# Patent

## Title
Biallelic markers derived from genomic regions carrying genes involved in arachidonic acid metabolism

## Abstract

The invention provides polynucleotides including biallelic markers derived from genes involved in arachidonic acid metabolism and from genomic regions flanking those genes. Primers hybridizing to regions flanking these biallelic markers are also provided. This invention also provides polynucleotides and methods suitable for genotyping a nucleic acid containing sample for one or more biallelic markers of the invention. Further, the invention provides methods to detect a statistical correlation between a biallelic marker allele and a phenotype and/or between a biallelic marker haplotype and a phenotype.

## Background
RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 10/170,097, filed Jun. 10, 2002 now U.S. Pat. No 6,794,143, which is a continuation of U.S. patent application Ser. No. 09/641,638, filed Aug. 16, 2000 (now U.S. Pat. No. 6,432,648); which is a continuation-in-part of both U.S. patent application Ser. No. 09/502,330 (abandoned), filed Feb. 11, 2000, and International Patent Application No. PCT/IB00/00184, filed Feb. 11, 2000, which are continuations-in-part of U.S. patent application Ser. No. 09/275,267, filed Mar. 23, 1999 (abandoned). U.S. patent application Ser. No. 09/502,330 (abandoned) and International Patent Application No. PCT/IB00/00184 are continuations-in-part of U.S. patent application Ser. No. 09/275,267, filed Mar. 23, 1999 (abandoned), and International Patent Application No. PCT/IB00/00184 claims benefit of U.S. Provisional Patent Application Ser. No. 60/133,200, filed May 7, 1999, and U.S. Provisional Patent Application Ser. No. 60/119,917, filed Feb. 12, 1999. Each of the above applications are hereby incorporated herein in their entirety including any sequence lists, figures, tables, or drawings.


SEQUENCE LISTING INFORMATION
The Sequence Listing for this application is on duplicate compact discs labeled “Copy 1” and “Copy 2.” Copy 1 and Copy 2 each contain only one file named “Sequence-List.txt” which was created on Jun. 10, 2002, and is 1,243 KB. The entire contents of each of the computer discs are incorporated herein by reference in their entireties.
FIELD OF THE INVENTION
The present invention is in the field of pharmacogenomics, and is primarily directed to biallelic markers that are located in or in the vicinity of genes, which have an impact on arachidonic acid metabolism and the uses of these markers. The present invention encompasses methods of establishing associations between these markers and diseases involving arachidonic acid metabolism such as inflammatory diseases as well as associations between these markers and treatment response to drugs acting on arachidonic acid metabolism. The present invention also provides means to determine the genetic predisposition of individuals to such diseases and means to predict responses to such drugs.
BACKGROUND OF THE INVENTION
The metabolites of arachidonic acid and related fatty acids, collectively termed eicosanoids, exhibit a wide range of biological activities affecting virtually every organ system in mammals. Eicosanoids are among the most important chemical mediators and modulators of the inflammatory reaction and contribute to a number of physiological and pathological processes (See Hardman J. G., Goodman, Gilman A., Limbird L. E.; Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th edition, McGraw-Hill, N.Y., 1996).
Physiology Pathophysiology and Pharmacological Importance of the Eicosanoids
The eicosanoids are extremely prevalent and have been detected in almost every tissue and body fluid. These lipids contribute to a number of physiological and pathological processes including inflammation, smooth muscle tone, hemostasis, thrombosis, parturition and gastrointestinal secretion. Once synthesized in response to a stimulus, the eicosanoids are not stored to any significant extent but are released immediately and act locally. After they act, they are quickly metabolized by local enzymes to inactive forms. Accordingly, the eicosanoids are categorized as autocrine agents or local hormones. They alter the activities of the cells in which they are synthesized and of adjoining cells. The nature of these effects may vary from one type of cell to another, in contrast with the more uniform actions of global hormones such as insulin, for example. Therefore, the eicosanoids, as local chemical messengers, exert a wide variety of effects in virtually every tissue and organ system.
The principal eicosanoids are the prostaglandins (PG), the thromboxanes (TX) and the leukotrienes (LT), though other derivatives of arachidonate, for example lipoxins, are also produced. They fall into different classes designated by letters and the main classes are further subdivided and designated by numbers.
Inflammatory and Immune Responses
Eicosanoids are lipid mediators of inflammation and play a central, often synergistic, role in numerous aspects of inflammatory responses and host defense. Prostaglandins and leukotrienes are released by a host of mechanical, thermal, chemical, bacterial, and other insults, and they contribute importantly to the genesis of the signs and symptoms of inflammation. The ability to mount an inflammatory response is essential for survival in the face of environmental pathogens and injury, although in some situations and diseases the inflammatory response may be exaggerated and sustained for no apparent beneficial reason. This is the case in numerous chronic inflammatory diseases and allergic inflammation. Acute allergic inflammation is characterized by increased blood flow, extravasation of plasma and recruitment of leukocytes. These events are triggered by locally released inflammatory mediators including eicosanoids and more particularly leukotrienes. The leukotrienes generally have powerful effects on vascular permeability and the leukotriene LTB4 is a potent chemoattractant for leukocytes and promotes exudation of plasma. The prostaglandins PGE2 and PGI2 markedly enhance edema formation and leukocyte infiltration in the inflamed region. Moreover, they potentiate the pain-producing activity of bradykinin.
The participation of arachidonic acid (AA) metabolism in inflammatory diseases such as rheumatoid arthritis, asthma and acute allergy is well established. Prostaglandins have been involved in inflammation, pain and fever. Pathological actions of leukotrienes are best understood in terms of their roles in immediate hypersensitivity and asthma. Lipoxygenases, e.g., 5-lipoxygenase (5-LO), 12-lipoxygenase (12-LO), 15-lipoxygenase A (15-LOA), and 15-lipoxygenase B (15-LOB), have been implicated in the pathogenesis of a variety of inflammatory conditions such as psoriasis and arthritis.
Cardiovascular System
The prostaglandins PGEs, PGF2 and PGD2 cause both vasodilation and vasoconstriction. Responses vary with concentration and vascular bed. Systemic blood pressure generally falls in response PGEs, and blood flow to most organs, including the heart, is increased. These effects are particularly striking in some hypertensive patients. Cardiac output is generally increased by prostaglandins of the E and F series. The importance of these vascular actions is emphasized by the participation of PGI2 and PGE2 in the hypotension associated with septic shock. The prostaglandins also have been implicated in the maintenance of patency of the ductus arteriosus. Thromboxane synthase (TXA2), also known as CYP5, is a potent vasoconstrictor. Leukotriene C4 synthase (LTC4) and the leokotriene LTD4 cause hypotension. The leukotrienes have prominent effects on the microvasculature. LTC4 and LTD4 appear to act on the endothelial lining of postcapillary venules to cause exudation of plasma; they are more potent than histamine in this regard. In higher concentrations, LTC4 and LTD4 constrict arterioles and reduce exudation of plasma.
Blood/Platelets
Prostanoids including prostaglandins and thromboxanes exhibit a wide variety of actions in various cells and tissues to maintain local homeostasis in the body. Eicosanoids modify the function of the formed elements of the blood. PGI2 controls the aggregation of platelets in vivo and contributes to the antithrombogenic properties of the intact vascular wall.
TXA2 is a major product of arachidonate metabolism in platelets and, as a powerful inducer of platelet aggregation and the platelet release reaction, is a physiological mediator of platelet aggregation. Pathways of platelet aggregation that are dependent on the generation of TXA2 are sensitive to the inhibitory action of aspirin, which inhibits the cyclooxygenase (COX) pathway. There has been considerable interest in the elucidation of the role played by prostaglandins and TXA2 in platelet aggregation and thrombosis and by PGI2 in the prevention of these events. The platelet thromboxane pathway is activated markedly in acute coronary artery syndromes and aspirin is beneficial in the secondary prevention of coronary and cerebrovascular diseases. PGI that is generated in the vessel wall may be the physiological antagonist of this system; it inhibits platelet aggregation and contributes to the nonthrombogenic properties of the endothelium. According to this concept, PGI2 and TXA2 represent biologically opposite poles of a mechanism for regulating platelet-vessel wall interaction and the formation of hemostatic plugs and intraarterial thrombi. There is interest in drugs which inhibit thromboxane synthase and modulate PGI2 production.
Smooth Muscle
Prostaglandins contract or relax many smooth muscles beside those of the vasculature. The leukotrienes contract most smooth muscles. In general, PGFs and PGD2 contract and PGEs relax bronchial and tracheal muscle. LTC4 and LTD4 are bronchoconstrictors. They act principally on smooth muscle in peripheral airways and are 1000 times more potent than histamine both in vitro and in vivo. They also stimulate bronchial mucus secretion and cause mucosal edema. A complex mixture of chemical messengers is released when sensitized lung tissue is challenged by the appropriate antigen. Various prostaglandins and leukotrienes are prominent components of this mixture. Response to the leukotrienes probably dominates during allergic constriction of the airway. Evidence for this conclusion is the ineffectiveness of inhibitors of cycloxygenase and of histaminergic antagonists in the treatment of human asthma and the protection afforded by leukotriene antagonists in antigen induced bronchoconstriction. A particularly important role for the cysteinyl-leukotrienes (LTC4, LTD4, and LTE4) has been suggested in pathogenesis of asthma, which is now recognized as a chronic inflammatory condition. They are potent spasmogens causing a contraction of bronchiolar muscle and an increase in mucus secretion.
Gastric and Intestinal Secretions
PGEs and PGI2 inhibit gastric acid secretion stimulated by feeding, histamine or gastrin. Mucus secretion in the stomach and small intestine is increased by PGEs. These effects help to maintain the integrity of the gastric mucosa and are referred to as the cytoprotectant properties of PGEs. Furthermore, PGEs and their analogs inhibit gastric damage caused by a variety of ulcerogenic agents and promote healing of duodenal and gastric ulcers. Cytoprotection is of therapeutic importance and PGE1 analogs are used for the prevention of gastric ulcers.
Kidney and Urine Formation
Prostaglandins modulate renal blood flow and may serve to regulate urine formation by both renovascular and tubular effects. Increased biosynthesis of prostaglandins has been associated with Bartter's syndrome, a rare disease, characterized by urinary wasting of K+.
Leukotrienes have been involved in the pathophysiology of glomerular immune injury.
Reproduction and Parturition
Much interest is attached to the possible involvement of prostaglandins in reproductive physiology. Lowered concentrations of prostaglandins in semen have been implicated in male infertility. Prostaglandins are also thought to contribute to the symptoms of primary dysmenorrhea. Inhibitors of cyclooxygenase are effective in relieving the symptoms of this condition. Elevated levels of prostaglandins are involved in onset of labor. Inhibitors of cyclooxygenase increase the length of gestation and interrupt premature labor.
Cancer Metastasis
Tumors in animals and certain spontaneous human tumors are accompanied by increased concentrations of local or circulating prostaglandins. Eicosanoids have been shown to be involved in various aspects of neoplasia including cell transformation, tumor promotion, tumor cell growth, and metastasis. Some studies have implicated platelet aggregation and the effects of prostaglandins and hydroxyeicosatetraenoic acid (12-HETE) in the hematogenous metastasis of tumors.
Many of the products of arachidonic acid metabolism are potent mediators of physiological responses and contribute to disorders of development, cellular function, tissue repair, and host defenses in a number of diseases.
Arachidonic Acid Metabolism and Biosynthesis of Eicosanoids
The primary source of eicosanoids in mammalian systems is the metabolic products of arachidonic acid. After stimulation by trauma, infection, or inflammation, translocated phospholipases, especially phospholipase A2, act on membrane phospholipids to liberate arachidonic acid. Once released, arachidonate is metabolized to oxygenated products by several distinct enzyme pathways, including cyclooxygenases, several lipoxygenases, and cytochrome P450s (CYP). The specific enzyme pathway involved determines, which products are formed.
Release of Arachidonic Acid from Cell Membranes and its Regulation
The eicosanoids are a family of substances produced from the polyunsaturated fatty acid arachidonic acid, which is present in plasma-membrane phospholipids. The first rate-limiting step in the biosynthesis of eicosanoids is the release of arachidonic acid from the membrane, a process that is mainly catalyzed by cytosolic phosholipase A2 (cPLA2). The synthesis of eicosanoids begins when a stimulus such as a hormone, a neurotransmitter, a drug or a toxic agent activates cytosolic phospholipase A2. This arachidonic acid specific phospholipase plays a major role in the cell signaling events that initiate the arachidonate cascade. One important trigger of arachidonate release and eicosanoid synthesis involves tissue injury and inflammation.
The activities of many enzymes are regulated by calmodulins (CAL) that serve as calcium sensors in eukaryotic cells. The binding of Ca2+ to multiple sites in calmodulin induces a major conformational change that converts it from an inactive to an active form. Activated calmodulin then binds to many enzymes and target proteins in the cell, modifying their activities and thereby regulating various metabolic pathways. Calmodulins are involved in a number of processes regulated by Ca2+ including smooth muscle contraction, neurotransmission, apoptosis, cell cycle progression and gene expression. Calmodulins also participate in the regulation of arachidonate release. They directly stimulate cytosolic phospholipase A2, whereas calmodulin antagonists inhibit enzyme activity and the release of arachidonic acid.
Annexins (ANX) are a family of multifunctional calcium and phospholipid-binding proteins, they belong to a family of proteins that interact with phospholipids in a Ca2+ dependant manner. Annexins have been implicated in the pathogenesis of benign and malignant neoplasms of different origins. Moreover, several annexins have also been involved in autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis and inflammatory bowl disease. Numerous physiological functions have been attributed to annexins including regulation of membrane traffic during exocytosis and endocytosis, mediation of cytoskeletal-membrane interactions, membrane receptor function, regulation of membrane-dependent enzymes, mitogenic signal transduction, transmembrane ion channel activity, cell-cell adhesion, antiinflammatory properties, inhibition of blood coagulation and inhibition of phospholipase A2. Annexins have been suggested as regulators of prostaglandin metabolism and of the arachidonate cascade as a result of their inhibitory effect on phospholipase A2. It is still a matter of debate as to whether inhibition of phospholipase A2 is the result of calcium-dependent sequestration of phospholipids (substrate depletion mechanism) or a direct effect of the annexins acting via protein-protein interactions. Calpactin I (light chain) is the cellular ligand of annexin II and induces its dimerization. Annexin II and calpactin I (CALPA) constitute a calcium binding complex composed of two light chains (calpactin I) and two heavy chains (annexin II). Calpactin I may function as regulator of annexin II phosphorylation.
The activities of phospholipase A2, annexins and calmodulins are common points of regulation in the formation of all eicosanoids.
Downstream of phospholipase A2, the varying eicosanoid-pathway enzymes found in particular cell types determine which eicosanoids are synthesized in response to particular stimuli.
Cyclooxygenase Pathway
This pathway initiated by cyclooxygenase (COX) leads ultimately to formation of the cyclic endoperoxides, prostaglandins (PG), and thromboxanes (TX).
There are two isoforms of the cyclooxygenase, COX-1 and COX-2. The former is constitutively expressed in most cells. In contrast, COX-2 is not normally present but may be induced by certain factors such as cytokines and growth factors. The cyclooxygenases have two distinct activities: an endoperoxidase synthase activity that oxygenates and cyclizes the unesterified precursor fatty acid to form the cyclic endoperoxide PGG and a peroxidase activity that converts PGG to PGH. PGG and PGH are chemically unstable, but they can be transformed enzymatically into a variety of products, including PGI, TXA2, PGE, PGF or PGD. Isomerases lead to the synthesis of PGE2 and PGD2, whereas PGI2 is formed from PGH2 through prostacyclin synthase. TXA2 is formed by thromboxane synthase. Although most tissues are able to synthesize the PGG and PGH intermediates from free arachidonate, the fate of these precursors varies in each tissue and depends on the complement of enzymes that are present and on their relative abundance. For example, lung and spleen are able to synthesize the whole range of products. In contrast, platelets contain thromboxane synthase as the principal enzyme that metabolizes PGH, while endothelial cells contain primarily prostacyclin synthase.

