow for the matching of the genetic backgrounds of case and control groups in case-controlled clinical studies. In a traditional case-control association study with any protein, RNA, DNA or analyte marker it is valuable if the genetic backgrounds of the case and control groups be matched. Using an racial panel may improve reliability of matching case and control group beyond what can be achieved through self-reporting of race. This would have the benefit of decreasing chance of finding false-positive associations and would lead to more accurate determination of the magnitude of risk of markers that showed true association.
Difference in ethnic or racial ancestry may also be considered as a risk marker (genetic, RNA, protein, other analyte). Therefore, the determination of race or ancestry using the panel of genetic markers may be useful to condition that risk in any given subject based on his/her relative ethnic makeup. In addition, analysis of case-control studies based on an ethnicity panel may lead to discoveries that certain markers ((genetic, RNA, protein, other analyte) may show race-dependent risks. Use of an ethnicity panel may improve the sensitivity of finding ethnic differences in risk, especially when compared to self-reporting.
Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the drawing and detailed description, and all such features are intended as aspects of the invention. Likewise, features of the invention described herein can be re-combined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specifically mentioned above as an aspect or embodiment of the invention. Also, only such limitations which are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention.
In addition to the foregoing, the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above. For example, although aspects of the invention may have been described by reference to a genus or a range of values for brevity, it should be understood that each member of the genus and each value within the range is intended as an aspect of the invention. Likewise, various aspects and features of the invention can be combined, creating additional aspects which are intended to be within the scope of the invention. Although the applicant(s) invented the full scope of the claims appended hereto, the claims appended hereto are not intended to encompass within their scope the prior art work of others. Therefore, in the event that statutory prior art within the scope of a claim is brought to the attention of the applicants by a Patent Office or other entity or individual, the applicant(s) reserve the right to exercise amendment rights under applicable patent laws to redefine the subject matter of such a claim to specifically exclude such statutory prior art or obvious variations of statutory prior art from the scope of such a claim. Variations of the invention defined by such amended claims also are intended as aspects of the invention.



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 <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 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. 6 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. 7 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. 8 shows the analysis of carry-over effect for CRP and SAA (on log-scale).
FIG. 9 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. 10 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. 11 shows LD and haplotypes in the LTA4H region. FIG. 11, left panel, 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 R2-values in the lower right triangle. Scales for both measures of LD are shown on the right. FIG. 11, right panel, depicts 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 35 (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 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-B 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-A correlates with HapA and HapB.
In addition, genetic analysis in Icelandic and North American cohorts (Examples 17) identified LTA4H haplotypes associated MI as demonstrated in Tables 42 and 44. HapK is defined by allele C of the SNPs SG12S16, allele G of the SNPs SG12S21, allele T of the SNPs SG12S23, allele A of the SNPs SG12S25, allele T of the SNPs SG12S26, allele T of the SNPs SG12S100, allele T of the SNPs SG12S28, allele C of the SNPs SG12S143, allele G of the SNPs SG12S144, and allele G of the SNPs SG12S221.
For Icelandic and European American cohorts, essentially the HapK association to MI can be obtained by analyzing the following 5 SNPs: allele A of the SNPs SG12S25, allele T of the SNPs SG12S26, allele C of the SNPs SG12S143, allele G of the SNPs SG12S144, and allele G of the SNPs SG12S221. However, for African American cohorts, the HapK association to MI can be obtained analyzing the following 7 SNPs: allele A of the SNPs SG12S25, allele T of the SNPs SG12S26, allele T of the SNPs SG12S100, allele T of the SNPs SG12S28, allele C of the SNPs SG12S143, allele G of the SNPs SG12S144, and allele G of the SNPs SG12S221. The addition of these two SNPs to the 5 SNPs that define HapK adds additional information to improve accuracy of determination of the carrier status of a tested individual for this haplotype. This extra information is more helpful in African-American cohorts than Caucasian since there is a greater diversity of haplotypes in African-Americans, although the 5-SNP HapK has good accuracy for determining carrier status in African-Americans. Single SNPs, such as SG12S551 with allele T and SG12S540 with allele A, have been defined that are highly correlated to HapK and can in large measure substitute for HapK in terms of defining MI risk.
HapL is defined by allele C of the SNPs SG12S16, allele G of the SNPs SG12S21, allele T of the SNPs SG12S23, allele A of the SNPs SG12S25, allele T of the SNPs SG12S26, allele T of the SNPs SG12S100, allele T of the SNPs SG12S28, allele T of the SNPs SG12S143, allele A of the SNPs SG12S144, and allele G of the SNPs SG12S221. HapQ is defined by allele C of the SNPs SG12S16, allele G of the SNPs SG12S21, allele T of the SNPs SG12S23, allele G of the SNPs SG12S25, allele T of the SNPs SG12S26, allele C of the SNPs SG12S100, allele T of the SNPs SG12S28, allele T of the SNPs SG12S143, allele A of the SNPs SG12S144, and allele G of the SNPs SG12S221.
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 21 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 37 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'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 5, 7, 8, 9, 10 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 40. 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 (now U.S. Pat. No. 7,507,531) may indicate one's increased risk for MI and ACS. Other representatibe at-risk haplotypes are shown in Table 41 and Table 42. 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: 22:1668-167). 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., >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 <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 1-df likelihood ratio statistic is used to evaluate the statistic significance.
To look for at-risk-haplotypes in the 1-1od 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 <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. BLT 1, 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'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'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:













AGENT TABLE I










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- fluorophenyl)methyl]-2thienyl]- 1methyl-2-propynyl]--N- hydroxurea
U.S. Pat. No. 5288751, U.S. Pat. No. 5288743, U.S. Pat. No. 5616596
Feb. 22, 1994 Apr. 01, 1997
5-LPO inhibitor


 


Abbott
A-81834




3-(3-(1,1-dimethylethylthio-5- (quinoline-2-ylmethoxy)-1-(4- chloromethylphenyl)indole-2-yl)- 2,2-dimethylpropionaldehyde oxime-0-2-acetic acid
WO9203132, U.S. Pat. No. 5459150
Mar. 5, 1992, Oct. 17, 1995
FLAP inhibitor


 


Abbott
A-86886




3-(3-(1,1-dimethylethylthio-5- (pyridin-2-ylmethoxy)-1-(4- chloromethylphenyl)indole-2-yl)- 2,2-dimethylpropionaldehyde oxime-0-2-acetic acid
WO9203132, U.S. Pat. No. 5459150
Mar. 5, 1992, Oct. 17, 1995
5-LPO inhibitor


 


Abbott
A-93178







FLAP inhibitor


 


AstraZeneca
AZD-4407





EP 623614
Sep. 11, 1994
5-LPO inhibitor


 


AstraZeneca
ZD-2138




6-((3-fluoro-5- (tetrahydro-4-methoxy-2H- pyran-4yl)phenoxy)methyl) 1-methyl-2(1H)- quinlolinone (alternatively NH can be N-methyl)
EP 466452

5-LPO inhibitor


 


Bayer
BAY-X-1005




(R)-(+)-alpha-cyclopentyl 4-(2-quinolinylmethoxy)- Benzeneacetic acid
U.S. Pat. No. 4970215 EP 344519, DE 19880531

FLAP inhibitor


 


Merck
MK-0591




1-((4- chlorophenyl)methyl)-3- ((1,1-dimethylethyl)thio) alpha, alpha-dimethyl-5- (2-quinolinylmethoxy)-1H- Indole-2-propanoic acid
EP 419049, U.S. Pat. No. 19890822

FLAP inhibitor


 


Merck
MK-866

(3[3-)4-chlorobenzyl)-3-t-butyl-


5-LPO inhibitor





thio-5-isopropylindol-2yl]2,2-








dimethyl-proanoic acid





 


Merck
MK-886




1-((4-chlorophenyl)methyl)-3- ((1,1dimethylethyl)thio)- alpha, alpha-dimethyl-5- (2-quinolinylmethoxy)-1H- Indole-2-propanoic acid
EP 419049, U.S. Pat. No. 19890822

5-LPO inhibitor


 


Pfizer
CJ-13610

4-(3-(4-(2-Methyl-


5-LPO inhibitor





imidazol-1-yl)-








phenylsulfanyl)-phenyl)-








tetrahydro-pyran-4-








carboxylic acid amide









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) >C═O, (f) >C═N—OR1, where R1 is hydrogen or C1-C6 alkyl, (g) −(CHR1)n (CO)(CHR2)m, where n and m are independently selected from an integer from one to six and R1 and R2 are independently selected from hydrogen and C1-C6-alkyl, (h) —(CHR1)nC═NOR2, where R1, R2 and n are as defined above; (i) —(CHR1)nON═CR2, where R1, R2 and n are as: defined above; (j) —(CHR1)n—O—(CHR2)m—, where R1, R2, n and m are as defined above, (k) —(CHR1)n—NR2 (CHR3)m—, where R1, R2, n and m are as defined above and R3 is selected from hydrogen and C1-C6-alkyl; (1) —(CHR1)n—S—CHR2)m—, where R1, R2, n and m are as defined above; and (m) —(CHR1)n—(SO2)—(CHR2)m—, where R1, R2, 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; R1 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, R2 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, —CONR5R6 wherein R5 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 R6 is selected from the group consisting of hydrogen and alkyl of from one to six carbon atoms, —COR6, and —OH; R3 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, R4 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; R1 is alkylthio of one to six carbon atoms; R6 is selected from the group consisting of hydrogen and alkyl of one to six carbon atoms; R7 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; R3 is phenylalkyl in which the alkyl portion is of one to six carbon atoms; R4 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:

Q1-X—Ar-Q2 

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-lmethoxy)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 R1 represents a group of the formula:




R2 and R3 are identical or different and represent hydrogen, lower alkyl, phenyl, benzyl or a group of the formula:




R4 represents hydrogen, lower alkyl, phenyl or benzyl, which can optionally be substituted by hydroxyl, carboxyl, lower alkoxycarbonyl, lower alkylthio, heteroaryl or carbamoyl, R5 represents hydrogen, lower alkyl, phenyl or benzyl, R6 represents a group of the formula —COR5 or —CO2 R5, R7 represents hydrogen, lower alkyl or phenyl, Y represents a group of the formula:




wherein R8 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 R9 and R10 are identical or different and denote hydrogen, lower alkyl or phenyl, or R9 and R10 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'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 '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-methylpropanoic 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-y]-2,2-dimethylpropanoic acid,
    3-[N-(4-chlorobenzyl)-3-(4-chlorobenzoyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimiethylpropanoic 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-l -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—, —CH2CH═CH—, —C(CH3)═CH—, —CH2CH═CHCH2—, 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—CH2—, —CH≡CH—CH(CH3)—, 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 moiety 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 —(CH2)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.














AGENT TABLE III












(Infor from









PDR)









Dosages



Approved St





(tablet


Resigeeter to
Marketed as
Stain Name
Chemical
Structure
Patent No
Patent Expiration
sizes)







AstraZeneca
CRESTOR
ROVUVASTATIN
bis[(E)-7-[4-(4-fluorophenyl)-6-isopropyl- 2-[methyl(methylsulfonyl)amino] pyrimidin-5-yl](3R, 5S)-3,5- dihydroxyhept-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-fluorophenyl)- 1-(1-methylethyl)-1H-indol-2-yl]-3,5- dihydroxy-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
ATORVASTATIN
[R-(R*, R*)]-2-(4-fluorophenyl)-(beta), [dgr]-dihydroxy-5-(1-methylethyl)-3- phenyl-4-[(phenylamino)carbonyl]-1H- pyrrole-1-heptanoic acid, calcium salt (2:1)trihydrate




4681893 4681893*PED 5273995 5273995*PED 5685104 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
MEVACOR
LOVASTATIN
[1S-[1(alpha)(R*), 3(alpha), 7(beta), 8(beta)(2S*, 4S*), 8a(beta)]]-1,2,3,7,8,8a-hexahydro-3,7- dimethyl-8-[2-(tetrahydro-4-hydroxy-6- oxo-2H-pyran-2-yl)ethyl]-1- naphthalenyl 2-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- hexahydro-3,7-dimethyl-8-[2-(tetrahydro- 4-hydroxy-6-oxo-2H-pyran-2-yl)-ethyl]- 1-naphthalenyl ester, [1S- [1(alpha), 3(alpha), 7(beta), 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
PRAVACOL
PRAVASTATIN
1-Naphthalene-heptanoic acid, 1,2,6,7,8,8a-hexahydro-(beta), (delta), 6- trihydroxy-2-methyl-8-(2-methyl-1- oxobutoxy)-, monosodium salt, [1S- [1(alpha)((beta)S*, (delta)S*), 2(alpha), 6(alpha), 8(beta)(R*), 8a(alpha)]]-.




4346227 4346227*PED 5035447 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
5 mg, 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'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, 15mg, 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 RNAas 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 & 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, serum sCD40L, 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, sCD40L 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, sCD40L, 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'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, intemucleotide 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 & 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 & 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 Tm 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 environmental 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 & 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 & 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 Tm 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 Tm 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 “GenechipsTM,” 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 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 antibody 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 <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 41 or Table 42, 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, SG13S114, 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 expression 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 expressions f 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 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 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 125I, 35S, 14C or 3H, 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., CytosensorTM) 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 MatchmakerTM 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 microtiter 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 agent; 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 as described in detail in International Application No. PCT/US2005/03312 (Publication No. WO2005/075022) incorporated herein by reference in its entirety. 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.
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
relativesa














All MI patients
713
296
863
1741


Males
575
248
724
1385


Females
140
56
108
366


Early onset
194
93
156
739





aGenotyped 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.
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. 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. 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. 6. 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















Phenotype
Marker
Allele
P value
RR
# Pat.
% Pat.
# Ctrl
% Ctrl


















All patients
SG13S106
G
0.0044
1.29
681
72.0
530
66.6



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 onset
SG13S100
A
0.0440
1.43
99
71.7
377
63.9





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 valuea
P valueb
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





aSingle test P values.


bP values adjusted for all the SNP haplotypes tested.


cMeasure 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, 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-valuea















MI (779)
0.158
1.80
0.135
0.0000023
0.005


Males (486)
0.169
1.95
0.158
0.00000091
NDb


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





aP value adjusted for the number of haplotypes tested.


bNot done.


cExcluding 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





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


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



















SG13S377
SG13S106
SG13S114
SG13S89
SG13S30
SG13S32
SG13S42
SG13S35





B4

G


G

A



B5

G


G

A



B6
G
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
p-val





Female MI
C
T
0
T
T
G
−2
6.38E−06



C
T
0


G
−2
2.74E−05




















N_aff
aff. frq
N_ctrl
ctrl. frq
rel_risk
PAR
info






Female MI
454
0.059
809
0.015
4.05
0.086
0.577




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.




















NCBI
NCBI








end


build34;
build34;






minor
start
position


start
stop
SNP/





allele
position in
in


on chr.
on chr.
marker/


public

minor
frequency
SEQ ID
SEQ ID


13 (bp)
13 (bp)
exon name
alias1
alias2
SNP
Variation
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
DG13S111















1 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 #'s: For SNP alleles A = 0, C = 1, G = 2, T = 3; for microsatellite alleles: the CEPH sample (Centre d'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
















DG13S175
















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<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 < 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 patients
541
0.8743
734
0.8501
1.2262
0.046


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 as described in detail in International Application No. PCT/US2005/03312 (Publication No. WO2005/075022) incorporated herein by reference in its entirety.
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 (>50 pg/ml (roughly the upper 50th percentile).























SG13S418
SG13S420
DG13S166
SG13S114
SG13S88
SG13S184
D13S1238





High LTE4










C
T
0
T
T
G
−2



C
T
0


G
−2


Low LTE4










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






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 (<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
D13S1238





High vs low LTE4
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





High vs low LTE4
1.61E−01
221
0.084
185
0.054
1.61
0.063
0.689



1.20E−01
220
0.13 
182
0.088
1.54
0.089
0.686





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 LTE-4 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 as described in detail in International Application No. PCT/US2005/03312 (Publication No. WO2005/075022) incorporated herein by reference in its entirety. 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. Moreover, 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's response to factors that stimulate inflammatory cells, resulting in increased leukotriene production and increased risk for MI.







TABLE 20







LTB4 levels after ionomycin stimulation of isolated neutrophilsa










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





aMean ± 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 MI
6.5E−04
1.82
297
0.16
614
0.09
0.72
G


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 MI

T
G

A



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 MI

T
G

A



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
















Strokea (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





aExcluding 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. & 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. 5), 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-valuea















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





aP 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. & Samani, N. J., Thromb Haemost 79, 980-4 (1998); Brouilette, S., Singh, R. K., Thompson, J. R., Goodall, A. H. & 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 Organization 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
AGCCAGTGTCCACAAGGAAG
GAGGGTGAGACACATCTCTGG
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
TGACACCATGTCTTACTGTTTGC
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
AGGAGTGTGGCAGCTTTGAG
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
CACTTTGCCAGTAGCCTTGA
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
CCTACTTTCCATCTCCTCCTTG
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)




 


DG13S1103
TTGCTGGATGCTGGTTTCTA
AAAGAGAGAGAGAAAGAGAAAG
28910502
28910765



(SEQ ID NO. 136)
AAAGA (SEQ ID NO. 137)




 


DG13S3147
AAAGTGGATGCAGTTGAGGTTT
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
GGGAAATGGAGCTGCTGTTA
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
CAAATTCCTCAATTGCAAAAT
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
GGATCCAATCGTACGCTACC
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
TTTAAGCCCTGTGGAATGTATTT
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
30411755



(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
31179791
31179953



(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
AAGATGCAATCTGGTTTCCAA
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
TGATGGATGCACCATGTTTA
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
TGCATTCTTTAAGTCCATGTC
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
AGCCCAAGGCTCTTGTGAG
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
CGATTGCCATGTCTCTTTGA
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
CATCTATCTTTGGATTCAGTGGTG
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
GCCACATTGCTATCAGCGTA
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
TGTCATAGGCTTGCGGTATTT
TTGGTAGGGTCCTTTCCTTT
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
ACCTTTCAAGCTTCCGGTTT
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
GATGCTTGCTTTGGGAGGTA
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
TACCCTCACCTTCCTCATGC
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
TGGGTCATTCTGGACATTCA
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
GGAACCAGTCATTTGGGTGT
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
GGTTTGGGAACCATTCTCCT
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
TGTTCCATCTCTCTACCCATGT
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



G (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
GAACCAGTGAGTTTTTATTAC
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
TTGCATGAAGTAAAGTATCCCTG
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
GGTAACTTGCCATTCTTCTACCA
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
TGTCTGTTGGCAACTTTCTGTC
41107773
41108117



(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 B4, one of the most potent chemokine mediators of arterial inflammation. The experiments described in Example 7 showed that MI patients make more LTB4 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 (now abandoned; published as U.S. 2005/0272051A1) 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 LTA4 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 (LTA4-OH gene)
A
SG12S25
C
SG12S223




5
GF (LTA4-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 LTA4 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's judgement, will interfere with the subject'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 <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 > 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/m2
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'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 (LTA4-OH)
13
(41%)
18
(56%)
19
(59%)
22
(69%)
13
(41%)
20
(65%)


GF carrier (LTA4-OH)
3
(9%)
9
(28%)
5
(16%)
6
(19%)
7
(22%)
3
(10%)


NA3/A3 & 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'l low'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'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's standard care, including all medications and treatment plan as prescribed by the subject's cardiologist prior to enrollment. The cross-over study design is summarized in FIG. 7. 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 measurements of the AB (drug/placebo) group with the measurements 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, LTB4 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 LTB4 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
Ref










ELISA method












Myeloperoxidase (MPO)
Assay Design,
Titerzyme EIA
# 900-115
A quantitative solid phase
14



Inc.


sandwich ELISA



LTB4
R&D
LTB4
# DE0275
A competitive binding
15






immunoassay



Amyloid A
Biosource
Human SAA kit
# KHA0012
A quantitative solid phase
16






sandwich ELISA



Cysteinyl Leukotriene
R&D
Cysteinyl
# DE3200
A competitive binding
17




Leukotriene

immunoassay



Nitrotyrosine
OxisResearch
Bioxytech Nitro
# 21055
A quantitative solid phase
18




tyrosine-EIA

sandwich ELISA



TNF-α
R&D
Quantikine HS
# HSTA00C
A quantitative solid phase
19




Human TNF-α

sandwich ELISA



IL6
R&D
Quantikine HS
# HS600B
A quantitative solid phase
20




Human IL-6

sandwich ELISA



IL12p40
R&D
Quantikine Human
# DP400
A quantitative solid phase
21




IL-12p40

sandwich ELISA



MCP-1
R&D
Quantikine Human
# DCP00
A quantitative solid phase
22




MCP-1

sandwich ELISA



ICAM
R&D
Parameter human
# BBE 1B
A quantitative solid phase
23




sICAM-1

sandwich ELISA



sE-Selectin
R&D
Parameter human
# BBE 2B
A quantitative solid phase
24




sEselectin

sandwich ELISA



sP-Selectin
R&D
Parameter human
# BBE 6
A quantitative solid phase
25




sPselectin

sandwich ELISA



VCAM
R&D
Parameter human
# BBE 3
A quantitative solid phase
26




sVCAM-1

sandwich ELISA



MMP 9
R&D
Quantikine Human
# DMP900
A quantitative solid phase
27




MMP-9(total)

sandwich ELISA



Oxidised LDL
Mercodia
Oxidised LDL Elisa
# 10-1143-01
A quantitative solid phase
28






sandwich ELISA



Lp-PLA2
Diadexus San
PLAC test

A quantitative solid phase
29



Fransisco, CA


sandwich ELISA








Other methods












Hs-CRP
Roche Hitachi
Hs-CRP
11972855
Immunoturbidimetric assay
30



912 analyser






LTB4 (MS)
LC/MS/MS
LTB4 assay

Mass spectrometer with
31






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 LTB4 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 LTB4 and MPO production amounted to 26% for LTB4 (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-PLA2 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-PLA2. In contrast, the effects of the 2 lower doses (250 mg/day and 500 mg/day) on Lp-PLA2 were not significant. Urine levels of LTE4 increased by 27% in response to the highest dosage of DG-031 (nominal p=0.00002)). Significant correlation was observed between the inhibition of LTB4 and MPO production in response to DG-031 (r=0.65, p<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-PLA2
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


LTB4 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


LTB4 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


LTE4 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 LTB4 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).













250 mg/day
500 mg/day
750 mg/day
500 & 750 mg/day
250, 500 & 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 [−0.011, 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-PLA2
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)


LTB4 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)


LTB4 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)


LTB4 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)


LTB4 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)


LTE4 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 < 0.00001)
(p < 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-PLA2 and N-tyrosine). No carry over effects were observed with LTB4 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% CI













CRP
0.017
−0.28
[−0.52, −0.05]


Amyloid A
0.051
−0.14
[−0.29, 0.00]


LTE4 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]


LTB4 in whole blood, corr. for wbc‡
0.45
−0.10
[−0.36, 0.16]


MPO in whole blood, corr. for wbc‡
0.93
0.01
[−0.13, 0.15]


LTB4 in whole blood, mass spec.§
0.45
0.19
[−0.33, 0.71]


LTB4 in whole blood, corr. for wbc,
0.64
0.12
[−0.40, 0.64]


m.s.§





‡corr. for wbc = corrected for white blood cell count


§m.s. = mass spec. = mass spectrometry






FIG. 9 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<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 arms 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 ALOX5AP, 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 & Rubinstein P Ann Hum Genet 51 (Pt 3):227-33, 1987: Terwilliger & 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)









This analysis was repeated in three additional North American cohorts from Philadelphia and two from Atlanta (referred to Emory cohort 1 and Emory cohort 2). A comparison of the data from European Americans and African Americans is provided as Table 33 below. The combined data for European-Americans (Caucasians) in the North American cohorts (Cleveland, Philadelphia and Atlanta), indicated that the allelic/haplotype frequence of HapA was 16.5% in patients and 15% in controls (P=0.04). The relative risk of MI in the total North American patients of European ancestry was 1.13. In African-Americans within the combined North American cohorts, the allelic/haplotype frequencies of HapA was 12.4% in patients and 9.5% in controls (P=0.06). The relative risk of MI in the total of African Americans was 1.4. Even though the frequency of HapA was less in the African American members of the cohort, the relative risk was higher. The results from this analysis indicate that African Americans are an especially promising target population for the anti-leukotriene therapies described herein. At risk individuals of black African origin that exhibits HapA or other risk factors described herein are specifically contemplated for prophylaxis therapy according to the invention described herein.














TABLE 33







p-val
r
#aff
aff. freq
#con
con.freq






















European








Americans


Philadelphia cohort
0.439
0.9076
723
0.159247
428
0.17265


Cleveland cohort
0.114
1.1998
627
0.163285
777
0.1399


Emory cohort 1
0.196
1.2259
236
0.16713
637
0.14067


Emory cohort 2
0.342
1.1855
292
0.179848
291
0.1561


African Americans


Philadelphia cohort
0.365
0.7228
105
0.085406
125
0.11441


Cleveland cohort
0.163
1.7542
53
0.157175
110
0.09609


Emory cohort 1
0.036
2.4673
39
0.169904
161
0.0766


Emory cohort 2
0.336
1.4262
69
0.145303
146
0.10651









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).
Analysis of HapB was also repeated in three additional North American cohorts from Philadelphia and two from Atlanta (referred to Emory cohort 1 and Emory cohort 2). A comparison of the data from European Americans and African Americans is provided as Table 34 below. The combined data for European-Americans (Caucasians) within the North American cohorts (Cleveland, Philadelphia and Atlanta), indicated that the allelic/haplotype frequence of HapB was 7.4% in patients and 6.2% in controls (P=0.03). The relative risk of MI in the total North American patients of European ancestry was 1.22. In African-Americans in the combined North American cohorts, the allelic/haplotype frequencies of HapB was 12.6% in patients and 14.4% in controls (P=0.37) The relative risk of MI in the total of African Americans was 0.857.














TABLE 34







p-val
r
#aff
aff. freq
#con
con.freq






















European








Americans


Philadelphia cohort
0.541
1.1176
724
0.077524
428
0.06994


Cleveland cohort
0.026
1.4371
627
0.08299
778
0.05924


Emory cohort 1
0.78
1.0853
236
0.058363
633
0.05403


Emory cohort 2
0.47
0.825
266
0.057363
296
0.0687


African Americans


Philadelphia cohort
0.645
1.1394
105
0.150802
126
0.13484


Cleveland cohort
0.604
0.8195
53
0.115501
109
0.13744


Emory cohort 1
0.429
0.6995
39
0.099588
160
0.13653


Emory cohort 2
0.126
0.588
62
0.102502
148
0.16264









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) also conferred risk of MI in Caucasians 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 35. 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 HapC4-B in different populations are shown in the Table 35. 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 35







Total Cleveland Cohort





















# of
Aff.
# of
Control







Haplotype
p-value
RR
Affected
Frequency
Controls
Frequency
Info
SG13S375
SG13S25
SG13S106
SG13S32





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








# of
Aff.
# of
Control







Haplotype
P-value
RR
Affected
Frequency
Controls
Frequency
Info
SG13S375
SG13S25
SG13S106
SG13S32










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









Analysis of HapC4A was repeated in four additional North American cohorts from Philadelphia and from Atlanta (referred to as Emory Cohort 1). A comparison of the data from European Americans and African Americans is provided as Table 36 below. The combined data for European-Americans (Caucasians) in the North American cohorts (Cleveland, Philadelphia and Atlanta), indicated that the allelic/haplotype frequence of HapC4A was 17.99% in patients and 17.1% in controls (P=0.18). The relative risk of MI in the total North American patients of European ancestry was 1.06. In African-Americans within the combined North American cohorts, the allelic/haplotype frequencies of HapC4A was 12.96% in patients and 10.62% in controls (P=0.14). The relative risk of MI in the total of African Americans was 1.25. Even though the frequency of HapC4A was less in the African American members of the cohort, the relative risk was higher. The results from this analysis indicate that African Americans are an especially promising target population for the anti-leukotriene therapies described herein. At risk individuals of black African origin that exhibits HapC4A or other risk factors described herein are specifically contemplated for prophylaxis therapy according to the invention described herein.














TABLE 36







p-val
r
#aff
aff. freq
#con
con.freq






















European Americans








Philadelphia
0.117
0.82
722
0.166
424
0.195


Cleveland
0.052
1.24
627
0.189
777
0.158


Emory 1
0.401
1.14
236
0.196
638
0.177


Emory 2
NA


African Americans


Philadelphia
0.512
0.79
105
0.099
126
0.121


Cleveland
0.327
1.50
53
0.144
110
0.100


Emory 1
0.086
2.02
39
0.181
158
0.098


Emory 2
NA









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=112) (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& Rubinstein P Ann Hum Genet 51 (Pt 3):227-33, 1987: Terwilliger & 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 37. 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 37







Association of HapA and HapB with ischemic stroke










HapA
HapB













Phenotype (n)
Frequency
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 37). 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 37, 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'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 AS, 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 41 and Table 42.
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 41.
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 38 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 39 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 40 shows SNPs that were found by sequencing the LTA4H gene. One of the SNPs, LTA4H—31334, is in the coding region. The polymorphism, A\G, 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 41 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, SG12S100, 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 39).
Table 42 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 42 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 42 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 among 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 38







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 39







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
0.5694
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 40