Lipoxygenase Pathways

Lipoxygenases are a family of cytosolic enzymes that catalyze the oxygenation of fatty acids to corresponding lipid hydroperoxides. Arachidonate is metabolized to HPETE (hydroperoxyeicosatetraenoic acid), which is then converted either enzymatically or non-enzymatically to 12-HETE (hydroxyeicosatetraenoic acid). HPETEs may further be converted to hepoxilins and lipoxins. Lipoxygenases differ in their specificity for placing the hydroperoxy group, and tissues differ in the lipoxygenases they contain. These enzymes are referred to as 12-, 15-, 5- and 8-lipoxygenases according to the oxygenation sites in arachidonic acid as substrate.
The lipoxygenases catalyze reactions and generate products of potential relevance to membrane remodeling, cell differentiation and inflammation. Products of the 15-LO pathway could contribute to the pathophysiology of allergic airway inflammation while products of the 12-LO pathway have been implicated in cancer metastasis, psoriasis and inflammation.
Various biological activities have been reported for the 12-lipoxygenase metabolites of arachidonic acid. As other eicosanoids, they are important chemical mediators and modulators of the inflammatory reaction. 12-HETE is the major arachidonic acid metabolite of 12-lipoxygenase and seems to be implicated in a wide-spectrum of biological activities such as stimulation of insulin secretion by pancreatic tissue, suppression of renin production, chemoattraction of leukocytes and initiation of growth-related signaling events, such as activation of oncogenes, protein kinase C, and mitogen-activated protein kinases. 12-lipoxygenase activity and 12-HETE production are also important determining factors in tumor cell metastasis and have been implicated in human prostate cancer and breast cancer (Honn et al., Cancer Metastasis Rev., 13:365-396, 1994, Gao et al., Adv. Exp. Med. Biol., 407:41-53, 1997; Natarajan et al., J. Clin. Endocr. Metab., 82:1790-1789, 1997,). Further, 12-HETE has also been implicated in inflammatory skin diseases such as psoriasis (Hussai et al., Am. J. Physiol., 266:243-253, 1994). As mentioned above, metabolism of arachidonic acid by 12-lipoxygenase further generates lipoxins and hepoxillins. Lipoxins play the role of both immunologic and hemodynamic regulators and a variety of biological activities have been reported for hepoxillins which are related to the release of intracellular calcium and the opening of potassium channels (Yamamoto et al., Pro. Lipid Res., 36:23-41, 1997).
The 5-lipoxygenase (5-LO) is perhaps the most important of these enzymes since it leads to the synthesis of leukotrienes. Activation of the 5-LO enzyme involves its docking to a protein termed 5-lipoxygenase-activating protein (FLAP). This binding activates the enzyme, results in its association with the cell membrane and increased synthesis of 5-HPETE and leukotrienes. Leukotriene A (LTA) synthase is associated with 5-lipoxygenase and promotes the rearrangement of 5-HPETE to an unstable intermediate LTA4; which may be transformed to LTB4 by leukotriene A4 hydrolase (LTA4H); alternatively, it may be conjugated with glutathione by LTC4 synthase to form LTC4. LTA4 hydrolase is a pivotal element in leukotriene biosynthesis. Omega-oxidation is regarded as the major pathway for the catabolism of LTB4. This reaction is catalyzed by LTB4 omega-hydroxylase (LTB4H3) also called CYP4F2. LTD4 is produced by the removal of glutamic acid from LTC4 and LTE4 results from the subsequent cleavage of glycine; the reincorporation of glutamic acid yields LTF4.
Epoxygenase Pathway
Arachidonate is metabolized to a variety of metabolites by enzymes that contain cytochrome P450. The epoxygenase pathway of the arachidonic acid cascade leads to the formation of epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs). CYP2J2 is a human cytochrome P450 arachidonic acid epoxygenase expressed in extrahepatic tissues and particularly in the intestine. In addition to the known effects on intestinal vascular tone, CYP2J2 products may be involved in the release of intestinal neuropeptides, control of intestinal motility and modulation of intestinal fluid/electrolyte transport.
Eicosanoid Receptors
The diversity of the effects of eicosanoids is explained by the existence of a number of distinct receptors that mediate their actions. All prostaglandin receptors identified to date are coupled to effector mechanisms through G proteins. Distinct receptors for leukotrienes also have been identified in different tissues, all of these appear to activate phospholipase C.
Therapeutic Agents Interacting with Arachidonic Acid Metabolism
Because of their involvement in so many disease states, there has been a considerable effort to develop effective inhibitors to the formation or action of the eicosanoids. The drugs that influence the eicosanoid pathways are the most commonly used drugs in the world today. Their major uses are to reduce pain, fever and inflammation. Several classes of drugs, most notably the nonsteroidal antiinflammatory drugs (NSAIDs) owe their therapeutic effects to blockade of the formation of eicosanoids. Selective inhibitors of arachidonic acid metabolism also have an important therapeutic value. Inhibition of cyclooxygenase (COX), the enzyme responsible for the biosynthesis of the prostaglandins and certain related autacoids, generally is thought to be a major facet of the mechanism of NSAIDs. Aspirin and newer, widely used drugs belong to the NSAIDs. All NSAIDs are antipyretic, analgesic and antiinflammatory but there are important differences in their activities and in their side effects. The reasons for such differences are not fully understood. Side effects of these drugs include gastrointestinal ulceration, disturbances in platelet function, changes in renal function and hypersensitivity reactions. It is now appreciated that there are two forms of cyclooxygenase (COX), inhibition of COX-2 is thought to mediate the antipyretic, analgesic and antiinflammatory action of NSAIDs, whereas the simultaneous inhibition of COX-1 may result in unwanted side effects. Efforts are under way to identify COX-2 specific agents. But, it is also possible that enhanced generation of lipoxygenase products, due to the diversion of arachidonic acid metabolism from the cyclooxygenase pathway towards the lipoxygenase pathways, contributes to some of the side effects. Effort is being devoted to a search for drugs that will produce more selective interventions by acting farther along the biosynthetic pathways. Several compounds have been described that selectively antagonize responses to TXA2 and to PGH2. Some are receptor antagonists others directly inhibit thromboxane synthase.
Advances in understanding the pathobiology of the inflammatory process has suggested several novel approaches for development of drugs to block this process. These include phospholipase A2 inhibitors. Glucocorticoids are thought to have an effect on arachidonic acid metabolism through the induction of lipocortin that inhibits phospholipase A2.
NSAIDs generally do not inhibit the formation of other eicosanoids such as the lipoxygenase-produced leukotrienes. Substantial evidence indicates that leukotrienes contribute to the inflammatory response through a variety of effects. Leukotrienes have been implicated as mediators of inflammation and immediate hypersensitivity reactions—in particular, human bronchial asthma—and thus considerable effort has been done to develop either inhibitors of the production or blockers of the action of the actions of these mediators. Various therapeutic approaches have been used including 5-lipoxygenase inhibitors, which block leukotriene formation, or cysteinyl leukotriene receptor antagonists, which block receptor function. LTC4 synthase is another key step in biosynthesis of leukotrienes and represents another possible site for therapeutic intervention. Drugs targeting leukotriene biosynthesis are being tested and used for their utility in the treatment of various inflammatory conditions.
Most of these drugs are efficacious in providing relief but all available agents have associated, and sometimes severe, toxicity. Certain individuals display intolerance to aspirin and to other drugs acting on arachidonic acid metabolism; this is manifest by symptoms that range from liver toxicity, gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock. The underlying mechanism for these severe side effects is not known. Moreover, while these agents have been highly useful for treatment of acute, self-limited inflammatory conditions; their ability to modify disease progression in chronic inflammatory settings remains an area of controversy. The complexity of the highly regulated pathways and enzymes that lead to the formation of the eicosanoids, has limited the precise identification of the metabolites and enzymes in the arachidonic acid cascade, which play the causal role in pathologies or in side effects to some drugs.
Pharmacogenomics and Arachidonic Acid Metabolism
The vast majority of common diseases, such as cancer, hypertension, diabetes and some inflammatory diseases are polygenic, meaning that they are caused by multiple genes. In addition, these diseases are modulated by environmental factors such as pollutants, chemicals and diet. This is why many diseases are called multifactorial; they result from a synergistic combination of factors, both genetic and environmental. Therapeutic management and drug development could be markedly improved by the identification of specific genetic polymorphisms that determine and predict patient susceptibility to diseases or patient responses to drugs.
To assess the origins of individual variations in disease susceptibility or drug response, pharmacogenomics uses the genomic technologies to identify polymorphisms within genes which are part of biological pathways involved in disease susceptibility, etiology, and development, or more specifically in drug response pathways responsible for a drug's efficacy, tolerance or toxicity. It can provide tools to refine the design of drug development by decreasing the incidence of adverse events in drug tolerance studies, by better defining patient subpopulations of responders and non-responders in efficacy studies and, by combining the results obtained therefrom, to further allow better enlightened individualized drug usage based on efficacy/tolerance prognosis. Pharmacogenomics can also provide tools to identify new targets for designing drugs and to optimize the use of already existing drugs, in order to either increase their response rate and/or exclude non-responders from corresponding treatment, or decrease their undesirable side effects and/or exclude from corresponding treatment patients with marked susceptibility to undesirable side effects. However, for pharmacogenomics to become clinically useful on a large scale, molecular tools and diagnostics tests must become available.
Inflammatory reactions, which are involved in numerous diseases, are highly relevant to pharmacogenomics both because they are at the core of many widespread serious diseases, and because targeting inflammation pathways to design new efficient drugs includes numerous risks of potentiating serious side effects. Arachidonic acid metabolism is particularly relevant since its products, the eicosanoids, are powerful inflammatory molecules and play a role in a number of physiological functions.
Genetic Analysis of Complex Traits
Until recently, the identification of genes linked with detectable traits has relied mainly on a statistical approach called linkage analysis. Linkage analysis is based upon establishing a correlation between the transmission of genetic markers and that of a specific trait throughout generations within a family. Linkage analysis involves the study of families with multiple affected individuals and is useful in the detection of inherited-traits, which are caused by a single gene, or possibly a very small number of genes. Linkage analysis has been successfully applied to map simple genetic traits that show clear Mendelian inheritance patterns and which have a high penetrance (the probability that a person with a given genotype will exhibit a trait). About 100 pathological trait-causing genes have been discovered using linkage analysis over the last 10 years.
But, linkage studies have proven difficult when applied to complex genetic traits. Most traits of medical relevance do not follow simple Mendelian monogenic inheritance. However, complex diseases often aggregate in families, which suggests that there is a genetic component to be found. Such complex traits are often due to the combined action of multiple genes as well as environmental factors. Such complex trait, include susceptibilities to heart disease, hypertension, diabetes, cancer and inflammatory diseases. Drug efficacy, response and tolerance/toxicity can also be considered as multifactoral traits involving a genetic component in the same way as complex diseases. Linkage analysis cannot be applied to the study of such traits for which no large informative families are available. Moreover, because of their low penetrance, such complex traits do not segregate in a clear-cut Mendelian manner as they are passed from one generation to the next. Attempts to map such diseases have been plagued by inconclusive results, demonstrating the need for more sophisticated genetic tools.
Knowledge of genetic variation in the arachidonic acid cascade is important for understanding why some people are more susceptible to disease involving arachidonic acid metabolites or respond differently to treatments targeting arachidonic acid metabolism. Ways to identify genetic polymorphism and to analyze how they impact and predict disease susceptibility and response to treatment are needed.
Although the genes involved in arachidonic acid metabolism represent major drug targets and are of high relevance to pharmaceutical research, we still have scant knowledge concerning the extent and nature of sequence variation in these genes and their regulatory elements. For example, the cDNA and part of the genomic sequence for human 12-lipoxygenase have been cloned and sequenced (Izumi et al., Proc. Natl. Acad. Sci. USA, 87:7477-7481, 1990; Funk et al., Proc. Natl. Acad. Sci. USA, 87:5638-5642, 1990; Yoshimoto et al., Biochem. Biophys. Res. Commun., 172:1230-1235, 1990, Yoshimoto, et al., J. Biol. Chem., 267:24805-24809, 1992). However, the complete genomic sequence of the 12-lipoxygenase, including its regulatory elements, have not been described.
In the cases where polymorphisms have been identified, the relevance of the variation is rarely understood. While polymorphisms hold promise for use as genetic markers in determining which genes contribute to multigenic or quantitative traits, suitable markers and suitable methods for exploiting those markers have not been found and brought to bare on the genes related to arachidonic acid metabolism.
SUMMARY OF THE INVENTION
The present invention is based on the discovery of a set of novel eicosanoid-related biallelic markers. See Table 7(A-B). These markers are located in the coding regions as well as non-coding regions adjacent to genes which express proteins associated with arachidonic acid metabolism. The position of these markers and knowledge of the surrounding sequence has been used to design polynucleotide compositions which are useful in determining the identity of nucleotides at the marker position, as well as more complex association and haplotyping studies which are useful in determining the genetic basis for disease states involving arachidonic acid metabolism. In addition, the compositions and methods of the invention find use in the identification of the targets for the development of pharmaceutical agents and diagnostic methods, as well as the characterization of the differential efficacious responses to and side effects from pharmaceutical agents acting on arachidonic acid metabolism.
The present invention further stems from the isolation and characterization of the genomic sequence of the 12-lipoxygenase gene including its regulatory regions and of the complete cDNA sequence encoding the 12-lipoxygenase enzyme. Oligonucleotide probes and primers hybridizing specifically with a genomic sequence of 12-lipoxygenase are also part of the invention. Furthermore, an object of the invention consists of recombinant vectors comprising any of the nucleic acid sequences described in the present invention, and in particular of recombinant vectors comprising the promoter region of 12-lipoxygenase or a sequence encoding the 12-lipoxygenase enzyme, as well as cell hosts comprising said nucleic acid sequences or recombinant vectors. The invention also encompasses methods of screening of molecules which, modulate or inhibit the expression of the 12-lipoxygenase gene. The invention is also directed to biallelic markers that are located within the 12-lipoxygenase genomic sequence, these biallelic markers representing useful tools in order to identify a statistically significant association between specific alleles of 12-lipoxygenase gene and one or several disorders related to asthma and/or hepatotoxicity.