Minor


NCBI Build34





Minor
allele


position
Marker name
Marker alias
IUPAC
Public SNP
Variation
allele
percentage






















94877218
SG12S432

R
rs2270318
A/G
A
25.3


94877441


indel
rs10708992
—/A


94878002


R
rs7966537
A/G


94878095


M
rs17024935
A/C
C
10.3


94878150


Y
rs10859995
C/T
C
42.1


94878253


R
rs17676451
A/G
A
9.9


94878559


Y
rs17024937
C/T
C
1.4


94878727


Y
rs17733203
C/T
T
8.6


94878752


S
rs11108359
C/G


94879491


R
rs11108360
A/G


94880727
SG12S433

R
rs2300560
A/G
A
35.9


94880770


S
rs7136523
C/G


94880966


K
rs11108361
G/T


94881186


Y
rs12298414
C/T


94881226


M
rs7136852
A/C


94881239
SG12S434

R
rs3819817
A/G
A
38


94881275


Y
rs10859996
C/T


94881395


Y
rs10492228
C/T
T
12.1


94881566


K
rs11108362
G/T


94881734


K
rs11108363
G/T


94882274
SG12S435

Y
rs3213737
C/T
T
39.8


94882599


Y
rs12301817
C/T


94883443


R
rs11108364
A/G
G
12


94883700
SG12S436

K
rs2072511
G/T
T
28.5


94883960


Y
rs11108365
C/T


94884133


W
rs2287057
A/T


94884140


K
rs7304917
G/T


94884144


S
rs2270317
C/G


94884167


W
rs12814960
A/T


94884256


M
rs4762262
A/C


94884722


K
rs11108366
G/T
T
14.8


94884775
SG12S437

R
rs2268520
A/G
G
25.3


94884783


S
rs7308555
C/G


94885068


S
rs2268519
C/G
G
9.9


94885112


R
rs7308827
A/G


94885134


R
rs12319274
A/G


94885170


R
rs2268518
A/G
G
34.5


94885285
SG12S438

S
rs2268517
C/G
G
9.36


94885406


Y
rs12307364
C/T


94885635


S
rs11108367
C/G


94886499


Y
rs10745747
C/T
T
18.2


94886568


Y
rs7316786
C/T


94886813


Y
rs17676826
C/T
C
25.4


94887010


R
rs4762263
A/G


94887137


S
rs12309712
C/G


94887170


Y
rs2287056
C/T


94887310


R
rs2287055
A/G


94887493


indel
rs3214368
—/A


94887827


Y
rs10859997
C/T


94887924


R
rs10859998
A/G


94888002


K
rs12318681
G/T


94888606


R
rs11108368
A/G
A
44.3


94888625


K
rs17024956
G/T
G
12.7


94888741


Y
rs17024962
C/T
T
12.7


94889326


Y
rs10492227
C/T
T
25.2


94889459
SG12S439

S
rs2241137
C/G
G
25.9


94889540
SG12S440

Y
rs2241136
C/T
T
18.9


94890077


R
rs17024973
A/G
A
1.4


94890089


Y
rs2302629
C/T
T
41.4


94890233


W
rs2302628
A/T
T
37.1


94890297


R
rs17677007
A/G
G
8.5


94890520


R
rs17024981
A/G
A
9.9


94890551


K
rs17024986
G/T
G
11.4


94890561


R
rs4595631
A/G


94890567


R
rs17024995
A/G
A
1.4


94890650


M
rs3741815
A/C


94891571


M
rs4762264
A/C


94891698


Y
rs4325387
C/T


94892369


K
rs11108369
G/T


94892620


Y
rs7970524
C/T
C
12


94892804


R
rs2070614
A/G
G
42.7


94892826


M
rs7134860
A/C
A
7


94892888


M
rs7134975
A/C
A
7.7


94892970


Y
rs7306086
C/T


94893073


R
rs7956014
A/G


94893802


Y
rs10859999
C/T
T
48.6


94893822


R
rs11108370
A/G


94893903


Y
rs11108371
C/T


94894461


Y
rs11835389
C/T


94894725


Y
rs11837995
C/T


94895550


Y
rs2367755
C/T


94895729


R
rs12302993
A/G


94895964


K
rs17025016
G/T
T
10


94896055
SG12S16
LTA4H_3645
Y
NN
C/T
T
22.64


94896115
SG12S56
LTA4H_3705
K
NN
G/T
G
4.14


94896261


M
rs11614411
A/C


94896339
SG12S57
LTA4H_3929
Y
NN
C/T
C
2.5


94896351
SG12S58
LTA4H_3941
S
NN
C/G
C
0.85


94896393
SG12S37
LTA4H_3983
W
NN
A/T
T
9.3


94896548


K
rs12298595
G/T


94896571


Y
rs12318335
C/T


94896705
SG12S59
LTA4H_4295
R
NN
A/G
A
4.5


94896786
SG12S60
LTA4H_4376
R
NN
A/G
A
2.87


94896832
SG12S61
LTA4H_4422
R
NN
A/G
G
1.56


94896897
SG12S29
LTA4H_4487
W
NN
A/T
T
4.26


94896985
SG12S17
LTA4H_4575
R
rs11108372
A/G
A
41.41


94897159


M
rs1050970
A/C
C
3


94897272


S
rs1803916
C/G


94897845
SG12S62
LTA4H_5435
Y
NN
C/T
C
1.17


94897991


S
rs11108373
C/G


94898791


R
rs17025023
A/G
A
6.3


94898807


Y
rs12322336
C/T


94898878
SG12S63
LTA4H_6468
Y
rs17025026
C/T
T
4.46


94898878
SG12S63

Y
rs17025026
C/T
T
12.7


94899057
SG12S64
LTA4H_6647
Y
NN
C/T
C
2.99


94899549
SG12S18
LTA4H_7139
W
rs17677343
A/T
A
21.72


94899839


indel
rs11398806
del/T


94900318
SG12S19
LTA4H_7908
indel
NN
indel


94900320


indel
rs11374431
del/A


94900639


K
rs17025028
G/T
G
14.1


94900639
SG12S65
LTA4H_8229
K
rs17677343
G/T
G
5.09


94900892
SG12S66
LTA4H_8482
R
NN
A/G
G
0.59


94901296
SG12S542/SG12S540
LTA4H_8886
Y
rs2540500
C/T
T
33.1


94901997
SG12S68
LTA4H_9587
indel
NN
indel


94902004


indel
rs5800240
del/T


94902169
SG12S69
LTA4H_9759
W
NN
A/T
A
0.88


94902337
SG12S70
LTA4H_9927
M
rs2540499
A/C
A
24.09


94902454
SG12S71
LTA4H_10044
Y
rs5020450
C/T
C
20.93


94902928
SG12S72
LTA4H_10518
Y
rs17025033
C/T
T
1.35


94903037
SG12S30
LTA4H_10627
W
rs2540498
A/T
A
22.36


94903300
SG12S73
LTA4H_10890
Y
rs2300559
C/T
C
2.33


94903618
SG12S20
LTA4H_11208
M
NN
A/C
C
39.08


94903720
SG12S21
LTA4H_11310
R
rs2660880
A/G
A
5.95


94903837


R
rs2300558
A/G


94905002
SG12S38
LTA4H_12592
Y
rs2110762
C/T
C
34.92


94905002


R
rs2270316
A/G


94905210


K
rs4462431
G/T


94905216
SG12S74
LTA4H_12806
Y
NN
C/T
T
0.8


94905667
SG12S22
LTA4H_13257
R
rs2072510
A/G
A
36.88


94905821
SG12S75
LTA4H_13411
Y
NN
C/T
T
1.39


94906078
SG12S23
LTA4H_13668
Y
rs6538697
C/T
C
7.06


94906362
SG12S31
LTA4H_13952
Y
rs7296106
C/T
T
5.67


94906457
SG12S76
LTA4H_14047
W
rs10492226
A/T
A
1.18


94906743
SG12S77
LTA4H_14333
W
rs2540497
A/T
A
24.77


94907375
SG12S78
LTA4H_14965
Y
NN
C/T
T
2.48


94907545
SG12S24
LTA4H_15135
Y
rs2660900
C/T
C
23.76


94907820


R
rs17025050
A/G
G
3.5


94907935
SG12S79
LTA4H_15525
S
NN
C/G
C
0.83


94907970


Y
rs12302496
C/T


94908408


Y
rs17025054
C/T
T
1.4


94908971
SG12S32
LTA4H_16561
R
rs2540496
A/G
A
31.11


94909012
SG12S80
LTA4H_16602
W
NN
A/T
A
0.74


94909191
SG12S39
LTA4H_16781
K
rs2540495
G/T
T
30.74


94909463


R
rs12306086
A/G


94909554
SG12S81
LTA4H_17144
R
rs12319438
A/G
G
4.12


94910164
SG12S82
LTA4H_17754
R
NN
A/G
A
0.4


94910246
SG12S83
LTA4H_17836
W
NN
A/T
T
1.21


94910273
SG12S84
LTA4H_17863
R
NN
A/G
A
2.82


94910771


S
rs11108374
C/G


94910810


S
rs4397945
C/G


94911126


Y
rs17403903
C/T


94911249


Y
rs11108375
C/T
T
6.9


94911444
SG12S543

W
rs2072512
A/T
A
45.2


94911540


Y
rs11108376
C/T


94911669
SG12S25
LTA4H_19259
R
rs1978331
A/G
G
31.68


94911781
SG12S85
LTA4H_19371
Y
NN
C/T
T
1.25


94912503


Y
rs11108377
C/T


94913865


R
rs12049939
A/G


94914296
SG12S40
LTA4H_21886
W
rs7959337
A/T
A
5.29


94914806


K
rs2540494
G/T


94915536


N
rs12721569
A/C/G/T
A
3.5


94915637


Y
rs12721570
C/T
T
1.7


94916236
SG12S86
LTA4H_23826
R
NN
A/G
G
4.71


94916322


Y
rs11108378
C/T


94916445
SG12S87
LTA4H_24035
Y
NN
C/T
T
1.27


94916452
SG12S88
LTA4H_24042
R
rs1990611
A/G
A
33.76


94916805
SG12S89
LTA4H_24395
R
rs7981011
A/G
G
4.91


94916919
SG12S26
LTA4H_24509
Y
rs17677715
C/T
C
17.16


94917444
SG12S90
LTA4H_25034
R
rs17025074
A/G
A
0.84


94918310


K
rs2540493
G/T


94918356


R
rs11108379
A/G


94918851
SG12S91
LTA4H_26441
Y
rs2660838
C/T
C
25


94919176
SG12S92
LTA4H_26766
Y
rs17677763
C/T
C
20.44


94919568


indel
rs3831819
—/TA


94919667
SG12S93
LTA4H_27257
R
rs2268516
A/G
A
2.44


94920127


R
rs2540492
A/G
G
47.4


94920368
SG12S94
LTA4H_27958
Y
rs2660839
C/T
C
31.82


94920656


Y
rs11108380
C/T


94920916


indel
rs5800241
—/T


94920977


R
rs2540491
A/G


94921393
SG12S544

R
rs17025079
A/G
A
7.1


94921441


M
rs7959838
A/C


94921763
SG12S41
LTA4H_29353
Y
NN
C/T
C
20.35


94921923
SG12S95
LTA4H_29513
R
rs4441106
A/G
G
7.07


94922111


Y
rs12299755
C/T


94922409
SG12S96
LTA4H_29999
R
rs763875
A/G
A
5.92


94922502
SG12S97
LTA4H_30092
Y
rs763876
C/T
T
2.1


94922681
SG12S98
LTA4H_30271
Y
rs763874
C/T
C
32.42


94923339


Y
rs12310175
C/T


94923421


Y
rs17025090
C/T
C
15.2


94923446
SG12S42
LTA4H_31036
Y
rs2660892
C/T
C
27.41


94923710


Y
rs12721566
C/T
T
9.3


94923744
SG12S55
LTA4H_31334
R
NN
A/G
A
0.27


94923759


Y
rs11556465
C/T


94924037
SG12S99
LTA4H_31627
R
NN
A/G
A
4.37


94924389


Y
rs2660893
C/T


94924541


Y
rs2660894
C/T


94924845
SG12S100
LTA4H_32435
Y
rs2247570
C/T
C
27.79


94924938
SG12S101
LTA4H_32528
R
NN
A/G
A
1.5


94925485


R
rs12721568
A/G
A
3.5


94925915
SG12S33
LTA4H_33505
Y
rs2660895
C/T
C
30.71


94925979


R
rs11611612
A/G


94926097


S
rs7312303
C/G


94926250


Y
rs2660896
C/T


94926316


Y
rs12308731
C/T


94926590
SG12S34
LTA4H_34180
Y
rs2247330
C/T
C
30.9


94926724
SG12S102
LTA4H_34314
R
rs2247323
A/G
G
31.85


94926915
SG12S103
LTA4H_34505
Y
rs2247313
C/T
T
32.74


94927010
SG12S104
LTA4H_34600
Y
rs2247309
C/T
C
32.74


94927133
SG12S27
LTA4H_34723
Y
rs2247304
C/T
C
25.57


94927140


M
rs12831920
A/C


94927693


M
rs7134753
A/C


94927696


W
rs7134754
A/T


94927900
SG12S35
LTA4H_35490
R
rs2660897
A/G
A
35.93


94927959
SG12S105
LTA4H_35549
Y
rs11108381
C/T
T
2.4


94928171


Y
rs7956370
C/T
T
9.2


94928465
SG12S28
LTA4H_36055
K
rs2660898
G/T
G
29.36


94928740
SG12S36
LTA4H_36330
Y
rs2540490
C/T
T
31


94928970
SG12S106
LTA4H_36560
Y
rs2540489
C/T
C
30.89


94929183
SG12S107
LTA4H_36773
Y
rs11108382
C/T
T
2.58


94929213
SG12S108
LTA4H_36803
R
rs2540488
A/G
A
26.28


94929669


Y
rs17025102
C/T
T
2.1


94929761
SG12S109
LTA4H_37351
Y
rs2300557
C/T
T
4.76


94929770
SG12S110
LTA4H_37360
W
rs2246990
A/T
A
28.57


94929936
SG12S111
LTA4H_37526
W
rs2300556
A/T
A
2.81


94930044
SG12S112
LTA4H_37634
M
NN
A/C
C
46.15


94930160


M
rs7298811
A/C
A
8.5


94930343
SG12S43
LTA4H_37933
K
rs2246973
G/T
G
32.93


94930357
SG12S113
LTA4H_37947
Y
rs2246972
C/T
T
33.54


94931246
SG12S114
LTA4H_38836
K
NN
G/T
T
7.55


94931694


K
rs11556466
G/T
T
3.33


94931694


K
rs2228060
G/T
T
2.9


94931845


Y
rs17525495
C/T
T
22


94931906


indel
rs17525488
del/A
\-
20.6


94932251


R
rs2540487
A/G


94932325


Y
rs7304178
C/T


94932351


Y
rs11108383
C/T


94932358


indel
rs3834483
—/A


94932888


K
rs2660899
G/T
T
30.1


94933142


K
rs11108384
G/T


94933194


S
rs10777767
C/G


94933475


S
rs2540486
C/G


94933547


R
rs11108385
A/G


94933939


R
rs2660891
A/G


94934007


indel
rs3834482
—/CCG


94934011


S
rs12581112
C/G


94934104


M
rs3759215
A/C


94934775
SG12S141

R
rs10777768
A/G


94934975
SG12S140

M
rs2660840
A/C
C
29.77


94935547


W
rs7966262
A/T
T
29.7


94935843


S
rs2540485
C/G


94935890


R
rs17025122
A/G
A
12.7


94936032


Y
rs2540484
C/T


94936244


Y
rs2160250
C/T


94936596


R
rs2540483
A/G


94936894


Y
rs2160249
C/T


94936993


Y
rs2110761
C/T


94937017


Y
rs2110760
C/T


94937070


Y
rs2110759
C/T


94937214


R
rs7971150
A/G


94937296
SG12S142

R
rs17025123
A/G
G
9.2


94937348
SG12S143

Y
rs2540482
C/T
C
17.02


94937435


S
rs2540481
C/G


94937520


Y
rs2540480
C/T


94937707


R
rs2540479
A/G


94937911


R
rs2887104
A/G


94937970


M
rs11108386
A/C


94938012


Y
rs11830502
C/T


94938018


R
rs11108387
A/G


94938027


R
rs2367870
A/G


94938134


R
rs2367871
A/G


94938212


R
rs10735339
A/G


94938212


Y
rs2540478
C/T


94938223


K
rs10860000
G/T


94938459


R
rs11615837
A/G


94939121


Y
rs7314867
C/T
T
17


94939817


R
rs10777769
A/G


94940022


Y
rs2540477
C/T


94940156


R
rs7954740
A/G


94940254


Y
rs2660841
C/T


94940371


W
rs2660842
A/T


94940394


R
rs2660843
A/G


94940429


Y
rs2367872
C/T


94940787


M
rs2660844
A/C


94941021
SG12S144

R
rs2660845
A/G
G
19.43


94941109


K
rs7966098
G/T


94941218


indel
rs11318627
del/G


94941392


K
rs11608655
G/T


94941458


R
rs10735340
A/G
G
40.8


94941488


R
rs2660846
A/G


94941741


R
rs11108388
A/G


94942862


Y
rs2660847
C/T


94942863


Y
rs2540476
C/T


94943081


M
rs2660848
A/C


94943193


Y
rs2660849
C/T


94943673


R
rs12424449
A/G


94943761
SG12S221

R
rs2540475
A/G
A
16.92


94944345


Y
rs10860001
C/T
T
19.1


94945277


Y
rs11108389
C/T


94946089
SG12S222

Y
rs2660850
C/T
C
15.47


94946133


R
rs12298458
A/G


94946358


K
rs12823598
G/T


94946381


K
rs12823759
G/T


94946826


R
rs12822954
A/G


94947165


K
rs2660851
G/T


94947830


Y
rs11108390
C/T


94948016
SG12S460

M
rs2660852
A/C
A
37.22


94948279


R
rs1002070
A/G
A
11.7


94948684


M
rs12579486
A/C


94949013


S
rs1123456
C/G


94949092


Y
rs12580856
C/T


94949200


Y
rs11108391
C/T


94949965
SG12S223

Y
rs2660875
C/T
C
43.79


94950315


Y
rs2540474
C/T


94950568
SG12S224

R
rs2540473
A/G
G
6.12


94951292


Y
rs11108392
C/T


94951298


Y
rs12820161
C/T


94951925


M
rs10745748
A/C


94951964


R
rs12425482
A/G
G
46.5


94952099


S
rs10745749
C/G


94952099


S
rs9737905
C/G


94952116


K
rs10745750
G/T


94952116


K
rs9738545
G/T


94952155


R
rs7294638
A/G


94952158


Y
rs7310911
C/T


94952847
SG12S225

R
rs2540472
A/G
A
5.63


94953483
SG12S226

S
rs2540471
C/G
C
37.7


94953648


K
rs7302955
G/T


94953798
SG12S227

R
rs17735342
A/G
G
12.5


94953801
SG12S228

Y
rs2660890
C/T
T
46.96


94953831
SG12S229

M
rs2660889
A/C


94953967


R
rs28374901
A/G


94954155
SG12S230

R
rs2660888
A/G
A
35.68


94954449
SG12S231

Y
rs4762661
C/T


94955089


R
rs7307954
A/G


94955162


R
rs12305408
A/G


94955164


R
rs12305409
A/G


94955310


K
rs2540470
G/T


94955354


R
rs2660887
A/G


94955396


Y
rs12368890
C/T


94955578


indel
rs11463945
del/T


94955864


M
rs17025133
A/C
A
11.3


94956808


R
rs11612148
A/G


94957324


R
rs2540469
A/G


94957363


Y
rs2660886
C/T


94957486


S
rs2540468
C/G


94958156
SG12S232

Y
NN
C/T


94958339
SG12S233

Y
rs2660885
C/T
T
15.18


94959833


M
rs11108393
A/C


94960744


R
rs4762662
A/G


94960854


R
rs4762663
A/G


94961810


K
rs759394
G/T
T
36.9


94962279


S
rs759393
C/G


94962304


R
rs759392
A/G


94962320


R
rs734592
A/G


94962388
SG12S234

R
rs5800242
A/G


94962388


indel
rs5800242
—/G


94962435
SG12S235

Y
rs759391
C/T


94962614


R
rs17025149
A/G
G
10.6


94963320
SG12S236

S
rs2540467
C/G


94963655
SG12S237

Y
rs2540466
C/T
T
37.05


94963774
SG12S238

Y
rs10492225
C/T
C
10.6


94964298
SG12S239

W
rs2660874
A/T


94964443


R
rs2660873
A/G


94964573


M
rs2540465
A/C


94964776


Y
rs12370699
C/T


94964783


W
rs28593423
A/T


94965433


R
rs2540464
A/G
A
37.8


94965472


Y
rs2052257
C/T
C
29.5


94965687


Y
rs12581811
C/T


94965875


W
rs11108394
A/T


94966090


Y
rs2540463
C/T


94966142


Y
rs7301759
C/T


94966155


indel
rs10564012
—/TT


94966160


indel
rs10583879
—/TT


94966184


indel
rs10564013
—/AC


94966209


indel
rs10680979
—/ACAC


94966242


K
rs2540462
G/T


94966584
SG12S240

W
rs2540461
A/T
A
24.1


94948279


R
rs1002070
A/G


94897159


M
rs1050970
A/C
















TABLE 41A





Haplotype association analysis including SNPs and microsatellite


markers across the LTA4H gene.


























DG
SG
SG
SG
SG
SG
SG
SG
SG
SG



12S1664
12S16
12S17
12S18
12S21
12S22
12S23
12S24
12S25
12S26










All MI vs controls


















0
C
A
T
G
G
T
T
A
T


short
0








T


form












 







MI males vs controls


















0
C
A
T
G
G
T
T
A
T


short
0








T


form












 







MI females vs controls


















0
C
A
T
G
G
T
T
A
T


short
0








T


form












 







Recurrent MI vs controls


















0
C
A
T
G
G
T
T
A
T


short
0








T


form





















DG
SG
SG
SG


#
aff.
#
con.



12S1666
12S100
12S28
12S144
p-val
r
aff
frq.
con
frq.












All MI vs controls




















0
T
T
A
1.67E−02
1.24
590
0.49
481
0.44



short
0


A
3.20E−03
1.32
590
0.5
480
0.43



form












 









MI males vs controls




















0
T
T
A
5.10E−03
1.34
361
0.51
481
0.44



short
0


A
1.50E−03
1.4
361
0.51
480
0.43



form












 









MI females vs controls




















0
T
T
A
3.80E−01
1.11
229
0.46
481
0.44



short
0


A
1.35E−01
1.2
229
0.47
480
0.43



form












 









Recurrent MI vs controls




















0
T
T
A
1.50E−02
1.51
88
0.54
481
0.44



short
0


A
2.40E−03
1.69
88
0.56
480
0.43



form
















TABLE 41B







Information on microsatellite markers that were used in the


haplotype association analysis shown in Table 41A.










Marker Name
DG12S1664
DG12S1666
DG12S1668





Chr
12
12
12


Cytoband
q23.1
q23.1
q23.1


Start in SEQ_ID_NO_718 (bp)
7855
38342
86595


NCBI_build33Start (Mb)
96.317853
96.34834
96.396593


Size
238
188
398


CEPH standard
245
193
398


(reference allele)





Polymorphism type
SNP
Microsatellite
Microsatellite


Polymorphism class
in-del
Di
Di


Heterozygosity ratio
0.23
0.52
0.72


Forward primer
GGAAGGAGGACACTTCTGGA
CACAGAAGCTGCAGTGGAAG
GCAGTTTAAGCTGTATGTATATGAGG



(SEQ ID NO: 721)
(SEQ ID NO: 723)
(SEQ ID NO: 725)


Reverse primer
GCTGTGAATGGCTAAACTTGG
CAAATGGAGGAGTCAAGACCA
TGAAAGCCATCACTGTAAGGA



(SEQ ID NO: 722)
(SEQ ID NO: 724)
(SEQ ID NO: 726)
















TABLE 42





Haplotype association analysis including SNPs and microsatellite


markers in the LTA4H gene region.





























SG
DG
SG
SG
SG
SG
SG
DG
SG
SG
SG
SG
SG



12S438
12S1664
12S16
12S21
12S23
12S25
12S26
12S1666
12S100
12S28
12S143
12S144
12S221










All MI vs controls




















Consecutive
C
0
C
G
T
A
T
0
T
T
T
A
G


Short
C
0













version















Protective
C

C



T



T




variant















 







MI males vs controls




















Consecutive
C
0
C
G
T
A
T
0
T
T
T
A
G


Short
C
0













version















Protective
C

C



T



T




variant















 







MI females vs controls




















Consecutive
C
0
C
G
T
A
T
0
T
T
T
A
G


Short
C
0













version















Protective
C

C



T



T




variant















 







Recurrent MI vs controls




















Consecutive
C
0
C
G
T
A
T
0
T
T
T
A
G


Short
C
0













version















Protective
C

C



T



T




variant















 







MI plus stroke or PAOD vs controls




















Consecutive
C
0
C
G
T
A
T
0
T
T
T
A
G


Short
C
0













version















Protective
C

C



T



T




variant

























SG
SG
SG
SG
SG
SG
DG

P-val

#
aff.
#
con.



12S222
12S223
12S225
12S226
12S233
12S237
12S1668
p-val
adj.
r
aff
frq.
con
frq.












All MI vs controls























Consecutive
C
C
G
G
C
T
0
6.2E−02

1.34
1560
0.051
953
0.039



Short
C


G


0
1.5E−03

1.63
1556
0.071
951
0.045



version

















Protective





C

7.5E−02

0.88
1557
0.290
951
0.317



variant
















 









MI males vs controls























Consecutive
C
C
G
G
C
T
0
2.2E−02

1.49
1096
0.051
953
0.035



Short
C


G


0
3.1E−03

1.66
1093
0.069
951
0.043



version

















Protective





C

6.3E−02

0.86
1094
0.283
951
0.314



variant
















 









MI females vs controls























Consecutive
C
C
G
G
C
T
0
4.3E−01

1.19
 464
0.046
953
0.039



Short
C


G


0
1.6E−02

1.60
 463
0.073
951
0.047



version

















Protective





C

3.1E−01

0.91
 463
0.301
951
0.322



variant
















 









Recurrent MI vs controls























Consecutive
C
C
G
G
C
T
0
7.7E−02

1.52
 273
0.060
953
0.040



Short
C


G


0
7.5E−02

1.54
 272
0.067
951
0.045



version

















Protective





C

9.8E−02

0.82
 273
0.274
951
0.316



variant
















 









MI plus stroke or PAOD vs controls























Consecutive
C
C
G
G
C
T
0
1.5E−03
0.007
1.97
 325
0.073
953
0.038



Short
C


G


0
2.4E−05
0.038
2.39
 325
0.099
951
0.044



version

















Protective





C

4.1E−05

0.61
 325
0.220
951
0.315



variant





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 June 1998 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 obtained 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 43





PCR Parameters


















RT Reaction





TaqMan RT Buffer
1X


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 Reverse
1.25 U/uL


Transcriptase


RNA
2 ng/uL



50 uL Reaction Volume


PCR Reaction


TaqMan Universal Master
1X
95° C.
10′


Mix









TaqManAssay (20X)
1X
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 1 deltaCT between duplicates were not used in our analysis. Quantity was obtained using the formula 2^-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<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 44





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. 11, left panel. All haplotypes with greater than 1% allelic frequency that were found in the Icelandic cohort are shown in FIG. 11, right panel.
Subjects from Iceland
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 described2. 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 changes30. 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 described31.
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 45. One SNP or SG12S100 (rs2247570) showed nominally significant association to MI (Table 46). 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 46).
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







TABLE 45







SNP allelic association to MI

















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 46







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


ethnicity
SG12S100
T
0.671
0.69
# 70
84
0.92
NS



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 47 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 48, 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 46: 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.










47a. Iceland-Caucasians (1555 patients/863 controls) Patients = All MI



















SG
SG
SG
SG
SG
SG
SG
SG
SG
SG

Frequency






















12S16
12S21
12S23
12S25
12S26
12S100
12S28
12S143
12S144
12S221
P
RR
Patients
Controls
Name





C
G
T
A
T
T
T
T
A
G
2.7E−01
1.07
0.497
0.480
HapL


 


C
G
T
A
T
T
T
C
G
G
3.8E−01
1.09
0.112
0.104
HapK


 


T
G
T
G
C
C
G
T
A
A
2.3E−01
1.15
0.090
0.079



 


C
G
C
G
T
T
G
T
A
G
5.8E−01
1.07
0.064
0.060



 


T
G
T
G
C
C
G
C
G
G
9.2E−01
0.99
0.058
0.059



 


T
A
T
G
T
C
G
T
A
A
4.7E−01
0.90
0.048
0.053



 


C
G
T
G
T
C
T
T
G
G
2.9E−02
0.70
0.031
0.044



 


T
G
T
G
C
C
G
T
A
G
1.0E−01
0.71
0.022
0.031



 


C
G
T
A
T
T
T
T
A
A
7.4E−01
0.92
0.018
0.019



 


C
G
T
G
T
C
T
T
A
G
1.1E−01
0.61
0.008
0.013
HapQ


 


C
A
T
G
T
C
G
T
A
A
9.1E−02
0.57
0.006
0.011



 


C
G
C
G
T
T
G
T
A
A
7.0E−01
1.13
0.012
0.011




















47b. Iceland-Caucasians (325 patients/863 controls) Patients = MI with additional CVD



















SG
SG
SG
SG
SG
SG
SG
SG
SG
SG

Frequency






















12S16
12S21
12S23
12S25
12S26
12S100
12S28
12S143
12S144
12S221
P
RR
Patients
Controls
Name





C
G
T
A
T
T
T
T
A
G
8.7E−01
0.98
0.477
0.480
HapL


 


C
G
T
A
T
T
T
C
G
G
9.1E−03
1.45
0.145
0.104
HapK


 


T
G
T
G
C
C
G
T
A
A
5.5E−01
1.11
0.086
0.079



 


C
G
C
G
T
T
G
T
A
G
8.6E−01
0.97
0.058
0.060



 


T
G
T
G
C
C
G
C
G
G
5.4E−01
0.88
0.051
0.059



 


T
A
T
G
T
C
G
T
A
A
8.9E−01
0.97
0.051
0.053



 


C
G
T
G
T
C
T
T
G
G
8.8E−03
0.48
0.021
0.044



 


T
G
T
G
C
C
G
T
A
G
9.6E−01
0.99
0.032
0.031



 


C
G
T
A
T
T
T
T
A
A
1.4E−01
0.51
0.010
0.019



 


C
G
T
G
T
C
T
T
A
G
4.1E−01
0.67
0.009
0.013
HapQ


 


C
A
T
G
T
C
G
T
A
A
2.6E−01
0.54
0.006
0.011



 


C
G
C
G
T
T
G
T
A
A
9.5E−01
0.97
0.011
0.011




















47c. Philadelphia-Caucasians (731 patients/434 controls)



















SG
SG
SG
SG
SG
SG
SG
SG
SG
SG

Frequency






















12S16
12S21
12S23
12S25
12S26
12S100
12S28
12S143
12S144
12S221
P
RR
Patients
Controls
Name





C
G
T
A
T
T
T
T
A
G
4.5E−02
0.83
0.363
0.406
HapL


 


C
G
T
A
T
T
T
C
G
G
1.5E−02
1.34
0.185
0.145
HapK


 


T
G
T
G
C
C
G
T
A
A
3.7E−01
0.88
0.098
0.110



 


C
G
C
G
T
T
G
T
A
G
8.9E−02
0.72
0.048
0.065



 


T
G
T
G
C
C
G
C
G
G
7.5E−01
0.94
0.048
0.051



 


T
A
T
G
T
C
G
T
A
A
3.3E−01
1.21
0.060
0.050



 


C
G
T
A
T
T
T
T
A
A
5.9E−01
1.17
0.030
0.026



 


C
G
C
G
T
T
G
T
A
A
1.3E−01
0.59
0.013
0.022



 


C
G
T
G
T
C
T
T
G
G
7.6E−01
1.10
0.023
0.021



 


T
G
T
G
C
C
G
T
A
G
8.8E−01
0.94
0.018
0.019



 


C
G
T
G
T
C
T
T
A
G
8.3E−02
1.75
0.027
0.016
HapQ


 


C
G
T
G
T
T
T
T
A
G
1.2E−01
0.47
0.007
0.014



 


C
G
T
G
T
T
T
T
G
G
3.7E−01
1.41
0.017
0.012




















47d. Cleveland-Caucasians (614 patients/583 controls)



















SG
SG
SG
SG
SG
SG
SG
SG
SG
SG

Frequency






















12S16
12S21
12S23
12S25
12S26
12S100
12S28
12S143
12S144
12S221
P
RR
Patients
Controls
Name





C
G
T
A
T
T
T
T
A
G
7.4E−01
0.97
0.380
0.386
HapL


 


C
G
T
A
T
T
T
C
G
G
8.8E−01
1.02
0.163
0.160
HapK


 


T
G
T
G
C
C
G
T
A
A
4.2E−01
1.12
0.100
0.091



 


C
G
C
G
T
T
G
T
A
G
7.9E−01
1.05
0.064
0.061



 


T
G
T
G
C
C
G
C
G
G
8.7E−01
0.97
0.057
0.058



 


T
A
T
G
T
C
G
T
A
A
7.8E−01
1.05
0.053
0.050



 


C
G
T
G
T
C
T
T
G
G
6.7E−01
0.90
0.030
0.033



 


C
G
T
G
T
C
T
T
A
G
9.8E−01
1.01
0.023
0.023
HapQ


 


C
G
T
A
T
T
T
T
A
A
9.1E−01
0.97
0.021
0.022



 


C
G
C
G
T
T
G
T
A
A
6.7E−01
0.86
0.016
0.019



 


T
G
T
G
C
C
G
T
A
G
6.5E−01
1.17
0.021
0.018



 


C
G
T
G
T
T
T
T
G
G
8.2E−01
0.92
0.013
0.014



 


C
G
T
A
T
T
T
C
A
G
3.9E−01
0.64
0.007
0.010




















47e. Philadelphia- Other or unknown ethnicity (73 patients/86 controls)



















SG
SG
SG
SG
SG
SG
SG
SG
SG
SG

Frequency






















12S16
12S21
12S23
12S25
12S26
12S100
12S28
12S143
12S144
12S221
P
RR
Patients
Controls
Name





C
G
T
A
T
T
T
T
A
G
7.7E−02
0.64
0.274
0.370
HapL


 


C
G
T
A
T
T
T
C
G
G
1.1E−02
2.04
0.283
0.162
HapK


 


C
G
T
G
T
C
T
T
A
G
3.2E−01
0.59
0.045
0.074
HapQ


 


T
A
T
G
T
C
G
T
A
A
2.5E−01
0.51
0.027
0.052



 


T
G
T
G
C
C
G
T
A
A
6.7E−01
1.24
0.059
0.048



 


C
G
C
G
T
T
G
T
A
G
9.0E−01
0.93
0.041
0.044



 


C
G
T
G
T
T
T
T
A
G
5.7E−01
0.69
0.027
0.039



 


C
G
T
G
T
C
T
T
G
G
1.8E−01
2.22
0.058
0.027



 


T
G
T
G
C
C
G
T
A
G
8.7E−01
0.84
0.021
0.025



 


C
G
C
G
T
C
G
C
G
G
9.4E−01
0.94
0.020
0.021



 


T
G
T
G
C
C
G
C
G
G
5.7E−02
3.88
0.063
0.017



 


C
G
C
G
T
T
G
T
A
A
1.8E−01
2.65
0.041
0.016



 


C
G
T
G
T
T
T
T
G
G
9.1E−02
0.00
0.000
0.014



 


T
G
T
G
T
T
T
C
G
G
1.2E−01
0.00
0.000
0.012



 


T
G
T
G
C
C
G
C
G
A
2.6E−01
0.00
0.000
0.010




















47f. Philadelphia-African Americans (103 patients/128 controls)



















SG
SG
SG
SG
SG
SG
SG
SG
SG
SG

Frequency






















12S16
12S21
12S23
12S25
12S26
12S100
12S28
12S143
12S144
12S221
P
RR
Patients
Controls
Name





C
G
T
G
T
C
T
T
A
G
3.4E−02
0.53
0.119
0.202
HapQ


 


C
G
T
G
T
T
T
T
A
G
4.8E−02
0.55
0.110
0.183



 


C
G
T
A
T
T
T
T
A
G
2.5E−02
1.77
0.235
0.148
HapL


 


C
G
C
G
T
T
G
T
A
G
4.7E−01
1.38
0.073
0.054



 


C
G
T
G
T
C
T
T
G
G
4.8E−01
1.34
0.070
0.054



 


C
G
T
G
T
C
T
C
G
G
1.9E−01
0.29
0.013
0.042



 


C
G
C
G
T
C
G
C
G
G
5.3E−01
0.65
0.026
0.039



 


C
G
T
A
T
C
T
C
G
A
5.4E−01
0.70
0.026
0.036



 


C
G
T
G
T
C
T
T
A
A
6.1E−02
0.18
0.006
0.033



 


C
G
C
G
T
C
G
T
A
G
3.6E−01
0.48
0.014
0.029



 


C
G
C
G
T
T
G
C
G
G
3.9E−01
0.48
0.014
0.029



 


C
G
T
A
T
T
T
T
A
A
9.0E−01
0.90
0.020
0.022



 


C
G
T
A
T
T
T
C
G
G
4.9E−04
5.48
0.105
0.021
HapK


 


C
G
T
G
T
T
T
C
G
G
5.9E−01
1.46
0.028
0.019



 


T
G
T
G
C
C
G
T
A
G
7.0E−01
0.70
0.012
0.012



 


C
G
T
A
T
T
T
C
G
A
7.6E−01
1.42
0.018
0.018



 


T
G
T
G
C
C
G
C
G
G
7.7E−01
0.74
0.009
0.009



 


C
G
T
G
T
T
T
T
G
G
3.0E−01
0.00
0.000
0.000



 


T
G
T
G
C
C
G
T
A
A
5.1E−01
0.17
0.002
0.002




















47g. Cleveland-African Americans (57patients/90controls)




















SG
SG
SG
SG
SG
SG
SG
SG
SG
SG

Frequency























12S16
12S21
12S23
12S25
12S26
12S100
12S28
12S143
12S144
12S221
P
RR
Patients
Controls
Name

























C
G
T
G
T
C
T
T
A
G
7.3E−02
0.51
0.116
0.206
HapQ



 


C
G
T
A
T
T
T
T
A
G
2.5E−01
1.51
0.196
0.140
HapL


 


C
G
T
G
T
T
T
T
A
G
8.3E−01
0.91
0.096
0.105



 


C
G
T
G
T
C
T
T
G
G
5.6E−02
0.33
0.031
0.087


 


C
G
T
G
T
T
T
C
G
G
4.4E−01
0.58
0.038
0.064


 


C
G
T
A
T
T
T
C
G
G
2.1E−02
2.89
0.153
0.059
HapK


 


C
G
C
G
T
T
G
T
A
G
1.9E−01
1.97
0.101
0.054



 


C
G
C
G
T
C
G
T
A
G
2.6E−01
1.92
0.083
0.045


 


C
G
T
G
T
C
T
C
G
G
8.8E−01
0.88
0.037
0.041


 


C
G
T
A
T
T
T
C
G
A
2.1E−02
0.00
0.000
0.041


 


T
G
T
G
C
C
G
T
A
A
4.0E−01
0.52
0.018
0.033


 


C
G
T
G
T
T
T
T
A
A
1.5E−01
0.00
0.000
0.014


 


C
G
C
G
T
C
G
C
G
G
4.9E−01
2.02
0.029
0.014


 


C
G
T
A
T
C
T
C
G
A
1.9E−01
0.00
0.000
0.013


 


C
G
T
A
T
T
T
T
A
A
5.1E−01
2.35
0.023
0.010


 


C
G
T
G
T
C
T
T
A
A
3.9E−01
3.26
0.031
0.010
















TABLE 48







The association of HapK to MI










Frequency












Cohorts (n)
Patients
Controls
RR
P-value





Iceland






Caucasians, all MI (1555/863)
0.112
0.104
1.09
NS


Early onset (725/863)
0.114
0.104
1.10
NS


MI and additional CVD (325/863)
0.145
0.104
1.45
0.0091


Philadelphia


Caucasians, all MI (731/434)
0.185
0.145
1.34
0.015


African Americans, all MI
0.105
0.021
5.28
0.00049


(103/128)


Other or undefined ethnicity, all
0.283
0.162
2.04
0.011


MI (73/86)


Cleveland


Caucasians, all MI (614/583)
0.163
0.160
1.02
NS


MI and additional CVD (147/601)
0.183
0.160
1.20
NS


African Americans, all MI (57/90)
0.153
0.059
2.89
0.021









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 45 (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 46 (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 45 (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 SG12S143 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 46). 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 47 (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 47.
Table 48 shows haplotype association results for the Icelandic at-risk haplotype HapK in the US cohorts. As shown in Table 48, 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 49). 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 48). 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 48).
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 47). 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 47).
Given the distinct haplotype diversity of African Americans, additional haplotypes for association to MI in this ethnic group were tested. Table 49 shows two haplotypes that in addition to HapK show association to MI in the African American cohorts. As shown in Table 49, 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 49, 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 49).
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 47). 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 47).







TABLE 49







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.
EXAMPLE 20
HapK is Associated with More Severe MI Phenotypes
Ten SNPs were tested for association to MI using 1553 MI cases and 863 population-based controls. No single SNP or haplotype defined by the ten SNPs were found to be significantly more common in all MI patients than in controls (Tables 49 and 50). Subsequently, HapK association to more severe MI phenotypes, i.e. early onset MI, or MI with other cardiovascular diseases (CVD), including peripheral vascular disease and/or stroke were tested. Early onset MI did not show significant association to any of the haplotypes. However, MI with additional CVD yielded an association to a HapK (Table 51). The frequency of HapK in MI patients that had additional CVD and in controls was 14.5% and 10.4%, respectively, which corresponds to a RR of 1.45 (P=0.0091) for each copy of HapK carried. This association remained significant (P=0.035) after adjusting for the number of haplotypes tested.