A first embodiment of the invention encompasses polynucleotides consisting of, consisting essentially of, or comprising a contiguous span of nucleotides of a sequence selected as an individual or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof, wherein said contiguous span is at least 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 75, 100, 200, 500, or 1000 nucleotides in length, to the extent that such a length is consistent with the lengths of the particular Sequence ID. The present invention also relates to polynucleotides hybridizing under stringent or intermediate conditions to a sequence selected as an individual or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof. In addition, the polynucleotides of the invention encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination: Said contiguous span may optionally include the eicosanoid-related biallelic marker in said sequence; Optionally either the original or the alternative allele of Table 9 may be specified as being present at said eicosanoid-related biallelic marker; Optionally either the first or the second allele of Tables 8 or 10 may be specified as being present at said eicosanoid-related biallelic marker; Optionally, said polynucleotide may consists of, or consist essentially of a contiguous span which ranges in length from 8, 10, 12, 15, 18 or 20 to 25, 35, 40, 50, 60, 70, or 80 nucleotides, or be specified as being 12, 15, 18, 20, 25, 35, 40, or 50 nucleotides in length and including an eicosanoid-related biallelic marker of said sequence, and optionally the original allele of Table 9 is present at said biallelic marker; Optionally, said biallelic marker may be within 6, 5, 4, 3, 2, or 1 nucleotides of the center of said polynucleotide or at the center of said polynucleotide; Optionally, the 3′ end of said contiguous span may be present at the 3′ end of said polynucleotide; Optionally, biallelic marker may be present at the 3′ end of said polynucleotide; Optionally, the 3′ end of said polynucleotide may be located within or at least 2, 4, 6, 8, 10, 12, 15, 18, 20, 25, 50, 100, 250, 500, or 1000 nucleotides upstream of an eicosanoid-related biallelic marker in said sequence, to the extent that such a distance is consistent with the lengths of the particular Sequence ID; Optionally, the 3′ end of said polynucleotide may be located 1 nucleotide upstream of an eicosanoid-related biallelic marker in said sequence; and Optionally, said polynucleotide may further comprise a label.
A second embodiment of the invention encompasses any polynucleotide of the invention attached to a solid support. In addition, the polynucleotides of the invention which are attached to a solid support encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said polynucleotides may be specified as attached individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the inventions to a single solid support; Optionally, polynucleotides other than those of the invention may attached to the same solid support as polynucleotides of the invention; Optionally, when multiple polynucleotides are attached to a solid support they may be attached at random locations, or in an ordered array; Optionally, said ordered array may be addressable.
A third embodiment of the invention encompasses the use of any polynucleotide for, or any polynucleotide for use in, determining the identity of one or more nucleotides at an eicosanoid-related biallelic marker. In addition, the polynucleotides of the invention for use in determining the identity of one or more nucleotides at an eicosanoid-related biallelic marker encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination. Optionally, said eicosanoid-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof; Optionally, said polynucleotide may comprise a sequence disclosed in the present specification; Optionally, said polynucleotide may consist of, or consist essentially of any polynucleotide described in the present specification; Optionally, said determining may be performed in a hybridization assay, sequencing assay, microsequencing assay, or an enzyme-based mismatch detection assay; Optionally, said polynucleotide may be attached to a solid support, array, or addressable array; Optionally, said polynucleotide may be labeled.
A fourth embodiment of the invention encompasses the use of any polynucleotide for, or any polynucleotide for use in, amplifying a segment of nucleotides comprising an eicosanoid-related biallelic marker. In addition, the polynucleotides of the invention for use in amplifying a segment of nucleotides comprising an eicosanoid-related biallelic marker encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said eicosanoid-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof; Optionally, said polynucleotide may comprise a sequence disclosed in the present specification; Optionally, said polynucleotide may consist of, or consist essentially of any polynucleotide described in the present specification; Optionally, said amplifying may be performed by a PCR or LCR. Optionally, said polynucleotide may be attached to a solid support, array, or addressable array. Optionally, said polynucleotide may be labeled.
A fifth embodiment of the invention encompasses methods of genotyping a biological sample comprising determining the identity of a nucleotide at an eicosanoid-related biallelic marker. In addition, the genotyping methods of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said eicosanoid-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof; Optionally, said method further comprises determining the identity of a second nucleotide at said biallelic marker, wherein said first nucleotide and second nucleotide are not base paired (by Watson & Crick base pairing) to one another; Optionally, said biological sample is derived from a single individual or subject; Optionally, said method is performed in vitro; Optionally, said biallelic marker is determined for both copies of said biallelic marker present in said individual's genome; Optionally, said biological sample is derived from multiple subjects or individuals; Optionally, said method further comprises amplifying a portion of said sequence comprising the biallelic marker prior to said determining step; Optionally, wherein said amplifying is performed by PCR, LCR, or replication of a recombinant vector comprising an origin of replication and said portion in a host cell; Optionally, wherein said determining is performed by a hybridization assay, sequencing assay, microsequencing assay, or an enzyme-based mismatch detection assay.
A sixth embodiment of the invention comprises methods of estimating the frequency of an allele in a population comprising genotyping individuals from said population for an eicosanoid-related biallelic marker and determining the proportional representation of said biallelic marker in said population. In addition, the methods of estimating the frequency of an allele in a population of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said eicosanoid-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof; Optionally, determining the frequency of a biallelic marker allele in a population may be accomplished by determining the identity of the nucleotides for both copies of said biallelic marker present in the genome of each individual in said population and calculating the proportional representation of said nucleotide at said eicosanoid-related biallelic marker for the population; Optionally, determining the frequency of a biallelic marker allele in a population may be accomplished by performing a genotyping method on a pooled biological sample derived from a representative number of individuals, or each individual, in said population, and calculating the proportional amount of said nucleotide compared with the total.
A seventh embodiment of the invention comprises methods of detecting an association between an allele and a phenotype, comprising the steps of a) determining the frequency of at least one eicosanoid-related biallelic marker allele in a case population, b) determining the frequency of said eicosanoid-related biallelic marker allele in a control population and; c) determining whether a statistically significant association exists between said genotype and said phenotype. In addition, the methods of detecting an association between an allele and a phenotype of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said eicosanoid-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof; Optionally, said control population may be a trait negative population, or a random population; Optionally, each of steps a) and b) is performed on a single pooled biological sample derived from each of said populations; Optionally, each of said steps a) and b) is performed on a single pooled biological sample derived from each of said populations; Optionally, each of said steps a) and b) is performed separately on biological samples derived from each individual in said populations; Optionally, said phenotype is a disease involving arachidonic acid metabolism, a response to an agent acting on arachidonic acid metabolism, or a side effects to an agent acting on arachidonic acid metabolism; Optionally, the identity of the nucleotides at the biallelic markers in everyone of the following sequences: SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300 is determined in steps a) and b).
An eighth embodiment of the present invention encompasses methods of estimating the frequency of a haplotype for a set of biallelic markers in a population, comprising the steps of: a) genotyping each individual in said population for at least one eicosanoid-related biallelic marker, b) genotyping each individual in said population for a second biallelic marker by determining the identity of the nucleotides at said second biallelic marker for both copies of said second biallelic marker present in the genome; and c) applying a haplotype determination method to the identities of the nucleotides determined in steps a) and b) to obtain an estimate of said frequency. In addition, the methods of estimating the frequency of a haplotype of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally said haplotype determination method is selected from the group consisting of asymmetric PCR amplification, double PCR amplification of specific alleles, the Clark method, or an expectation maximization algorithm; Optionally, said second biallelic marker is an eicosanoid-related biallelic marker in a sequence selected from the group consisting of the biallelic markers of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof; Optionally, the identity of the nucleotides at the biallelic markers in everyone of the sequences: SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300 is determined in steps a) and b).
A ninth embodiment of the present invention encompasses methods of detecting an association between a haplotype and a phenotype, comprising the steps of: a) estimating the frequency of at least one haplotype in a case population according to a method of estimating the frequency of a haplotype of the invention; b) estimating the frequency of said haplotype in a control population according to the method of estimating the frequency of a haplotype of the invention; and c) determining whether a statistically significant association exists between said haplotype and said phenotype. In addition, the methods of detecting an association between a haplotype and a phenotype of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said eicosanoid-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof; Optionally, said control population may be a trait negative population, or a random population; Optionally, said phenotype is a disease involving arachidonic acid metabolism, a response to an agent acting on arachidonic acid metabolism, or a side effects to an agent acting on arachidonic acid metabolism; Optionally, the identity of the nucleotides at the biallelic markers in everyone of the following sequences: SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300 is included in the estimating steps a) and b).
A tenth embodiment of the present invention is a method of administering a drug or a treatment comprising the steps of: a) obtaining a nucleic acid sample from an individual; b) determining the identity of the polymorphic base of at least one eicosanoid-related biallelic marker or 12-LO-related biallelic marker according to the methods taught herein which is associated with a positive response to said drug or treatment, or at least one eicosanoid-related marker or 12-LO-related biallelic marker or which is associated with a negative response to said drug or treatment; and c) administering said drug or treatment to said individual if said nucleic acid sample contains at least one biallelic marker associated with a positive response to said drug or treatment, or if said nucleic acid sample lacks at least one biallelic marker associated with a negative response to said drug or treatment. In addition, the methods of the present invention for administering a drug or a treatment encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, said eicosanoid-related biallelic marker or 12-LO-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof; or optionally, the administering step comprises administering the drug or the treatment to the individual if the nucleic acid sample contains said biallelic marker associated with a positive response to the treatment or the drug and the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug.
An eleventh embodiment of the present invention is a method of selecting an individual for inclusion in a clinical trial of a treatment or drug comprising the steps of: a) obtaining a nucleic acid sample from an individual; b) determining the identity of the polymorphic base of at least one eicosanoid-related biallelic marker or 12-LO-related biallelic marker which is associated with a positive response to the treatment or the drug, or at least one eicosanoid-related biallelic marker or 12-LO-related biallelic marker which is associated with a negative response to the treatment or the drug in the nucleic acid sample, and c) including the individual in the clinical trial if the nucleic acid sample contains said eicosanoid-related biallelic marker or 12-LO-related biallelic marker associated with a positive response to the treatment or the drug or if the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug. In addition, the methods of the present invention for selecting an individual for inclusion in a clinical trial of a treatment or drug encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said eicosanoid-related biallelic marker or 12-LO-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof, preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof, or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof.
Additional embodiments are set forth in the Detailed Description of the Invention and in the Examples.
BRIEF DESCRIPTION OF THE TABLES
Table 1 contains the first five markers listed in the sequence listing and their corresponding SEQ ID numbers.
Tables 2A-C are a list of 12-LO-related biallelic markers.
Table 3 is a listing of currently available forensic testing systems and their characteristics as compared to the method of the invention.
Table 4 sets forth the number of biallelic markers (VNTRs) needed to obtain, in mean, a ratio of at least 106 or 108.
Table 5 provides an indication of the descriminatory potential of the systems of the invention.
Table 6 is a listing of probabilities for several different types of relationships and likelihood ratios.
Table 7A is a chart containing a list of all of the eicosanoid-related biallelic markers for each gene with an indication of the gene for which the marker is in closest physical proximity, an indication of whether the markers have been validated by microsequencing (with a Y indicating that the markers have been validated by microsequencing and an N indicating that it has not), and an indication of the identity and frequency of the least common allele determined by genotyping (with a blank left to indicate that the frequency has not yet been reported for some markers). The frequencies were determined from DNA samples collected from a random US Caucasian population. When the marker was determined to be homozygous at the particular location for the random US Caucasian population, the homozygous bases were recorded in the “Genotyping Least Common Allele Frequency” column of Table 7A. For example, Seq. ID No. 16 was determined to be homozygous G/G at the biallelic marker position 478 in the US control population, therefore G/G was recorded in the “Genotyping Least Common Allele Frequency” column.
Table 7B contains all of the eicosanoid-related biallelic markers provided in Table 7A; however, they are provided in shorter, easier to search sequences of 47 nucleotides. Accordingly, Table 7A begins with SEQ ID No. 1 and ends with SEQ ID No. 654, while Table 7B begins with SEQ ID No. 655 and ends with SEQ ID No. 1604 (SEQ ID Nos. 651-654 correspond to the genomic and protein sequences of the invention and are not repeated in Table 7B). Table 1 contains the first five markers listed in the sequence listing and their corresponding SEQ ID numbers in Tables 7A and 7B to illustrate the relationship between Tables 7A and 7B:











TABLE 1







BIALLELIC

BIALLELIC




MARKER
SEQ ID
MARKER



SEQ ID NO.
POSITION
NO. IN
POSITION


BIALLELIC
IN TABLE
IN SEQ
TABLE
IN SEQ


MARKER ID
7A
ID NO.
7B
ID NO.







10-253-118
1
478
655
24


10-253-298
2
478
656
24


10-253-315
3
478
657
24


10-499-155
4
478
658
24


10-520-256
5
478
659
24









Table 7B is the same as Table 7A in that it is a list of all of the eicosanoid-related biallelic markers for each gene with an indication of the gene for which the marker is in closest physical proximity, an indication of whether the markers have been validated by microsequencing (with a Y indicating that the markers have been validated by microsequencing and an N indicating that it has not), and an indication of the identity and frequency of the least common allele determined by genotyping (with a blank left to indicate that the frequency has not yet been reported for some markers). However, the “Biallelic Marker Position in SEQ ID No.” for all of the eicosanoid-related biallelic markers provided in Table 7B is position 24 (representing the midpoint of the 47mers that make up Table 7B). The frequencies were determined from DNA samples collected from a random US Caucasian population. When the marker was determined to be homozygous at the particular location for the random US Caucasian population, the homozygous bases were recorded in the “Genotyping Least Common Allele Frequency” column of Table 7B. For example, Seq. ID No. 670 was determined to be homozygous G/G at the biallelic marker position 24 in the US control population, therefore G/G was recorded in the “Genotyping Least Common Allele Frequency” column.

Tables 8, 9, and 10 are charts containing lists of the eicosanoid-related biallelic markers. Each marker is described by indicating its SEQ ID, the biallelic marker ID, and the two most common alleles. Table 8 is a chart containing a list of biallelic markers surrounded by preferred sequences. In the column labeled, “POSITION RANGE OF PREFERRED SEQUENCE” of Table 8 regions of particularly preferred sequences are listed for each SEQ ID, which contain an eicosanoid-related biallelic marker, as well as particularly preferred regions of sequences that do not contain an eicosanoid-related biallelic marker but, which are in sufficiently close proximity to an eicosanoid-related biallelic marker to be useful as amplification or sequencing primers.
Table 11 is a chart listing particular sequences that are useful for designing some of the primers and probes of the invention. Each sequence is described by indicating its Sequence ID and the positions of the first and last nucleotides (position range) of the particular sequence in the Sequence ID.
Table 12 is a chart listing microsequencing primers which have been used to genotype eicosanoid-related biallelic markers (indicated by an *) and other preferred microsequencing primers for use in genotyping eicosanoid-related biallelic markers. Each of the primers which falls within the strand of nucleotides included in the Sequence Listing are described by indicating their Sequence ID number and the positions of the first and last nucleotides (position range) of the primers in the Sequence ID. Since the sequences in the Sequence Listing are single stranded and half the possible microsequencing primers are composed of nucleotide sequences from the complementary strand, the primers that are composed of nucleotides in the complementary strand are described by indicating their SEQ ID numbers and the positions of the first and last nucleotides to which they are complementary (complementary position range) in the Sequence ID.
Table 13 is a chart listing amplification primers which have been used to amplify polynucleotides containing one or more eicosanoid-related biallelic markers. Each of the primers which falls within the strand of nucleotides included in the Sequence Listing are described by indicating their Sequence ID number and the positions of the first and last nucleotides (position range) of the primers in the Sequence ID. Since the sequences in the Sequence Listing are single stranded and half the possible amplification primers are composed of nucleotide sequences from the complementary strand, the primers that are composed of nucleotides in the complementary strand are defined by the SEQ ID numbers and the positions of the first and last nucleotides to which they are complementary (complementary position range) in the Sequence ID.
Table 14 is a chart listing preferred probes useful in genotyping eicosanoid-related biallelic markers by hybridization assays. The probes are 25-mers with an eicosanoid-related biallelic marker in the center position, and described by indicating their Sequence ID number and the positions of the first and last nucleotides (position range) of the probes in the Sequence ID. The probes complementary to the sequences in each position range in each Sequence ID are also understood to be a part of this preferred list even though they are not specified separately.
Table 15 is a table showing the results of the association study between biallelic marker haplotypes from the FLAP gene and asthma.
Table 16 is a table showing the results of the permutation test confirming the statistical significance of the association between asthma and biallelic marker haplotypes from the FLAP gene.
Table 17 is a table showing the results of the association study between 12 biallelic marker haplotypes from the 12-LO gene and asthma.
Table 18A is a table showing the results of allele frequency analysis between seventeen 12-LO biallelic markers and asthma.
Table 18B is a table showing the results of the association study between seventeen 12-LO biallelic marker haplotypes from the 12-LO gene and asthma.
Table 19 is a table showing the results of the association study between 12 biallelic marker haplotypes from the 12-LO gene and hepatotoxicity upon treatment with zileuton.
Table 20A is a table showing the results of the allele frequency analysis between seventeen 12-LO biallelic markers and hepatotoxicity upon treatment with zileuton.
Table 20B is a table showing the results of the association study between seventeen 12-LO biallelic marker haplotypes from the 12-LO gene and hepatotoxicity upon treatment with zileuton.
Table 21 is a table showing a summary of the association study results, permutation tests confirming the statistical significance of the association between asthma and biallelic marker haplotypes from the 12-LO gene, and permutation tests confirming the statistical significance of the association between secondary effects upon treatment with zileuton and biallelic marker haplotypes from the 12-LO gene.
Table 22 is a table showing a summary of the association study results, permutation tests confirming the statistical significance of the association between asthma and additional biallelic marker haplotypes from the 12-LO gene, and permutation tests confirming the statistical significance of the association between secondary effects upon treatment with zileuton and biallelic marker haplotypes from the 12-LO gene.
Table 23 is a chart containing a list of preferred 12-LO-related biallelic markers with an indication of the frequency of the least common allele determined by genotyping. Frequencies were determined in a random US Caucasian population, in an asthmatic population showing no side effects upon treatment with ZyfloTM (ALT−) and in an asthmatic population showing elevated alanine aminotransferase levels upon treatment with ZyfloTM (ALT+).