TABLE 50







SNP allelic association to MI










European Americans
African Americans














SNP (variation)
Iceland
Philadelphia
Cleveland
Atlanta
Philadelphia
Cleveland
Atlanta





SG12S16 (T/C)









Frq. in patients (n)
0.233 (1515)
0.239 (545)
0.250 (619)
0.274 (234)
0.043 (82)
0.019 (52)
0.013 (39)


Frq. in controls (n)
0.233 (807)
0.238 (332)
0.239 (782)
0.253 (419)
0.062 (97)
0.045 (111)
0.042 (120)


RR/P-value
1.00/0.98
1.01/0.94
1.06/0.49
1.11/0.42
0.68/0.42
0.42/0.22
0.30/0.18


rs2660880 (A/G)


Frq. in patients (n)
0.062 (1505)
0.073 (701)
0.066 (599)
0.045 (223)
0.020 (99)
0.010 (49)
0.000 (38)


Frq. in controls (n)
0.071 (829)
0.061 (418)
0.070 (768)
0.056 (516)
0.012 (123)
0.005 (108)
0.015 (136)


RR/P-value
0.87/0.25
1.22/0.26
0.93/0.65
0.79/0.36
1.67/0.50
2.22/0.58
0.00/0.16


rs6538697 (T/C)


Frq. in patients (n)
0.926 (1425)
0.933 (711)
0.915 (610)
0.942 (233)
0.847 (101)
0.765 (51)
0.859 (39)


Frq. in controls (n)
0.928 (822)
0.910 (422)
0.914 (776)
0.916 (528)
0.848 (125)
0.849 (109)
0.826 (144)


RR/P-value
0.98/0.84
1.38/0.045
1.01/0.97
1.50/0.068
0.99/0.97
0.58/0.073
1.28/0.49


rs1978331 (A/G)


Frq. in patients (n)
0.635 (1344)
0.605 (715)
0.583 (617)
0.606 (227)
0.373 (102)
0.340 (50)
0.294 (34)


Frq. in controls (n)
0.613 (815)
0.598 (419)
0.588 (782)
0.596 (537)
0.240 (125)
0.266 (109)
0.219 (146)


RR/P-value
1.10/0.15
1.03/0.74
0.98/0.80
1.04/0.72
1.88/0.002
1.42/0.18
1.48/0.20


rs17677715 (T/C)


Frq. in patients (n)
0.830 (1436)
0.832 (695)
0.814 (619)
0.776 (230)
0.965 (99)
0.990 (52)
0.986 (35)


Frq. in controls (n)
0.827 (812)
0.812 (413)
0.824 (780)
0.803 (539)
0.959 (121)
0.959 (110)
0.952 (146)


RR/P-value
1.02/0.81
1.15/0.22
0.94/0.52
0.85/0.23
1.18/0.75
4.39/0.094
3.47/0.16


rs2247570 (T/C)


Frq. in patients (n)
0.723 (1496)
0.700 (686)
0.691 (595)
0.676 (236)
0.622 (98)
0.649 (47)
0.462 (39)


Frq. in controls (n)
0.696 (805)
0.710 (405)
0.696 (759)
0.698 (497)
0.525 (122)
0.523 (107)
0.475 (140)


RR/P-value
1.14/0.052
0.95/0.62
0.97/0.76
0.90/0.39
1.49/0.039
1.68/0.040
0.95/0.83


rs2660898 (T/G)


Frq. in patients (n)
0.694 (1374)
0.686 (708)
0.663 (616)
0.665 (233)
0.797 (101)
0.735 (51)
0.842 (38)


Frq. in controls (n)
0.678 (800)
0.664 (421)
0.665 (785)
0.654 (515)
0.798 (124)
0.806 (111)
0.755 (141)


RR/P-value
1.07/0.29
1.10/0.28
0.99/0.92
1.05/0.68
0.99/0.97
0.67/0.15
1.73/0.098


rs2540482 (C/T)


Frq. in patients (n)
0.174 (1472)
0.260 (713)
0.249 (606)
0.244 (232)
0.299 (102)
0.265 (49)
0.303 (33)


Frq. in controls (n)
0.173 (833)
0.208 (420)
0.232 (762)
0.235 (531)
0.230 (124)
0.303 (109)
0.241 (147)


RR/P-value
1.00/0.95
1.34/0.005
1.10/0.29
1.05/0.73
1.43/0.096
0.83/0.47
1.37/0.31


rs2660845 (G/A)


Frq. in patients (n)
0.210 (1414)
0.298 (694)
0.292 (577)
0.273 (225)
0.345 (97)
0.357 (49)
0.329 (38)


Frq. in controls (n)
0.214 (807)
0.248 (412)
0.286 (753)
0.257 (514)
0.298 (124)
0.379 (103)
0.301 (138)


RR/P-value
0.98/0.76
1.29/0.010
1.03/0.74
1.09/0.51
1.24/0.29
0.91/0.72
1.14/0.64


rs2540475 (G/A)


Frq. in patients (n)
0.822 (1438)
0.777 (716)
0.790 (616)
0.794 (223)
0.883 (98)
0.882 (51)
0.857 (35)


Frq. in controls (n)
0.821 (773)
0.770 (424)
0.793 (786)
0.802 (534)
0.864 (125)
0.858 (109)
0.854 (144)


RR/P-value
1.00/0.99
1.04/0.72
0.98/0.82
0.95/0.70
1.31/0.36
1.24/0.54
0.95/1.02









Shown in Table 50 are the genotyped SNPs (variation shown in brackets) and the frequency of the underlined allele, in the Icelandic, and the European and African American cohorts. 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. A public name for SNP SG12S16 is not available. The basepair position on chromosome 12 for this SNPs in the human genome assembly build 34 (National Center for Biotechnology Information (NCBI)) is 94896055.







TABLE 51







Frequency of haplotypes derived from HapK SNPs










Philadelphia:












Iceland
European














All MI
MI patients
Americans



Ctrl
patients
with 2CVD
Ctrl



(863)
(1553)
(325)
(430)















Haplotype
Frq
Frq
RR
P-val
Frq
RR
P-val
Frq



















CATGTCGTAA
1.1%
0.6%
0.57
0.094
0.6%
0.54
0.261
—



 


CGCGTCGCAG
—
—
—
—
—
—
—
—


 


CGCGTCGCGG
—
—
—
—
—
—
—
—


 


CGCGTCGTAG
—
—
—
—
—
—
—
—


 


CGCGTTGCAG
—
—
—
—
—
—
—
—


 


CGCGTTGCGG
—
—
—
—
—
—
—
—


 


CGCGTTGTAA
1.1%
1.1%
1.02
0.953
1.1%
0.97
0.947
2.2%


 


CGCGTTGTAG
6.0%
6.5%
1.09
0.519
5.8%
0.97
0.864
6.5%


 


CGCGTTGTGG
—
—
—
—
—
—
—
—


 


CGCGTTTTAA
—
—
—
—
—
—
—
—


 


CGTATCTCGA
—
—
—
—
—
—
—
—


 


CGTATCTCGG
—
—
—
—
—
—
—
—


 


CGTATCTTAG
—
—
—
—
—
—
—
—


 


CGTATTGCGG
—
—
—
—
—
—
—
—


 


CGTATTGTAG
—
—
—
—
—
—
—
—


 


CGTATTTCAG
—
—
—
—
—
—
—
0.4%


 


CGTATTTCGA
—
—
—
—
—
—
—
—


 


CGTATTTCGG
10.4%
11.3%
1.10
0.357
14.5%
1.45
0.009
14.3%


 


CGTATTTTAA
1.9%
1.7%
0.87
0.594
1.0%
0.51
0.141
2.6%


 


CGTATTTTAG
48.0%
49.7%
1.07
0.274
47.7%
0.98
0.871
40.6%


 


CGTGTCGTAG
—
—
—
—
—
—
—
—


 


CGTGTCGTGG
—
—
—
—
—
—
—
—


 


CGTGTCTCAG
—
—
—
—
—
—
—
—


 


CGTGTCTCGA
—
—
—
—
—
—
—
—


 


CGTGTCTCGG
—
—
—
—
—
—
—
—


 


CGTGTCTTAA
—
—
—
—
—
—
—
—


 


CGTGTCTTAG
1.3%
0.8%
0.61
0.118
0.9%
0.67
0.407
1.6%


 


CGTGTCTTGA
—
—
—
—
—
—
—
—


 


CGTGTCTTGG
4.4%
3.0%
0.68
0.018
2.1%
0.48
0.009
1.9%


 


CGTGTTGTAA
—
—
—
—
—
—
—
—


 


CGTGTTTCAG
—
—
—
—
—
—
—
—


 


CGTGTTTCGA
—
—
—
—
—
—
—
—


 


CGTGTTTCGG
—
—
—
—
—
—
—
—


 


CGTGTTTTAA
—
—
—
—
—
—
—
—


 


CGTGTTTTAG
—
—
—
—
—
—
—
1.5%


 


CGTGTTTTGG
0.4%
1.0%
2.29
0.034
1.1%
2.55
0.093
1.2%


 


TATGTCGTAA
5.3%
4.7%
0.89
0.404
5.1%
0.97
0.891
4.9%


 


TATGTCGTAG
0.7%
0.7%
1.04
0.927
1.4%
2.06
0.153
—


 


TGTGCCGCGG
5.9%
5.6%
0.95
0.730
5.1%
0.88
0.537
5.0%


 


TGTGCCGTAA
7.9%
9.2%
1.18
0.139
8.6%
1.11
0.555
11.1%


 


TGTGCCGTAG
3.1%
2.4%
0.74
0.159
3.2%
0.99
0.963
1.9%














Philadelphia:
Philadelphia:




European
African-










Americans
Americans












All MI

All MI




patients
Ctrl
patients



(728)
(127)
(105)
















Haplotype
Frq
RR
P-val
Frq
Frq
RR
P-val





















CATGTCGTAA
—
—
—
—
—
—
—




 



CGCGTCGCAG
—
—
—
—
—
—
—



 



CGCGTCGCGG
—
—
—
4.8%
2.8%
0.56
0.368



 



CGCGTCGTAG
—
—
—
3.0%
1.8%
0.58
0.522



 



CGCGTTGCAG
—
—
—
—
—
—
—



 



CGCGTTGCGG
—
—
—
3.5%
0.0%
0.00
0.033



 



CGCGTTGTAA
1.3%
0.58
0.125
0.2%
1.3%
7.64
0.393



 



CGCGTTGTAG
4.8%
0.72
0.093
5.1%
7.7%
1.55
0.366



 



CGCGTTGTGG
—
—
—
—
—
—
—



 



CGCGTTTTAA
—
—
—
—
—
—
—



 



CGTATCTCGA
—
—
—
3.4%
2.2%
0.63
0.470



 



CGTATCTCGG
—
—
—
—
—
—
—



 



CGTATCTTAG
—
—
—
—
—
—
—



 



CGTATTGCGG
—
—
—
—
—
—
—



 



CGTATTGTAG
—
—
—
—
—
—
—



 



CGTATTTCAG
1.1%
2.89
0.080
—
—
—
—



 



CGTATTTCGA
—
—
—
1.4%
1.4%
1.01
0.994



 



CGTATTTCGG
18.6%
1.37
0.010
1.7%
10.3%
6.50
0.00013



 



CGTATTTTAA
3.0%
1.19
0.549
2.2%
1.5%
0.69
0.704



 



CGTATTTTAG
36.4%
0.84
0.049
14.4%
22.7%
1.74
0.030



 



CGTGTCGTAG
—
—
—
—
—
—
—



 



CGTGTCGTGG
—
—
—
—
—
—
—



 



CGTGTCTCAG
—
—
—
0.5%
1.4%
2.79
0.391



 



CGTGTCTCGA
—
—
—
—
—
—
—



 



CGTGTCTCGG
—
—
—
3.6%
2.9%
0.79
0.757



 



CGTGTCTTAA
—
—
—
3.5%
0.6%
0.18
0.049



 



CGTGTCTTAG
2.7%
1.75
0.085
19.9%
10.5%
0.48
0.014



 



CGTGTCTTGA
—
—
—
—
—
—
—



 



CGTGTCTTGG
2.3%
1.24
0.493
6.3%
6.9%
1.10
0.814



 



CGTGTTGTAA
—
—
—
—
—
—
—



 



CGTGTTTCAG
—
—
—
0.0%
2.5%
17338
0.009



 



CGTGTTTCGA
—
—
—
—
—
—
—



 



CGTGTTTCGG
—
—
—
2.1%
3.7%
1.84
0.504



 



CGTGTTTTAA
—
—
—
—
—
—
—



 



CGTGTTTTAG
0.7%
0.47
0.111
18.1%
10.7%
0.54
0.045



 



CGTGTTTTGG
1.7%
1.40
0.370
1.1%
0.0%
0.00
0.194



 



TATGTCGTAA
6.0%
1.23
0.289
0.8%
1.9%
2.47
0.282



 



TATGTCGTAG
—
—
—
—
—
—
—



 



TGTGCCGCGG
4.7%
0.93
0.724
1.2%
1.4%
1.19
0.848



 



TGTGCCGTAA
9.8%
0.87
0.322
1.1%
0.1%
0.13
0.452



 



TGTGCCGTAG
1.8%
0.92
0.829
1.7%
1.2%
0.73
0.732










Table 51 shows are all observed haplotypes with frequency greater than 1% in the population controls for each indicated cohort, and the allelic frequency (Frq) in patients and controls (Ctrl). Also shown is the relative risk (RR) and the two sided P-value for the association to MI. The SNPs defining the haplotypes are SG12S16, rs2660880, rs6538697, rs1978331, rs17677715, rs2247570, rs2660898, rs2540482, rs2660845, and rs2540475, respectively. HapK is colored red. All SNPs are reported on the plus strand.
Table 52 shows the frequency of HapK in patients and controls, with the corresponding numbers (n) of subjects (patients/controls), the relative risk (RR), and the P-values, in the Icelandic and American cohorts. The results are shown for European Americans and African Americans, defined by their self reported ethnicity (sre). Within each self-reported group, results are shown for those who have a genetically detected ancestry (gda) of at least 20% European and African, respectively. Further, results adjusted for admixed ancestry in each self-reported group are shown. MI and additional CVD refer to those MI patients who also have either peripheral vascular disease or who have suffered a stroke. Previous history of stroke or peripheral vascular disease was not available for the subjects from Philadelphia. The P-values are two-sided for Icelanders, but one-sided in all the other cases since we are specifically testing the excess of HapK in patients relative to controls:







TABLE 52







The Association of HapK to MI










Frequency of HapK












Cohorts (n)
Patients
Controls
RR
P-value














Icelanders






All MI (1553/863)
0.113
0.104
1.1
0.36


MI and additional CVD
0.145
0.104
1.45
0.0091


(325/863)


European Americans


Philadelphia


All MI sre (728/430)
0.186
0.143
1.37
0.0051


All MI gda (724/430)
0.186
0.143
1.37
0.0051


All MI admix adj


1.36
0.0048


Cleveland


All MI sre (627/792)
0.166
0.151
1.12
0.15


All MI gda (626/792)
0.166
0.151
1.11
0.16


All MI admix adj


1.12
0.15


MI and additional CVD sre
0.193
0.151
1.34
0.046


(144/792)


MI and additional CVD admix


1.34
0.044


adj


Atlanta


All MI sre (236/553)
0.135
0.143
0.94
0.64


All MI gda (236/553)
0.135
0.143
0.94
0.64


All MI admix adj


0.94
0.63


MI and additional CVD sre
0.173
0.143
1.25
0.25


(39/553)


MI and additional CVD admix


1.24
0.26


adj


African Americans


Philadelphia


All MI sre (105/127)
0.103
0.017
6.5
0.000067


All MI gda (100/126)
0.104
0.018
6.45
0.000088


All MI admix adj


6.34
0.00010


Cleveland


All MI sre (53/111)
0.122
0.072
1.78
0.11


All MI gda (52/111)
0.112
0.072
1.61
0.17


All MI admix adj


1.75
0.11


MI and additional CVD sre
0.152
0.072
2.31
0.14


(13/111)


MI and additional CVD admix


2.27
0.16


adj


Atlanta


All MI sre (39/149)
0.075
0.015
5.21
0.018


All MI gda (38/146)
0.071
0.016
4.71
0.025


All MI admix adj


5.08
0.019


MI and additional CVD sre
0.202
0.015
16.36
0.0039


(8/149)


MI and additional CVD admix


16.67
0.0035


adj









EXAMPLE 21
HapK Identifies Ethnic-specific Higher Risk of Myocardial Infarction
Further analysis of the replication study presented in Example 18 on three independent MI cohorts from the United States revealed that HapK is associated with high risk of MI in African American subjects. All three cohorts contained both self-reported European Americans and African Americans (see Table 52 above for breakdown), who were analyzed separately. Table 52 shows association results for HapK, in each of the US cohorts. P-values reported for all the replication analyses are one-sided, as we tested only HapK for increased risk. An excess of HapK was detected in European American patients from Philadelphia (RR=1.37, P=0.0051) and Cleveland (RR=1.12, NS), but not from Atlanta (Table 52). Table 53 shows that an association of HapK to MI in European Americans was replicated both when the three cohorts were simply combined (RR=1.19, P=0.006) and when a Mantel-Haenszel-like analysis (Falush et al., Genetics 164, 1567-87, 2003) was performed to adjust for differences in HapK frequency between controls in the three cohorts (RR=1.16, P=0.019). Interestingly, as in Iceland, the risk of HapK was greater in the subgroup of MI patients that had a history of stroke or peripheral vascular disease (Table 52), with the combined cohort adjusted analysis yielding a RR of 1.31 (P=0.037, Table 53).
The results displayed in Table 53 describe the association of HapK to MI in combined groups of self-reported European and African Americans respectively, from Philadelphia, Cleveland, and Atlanta. The haplotype frequencies, the resulting relative risk (RR), and the P-values are shown, first, without any population adjustment, second, adjusting for different cohort or population frequencies (coh adj), and third, adjusting for the admixture of African and European ancestries within each ethnic group (admix adj). All P-values are one-sided. PAR is the population attributable risk. For the severe phenotype MI and additional CVD, the data for only the Cleveland and Atlanta cohorts were combined, as this information was not available for the subjects from Philadelphia. For Icelanders and the European Americans, instead of using all 10 SNPs of HapK, essentially the same association results could be obtained using 5 SNPs: SG12S25 (rs1978331), SG12S26 (rs17677715), SG12S143 (rs2540482), SG12S144 (rs2660845) and SG12S221 (rs2540475). For the African American Americans, because of the complications of admixture, in addition to the 5 SNPs mentioned it is preferred to add another 2 SNPs: SG12S100(rs2247570) and SG12S25 (rs2660898) to measure HapK







TABLE 53







The association of HapK to MI in combined American cohorts










Frequency of HapK













Ethnic groups (n)
Patients
Controls
RR (95% CI)
P-value
PAR















European Americans







All MI (1591/1775)
0.171
0.148
1.19 (1.04, 1.36)
0.006


All MI coh adj


1.16 (1.01, 1.34)
0.019


All MI coh adj, admix adj


1.16 (1.01, 1.33)
0.017
0.046


MI and additional CVD (183/1345)b
0.192
0.15
1.35 (1.00, 1.81)
0.026


MI and additional CVD coh adj


1.31 (0.97, 1.78)
0.037


MI and additional CVD coh adj,


1.32 (0.98, 1.78)
0.035
0.089


admix adj


African Americans


All MI (197/387)
0.105
0.032
3.52 (1.96, 6.29)
0.000012


All MI coh adj


3.57 (1.94, 6.57)
0.000022


All MI coh adj, admix adj


3.50 (1.90, 6.43)
0.000029
0.144


MI and additional CVD (21/260)b
0.176
0.041
 4.94 (1.58, 15.43)
0.003


MI and additional CVD coh adj


 4.39 (1.32, 14.64)
0.008


MI and additional CVD coh adj,


 4.17 (1.21, 14.30)
0.012
0.219


admix adj









Strikingly, while HapK is less frequent in the general population of African Americans (Table 52), its association to MI was much stronger in this group, with the RR estimated as 6.50, 1.78, and 5.21 for the cohorts from Philadelphia, Cleveland and Atlanta, respectively (Table 52). The estimated RR in Cleveland is substantially less than in the other two cohorts mainly because the control frequency of HapK is higher there. The RR conferred by HapK in the combined group of all African Americans with cohort adjustment was estimated to be 3.57 (P=0.000022). Its confidence interval did not overlap with that of the European Americans (Table 53), demonstrating that RRs of HapK in these two groups are significantly different (P<0.001).
As HapK is much more frequent in European than African Americans, the possibility existed that the greater RR of MI in African Americans is in part attributable to greater European ancestry in patients than in controls. This could be caused either by a bias in data collection (e.g. differences in the recruitment of the patient and control groups), or because European ancestry is by itself a risk factor for MI in African Americans or a close correlate of such a risk factor.
To investigate this further, a set of 75 unlinked microsatellite markers was used to genetically estimate ancestry of the study cohorts from all thee US cohorts, 364 Icelanders and the 90 Nigerian Yorubans used in the HapMap project (The International HapMap Project. Nature 426, 789-96, 2003) The unlinked microsatellite markers were selected from about 2000 microsatellites genotyped in a previously described multi-ethnic cohort of 35 European Americans, 88 African Americans, 34 Chinese, and 29 Mexican Americans (Smith et al. Am J Hum Genet 74, 1001-13,2004). The microsatellite markers used in this analysis are set out in Table 54. The selected set of microsatellite markers showed the most significant differences between the European Americans, African Americans, and Asians, and also had good quality and yield. The markers were tested in 364 Icelanders, 35 European Americans and 88 African Americans. The markers in bold were used by Tang et al. (Am. J. Hum. Gen. 76: 268-75, 2005) to determine ethnicity and are described at the Marshfield Clinic Research Foundation web site (Screening Set 8).
The allele numbers set out in Table 54 refer to microsatellite alleles. The CEPH sample (Center d'Etudes du Polymorphisme Humain, genomics repository) was 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, allele 1 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.