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram showing the genomic structure of the FLAP gene and the positions of biallelic markers in close proximity of this gene.
FIG. 2 is a graph showing the results of the single point association study between biallelic markers from the FLAP gene and asthma.
FIG. 3 is a diagram showing the genomic structure of the 12-lipoxygenase gene and the positions of biallelic markers in close proximity of this gene.



DETAILED DESCRIPTION OF THE INVENTION
Advantages of the Biallelic Markers of the Present Invention
The eicosanoid-related biallelic markers of the present invention offer a number of important advantages over other genetic markers such as RFLP (Restriction fragment length polymorphism) and VNTR (Variable Number of Tandem Repeats) markers.
The first generation of markers, were RFLPs, which are variations that modify the length of a restriction fragment. But methods used to identify and to type RFLPs are relatively wasteful of materials, effort, and time. The second generation of genetic markers were VNTRs, which can be categorized as either minisatellites or microsatellites. Minisatellites are tandemly repeated DNA sequences present in units of 5-50 repeats which are distributed along regions of the human chromosomes ranging from 0.1 to 20 kilobases in length. Since they present many possible alleles, their informative content is very high. Minisatellites are scored by performing Southern blots to identify the number of tandem repeats present in a nucleic acid sample from the individual being tested. However, there are only 104 potential VNTRs that can be typed by Southern blotting. Moreover, both RFLP and VNTR markers are costly and time-consuming to develop and assay in large numbers.
Single nucleotide polymorphism or biallelic markers can be used in the same manner as RFLPs and VNTRs but offer several advantages. Single nucleotide polymorphisms are densely spaced in the human genome and represent the most frequent type of variation. An estimated number of more than 107 sites are scattered along the 3×109 base pairs of the human genome. Therefore, single nucleotide polymorphism occur at a greater frequency and with greater uniformity than RFLP or VNTR markers which means that there is a greater probability that such a marker will be found in close proximity to a genetic locus of interest. Single nucleotide polymorphisms are less variable than VNTR markers but are mutationally more stable.
Also, the different forms of a characterized single nucleotide polymorphism, such as the biallelic markers of the present invention, are often easier to distinguish and can therefore be typed easily on a routine basis. Biallelic markers have single nucleotide based alleles and they have only two common alleles, which allows highly parallel detection and automated scoring. The biallelic markers of the present invention offer the possibility of rapid, high-throughput genotyping of a large number of individuals.
Biallelic markers are densely spaced in the genome, sufficiently informative and can be assayed in large numbers. The combined effects of these advantages make biallelic markers extremely valuable in genetic studies. Biallelic markers can be used in linkage studies in families, in allele sharing methods, in linkage disequilibrium studies in populations, in association studies of case-control populations. An important aspect of the present invention is that biallelic markers allow association studies to be performed to identify genes involved in complex traits. Association studies examine the frequency of marker alleles in unrelated case- and control-populations and are generally employed in the detection of polygenic or sporadic traits. Association studies may be conducted within the general population and are not limited to studies performed on related individuals in affected families (linkage studies). Biallelic markers in different genes can be screened in parallel for direct association with disease or response to a treatment. This multiple gene approach is a powerful tool for a variety of human genetic studies as it provides the necessary statistical power to examine the synergistic effect of multiple genetic factors on a particular phenotype, drug response, sporadic trait, or disease state with a complex genetic etiology.
Candidate Genes of the Present Invention
Different approaches can be employed to perform association studies: genome-wide association studies, candidate region association studies and candidate gene association studies. Genome-wide association studies rely on the screening of genetic markers evenly spaced and covering the entire genome. Candidate region association studies rely on the screening of genetic markers evenly spaced covering a region identified as linked to the trait of interest. The candidate gene approach is based on the study of genetic markers specifically derived from genes potentially involved in a biological pathway related to the trait of interest. In the present invention, genes involved in arachidonic acid metabolism have been chosen as candidate genes. This metabolic pathway leads to the biosynthesis of eicosanoids, which are chemical mediators that play an important role in a number of inflammatory diseases, moreover, these pathways are important drug targets and genetic polymorphisms in these genes are highly relevant in the response to a number of drugs. The candidate gene analysis clearly provides a short-cut approach to the identification of genes and gene polymorphisms related to a particular trait when some information concerning the biology of the trait is available as is the case for arachidonic acid metabolism. However, it should be noted that all of the biallelic markers disclosed in the instant application can be employed as part of genome-wide association studies or as part of candidate region association studies and such uses are specifically contemplated in the present invention and claims. All of the markers are known to be in close proximity to the genes with which they are listed in Table 7. For a portion of the markers, the precise position of the marker with respect to the various coding and non-coding elements of the genes has also been determined.
Definitions
As used interchangeably herein, the terms “oligonucleotides”, “nucleic acids” and “polynucleotides” include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form. The term “nucleotide” as used herein as an adjective to describe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form. The term “nucleotide” is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a molecule, or individual unit in a larger nucleic acid molecule, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide. Although the term “nucleotide” is also used herein to encompass “modified nucleotides” which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar, for examples of analogous linking groups, purine, pyrimidines, and sugars see for example PCT publication No. WO 95/04064. However, the polynucleotides of the invention are preferably comprised of greater than 50% conventional deoxyribose nucleotides, and most preferably greater than 90% conventional deoxyribose nucleotides. The polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art.
Throughout the present specification, the expression “nucleotide sequence” may be employed to designate indifferently a polynucleotide or a nucleic acid. More precisely, the expression “nucleotide sequence” encompasses the nucleic material itself and is thus not restricted to the sequence information (i.e. the succession of letters chosen among the four base letters) that biochemically characterizes a specific DNA or RNA molecule.
The term “polypeptide” refers to a polymer of amino without regard to the length of the polymer; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not specify or exclude prost-expression modifications of polypeptides, for example, polypeptides which include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide. Also included within the definition are polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
The term “recombinant polypeptide” is used herein to refer to polypeptides that have been artificially designed and which comprise at least two polypeptide sequences that are not found as contiguous polypeptide sequences in their initial natural environment, or to refer to polypeptides which have been expressed from a recombinant polynucleotide.
As used herein, the term “isolated” requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated. Specifically excluded from the definition of “isolated” are: naturally occurring chromosomes (e.g., chromosome spreads) artificial chromosome libraries, genomic libraries, and cDNA libraries that exist either as an in vitro nucleic acid preparation or as a transfected/transformed host cell preparation, wherein the host cells are either an in vitro heterogeneous preparation or plated as a heterogeneous population of single colonies. Also specifically excluded are the above libraries wherein the 5′ EST makes up less than 5% of the number of nucleic acid inserts in the vector molecules. Further specifically excluded are whole cell genomic DNA or whole cell RNA preparations (including said whole cell preparations which are mechanically sheared or enzymaticly digested). Further specifically excluded are the above whole cell preparations as either an in vitro preparation or as a heterogeneous mixture separated by electrophoresis (including blot transfers of the same) wherein the polynucleotide of the invention have not been further separated from the heterologous polynucleotides in the electrophoresis medium (e.g., further separating by excising a single band from a heterogeneous band population in an agarose gel or nylon blot).
As used herein, the term “purified” does not require absolute purity; rather, it is intended as a relative definition. Individual 5′ EST clones isolated from a cDNA library have been conventionally purified to electrophoretic homogeneity. The sequences obtained from these clones could not be obtained directly either from the library or from total human DNA. The cDNA clones are not naturally occurring as such, but rather are obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The conversion of mRNA into a cDNA library involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection. Thus, creating a cDNA library from messenger RNA and subsequently isolating individual clones from that library results in an approximately 104-106 fold purification of the native message. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. Alternatively, purification may be expressed as “at least” a percent purity relative to heterologous polynucleotides (DNA, RNA or both). As a preferred embodiment, the polynucleotides of the present invention are at least; 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 96%, 98%, 99%, or 100% pure relative to heterologous polynucleotides. As a further preferred embodiment the polynucleotides have an “at least” purity ranging from any number, to the thousandth position, between 90% and 100% (e.g., 5′ EST at least 99.995% pure) relative to heterologous polynucleotides. Additionally, purity of the polynucleotides may be expressed as a percentage (as described above) relative to all materials and compounds other than the carrier solution. Each number, to the thousandth position, may be claimed as individual species of purity. The term “primer” denotes a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence. A primer serves as an initiation point for nucleotide polymerization catalyzed by DNA polymerase, RNA polymerase or reverse transcriptase.
The term “probe” denotes a defined nucleic acid segment (or nucleotide analog segment, e.g., polynucleotide as defined herein) which can be used to identify a specific polynucleotide sequence present in samples, said nucleic acid segment comprising a nucleotide sequence complementary of the specific polynucleotide sequence to be identified.
The term “disease involving arachidonic acid metabolism” refers to a condition linked to disturbances in expression, production or cellular response to eicosanoids such as prostaglandins, thromboxanes, prostacyclins, leukotrienes or hydroperoxyeicosaetrenoic acids. A disease involving arachidonic acid metabolism further refers to a condition involving one or several enzymes of the distinct enzyme systems contributing to arachidonate metabolism including particularly the cyclooxygenase pathway and the lipoxygenase pathway and the arachadonic acid metabolites of such systems including 12-HETE, 12-HPETE, lipoxins and hepoxolins. “Diseases involving arachidonic acid metabolism” also include chronic inflammatory diseases, acute allergic inflammation and inflammatory conditions such as pain, fever, hypersensitivity, asthma, psoriasis and arthritis. “Diseases involving arachidonic acid metabolism” also include disorders in platelet function, blood pressure, thrombosis, renal function, host defense mechanism, hemostasis, smooth muscle tone, male infertility, primary dysmenorrhea, disorders in parturition, and disorders in tissue injury repair, as well as disorders in cellular function and development. “Diseases involving arachidonic acid metabolism” also include diseases such as gastrointestinal ulceration, coronary and cerebrovascular syndromes, glomerular immune injury and cancer.