TABLE 54







Microsatellite markers used for the evaluation of the European and


African ancestry















frq.
frq, Eur.
frq.


Chrom
Marker
Allele
Icelanders
Am.
Affr. Am.















C01
D1S493
0
0.176
0.206
0.465


C01
D1S493
−2
0.117
0.074
0.116


C01
D1S493
2
0.041
0.074
0.093


C01
D1S493
−4
0.004
0.000
0.006


C01
D1S493
4
0.651
0.618
0.215


C01
D1S493
6
0.011
0.029
0.105


C01
D1S2866
0
0.563
0.636
0.163


C01
D1S2866
−10
0.000
0.000
0.006


C01
D1S2866
−1
0.009
0.000
0.000


C01
D1S2866
−16
0.000
0.000
0.047


C01
D1S2866
−2
0.289
0.258
0.215


C01
D1S2866
2
0.025
0.046
0.035


C01
D1S2866
−4
0.049
0.061
0.308


C01
D1S2866
4
0.009
0.000
0.012


C01
D1S2866
−6
0.055
0.000
0.204


C01
D1S2866
6
0.002
0.000
0.006


C01
D1S2866
8
0.000
0.000
0.006


C01
D1S2630
0
0.694
0.743
0.238


C01
D1S2630
10
0.000
0.000
0.035


C01
D1S2630
12
0.021
0.014
0.012


C01
D1S2630
2
0.000
0.000
0.058


C01
D1S2630
4
0.039
0.014
0.116


C01
D1S2630
6
0.216
0.186
0.238


C01
D1S2630
8
0.030
0.043
0.302


C01
D1S466
0
0.209
0.086
0.084


C01
D1S466
10
0.007
0.014
0.042


C01
D1S466
−12
0.000
0.000
0.006


C01
D1S466
12
0.000
0.000
0.018


C01
D1S466
14
0.005
0.014
0.006


C01
D1S466
16
0.000
0.000
0.006


C01
D1S466
−2
0.021
0.029
0.000


C01
D1S466
2
0.045
0.014
0.259


C01
D1S466
−22
0.003
0.000
0.000


C01
D1S466
−4
0.420
0.543
0.066


C01
D1S466
4
0.144
0.157
0.235


C01
D1S466
−6
0.007
0.029
0.145


C01
D1S466
6
0.080
0.057
0.060


C01
D1S466
−8
0.005
0.000
0.018


C01
D1S466
8
0.055
0.057
0.054


C01
D1S1660
0
0.201
0.229
0.294


C01
D1S1660
12
0.170
0.229
0.118


C01
D1S1660
16
0.021
0.029
0.053


C01
D1S1660
20
0.002
0.000
0.006


C01
D1S1660
−4
0.050
0.029
0.100


C01
D1S1660
4
0.251
0.186
0.177


C01
D1S1660
−8
0.010
0.000
0.135


C01
D1S1660
8
0.296
0.300
0.118


C01
D1S2847
0
0.685
0.661
0.386


C01
D1S2847
−2
0.030
0.065
0.048


C01
D1S2847
2
0.050
0.032
0.006


C01
D1S2847
−4
0.016
0.000
0.386


C01
D1S2847
4
0.000
0.016
0.000


C01
D1S2847
−6
0.220
0.226
0.175


C02
D2S1400
0
0.272
0.386
0.114


C02
D2S1400
12
0.000
0.000
0.102


C02
D2S1400
16
0.000
0.000
0.011


C02
D2S1400
20
0.014
0.000
0.051


C02
D2S1400
24
0.000
0.000
0.068


C02
D2S1400
28
0.000
0.000
0.051


C02
D2S1400
32
0.000
0.000
0.011


C02
D2S1400
36
0.000
0.000
0.006


C02
D2S1400
−4
0.301
0.329
0.307


C02
D2S1400
4
0.007
0.000
0.074


C02
D2S1400
−8
0.403
0.286
0.074


C02
D2S1400
8
0.003
0.000
0.131


C02
D2S166
0
0.587
0.618
0.244


C02
D2S166
−14
0.006
0.000
0.000


C02
D2S166
−2
0.112
0.162
0.097


C02
D2S166
2
0.190
0.118
0.148


C02
D2S166
−4
0.003
0.000
0.011


C02
D2S166
4
0.101
0.103
0.386


C02
D2S166
6
0.001
0.000
0.091


C02
D2S166
8
0.000
0.000
0.023


C02
D2S1777
0
0.618
0.583
0.319


C02
D2S1777
−12
0.000
0.000
0.007


C02
D2S1777
12
0.007
0.000
0.174


C02
D2S1777
16
0.000
0.000
0.076


C02
D2S1777
−20
0.002
0.000
0.000


C02
D2S1777
−4
0.094
0.083
0.090


C02
D2S1777
4
0.202
0.217
0.083


C02
D2S1777
−8
0.002
0.000
0.014


C02
D2S1777
8
0.076
0.117
0.236


C02
D2S2972
0
0.033
0.063
0.220


C02
D2S2972
−12
0.000
0.000
0.073


C02
D2S2972
12
0.170
0.094
0.043


C02
D2S2972
−16
0.000
0.000
0.006


C02
D2S2972
16
0.120
0.125
0.006


C02
D2S2972
20
0.014
0.031
0.012


C02
D2S2972
24
0.004
0.000
0.000


C02
D2S2972
−4
0.000
0.000
0.055


C02
D2S2972
4
0.426
0.453
0.396


C02
D2S2972
−8
0.001
0.016
0.092


C02
D2S2972
8
0.2
0.219
0.098


C02
D2S1353
0
0.218
0.343
0.163


C02
D2S1353
−12
0.103
0.114
0.064


C02
D2S1353
12
0.004
0.000
0.000


C02
D2S1353
−15
0.003
0.000
0.006


C02
D2S1353
−3
0.214
0.186
0.204


C02
D2S1353
3
0.127
0.057
0.070


C02
D2S1353
−6
0.207
0.200
0.366


C02
D2S1353
6
0.057
0.057
0.017


C02
D2S1353
−9
0.034
0.000
0.070


C02
D2S1353
9
0.034
0.043
0.041


C02
D2S125
0
0.278
0.258
0.222


C02
D2S125
−10
0.000
0.000
0.019


C02
D2S125
10
0.001
0.030
0.000


C02
D2S125
−2
0.106
0.136
0.093


C02
D2S125
2
0.100
0.091
0.210


C02
D2S125
−4
0.156
0.121
0.210


C02
D2S125
4
0.034
0.046
0.043


C02
D2S125
−6
0.192
0.197
0.117


C02
D2S125
6
0.108
0.106
0.056


C02
D2S125
−8
0.001
0.015
0.025


C02
D2S125
8
0.023
0.000
0.006


C03
D3S4559
0
0.096
0.167
0.059


C03
D3S4559
−2
0.385
0.300
0.294


C03
D3S4559
2
0.004
0.000
0.279


C03
D3S4559
−4
0.037
0.033
0.221


C03
D3S4559
4
0.474
0.500
0.132


C03
D3S4559
6
0.004
0.000
0.015


C03
D3S1515
0
0.528
0.414
0.802


C03
D3S1515
2
0.468
0.586
0.116


C03
D3S1515
4
0.003
0.000
0.000


C03
D3S1515
6
0.001
0.000
0.000


C03
D3S1515
8
0.000
0.000
0.081


C03
D3S1583
0
0.765
0.843
0.155


C03
D3S1583
10
0.000
0.000
0.069


C03
D3S1583
12
0.016
0.000
0.098


C03
D3S1583
14
0.005
0.014
0.035


C03
D3S1583
16
0.029
0.000
0.218


C03
D3S1583
18
0.045
0.014
0.149


C03
D3S1583
20
0.071
0.057
0.092


C03
D3S1583
22
0.003
0.014
0.081


C03
D3S1583
2
0.022
0.014
0.006


C03
D3S1583
24
0.026
0.014
0.075


C03
D3S1583
26
0.017
0.029
0.023


C03
D3S1583
28
0.002
0.000
0.000


C03
D3S3653
0
0.413
0.397
0.103


C03
D3S3653
10
0.001
0.015
0.006


C03
D3S3653
14
0.000
0.000
0.017


C03
D3S3653
−2
0.017
0.000
0.006


C03
D3S3653
2
0.361
0.412
0.247


C03
D3S3653
−4
0.001
0.000
0.000


C03
D3S3653
4
0.134
0.103
0.316


C03
D3S3653
6
0.023
0.000
0.201


C03
D3S3653
8
0.050
0.074
0.103


C03
D3S1276
0
0.371
0.288
0.361


C03
D3S1276
−10
0.000
0.000
0.017


C03
D3S1276
10
0.000
0.000
0.017


C03
D3S1276
12
0.000
0.000
0.017


C03
D3S1276
−2
0.010
0.046
0.017


C03
D3S1276
2
0.403
0.455
0.128


C03
D3S1276
−4
0.044
0.015
0.035


C03
D3S1276
4
0.112
0.121
0.099


C03
D3S1276
6
0.033
0.046
0.041


C03
D3S1276
−8
0.000
0.000
0.180


C03
D3S1276
8
0.027
0.030
0.087


C03
D3S3045
0
0.291
0.258
0.226


C03
D3S3045
−12
0.064
0.065
0.043


C03
D3S3045
12
0.000
0.016
0.061


C03
D3S3045
−16
0.106
0.161
0.049


C03
D3S3045
16
0.003
0.000
0.000


C03
D3S3045
−4
0.204
0.194
0.220


C03
D3S3045
4
0.175
0.242
0.281


C03
D3S3045
−8
0.105
0.048
0.031


C03
D3S3045
8
0.051
0.016
0.092


C03
D3S4011
0
0.858
0.829
0.190


C03
D3S4011
3
0.001
0.000
0.017


C03
D3S4011
6
0.138
0.157
0.776


C03
D3S4011
7
0.000
0.000
0.017


C03
D3S4011
9
0.003
0.014
0.000


C04
D4S403
0
0.238
0.186
0.035


C04
D4S403
10
0.407
0.386
0.273


C04
D4S403
12
0.013
0.000
0.099


C04
D4S403
14
0.069
0.086
0.023


C04
D4S403
16
0.002
0.000
0.017


C04
D4S403
18
0.000
0.000
0.023


C04
D4S403
2
0.040
0.014
0.023


C04
D4S403
−4
0.000
0.000
0.006


C04
D4S403
4
0.021
0.043
0.140


C04
D4S403
6
0.054
0.114
0.122


C04
D4S403
8
0.156
0.171
0.238


C04
D4S396
0
0.549
0.529
0.364


C04
D4S396
10
0.004
0.014
0.000


C04
D4S396
−2
0.012
0.000
0.006


C04
D4S396
2
0.001
0.000
0.358


C04
D4S396
−4
0.001
0.000
0.000


C04
D4S396
4
0.007
0.014
0.040


C04
D4S396
6
0.367
0.343
0.227


C04
D4S396
8
0.058
0.100
0.006


C04
D4S2460
0
0.435
0.343
0.296


C04
D4S2460
−10
0.000
0.000
0.006


C04
D4S2460
−2
0.032
0.000
0.347


C04
D4S2460
2
0.245
0.286
0.142


C04
D4S2460
−4
0.215
0.300
0.102


C04
D4S2460
4
0.052
0.057
0.063


C04
D4S2460
−6
0.002
0.014
0.028


C04
D4S2460
−8
0.019
0.000
0.017


C04
D4S2913
0
0.177
0.129
0.068


C04
D4S2913
−10
0.035
0.043
0.011


C04
D4S2913
10
0.002
0.014
0.000


C04
D4S2913
−2
0.021
0.029
0.449


C04
D4S2913
2
0.322
0.286
0.097


C04
D4S2913
−4
0.034
0.014
0.040


C04
D4S2913
4
0.027
0.029
0.046


C04
D4S2913
−6
0.341
0.414
0.261


C04
D4S2913
6
0.043
0.043
0.006


C04
D4S2913
−8
0.000
0.000
0.011


C04
D4S2913
8
0.000
0.000
0.011


C04
D4S1579
0
0.508
0.571
0.506


C04
D4S1579
10
0.045
0.086
0.006


C04
D4S1579
12
0.030
0.014
0.006


C04
D4S1579
14
0.000
0.014
0.006


C04
D4S1579
−2
0.001
0.000
0.031


C04
D4S1579
2
0.035
0.029
0.306


C04
D4S1579
−4
0.001
0.000
0.000


C04
D4S1579
4
0.018
0.014
0.075


C04
D4S1579
−6
0.013
0.000
0.000


C04
D4S1579
6
0.058
0.057
0.019


C04
D4S1579
8
0.291
0.214
0.044


C04
D4S3014
0
0.097
0.029
0.024


C04
D4S3014
10
0.000
0.000
0.113


C04
D4S3014
12
0.000
0.000
0.018


C04
D4S3014
14
0.000
0.000
0.006


C04
D4S3014
16
0.000
0.000
0.024


C04
D4S3014
−2
0.000
0.000
0.006


C04
D4S3014
2
0.085
0.015
0.191


C04
D4S3014
−4
0.077
0.118
0.101


C04
D4S3014
4
0.597
0.794
0.232


C04
D4S3014
−6
0.000
0.000
0.036


C04
D4S3014
6
0.136
0.015
0.196


C04
D4S3014
8
0.009
0.029
0.054


C04
D4S408
0
0.161
0.167
0.262


C04
D4S408
10
0.004
0.000
0.006


C04
D4S408
12
0.169
0.197
0.066


C04
D4S408
14
0.011
0.015
0.000


C04
D4S408
16
0.000
0.015
0.000


C04
D4S408
−2
0.389
0.424
0.143


C04
D4S408
2
0.176
0.121
0.268


C04
D4S408
−4
0.000
0.000
0.036


C04
D4S408
4
0.079
0.061
0.155


C04
D4S408
6
0.010
0.000
0.036


C04
D4S408
8
0.001
0.000
0.030


C05
D5S1967
0
0.093
0.157
0.063


C05
D5S1967
10
0.000
0.000
0.006


C05
D5S1967
2
0.032
0.000
0.210


C05
D5S1967
4
0.010
0.000
0.091


C05
D5S1967
6
0.828
0.786
0.438


C05
D5S1967
8
0.038
0.057
0.193


C05
D5S1725
0
0.049
0.059
0.082


C05
D5S1725
−12
0.013
0.000
0.000


C05
D5S1725
12
0.105
0.147
0.153


C05
D5S1725
16
0.019
0.000
0.006


C05
D5S1725
−4
0.037
0.074
0.071


C05
D5S1725
4
0.166
0.191
0.271


C05
D5S1725
−8
0.427
0.338
0.088


C05
D5S1725
8
0.185
0.191
0.329


C05
DG5S802
0
0.651
0.729
0.227


C05
DG5S802
−10
0.000
0.000
0.012


C05
DG5S802
10
0.007
0.000
0.035


C05
DG5S802
−12
0.000
0.014
0.000


C05
DG5S802
12
0.002
0.000
0.017


C05
DG5S802
14
0.002
0.000
0.012


C05
DG5S802
16
0.000
0.000
0.006


C05
DG5S802
−2
0.020
0.014
0.099


C05
DG5S802
2
0.131
0.186
0.070


C05
DG5S802
−4
0.002
0.000
0.012


C05
DG5S802
4
0.018
0.000
0.070


C05
DG5S802
−6
0.000
0.014
0.058


C05
DG5S802
6
0.153
0.014
0.047


C05
DG5S802
−8
0.009
0.029
0.297


C05
DG5S802
8
0.004
0.000
0.041


C05
D5S408
0
0.185
0.250
0.178


C05
D5S408
10
0.174
0.162
0.190


C05
D5S408
12
0.058
0.059
0.040


C05
D5S408
14
0.039
0.029
0.017


C05
D5S408
16
0.010
0.015
0.046


C05
D5S408
20
0.000
0.000
0.006


C05
D5S408
−2
0.021
0.000
0.063


C05
D5S408
2
0.011
0.015
0.012


C05
D5S408
22
0.000
0.000
0.006


C05
D5S408
4
0.008
0.000
0.012


C05
D5S408
6
0.142
0.074
0.213


C05
D5S408
8
0.352
0.397
0.218


C06
D6S1017
0
0.424
0.544
0.229


C06
D6S1017
−12
0.000
0.000
0.247


C06
D6S1017
13
0.041
0.029
0.029


C06
D6S1017
17
0.000
0.015
0.000


C06
D6S1017
−4
0.001
0.000
0.077


C06
D6S1017
5
0.050
0.015
0.059


C06
D6S1017
−8
0.283
0.132
0.265


C06
D6S1017
9
0.200
0.265
0.094


C06
D6S405
0
0.551
0.643
0.180


C06
D6S405
10
0.289
0.186
0.517


C06
D6S405
12
0.022
0.014
0.087


C06
D6S405
14
0.000
0.000
0.017


C06
D6S405
16
0.000
0.000
0.006


C06
D6S405
2
0.002
0.000
0.006


C06
D6S405
−4
0.002
0.000
0.000


C06
D6S405
4
0.000
0.000
0.006


C06
D6S405
6
0.000
0.000
0.006


C06
D6S405
8
0.134
0.157
0.174


C06
D6S446
0
0.222
0.171
0.172


C06
D6S446
−2
0.433
0.557
0.506


C06
D6S446
2
0.050
0.043
0.144


C06
D6S446
4
0.118
0.043
0.040


C06
D6S446
6
0.143
0.157
0.132


C06
D6S446
8
0.034
0.029
0.006


C07
D7S1802
0
0.310
0.329
0.302


C07
D7S1802
−10
0.002
0.000
0.000


C07
D7S1802
−12
0.005
0.000
0.047


C07
D7S1802
12
0.014
0.014
0.023


C07
D7S1802
−14
0.003
0.000
0.000


C07
D7S1802
16
0.007
0.000
0.006


C07
D7S1802
−4
0.337
0.471
0.209


C07
D7S1802
4
0.176
0.143
0.326


C07
D7S1802
−8
0.048
0.000
0.006


C07
D7S1802
8
0.098
0.043
0.081


C07
D7S559
0
0.129
0.157
0.230


C07
D7S559
13
0.000
0.000
0.012


C07
D7S559
14
0.000
0.000
0.006


C07
D7S559
15
0.015
0.000
0.000


C07
D7S559
19
0.000
0.000
0.006


C07
D7S559
20
0.000
0.000
0.006


C07
D7S559
21
0.000
0.000
0.006


C07
D7S559
−2
0.210
0.229
0.103


C07
D7S559
2
0.290
0.329
0.305


C07
D7S559
22
0.000
0.000
0.029


C07
D7S559
24
0.000
0.000
0.006


C07
D7S559
26
0.000
0.000
0.052


C07
D7S559
−4
0.087
0.086
0.115


C07
D7S559
4
0.060
0.057
0.035


C07
D7S559
−6
0.162
0.086
0.040


C07
D7S559
6
0.002
0.014
0.012


C07
D7S559
−8
0.010
0.000
0.000


C07
D7S559
8
0.035
0.043
0.040


C08
DG8S156
0
0.561
0.516
0.440


C08
DG8S156
12
0.000
0.000
0.048


C08
DG8S156
−3
0.000
0.000
0.006


C08
DG8S156
3
0.001
0.016
0.078


C08
DG8S156
−6
0.031
0.047
0.030


C08
DG8S156
6
0.386
0.375
0.295


C08
DG8S156
9
0.021
0.047
0.102


C08
D8S1719
0
0.119
0.143
0.122


C08
D8S1719
−2
0.757
0.786
0.256


C08
D8S1719
2
0.083
0.057
0.134


C08
D8S1719
−4
0.021
0.000
0.355


C08
D8S1719
4
0.020
0.014
0.029


C08
D8S1719
−6
0.000
0.000
0.047


C08
D8S1719
6
0.002
0.000
0.029


C08
D8S1719
−8
0.000
0.000
0.017


C08
D8S1719
8
0.000
0.000
0.012


C08
D8S1831
0
0.506
0.645
0.149


C08
D8S1831
10
0.076
0.065
0.081


C08
D8S1831
−12
0.013
0.000
0.000


C08
D8S1831
12
0.002
0.000
0.058


C08
D8S1831
14
0.002
0.000
0.006


C08
D8S1831
−2
0.026
0.032
0.017


C08
D8S1831
2
0.229
0.145
0.207


C08
D8S1831
−4
0.002
0.016
0.000


C08
D8S1831
4
0.024
0.016
0.149


C08
D8S1831
6
0.005
0.000
0.109


C08
D8S1831
8
0.116
0.081
0.224


C08
D8S1746
0
0.521
0.557
0.105


C08
D8S1746
10
0.043
0.014
0.017


C08
D8S1746
12
0.003
0.000
0.006


C08
D8S1746
16
0.000
0.000
0.029


C08
D8S1746
2
0.192
0.214
0.238


C08
D8S1746
4
0.178
0.171
0.151


C08
D8S1746
6
0.061
0.043
0.326


C08
D8S1746
8
0.001
0.000
0.128


C09
D9S1839
0
0.507
0.614
0.192


C09
D9S1839
10
0.016
0.014
0.058


C09
D9S1839
12
0.000
0.000
0.099


C09
D9S1839
14
0.000
0.000
0.076


C09
D9S1839
−2
0.012
0.000
0.000


C09
D9S1839
2
0.004
0.014
0.035


C09
D9S1839
22
0.003
0.000
0.000


C09
D9S1839
4
0.107
0.114
0.017


C09
D9S1839
6
0.012
0.029
0.058


C09
D9S1839
8
0.339
0.214
0.459


C09
D9S1839
9
0.000
0.000
0.006


C09
D9S1777
0
0.690
0.657
0.205