The term “agent acting on arachidonic acid metabolism” refers to a drug or a compound modulating the activity or concentration of an enzyme or regulatory molecule involved in arachidonic acid metabolism, including but not limited to cyclooxygenase, prostacyclin synthase, thromboxane synthase, lipoxygenases, 5-lipoxygenase and 5-lipoxygenase activating protein. “Agent acting on arachidonic acid metabolism” further refers to non-steroidal antiinflammatory drugs (NSAIDs), eicosanoid receptor antagonists, eicosanoid analogs, COX-1 inhibitors, COX-2 inhibitors, thromboxane synthase inhibitors, 5-lipoxygenase inhibitors and 5-lipoxygenase activating protein inhibitors. “Agent acting on arachidonic acid metabolism” also refers to compounds modulating the formation and action of eicosanoids such as prostaglandins, prostacyclins, thromboxanes, leukotrienes or hydroperoxyeicosaetrenoic acids.
The terms “response to an agent acting on arachidonic acid metabolism” refer to drug efficacy, including but not limited to ability to metabolize a compound, to the ability to convert a pro-drug to an active drug, and to the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.
The terms “side effects to an agent acting on arachidonic acid metabolism” refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors. “Side effects to an agent acting on arachidonic acid metabolism” include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock.
The terms “trait” and “phenotype” are used interchangeably herein and refer to any visible, detectable or otherwise measurable property of an organism such as symptoms of, or susceptibility to a disease for example. Typically the terms “trait” or “phenotype” are used herein to refer to symptoms of, or susceptibility to a disease involving arachidonic acid metabolism; or to refer to an individual's response to an agent acting on arachidonic acid metabolism; or to refer to symptoms of, or susceptibility to side effects to an agent acting on arachidonic acid metabolism.
The terms “agent acting on 5-lipoxygenase” refers to a drug or a compound modulating the activity or concentration of the 5-lipoxygenase enzyme such as 5-lipoxygenase inhibitors. “Agent acting on 5-lipoxygenase” also refers to compounds modulating the formation and action of leukotrienes.
The terms “side effects to an agent acting on 5-lipoxygenase” include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities.
The term “allele” is used herein to refer to variants of a nucleotide sequence. A biallelic polymorphism has two forms. Typically the first identified allele is designated as the original allele whereas other alleles are designated as alternative alleles. Diploid organisms may be homozygous or heterozygous for an allelic form.
The term “heterozygosity rate” is used herein to refer to the incidence of individuals in a population, which are heterozygous at a particular allele. In a biallelic system the heterozygosity rate is on average equal to 2Pa(1−Pa), where Pa is the frequency of the least common allele. In order to be useful in genetic studies a genetic marker should have an adequate level of heterozygosity to allow a reasonable probability that a randomly selected person will be heterozygous.
The term “genotype” as used herein refers the identity of the alleles present in an individual or a sample. In the context of the present invention a genotype preferably refers to the description of the biallelic marker alleles present in an individual or a sample. The term “genotyping” a sample or an individual for a biallelic marker consists of determining the specific allele or the specific nucleotide carried by an individual at a biallelic marker.
The term “mutation” as used herein refers to a difference in DNA sequence between or among different genomes or individuals which has a frequency below 1%.
The term “haplotype” refers to a combination of alleles present in an individual or a sample. In the context of the present invention a haplotype preferably refers to a combination of biallelic marker alleles found in a given individual and which may be associated with a phenotype.
The term “polymorphism” as used herein refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals. “Polymorphic” refers to the condition in which two or more variants of a specific genomic sequence can be found in a population. A “polymorphic site” is the locus at which the variation occurs. A single nucleotide polymorphism is a single base pair change. Typically a single nucleotide polymorphism is the replacement of one nucleotide by another nucleotide at the polymorphic site. Deletion of a single nucleotide or insertion of a single nucleotide, also give rise to single nucleotide polymorphisms. In the context of the present invention “single nucleotide polymorphism” preferably refers to a single nucleotide substitution. Typically, between different genomes or between different individuals, the polymorphic site may be occupied by two different nucleotides.
The terms “biallelic polymorphism” and “biallelic marker” are used interchangeably herein to refer to a polymorphism having two alleles at a fairly high frequency in the population, preferably a single nucleotide polymorphism. A “biallelic marker allele” refers to the nucleotide variants present at a biallelic marker site. Typically the frequency of the less common allele of the biallelic markers of the present invention has been validated to be greater than 1%, preferably the frequency is greater than 10%, more preferably the frequency is at least 20% (i.e. heterozygosity rate of at least 0.32), even more preferably the frequency is at least 30% (i.e. heterozygosity rate of at least 0.42). A biallelic marker wherein the frequency of the less common allele is 30% or more is termed a “high quality biallelic marker.”
The location of nucleotides in a polynucleotide with respect to the center of the polynucleotide are described herein in the following manner. When a polynucleotide has an odd number of nucleotides, the nucleotide at an equal distance from the 3′ and 5′ ends of the polynucleotide is considered to be “at the center” of the polynucleotide, and any nucleotide immediately adjacent to the nucleotide at the center, or the nucleotide at the center itself is considered to be “within 1 nucleotide of the center.” With an odd number of nucleotides in a polynucleotide any of the five nucleotides positions in the middle of the polynucleotide would be considered to be within 2 nucleotides of the center, and so on. When a polynucleotide has an even number of nucleotides, there would be a bond and not a nucleotide at the center of the polynucleotide. Thus, either of the two central nucleotides would be considered to be “within 1 nucleotide of the center” and any of the four nucleotides in the middle of the polynucleotide would be considered to be “within 2 nucleotides of the center”, and so on. For polymorphisms which involve the substitution, insertion or deletion of 1 or more nucleotides, the polymorphism, allele or biallelic marker is “at the center” of a polynucleotide if the difference between the distance from the substituted, inserted, or deleted polynucleotides of the polymorphism and the 3′ end of the polynucleotide, and the distance from the substituted, inserted, or deleted polynucleotides of the polymorphism and the 5′ end of the polynucleotide is zero or one nucleotide. If this difference is 0 to 3, then the polymorphism is considered to be “within 1 nucleotide of the center.” If the difference is 0 to 5, the polymorphism is considered to be “within 2 nucleotides of the center.” If the difference is 0 to 7, the polymorphism is considered to be “within 3 nucleotides of the center,” and so on. For polymorphisms which involve the substitution, insertion or deletion of 1 or more nucleotides, the polymorphism, allele or biallelic marker is “at the center” of a polynucleotide if the difference between the distance from the substituted, inserted, or deleted polynucleotides of the polymorphism and the 3′ end of the polynucleotide, and the distance from the substituted, inserted, or deleted polynucleotides of the polymorphism and the 5′ end of the polynucleotide is zero or one nucleotide. If this difference is 0 to 3, then the polymorphism is considered to be “within 1 nucleotide of the center.” If the difference is 0 to 5, the polymorphism is considered to be “within 2 nucleotides of the center.” If the difference is 0 to 7, the polymorphism is considered to be “within 3 nucleotides of the center,” and so on.
A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell required to initiate the specific transcription of a gene.
As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. More precisely, two DNA molecules (such as a polynucleotide containing a promoter region and a polynucleotide encoding a desired polypeptide or polynucleotide) are said to be “operably linked” if the nature of the linkage between the two polynucleotides does not (1) result in the introduction of a frame-shift mutation or (2) interfere with the ability of the polynucleotide containing the promoter to direct the transcription of the coding polynucleotide.
The term “upstream” is used herein to refer to a location, which is toward the 5′ end of the polynucleotide from a specific reference point.
The terms “base paired” and “Watson & Crick base paired” are used interchangeably herein to refer to nucleotides which can be hydrogen bonded to one another be virtue of their sequence identities in a manner like that found in double-helical DNA with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds (See Stryer, L., Biochemistry, 4th edition, 1995).
The terms “complementary” or “complement thereof” are used herein to refer to the sequences of polynucleotides which is capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety of the complementary region. This term is applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind.
As used herein, the term “non-human animal” refers to any non-human vertebrate, birds and more usually mammals, preferably primates, farm animals such as swine, goats, sheep, donkeys, and horses, rabbits or rodents, more preferably rats or mice. As used herein, the term “animal” is used to refer to any vertebrate, preferable a mammal. Both the terms “animal” and “mammal” expressly embrace human subjects unless preceded with the term “non-human.”
As used herein, the term “antibody” refers to a polypeptide or group of polypeptides which are comprised of at least one binding domain, where an antibody binding domain is formed from the folding of variable domains of an antibody molecule to form three-dimensional binding spaces with an internal surface shape and charge distribution complementary to the features of an antigenic determinant of an antigen., which allows an immunological reaction with the antigen. Antibodies include recombinant proteins comprising the binding domains, as wells as fragments, including Fab, Fab′, F(ab)2, and F(ab′)2 fragments.
As used herein, an “antigenic determinant” is the portion of an antigen molecule, in this case a 12-LO polypeptide, that determines the specificity of the antigen-antibody reaction. An “epitope” refers to an antigenic determinant of a polypeptide. An epitope can comprise as few as 3 amino acids in a spatial conformation which, is unique to the epitope. Generally an epitope consists of at least 6 such amino acids, and more usually at least 8-10 such amino acids. Methods for determining the amino acids which make up an epitope include x-ray crystallography, 2-dimensional nuclear magnetic resonance, and epitope mapping e.g. the Pepscan method described by H. Mario Geysen et al. 1984. Proc. Natl. Acad. Sci. U.S.A. 81:3998-4002; PCT Publication No. WO 84/03564; and PCT Publication No. WO 84/03506, the disclosures of which are incorporated herein by reference in their entireties.
As used herein the term “eicosanoid-related biallelic marker” relates to a set of biallelic markers in linkage disequilibrium with all of the genes disclosed in Table 7(A-B) with the exception of FLAP. All of these genes express proteins that are related to eicosanoid metabolism. The term eicosanoid-related biallelic marker encompasses all of the biallelic markers disclosed in Table 7(A-B), preferably the biallelic markers found in SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300. The preferred eicosanoid-related biallelic marker alleles of the present invention include each one the alleles described in Tables 7, 8, 9, and 10 individually or in groups consisting of all the possible combinations of the alleles included in Tables 7(A-B), 8, 9, and 10, preferably the biallelic markers found in SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300.