C09
D9S1777
10
0.011
0.000
0.011


C09
D9S1777
18
0.001
0.000
0.006


C09
D9S1777
−2
0.000
0.000
0.483


C09
D9S1777
2
0.204
0.200
0.011


C09
D9S1777
4
0.001
0.000
0.023


C09
D9S1777
6
0.076
0.143
0.250


C09
D9S1777
8
0.013
0.000
0.011


C09
D9S1777
−9
0.004
0.000
0.000


C10
D10S189
0
0.434
0.543
0.358


C10
D10S189
10
0.004
0.000
0.000


C10
D10S189
−2
0.213
0.157
0.091


C10
D10S189
2
0.004
0.000
0.102


C10
D10S189
4
0.224
0.229
0.398


C10
D10S189
6
0.121
0.057
0.051


C10
D10S189
8
0.000
0.014
0.000


C10
D10S2327
0
0.570
0.586
0.193


C10
D10S2327
12
0.124
0.157
0.296


C10
D10S2327
16
0.086
0.029
0.239


C10
D10S2327
20
0.163
0.186
0.165


C10
D10S2327
24
0.019
0.014
0.046


C10
D10S2327
28
0.002
0.000
0.000


C10
D10S2327
4
0.023
0.029
0.023


C10
D10S2327
8
0.015
0.000
0.040


C10
D10S1698
0
0.834
0.829
0.369


C10
D10S1698
16
0.056
0.043
0.179


C10
D10S1698
18
0.000
0.000
0.018


C10
D10S1698
20
0.068
0.086
0.089


C10
D10S1698
22
0.042
0.043
0.232


C10
D10S1698
24
0.000
0.000
0.054


C10
D10S1698
−2
0.000
0.000
0.006


C10
D10S1698
2
0.000
0.000
0.030


C10
D10S1698
4
0.000
0.000
0.024


C10
D10S212
0
0.564
0.529
0.627


C10
D10S212
−2
0.007
0.000
0.025


C10
D10S212
2
0.062
0.044
0.101


C10
D10S212
−4
0.107
0.103
0.057


C10
D10S212
4
0.117
0.177
0.063


C10
D10S212
−6
0.112
0.132
0.063


C10
D10S212
6
0.030
0.015
0.063


C11
D11S4130
0
0.343
0.343
0.171


C11
D11S4130
−2
0.051
0.000
0.324


C11
D11S4130
2
0.486
0.486
0.318


C11
D11S4130
−4
0.119
0.171
0.114


C11
D11S4130
4
0.001
0.000
0.057


C11
D11S4130
6
0.000
0.000
0.017


C11
D11S1321
0
0.509
0.529
0.128


C11
D11S1321
−10
0.000
0.000
0.007


C11
D11S1321
10
0.015
0.015
0.054


C11
D11S1321
−2
0.000
0.000
0.047


C11
D11S1321
2
0.073
0.059
0.135


C11
D11S1321
3
0.000
0.000
0.034


C11
D11S1321
−4
0.000
0.000
0.061


C11
D11S1321
4
0.019
0.015
0.142


C11
D11S1321
6
0.341
0.368
0.291


C11
D11S1321
8
0.043
0.015
0.101


C11
D11S4206
0
0.401
0.324
0.223


C11
D11S4206
−2
0.112
0.088
0.157


C11
D11S4206
2
0.280
0.397
0.054


C11
D11S4206
−4
0.000
0.015
0.048


C11
D11S4206
4
0.006
0.000
0.000


C11
D11S4206
−6
0.029
0.015
0.012


C11
D11S4206
−8
0.172
0.162
0.506


C11
D11S1320
0
0.576
0.606
0.363


C11
D11S1320
10
0.000
0.000
0.006


C11
D11S1320
12
0.001
0.000
0.000


C11
D11S1320
−2
0.030
0.030
0.238


C11
D11S1320
2
0.060
0.061
0.054


C11
D11S1320
−4
0.000
0.000
0.018


C11
D11S1320
4
0.296
0.242
0.185


C11
D11S1320
−6
0.010
0.000
0.000


C11
D11S1320
6
0.020
0.046
0.131


C11
D11S1320
8
0.006
0.015
0.006


C12
D12S1723
0
0.082
0.100
0.392


C12
D12S1723
10
0.098
0.100
0.108


C12
D12S1723
12
0.011
0.043
0.054


C12
D12S1723
14
0.000
0.000
0.024


C12
D12S1723
16
0.000
0.000
0.054


C12
D12S1723
18
0.000
0.000
0.024


C12
D12S1723
20
0.000
0.000
0.042


C12
D12S1723
−2
0.027
0.029
0.102


C12
D12S1723
2
0.008
0.000
0.042


C12
D12S1723
22
0.000
0.000
0.012


C12
D12S1723
4
0.466
0.500
0.096


C12
D12S1723
6
0.289
0.214
0.048


C12
D12S1723
8
0.018
0.014
0.000


C13
D13S894
0
0.058
0.091
0.082


C13
D13S894
12
0.432
0.561
0.388


C13
D13S894
16
0.155
0.121
0.141


C13
D13S894
20
0.033
0.030
0.071


C13
D13S894
4
0.035
0.030
0.112


C13
D13S894
7
0.006
0.000
0.000


C13
D13S894
8
0.281
0.167
0.206


C13
D13S152
0
0.318
0.294
0.256


C13
D13S152
10
0.161
0.074
0.091


C13
D13S152
12
0.006
0.015
0.000


C13
D13S152
−2
0.000
0.000
0.063


C13
D13S152
2
0.050
0.074
0.125


C13
D13S152
−4
0.000
0.000
0.011


C13
D13S152
4
0.224
0.250
0.193


C13
D13S152
6
0.116
0.191
0.193


C13
D13S152
8
0.125
0.103
0.068


C14
D14S741
0
0.505
0.471
0.455


C14
D14S741
−12
0.012
0.029
0.080


C14
D14S741
−14
0.087
0.071
0.000


C14
D14S741
−16
0.011
0.000
0.006


C14
D14S741
−4
0.194
0.243
0.273


C14
D14S741
4
0.180
0.157
0.142


C14
D14S741
−8
0.002
0.000
0.040


C14
D14S741
8
0.011
0.029
0.006


C14
D14S588
0
0.407
0.500
0.230


C14
D14S588
−12
0.000
0.000
0.006


C14
D14S588
13
0.086
0.029
0.029


C14
D14S588
17
0.006
0.014
0.023


C14
D14S588
22
0.000
0.000
0.006


C14
D14S588
−4
0.226
0.200
0.172


C14
D14S588
5
0.150
0.186
0.115


C14
D14S588
−8
0.002
0.000
0.264


C14
D14S588
9
0.124
0.071
0.155


C14
D14S617
0
0.075
0.143
0.115


C14
D14S617
−12
0.002
0.000
0.190


C14
D14S617
12
0.274
0.257
0.218


C14
D14S617
16
0.187
0.257
0.126


C14
D14S617
20
0.030
0.043
0.040


C14
D14S617
24
0.005
0.043
0.006


C14
D14S617
−4
0.092
0.086
0.063


C14
D14S617
4
0.003
0.000
0.000


C14
D14S617
−8
0.273
0.143
0.161


C14
D14S617
8
0.061
0.029
0.081


C15
D15S659
0
0.027
0.000
0.052


C15
D15S659
12
0.048
0.086
0.035


C15
D15S659
16
0.031
0.071
0.047


C15
D15S659
20
0.177
0.171
0.291


C15
D15S659
24
0.191
0.157
0.297


C15
D15S659
28
0.106
0.086
0.081


C15
D15S659
32
0.035
0.014
0.017


C15
D15S659
36
0.001
0.000
0.012


C15
D15S659
−4
0.001
0.000
0.000


C15
D15S659
4
0.203
0.171
0.070


C15
D15S659
−8
0.000
0.000
0.006


C15
D15S659
8
0.179
0.243
0.093


C15
D15S1507
0
0.375
0.429
0.247


C15
D15S1507
12
0.148
0.157
0.282


C15
D15S1507
16
0.029
0.029
0.121


C15
D15S1507
20
0.002
0.000
0.006


C15
D15S1507
−4
0.013
0.000
0.000


C15
D15S1507
4
0.261
0.243
0.167


C15
D15S1507
−8
0.000
0.000
0.017


C15
D15S1507
8
0.172
0.143
0.161


C16
D16S404
0
0.161
0.191
0.213


C16
D16S404
−14
0.036
0.029
0.115


C16
D16S404
−2
0.007
0.029
0.046


C16
D16S404
2
0.230
0.265
0.086


C16
D16S404
−4
0.230
0.147
0.397


C16
D16S404
4
0.250
0.235
0.063


C16
D16S404
−6
0.014
0.015
0.029


C16
D16S404
6
0.071
0.074
0.040


C16
D16S404
8
0.000
0.015
0.012


C16
D16S403
0
0.117
0.186
0.256


C16
D16S403
−10
0.000
0.000
0.012


C16
D16S403
10
0.011
0.014
0.006


C16
D16S403
12
0.013
0.000
0.000


C16
D16S403
14
0.004
0.000
0.000


C16
D16S403
16
0.001
0.000
0.000


C16
D16S403
−2
0.275
0.314
0.180


C16
D16S403
2
0.133
0.171
0.308


C16
D16S403
−4
0.050
0.043
0.006


C16
D16S403
4
0.148
0.071
0.099


C16
D16S403
−6
0.053
0.014
0.012


C16
D16S403
6
0.137
0.129
0.099


C16
D16S403
−8
0.000
0.000
0.006


C16
D16S403
8
0.058
0.057
0.017


C16
D16S3253
0
0.340
0.357
0.331


C16
D16S3253
−12
0.009
0.018
0.029


C16
D16S3253
−16
0.008
0.018
0.221


C16
D16S3253
16
0.005
0.000
0.007


C16
D16S3253
−20
0.000
0.000
0.007


C16
D16S3253
−4
0.100
0.232
0.052


C16
D16S3253
4
0.347
0.268
0.125


C16
D16S3253
−8
0.187
0.089
0.228


C16
D16S3253
8
0.005
0.018
0.000


C17
D17S745
0
0.375
0.371
0.177


C17
D17S745
12
0.000
0.000
0.018


C17
D17S745
−2
0.001
0.014
0.218


C17
D17S745
2
0.077
0.000
0.418


C17
D17S745
−4
0.001
0.000
0.000


C17
D17S745
4
0.518
0.614
0.171


C17
D17S745
6
0.027
0.000
0.000


C17
D17S1303
0
0.191
0.150
0.150


C17
D17S1303
12
0.455
0.483
0.425


C17
D17S1303
16
0.154
0.200
0.231


C17
D17S1303
20
0.014
0.000
0.000


C17
D17S1303
24
0.003
0.000
0.000


C17
D17S1303
−4
0.000
0.000
0.019


C17
D17S1303
4
0.065
0.067
0.044


C17
D17S1303
8
0.117
0.100
0.131


C17
D17S1799
0
0.506
0.586
0.415


C17
D17S1799
−2
0.000
0.000
0.011


C17
D17S1799
2
0.102
0.086
0.415


C17
D17S1799
4
0.375
0.329
0.131


C17
D17S1799
6
0.009
0.000
0.028


C17
D17S1799
8
0.009
0.000
0.000


C17
D17S784
0
0.324
0.339
0.309


C17
D17S784
10
0.003
0.000
0.020


C17
D17S784
−14
0.000
0.000
0.013


C17
D17S784
−2
0.134
0.081
0.112


C17
D17S784
2
0.055
0.129
0.151


C17
D17S784
−4
0.308
0.339
0.105


C17
D17S784
4
0.015
0.016
0.112


C17
D17S784
−6
0.095
0.032
0.079


C17
D17S784
6
0.000
0.016
0.059


C17
D17S784
−8
0.067
0.048
0.026


C17
D17S784
8
0.000
0.000
0.013


C18
D18S843
0
0.167
0.214
0.115


C18
D18S843
12
0.004
0.000
0.000


C18
D18S843
−3
0.045
0.043
0.052


C18
D18S843
3
0.348
0.329
0.333


C18
D18S843
−6
0.000
0.000
0.006


C18
D18S843
6
0.327
0.300
0.299


C18
D18S843
9
0.110
0.114
0.195


C18
D18S464
0
0.706
0.677
0.224


C18
D18S464
−2
0.083
0.118
0.374


C18
D18S464
2
0.111
0.118
0.058


C18
D18S464
−4
0.035
0.029
0.259


C18
D18S464
4
0.008
0.029
0.006


C18
D18S464
−6
0.058
0.029
0.069


C18
D18S464
6
0.000
0.000
0.006


C18
D18S464
−8
0.000
0.000
0.006


C18
D18S466
0
0.513
0.529
0.460


C18
D18S466
−2
0.411
0.414
0.494


C18
D18S466
2
0.074
0.057
0.046


C18
D18S466
4
0.001
0.000
0.000


C19
D19S534
0
0.742
0.800
0.309


C19
D19S534
10
0.000
0.000
0.012


C19
D19S534
12
0.000
0.000
0.049


C19
D19S534
−2
0.040
0.071
0.019


C19
D19S534
2
0.090
0.057
0.346


C19
D19S534
−4
0.000
0.000
0.006


C19
D19S534
4
0.123
0.071
0.216


C19
D19S534
6
0.004
0.000
0.031


C19
D19S534
8
0.000
0.000
0.012


C19
D19S113
0
0.108
0.114
0.035


C19
D19S113
10
0.000
0.000
0.047


C19
D19S113
12
0.000
0.000
0.012


C19
D19S113
2
0.254
0.200
0.118


C19
D19S113
4
0.391
0.457
0.159


C19
D19S113
6
0.225
0.200
0.165


C19
D19S113
8
0.022
0.029
0.465


C19
D19S254
0
0.307
0.353
0.087


C19
D19S254
12
0.013
0.029
0.012


C19
D19S254
16
0.101
0.088
0.355


C19
D19S254
20
0.347
0.368
0.279


C19
D19S254
24
0.082
0.059
0.099


C19
D19S254
28
0.071
0.029
0.058


C19
D19S254
33
0.005
0.029
0.017


C19
D19S254
37
0.006
0.000
0.000


C19
D19S254
4
0.006
0.000
0.017


C19
D19S254
−8
0.021
0.000
0.006


C19
D19S254
8
0.042
0.044
0.070


C20
D20S103
0
0.366
0.286
0.199


C20
D20S103
−10
0.000
0.000
0.006


C20
D20S103
−2
0.338
0.400
0.421


C20
D20S103
2
0.017
0.014
0.063


C20
D20S103
−4
0.192
0.229
0.131


C20
D20S103
4
0.000
0.000
0.006


C20
D20S103
−6
0.003
0.014
0.068


C20
D20S103
6
0.006
0.000
0.011


C20
D20S103
−8
0.079
0.057
0.097


C20
D20S878
0
0.596
0.636
0.187


C20
D20S878
−2
0.044
0.015
0.121


C20
D20S878
2
0.094
0.061
0.078


C20
D20S878
−4
0.176
0.152
0.211


C20
D20S878
4
0.009
0.015
0.145


C20
D20S878
−6
0.000
0.030
0.199


C20
D20S878
6
0.059
0.076
0.012


C20
D20S878
−8
0.023
0.000
0.048


C20
D20S878
8
0.000
0.015
0.000


C21
D21S1884
0
0.532
0.514
0.398


C21
D21S1884
10
0.000
0.000
0.091


C21
D21S1884
2
0.012
0.000
0.222


C21
D21S1884
−4
0.020
0.014
0.034


C21
D21S1884
4
0.042
0.043
0.034


C21
D21S1884
6
0.390
0.400
0.136


C21
D21S1884
8
0.004
0.029
0.085


C22
D22S420
0
0.215
0.186
0.256


C22
D22S420
−14
0.000
0.000
0.023


C22
D22S420
−16
0.000
0.000
0.006


C22
D22S420
−2
0.380
0.343
0.233


C22
D22S420
2
0.241
0.329
0.122


C22
D22S420
−4
0.041
0.057
0.122


C22
D22S420
4
0.078
0.057
0.047


C22
D22S420
−6
0.009
0.014
0.081


C22
D22S420
6
0.022
0.014
0.023


C22
D22S420
−8
0.013
0.000
0.087


C22
D22S420
8
0.002
0.000
0.000


C22
D22S689
0
0.349
0.279
0.256


C22
D22S689
−12
0.023
0.000
0.006


C22
D22S689
12
0.054
0.059
0.070


C22
D22S689
−16
0.005
0.000
0.000


C22
D22S689
16
0.009
0.000
0.006


C22
D22S689
−20
0.000
0.000
0.035


C22
D22S689
−4
0.087
0.088
0.076


C22
D22S689
4
0.319
0.368
0.297


C22
D22S689
6
0.000
0.000
0.012


C22
D22S689
−8
0.020
0.074
0.116


C22
D22S689
8
0.136
0.132
0.128









The Structure software (Falush et al., Genetics 164, 1567-87, 2003; Pritchard et al., Genetics 155, 945-59, 2000; both incorporated by reference herein in their entirety) was then applied to these data to estimate the distribution of European ancestry of individuals according to disease status and self-reported ethnicity (Table 55). Structure infers the allele frequencies of K ancestral populations on the basis of multilocus genotypes from a set of individuals and a user-specified value of K, and assigns a proportion of ancestry from each of the inferred K populations to each individual. The analysis of the data set was run using the admixture model, where the ancestry prior α was allowed to vary among populations. This is an important option when genetic material from the K inferred ancestral populations (in this case the African and European ancestral populations) are not equally represented in the dataset. This is clearly the case in the present data, which contains 3366 self-reported European Americans, 584 self-reported African Americans, 364 Icelanders and 87 Nigerians. The Structure program was run multiple times for each K in the range 2 to 5. The Icelanders and European Americans data was used to identify the European ancestry component in the African Americans and the Nigerians to identify the African ancestry component. Based on these runs, there is evidence to indicate that K=3 provides the best estimates of European ancestry in African Americans. First, these estimates correspond closely to independent group estimates based on Long's weighted least-squares admixture estimator ((Long et al. Genetics 127, 417-28, 1991). Second, K=3 results indicated the existence of clearly defined African and European ancestral gene pools and a third gene pool, that contributeed a small amount (1-2%) to European and African Americans, but nothing to Nigerians and Icelanders. An independent Structure analysis that also included Native American and East Asian reference samples indicated that this third component represents Asian ancestry. When K>3 the European component becomes divided equally among the additional ancestral gene pools, whereas the African and Asian components remained stable in single components. Thus, K>3 did not provide any additional resolution to the data. The estimates of European ancestry for African American individuals were strongly correlated between different runs of Structure, regardless of the value of K. Thus, the average Spearman's rank correlation between runs was 0.987, with a minimum of 0.964. The statistical significance of the difference in mean European ancestry between African American patients and controls was evaluated by reference to a null distribution derived from 10000 randomized datasets.
The data includes group estimates of European ancestry obtained by applying a weighted least-squares (WLS) estimator (Long et al. Genetics 127, 417-28, 1991) to a subset of the microsatellite alleles that exhibited the greatest differences in frequency between European and African populations (in accordance with the teachings in Parra et al. Am J Hum Genet 63, 1839-51, 1998). Overall, there is a close correspondence between self-reported ethnicity and the estimated ancestry derived from the genetic markers and also between the estimated individual ancestry (Structure) and group ancestry (WLS). In particular, the almost perfect assignment of African ancestry to the Nigerian Yorubans and European ancestry to the Icelanders indicates that the admixture estimates of the American cases and controls are reliable.
Long's weighted least-squares measure of admixture was calculated using alleles from the set of 75 microsatellite markers. Frequencies from Icelanders and Nigerians were used as representative of the ancestral allele frequencies of the European and African gene pools, respectively. In line with Parra et al., only 16 loci with alleles exhibiting large differences in frequency (δ=0.5) between the two parental populations were used. In the case of the African American cohorts, the WLS admixture statistic was calculated using the European American controls from the same city as representatives of the ancestral European gene pool. In each case, the estimate of European ancestry was higher by about 0.01. This is likely to be the result of the small fraction of African alleles present in the European Americans and indicates that Icelanders serve effectively as representatives of the European component of the European American gene pool. The WLS admixture statistic was also calculated using alleles of all the 75 microsatellite markers, yielding estimates of European ancestry in African Americans that tended to be a little higher than those reported above (by 0.01-0.02).
Estimates of genetic ancestry were obtained from the Structure software, using the parameters and data described above. We note that the output from Structure does not label the ancestral admixture proportions as either “European” or “African”, but rather as “inferred clusters” 1 and 2. However, the distribution of ancestry from these inferred clusters in Icelanders, Nigerians and the American cohorts suggest a relatively straightforward correspondence with the labels “African” and “European” ancestry







TABLE 55







Distribution of genetically determined European ancestry in MI case-controlled cohorts









Distribution of estimated



individual European ancestryb















Self-

WLS group estimate of



25-75



Reported
Disease
European ancestry

Std.

percentile


Cohort
Ethnicity
status
(Std. Err.)a
Mean
Deviation
Median
range

















Yoruban Nigerians
African
N/A
N/A
0.036
0.024
0.03
0.019-0.043


Iceland
European
N/A
N/A
0.991
0.015
0.994
0.990-0.996


All American
Eur. Am.
Patients
 0.98 (0.0083)
0.965
0.083
0.991
0.977-0.995


All American
Eur. Am.
Controls
0.979 (0.0079)
0.969
0.07
0.992
0.979-0.995


Philadelphia
Eur. Am.
Patients
0.974 (0.0101)
0.955
0.101
0.99
0.971-0.995


Philadelphia
Eur. Am.
Controls
0.969 (0.009)
0.959
0.09
0.991
0.969-0.995


Cleveland
Eur. Am.
Patients
0.982 (0.0079)
0.971
0.068
0.991
0.980-0.995


Cleveland
Eur. Am.
Controls
0.981 (0.0081)
0.972
0.06
0.991
0.979-0.995


Atlanta
Eur. Am.
Patients
0.995 (0.0075)
0.981
0.038
0.991
0.984-0.994


Atlanta
Eur. Am.
Controls
0.982 (0.0092)
0.973
0.066
0.993
0.983-0.995


All American
Afr. Am.
Patients
0.243 (0.0138)
0.223
0.184
0.178
0.108-0.282


All American
Afr. Am.
Controls
0.213 (0.016)
0.199
0.145
0.174
0.094-0.267


Philadelphia
Afr. Am.
Patients
0.252 (0.0178)
0.235
0.195
0.188
0.121-0.288


Philadelphia
Afr. Am.
Controls
0.213 (0.0217)
0.186
0.137
0.157
0.082-0.257


Cleveland
Afr. Am.
Patients
0.232 (0.0222)
0.21
0.174
0.16
0.096-0.282


Cleveland
Afr. Am.
Controls
0.239 (0.0219)
0.223
0.136
0.191
0.127-0.281


Atlanta
Afr. Am.
Patients
0.226 (0.0246)
0.206
0.166
0.167
0.098-0.283


Atlanta
Afr. Am.
Controls
0.198 (0.0128)
0.193
0.155
0.161
0.086-0.252









African American MI patients have, on average, a slightly greater European ancestry than the African American controls in the Philadelphia and Atlanta cohorts (Table 55). When all three cohorts are combined, the African American patients and controls have on average 22.3% and 19.9% European ancestry, respectively (one-sided P=0.046). This difference can largely be accounted for by a handful of individuals who are reported as African Americans, but have a relatively high European ancestry. Potentially misclassified individuals were corrected for by excluding from the study self-reported African Americans with <20% African genetic ancestry according to the Structure results (seven patients and four controls). The result was a notable reduction in the difference between patients (20%) and controls (19.2%). Tables 52 and 53 show that controlling for ancestry, whether by excluding potentially misclassified individuals or by using individual European ancestry estimates as covariate (Pritchard et al. Am J Hum Genet 67, 170-81, 2000), referred to as admixture adjustment, has a negligible effect on the RR and statistical significance of HapK's association to MI in African Americans. Thus, the higher RR of HapK in African Americans is not simply a consequence of differences in European ancestry between patients and controls.
The African American carriers of HapK have, on average, more European ancestry than those who do not carry HapK, 28.9% against 19.8% (two-sided P=0.00008). This is consistent with the observation that HapK is not found in the Nigerian HapMap sample while it is relatively common in Iceland and the CEPH CEU samples used in the HapMap project (See Table 51). Although HapK is also common in the Asian HapMap samples, the Structure-based estimate of Asian ancestry in African Americans is small ( ̃1%), supporting the hypothesis that HapK observed in African Americans are mostly of European origin. Furthermore, we could detect no difference in Asian ancestry between African American patients and controls or between HapK carriers and non-carriers.
The LTA4H gene resides within a single linkage disequilibrium (LD) block in both European and African populations and is the only known gene within that block. To identify a single causal variant captured by HapK, a 75 kb region encompassing the LD block containing LTA4H in a number of pooled DNA samples of Icelandic patients and controls (some of which contained only HapK carriers) was sequenced. In addition, the correlation of HapK with other SNPs in the HapMap database (Phase I, version 16c.1) was examined. The best single SNP surrogate of HapK identified through both of these approaches was rs2660899 or SG12S551 (R2=0.7 in the CEU samples). This SNP was genotyped in the Philadelphia cohort, where HapK exhibited the strongest effect. While its T allele conferred a RR of 1.31 (P=0.008) in European Americans, it did not capture the RR conferred by HapK in this African American cohort. Thus, rs2660899 can be ruled out as a sole causal variant captured by HapK. However, this SNP may be used as a surrogate for HapK in defining carrier status and MI risk due to HapK.
In theory, the observed association of MI to HapK could be the result of association to a causal variant in the neighbourhood of LTA4H but outside the LD block. This scenario might explain the high RR observed in the recently admixed African Americans, potentially boosted by strong admixture-derived LD, and the modest RR in the non-admixed groups of European Americans and Icelanders. However, given the existing patterns of LD in European and African populations, the kind of admixture found in African Americans, which was examined by creating a 4:1 mixture of haplotypes from the Yoruban and CEPH HapMap samples, is not expected to produce a correlation between HapK and any known SNP outside of the LTA4H LD block with R2>0.25. Given that the observed effect of HapK on MI is very strong in African Americans, it is implausible that the association is the consequence of a variant only loosely correlated with HapK. Moreover, an analysis of five markers with significant allele frequency differences between African and European Americans controls that were located just outside the LTA4H LD block, revealed none that were associated with HapK or uncovered differences between African American patients and controls (Table 56). Thus, the difference in local ancestry between African American patients and controls appears to be limited to HapK.







TABLE 56







Markers used to evaluate the local ancestry arount LTA4H


















Eur. Am.
Afr. Am.
Afr. Am.


Icelandic
HapMap
HapMap


Marker (allele)
Location
controls (n)
controls (n)
patients (n)
P-val 1
P-val 2
controls
CEU (n)
YRI (n)





*DG12S1668 (10)
94966113
0.238 (1105)
0.072 (215)
0.072 (145)
1.60E−17
0.85
0.218 (808)
0.25 (56)
   0 (54)


rs2660885 (C)
94958339
0.805 (1720)
0.644 (374)
0.629 (190)
6.40E−20
0.65
0.854 (833)
0.85 (60)
0.442 (60)


rs2660875 (T)
94949965
0.535 (1725)
 0.4 (380)
 0.38 (183)
1.50E−11
0.41
0.496 (795)
NA
NA


rs2660850 (T)
94946089
0.827 (1742)
 0.68 (381)
0.696 (189)
1.40E−18
0.58
0.866 (830)
NA
NA


**DG12S1664 (0)
94887493
0.873 (926) 
0.721 (201)
0.758 (126)
7.60E−13
0.28
0.856 (825)
0.873 (55) 
0.509 (55)









Table 56 displays the allelic frequencies of markers flanking the LTA4H gene with very significant differences between European American and African American population controls, characterized by the “P-values 1.” The “P-vaules 2” measure the differences between African American patients and African American controls adjusted for cohort and ancestry, all non-significant. The markers used in this analysis of DG 12S1668 are “forward primer”—GCAGTTTAAGCTGTATGTAT ATGAGG (SEQ ID NO: 727), and “reverse primer”—TGAAAGCCATCACTGTAAGGA (SEQ ID NO: 728). The markers used in the analysis of DG12S1664 are: “forward primer”—GGAAGGAGGACACTTCTGGA (SEQ ID NO: 729) and “reverse primer”—GCTGTGAATGGCTAAACTTGG (SEQ ID NO: 730). As shown in Table 57, the location of the primers are the basepair position on chromosome 12 in the human genome assembly build 34 (National Center for Biotechnology Information (NCBI)).
It is striking to discover a genetic variant that confers such different risks of MI in African Americans and populations of European descent. This suggests a strong interaction between HapK and other genetic variants, and/or non-genetic risk factors, such as lifestyle, that are more common in African Americans than in European Americans and Icelanders. These results emphasize that although genetic differences between human continental groups are small (Bamshad et al., Nat Rev Genet 5, 598-609, 2004; Jorde et al., Nat Genet 36, S28-33, 2004), some of these differences may nonetheless contribute to ethnicity-based health disparities (Royal, et al., Nat Genet 36, S5-7, 2004), whether through frequencies of risk alleles and/or risk conferred by such alleles. A strong correspondence between self-reported ethnicity and genetically estimated ancestry had been found. However, ancestry is a quantifiable trait, particularly in heterogeneous or recently admixed populations such as African Americans, which needs to be assessed in order to reliably interpret interactions between ancestry, genes, and environment in the pathogenesis of disease (Reiner et al. Am J Hum Genet 76, 463-77, 2005; Barnholtz-Sloan et al., Cancer Epidemiol Biomarkers Prev 14, 1545-51, 2005; Hoggart et al. Am J Hum Genet 72, 1492-1504, 2003).
The results from this analysis indicate that African Americans are an especially promising target population for the anti-leukotriene therapies described herein. At risk individuals of black African origin that exhibits HapK or other risk factors described herein are specifically contemplated for prophylaxis therapy according to the invention described herein. In addition, HapK or its surrogates may be an especially promising diagnostic marker for defining risk for cardiovascular diseases such as MI and stroke, especially in African-Americans, that may lead to more aggressive treatment of this and other cardiovascular risk factors and modification of lifestyle.

## Claims

1. A method of detecting an LTA4H haplotype associated with myocardial infarction (MI) risk in a human individual and assessing susceptibility to MI in said human individual, comprising
assaying a nucleic acid-containing sample obtained from the human individual to detect therein the presence of an LTA4H haplotype associated with MI risk, wherein the LTA4H haplotype comprises polymorphisms SG12S16 having allele C, SG12S21 having allele G, SG12S23 having allele T, SG12S25 having allele A, SG12S26 having allele T, SG12S100 having allele T, SG12S28 having allele T, SG12S143 having allele C, SG12S144 having allele G, and SG12S221 having allele G,
wherein the presence of the LTA4H haplotype in the nucleic acid-containing sample identifies the individual as having elevated susceptibility to MI.