As used herein the term “12-LO-related biallelic marker” and “12-lipoxygenase-related biallelic marker” are used interchangeably herein to relate to all biallelic markers in linkage disequilibrium with the biallelic markers of the 12-lipoxygenase gene. The term 12-LO-related biallelic marker includes both the genic and non-genic biallelic markers described in Table 2(a-c).
The term “non-genic” is used herein to describe 12-LO-related biallelic markers, as well as polynucleotides and primers which occur outside the nucleotide positions shown in the human 12-LO genomic sequence of SEQ ID No. 651. The term “genic” is used herein to describe 12-LO-related biallelic markers as well as polynucleotides and primers which do occur in the nucleotide positions shown in the human 12-LO genomic sequence of SEQ ID No 651.
The term “sequence described in Table 7(A-B)” is used herein to refer to the entire collection of nucleotide sequences or any individual sequence defined in Table 7(A-B). The SEQ ID that contains each “sequence described in Table 7(A-B)” is provided in the column labeled, “SEQ ID NO.” The column labeled “Gene” indicates the gene for which the marker is in closest physical proximity, an indication of whether the markers have been validated by microsequencing (with a Y indicating that the markers have been validated by microsequencing and an N indicating that it has not), and an indication of the identity and frequency of the least common allele determined by genotyping (with a blank left to indicate that the frequency has not yet been reported for some markers). The frequencies were determined from DNA samples collected from a random US Caucasian population.
The term “sequence described in Table 7B” is used herein to refer to the entire collection of nucleotide sequences or any individual sequence defined in Table 7B. The SEQ ID that contains each “sequence described in Table 7B” is provided in the column labeled, “SEQ ID NO.” The column labeled “Gene” indicates the gene for which the marker is in closest physical proximity, an indication of whether the markers have been validated by microsequencing (with a Y indicating that the markers have been validated by microsequencing and an N indicating that it has not), and an indication of the identity and frequency of the least common allele determined by genotyping (with a blank left to indicate that the frequency has not yet been reported for some markers). The frequencies were determined from DNA samples collected from a random US Caucasian population. The “Biallelic Marker location in SEQ ID No.” indicates the biallelic marker location within the 47 nucleotide sequence. In Table 7B, this location is 24 for all of the markers.
The term “sequence described in Table 8” is used herein to refer to the entire collection of nucleotide sequences or any individual sequence defined in Table 8. The SEQ If) that contains each “sequence described in Table 8” is provided in the column labeled, “SEQ ID NO.” The range of nucleotide positions within the Sequence if) of which each sequence consists is provided in the same row as the Sequence ID in a column labeled, “POSITION RANGE OF PREFERRED SEQUENCE”. It should be noted that some of the Sequence ID numbers have multiple sequence ranges listed, because they contain multiple “sequences described in Table 8.” Unless otherwise noted the term “sequence described in Table 8” is to be construed as encompassing sequences that contain either of the two alleles listed in the columns labeled, “1ST ALLELE” and “2ND ALLELE” at the position identified in field <222> of the allele feature in the appended Sequence Listing for each Sequence ID) number referenced in Table 8. For all inventions which relate to biallelic markers or sequences described in Table 8, a preferred set of markers or sequences excludes Sequence if) Nos. 1-10, 19, 23-25, and 647-650.
The term “sequence described in Table 9” is used herein to refer to the entire collection of nucleotide sequences or any individual sequence defined in Table 9. Unless otherwise noted, the “sequences described in Table 9” consist of the entire sequence of each Sequence ID provided in the column labeled, “SEQ ID NO.” Also unless otherwise noted the term “sequence described in Table 9” is to be construed as encompassing sequences that contain either of the two alleles listed in the columns labeled, “ORIGINAL ALLELE” and “ALTERNATIVE ALLELE” at the position identified in field <222> of the allele feature in the appended Sequence Listing for each Sequence ID) number referenced in Table 9. For all inventions which relate to biallelic markers or sequences described in Table 9, a preferred set of markers or sequences excludes Sequence ID Nos. 11-18 and 20-21.
The term “sequence described in Table 10” is used herein to refer to the entire collection of nucleotide sequences or any individual sequence defined in Table 10. Unless otherwise noted, the “sequences described in Table 10” consist of the entire sequence of each Sequence ID provided in the column labeled, “SEQ ID NO.” Also unless otherwise noted the term “sequence described in Table 10” is to be construed as encompassing sequences that contain either of the two alleles listed in the columns labeled, “1ST ALLELE” and “2ND ALLELE” at the position identified in field <222> of the allele feature in the appended Sequence Listing for each Sequence ID number referenced in Table 10. For all inventions which relate to biallelic markers or sequences described in Table 8, a preferred set of markers or sequences excludes Sequence ID No. 22.
The term “sequence described in Table 11” is used herein to refer to the entire collection of nucleotide sequences or any individual sequence defined in Table 11. The SEQ ID that contains each “sequence described in Table 11” is provided in the column labeled, “SEQ ID NO.” The range of nucleotide positions within the Sequence ID of which each sequence consists is provided in the same row as the Sequence ID in a column labeled, “POSITION RANGE OF PREFERRED SEQUENCE”. It should be noted that some of the Sequence ID numbers have multiple sequence ranges listed, because they contain multiple “sequences described in Table 11.”
The term “sequence described in Table 12” is used herein to refer to the entire collection of nucleotide sequences or any individual sequence defined in Table 12. The SEQ ID that contains each “sequence described in Table 12” is provided in the column labeled “SEQ ID.” The range of nucleotide positions within the Sequence I) of which half of the sequences consists is provided in the same row as the Sequence ID in a column labeled, “POSITION RANGE OF MICROSEQUENCING PRIMERS.” The remaining half of the sequences described in Table 12 are complementary to the range of nucleotide positions within the Sequence ID provided in the same row as the Sequence ID in a column labeled, “COMPLEMENTARY POSITION RANGE OF MICROSEQUENCING PRIMERS.” For all inventions which relate to biallelic markers or sequences described in Table 12, a more preferred set of markers or sequences consists of those markers or sequences found in SEQ ID Nos. 26-70, 72-418, 425-489, 491-530, 532-539, 541-646, and 651-652.
The term “sequence described in Table 13” is used herein to refer to the entire collection of nucleotide sequences or any individual sequence defined in Table 13. The SEQ ID that contains each “sequence described in Table 13” is provided in the column labeled, “SEQ ID.” The range of nucleotide positions within the Sequence ID of which half of the sequences consists is provided in the same row as the Sequence ID in a column labeled, “POSITION RANGE OF AMPLIFICATION PRIMERS.” The remaining half of the sequences described in Table 13 are complementary to the range of nucleotide positions within the Sequence ID provided in the same row as the Sequence ID in a column labeled, “COMPLEMENTARY POSITION RANGE OF AMPLIFICATION PRIMERS.” For all inventions which relate to biallelic markers or sequences described in Table 13, a more preferred set of markers or sequences consists of those markers or sequences found in SEQ ID Nos. 26-70, 72-418, 425-489, 491-530, 532-539, 541-646.
The term “sequence described in Table 13” is used herein to refer to the entire collection of nucleotide sequences or any individual sequence defined in Table 13. The SEQ ID that contains each “sequence described in Table 13” is provided in the column labeled, “SEQ ID”. The range of nucleotide positions within the Sequence ID of which each sequence consists is provided in the same row as the Sequence ID in a column labeled, “POSITION RANGE OF PROBES”. The sequences which are complementary to the ranges listed in the column labeled, “POSITION RANGE OF PROBES” are also encompassed by the term, “sequence described in Table 13.” Unless otherwise noted the term “sequence described in Table 13” is to be construed as encompassing sequences that contain either of the two alleles listed in the allele feature in the appended Sequence Listing for each Sequence ID number referenced in Table 13. For all inventions which relate to biallelic markers or sequences described in Table 13, a more preferred set of markers or sequences consists of those markers or sequences found in SEQ ID Nos. 26-70, 72-418, 425-489, 491-530, 532-539, 541-646, and 651-652.
The terms “biallelic marker described in Table” and “allele described in Table” are used herein to refer to any or all alleles which are listed in the allele feature in the appended Sequence Listing for each Sequence ID number referenced in the particular Table being mentioned.
The following abbreviations are used in this disclosure: the LTB4H2 gene is abbreviated LTB4H2; leukotriene B4-12-OH dehydrogenase is abbreviated LTB4-12OH; leukotriene B4 receptor is abbreviated LTB4R; PGD-synthase is abbreviated PGDS; and PG-15-OH dehydrogenase is abbreviated PG 15OH.
Variants and Fragments
The invention also relates to variants and fragments of the polynucleotides described herein, particularly of a 12-LO gene containing one or more biallelic markers according to the invention.
Variants of polynucleotides, as the term is used herein, are polynucleotides that differ from a reference polynucleotide. A variant of a polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms. Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical. Variants of polynucleotides according to the invention include, without being limited to, nucleotide sequences which are at least 95% identical, preferably at least 99% identical, more particularly at least 99.5% identical, and most preferably at least 99.8% identical to a polynucleotide selected from the group consisting of the polynucleotides of a sequence from any sequence in the Sequence Listing as well as sequences which are complementary thereto or to any polynucleotide fragment of at least 8 consecutive nucleotides of a sequence from any sequence in the Sequence Listing. Nucleotide changes present in a variant polynucleotide may be silent, which means that they do not alter the amino acids encoded by the polynucleotide. However, nucleotide changes may also result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. In the context of the present invention, particularly preferred embodiments are those in which the polynucleotides encode polypeptides which retain substantially the same biological function or activity as the mature 12-LO protein, or those in which the polynucleotides encode polypeptides which maintain or increase a particular biological activity, while reducing a second biological activity. A polynucleotide fragment is a polynucleotide having a sequence that is entirely the same as part but not all of a given nucleotide sequence, preferably the nucleotide sequence of a 12-LO gene, and variants thereof. The fragment can be a portion of an exon or of an intron of a 12-LO gene. It can also be a portion of the regulatory regions of the 12-LO gene preferably of the promoter sequence of the 12-LO gene. Such fragments may be “free-standing”, i.e. not part of or fused to other polynucleotides, or they may be comprised within a single larger polynucleotide of which they form a part or region. Indeed, several of these fragments may be present within a single larger polynucleotide.
Identity Between Nucleic Acids and Polypeptides
The terms “percentage of sequence identity” and “percentage homology” are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Homology is evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. 85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol. 215(3):403-410,1990; Thompson et al., Nucleic Acids Res. 22(2):4673-4680, 1994; Higgins et al., Methods Enzymol. 266:383-402, 1996; Altschul et al., Nature Genetics 3:266-272, 1993, the disclosures of which are incorporated herein by reference in their entireties). In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool (“BLAST”) which is well known in the art (See, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2267-2268, 1990; Altschul et al., J. Mol. Biol. 215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272, 1993; Altschul et al., Nuc. Acids Res. 25:3389-3402, 1997, the disclosures of which are incorporated herein by reference in their entireties). In particular, five specific BLAST programs are used to perform the following task:

    (1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database;
    (2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database;
    (3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database;
    (4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and
    (5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.


The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as “high-scoring segment pairs,” between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., Science 256:1443-1445, 1992; Henikoff and Henikoff, Proteins 17:49-61, 1993, the disclosures of which are incorporated herein by reference in their entireties). Less preferably, the PAM or PAM250 matrices may also be used (See, e.g., Schwartz and Dayhoff, eds., Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation, 1978, the disclosure of which is incorporated herein by reference in its entirety). The BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user-specified percent homology. Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2267-2268, 1990, the disclosure of which is incorporated herein by reference in its entirety).
Stringent Hybridization Conditions
By way of example and not limitation, procedures using conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C., the preferred hybridization temperature, in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20×106 cpm of 32P-labeled probe. Alternatively, the hybridization step can be performed at 65° C. in the presence of SSC buffer, 1×SSC corresponding to 0.15M NaCl and 0.05 M Na citrate. Subsequently, filter washes can be done at 37° C. for 1 h in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1×SSC at 50° C. for 45 min. Alternatively, filter washes can be performed in a solution containing 2×SSC and 0.1% SDS, or 0.5×SSC and 0.1% SDS, or 0.1×SSC and 0.1% SDS at 68° C. for 15 minute intervals. Following the wash steps, the hybridized probes are detectable by autoradiography. Other conditions of high stringency which may be used are well known in the art and as cited in Sambrook et al., 1989; and Ausubel et al., 1989. These hybridization conditions are suitable for a nucleic acid molecule of about 20 nucleotides in length. There is no need to say that the hybridization conditions described above are to be adapted according to the length of the desired nucleic acid, following techniques well-known to one skilled in the art. The suitable hybridization conditions may for example be adapted according to the teachings disclosed in the book of Hames and Higgins (Nucleic Acid Hybridization: A Practical Approach, IRL Press, Oxford, 1985) or in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989), the disclosures of which are incorporated herein by reference in their entireties.
I. Biallelic Markers and Polynucleotides Comprising Biallelic Markers
A. Polynucleotides of the Present Invention
The present invention encompasses polynucleotides for use as primers and probes in the methods of the invention. These polynucleotides may consist of, consist essentially of, or comprise a contiguous span of nucleotides of a sequence from any sequence in the Sequence Listing as well as sequences which are complementary thereto (“complements thereof”). The “contiguous span” may be at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 nucleotides in length, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID. It should be noted that the polynucleotides of the present invention are not limited to having the exact flanking sequences surrounding the polymorphic bases, which are enumerated in the Sequence Listing. Rather, it will be appreciated that the flanking sequences surrounding the biallelic markers, or any of the primers of probes of the invention which, are more distant from the markers, may be lengthened or shortened to any extent compatible with their intended use and the present invention specifically contemplates such sequences. It will be appreciated that the polynucleotides referred to in the Sequence Listing may be of any length compatible with their intended use. Also the flanking regions outside of the contiguous span need not be homologous to native flanking sequences which actually occur in human subjects. The addition of any nucleotide sequence, which is compatible with the nucleotides intended use is specifically contemplated. The contiguous span may optionally include the eicosanoid-related biallelic marker in said sequence. Biallelic markers generally consist of a polymorphism at one single base position. Each biallelic marker therefore corresponds to two forms of a polynucleotide sequence which, when compared with one another, present a nucleotide modification at one position. Usually, the nucleotide modification involves the substitution of one nucleotide for another. Optionally either the original or the alternative allele of the biallelic markers disclosed in Table 9, or the first or second allele disclosed in Tables 8 and 10 may be specified as being present at the eicosanoid-related biallelic marker. Optionally, the biallelic markers may be specified as 12-214-85, 12-215-272, 12-221-163, 12-225-82, 10-234-179, 10-235-272, 10-251-342, 10-395-367, 12-730-58, 12-735-208, 12-739-22, 12-540-363, 12-550-206, 10-207-410, 10-171-254, 12-94-110, 12-834-290, 10-55-115, 12-857-122, 12-872-175, 12-882-40, 12-888-234, 12-278-353, 12-283-386, 12-44-181, 10-343-231, 10-349-216, 10-509-295, 10-511-337, 10-349-216, 10-343-231, 10-13-396, 12-570-62, 10-474-320, 10-510-173 and 10-342-301 which consist of more complex polymorphisms including insertions/deletions of at least one nucleotide. Optionally either the original or the alternative allele of these biallelic markers may be specified as being present at the eicosanoid-related biallelic marker. Preferred polynucleotides may consist of, consist essentially of, or comprise a contiguous span of nucleotides of a sequence from SEQ ID No 571-595, 600, 606, 613, 620, 628, and 638-639; or more preferably from SEQ ID No 1225-1249, 1254, 1260, 1267, 1274, 1282, 1292 and 1293 as well as sequences which are complementary thereto. The “contiguous span” may be at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 nucleotides in length, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID. The contiguous span may optionally comprise a biallelic marker selected from the group consisting of biallelic markers 12-214-85, 12-215-272, 12-221-163, 12-225-82, 10-234-179, 10-235-272, 10-251-342, 10-395-367, 12-730-58, 12-735-208, 12-739-22, 12-540-363, 12-550-206, 10-207-410, 10-171-254, 12-94-110, 12-834-290, 10-55-115, 12-857-122, 12-872-175, 12-882-40, 12-888-234, 12-278-353, 12-283-386, 12-44-181, 10-343-231, 10-349-216, 10-509-295, 10-511-337, 10-349-216, 10-343-231, 10-13-396, 12-570-62, 10-474-320, 10-510-173 and 10-342-301.
The invention also relates to polynucleotides that hybridize, under conditions of high or intermediate stringency, to a polynucleotide of a sequence from any sequence in the Sequence Listing as well as sequences, which are complementary thereto. Preferably such polynucleotides are at least 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 nucleotides in length, to the extent that a polynucleotide of these lengths is consistent with the lengths of the particular Sequence ID. Preferred polynucleotides comprise an eicosanoid-related biallelic marker. Optionally either the original or the alternative allele of the biallelic markers disclosed in Table 10 may be specified as being present at the eicosanoid-related biallelic marker. Conditions of high and intermediate stringency are further described in III.C.4 “Methods of Genotyping DNA Samples for Biallelic Markers-Hybridization assay methods.”
The preferred polynucleotides of the invention include the sequence ranges included in any one the sequence ranges of Tables 8, 11, and 14 individually or in groups consisting of all the possible combinations of the ranges of included in Tables 8, 11, and 14. The preferred polynucleotides of the invention also include fragments of at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 consecutive nucleotides of the sequence ranges included in any one of the sequence ranges of Tables 8, 11, and 14 to the extent that fragments of these lengths are consistent with the lengths of the particular sequence range. The preferred polynucleotides of the invention also include fragments of at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 consecutive nucleotides of the sequence complementary to the sequence ranges included in any one of the sequence ranges of Tables 8, 11, and 14 to the extent that fragments of these lengths are consistent with the lengths of the particular sequence range.
Particularly preferred polynucleotides of the invention include isolated, purified or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 651, wherein said contiguous span comprises at least 1, 2, 3, 4, 5 or 10 of the following nucleotide positions of SEQ ID No. 651: 1 to 2584, 4425 to 5551, 5634 to 5757, 5881 to 5995, 6100 to 6348, 6510 to 7378, 7523 to 8644, 8855 to 12253, 12341 to 12853, 13024 to 13307, 13430 to 16566, 16668 to 16774, 16946 to 17062, 17555 to 20674; and the complements thereof. Other particularly preferred polynucleotides of the invention include isolated, purified or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides from a sequence of SEQ ID No. 651 and the complements thereof; wherein said contiguous span comprises at least one nucleotide positions selected from the group consisting of: a C at position 3355, a G at position 3488, a G at position 3489, and a G at position 3708 of SEQ ID No. 651.
Additional preferred polynucleotides of the invention include isolated, purified or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides from a sequence of SEQ ID No. 652, wherein said contiguous span comprises a T at position 1205 of SEQ ID No. 652 or nucleotide positions 2151 to 2157 of SEQ ID No. 652; and the complements thereof.
The present invention further embodies isolated, purified, and recombinant polynucleotides which encode polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No. 653, wherein said contiguous span comprises at least one amino acid position selected from the group consisting of the following: an His residue et amino acid position 189, an His residue at amino acid position 225, a Cys residue at amino acid position 243, an Arg residue at amino acid position 261, an Asn residue at amino acid position 322, an Arg residue at amino acid position 337, a Asn residue at amino acid position 362, an Asn at amino acid position 568 and a Lys residue at amino acid position 574. The present invention further provides isolated, purified, and recombinant polynucleotides which encode polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No. 653, wherein said contiguous span comprises at least one of amino acid positions 110-131 of SEQ ID No. 653.
Particularly preferred polynucleotides of the present invention include purified, isolated or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of a sequence selected from the group consisting of SEQ ID Nos. 26-68, 614-646, and 651-652; or more preferably from SEQ ID No 651-652, 680-722, and 1268-1300, or the complements thereof, wherein said span includes a 12-lipoxygenase-related biallelic marker. Optionally said biallelic marker is selected from the biallelic markers described in Table 2(a-c) and even more preferably said biallelic marker is selected from biallelic markers: 12-197-244, 12-208-35, 12-226-167, 12-206-366, 10-346-141, 10-347-111, 10-347-165, 10-347-203, 10-347-220, 10-349-97, 10-349-224, 10-341-116, 12-196-119, 12-214-129, 12-216-421, 12-219-230, and 12-223-207. Optionally either allele of the biallelic markers described above in the definition of 12-lipoxygenase-related biallelic marker is specified as being present at the 12-lipoxygenase-related biallelic marker.
Particularly preferred polynucleotides of the present invention include purified, isolated or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of a sequence of SEQ ID No. 651 and the complements thereof; wherein said contiguous span comprises a least one nucleotide positions selected from the group consisting of: a T at position 2323, a C at position 2341, an A at position 2623, an A at position 2832, a C at position 2844, an A at position 2934, an A at position 2947, a G at position 3802, a G at position 4062, a C at position 4088, a T at position 4109, a T at position 4170, an A at position 6019, a C at position 6375, a C at position 6429, an A at position 6467, a G at position 6484, an A at position 8658, a G at position 8703, an A at position 8777, a G at position 8785, a G at position 13341, an A at position 16836, an A at position 16854, and a T at position 17355 of SEQ ID No. 651.
Particularly preferred polynucleotides of the present invention include purified, isolated or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of a sequence of SEQ ID No. 652 and the complements thereof; wherein said contiguous span comprises a least one nucleotide position selected from the group consisting of: G at position 366, an A at position 605, a C at position 712, a T at position 766, an A at position 804, a G at position 821, an A at position 1004, a G at position 1049, an A at position 1123, a G at position 1131, a G at position 1491, an A at position 1742, an A at position 1760, an A at position 1941, and a T at position 2144 of SEQ ID No. 652.
Table 2(a-c) contains a list of preferred 12-LO-related biallelic markers. Each marker is described by indicating its Marker ID, the position of the marker in the SEQ ID and the two most common alleles.







TABLE 2a







NON-GENOMIC BIALLELIC MARKERS










POSITION OF
POSITION OF



BIALLELIC MARKER
BIALLELIC MARKER


BIALLELIC
IN SEQ ID (FIG. 2A)
IN SEQ ID (FIG. 2B)












MARKER ID
ALLELES
SEQ ID No.
Position
SEQ ID No
Position















12-196-119
C/T
44
119
698
24


12-197-244
C/T
45
243
699
24


12-198-128
A/G
46
128
700
24


12-208-35
A/T
48
35
702
24


12-214-129
C/T
49
129
703
24


12-214-151
G/C
50
151
704
24


12-214-360
C/G
51
358
705
24


12-214-85
Deletion CCTAT
571
85
1225
24


12-215-272
Deletion T
572
271
1226
24


12-215-467
G/T
52
466
706
24


12-216-421
A/G
53
418
707
24


12-219-230
A/G
54
229
708
24


12-219-256
C/T
55
255
709
24


12-221-163
GTCCTA/T
573
163
1227
24


12-221-302
A/C
57
302
711
24


12-223-179
A/G
58
179
712
24


12-223-207
C/T
59
207
713
24


12-225-541
C/T
60
540
714
24


12-225-82
Deletion T
574
82
1228
24


12-226-167
C/G
61
166
715
24


12-226-458
C/T
62
455
716
24


12-229-332
G/C
63
332
717
24


12-229-351
G/C
64
351
718
24


12-230-364
C/T
65
364
719
24


12-231-100
C/T
66
99
720
24


12-231-148
C/T
67
147
721
24


12-231-266
C/T
68
265
722
24

















TABLE 2b







BIALLELIC MARKERS IN GENOMIC SEQUENCE



(SEQ ID No. 651)


 










BIALLELIC

POSITION OF BIALLELIC



MARKER ID
ALLELES
MARKER IN SEQ ID













10-508-191
C/T
1128



 


10-508-245
C/T
1182


 


10-510-173
ATTTA/TTTTTT
1827


 


10-511-62
C/T
2048


 


10-511-337
Insertion of T
2323


 


10-512-36
G/C
2341


 


10-512-318
A/G
2623


 


10-513-250
A/G
2832


 


10-513-262
C/T
2844


 


10-513-352
A/G
2934


 


10-513-365
A/G
2947


 


12-206-81
A/G
3802


 


10-343-231
Deletion of C
4062


 


12-206-366
C/T
4088


 


10-343-278
C/T
4109


 


10-343-339
G/T
4170


 


10-346-23
A/G
5903


 


10-346-141
A/G
6019


 


10-346-263
G/C
6141


 


10-346-305
C/T
6183


 


10-347-74
A/G
6338


 


10-347-111
G/C
6375


 


10-347-165
C/T
6429


 


10-347-203
A/G
6467


 


10-347-220
A/G
6484


 


10-347-271
A/T
6534


 


10-347-348
A/G
6611


 


10-348-391
A/G
7668


 


10-349-47
C/T
8608


 


10-349-97
A/G
8658


 


10-349-142
G/C
8703


 


10-349-216
Deletion of CTG
8777


 


10-349-224
G/T
8785


 


10-349-368
C/T
8926


 


10-350-72
C/T
12171


 


10-350-332
C/T
12429


 


10-507-170
A/G
13341


 


10-507-321
A/C
13492


 


10-507-353
C/T
13524


 


10-507-364
C/T
13535


 


10-507-405
C/T
13576


 


12-220-48
G/A
15194


 


10-339-32
C/T
16468


 


10-339-124
C/T
16559


 


10-340-112
A/C
16836


 


10-340-130
A/T
16854


 


10-340-238
A/G
16962


 


10-341-116
A/G
17152


 


10-341-319
C/T
17355


 


10-342-301
Insertion of A
17623


 


10-342-373
C/T
17695

















TABLE 2c







BIALLELIC MARKERS IN 12-LO cDNA



(SEQ ID No 652)


 














POSITION OF




BIALLELIC

BIALLELIC MARKER



MARKER ID
ALLELES
IN SEQ ID
















10-343-231
Deletion of C
366




 



10-346-141
A/G
605



 



10-347-111
G/C
712



 



10-347-165
C/T
766



 



10-347-203
A/G
804



 



10-347-220
A/G
821



 



10-349-142
G/C
1049



 



10-349-216
Deletion of CTG
1123



 



10-349-224
G/T
1131



 



10-507-170
A/G
1491



 



10-340-112
A/C
1742



 



10-340-130
A/T
1760



 



10-341-116
A/G
1941



 



10-341-319
C/T
2144










The primers of the present invention may be designed from the disclosed sequences for any method known in the art. A preferred set of primers is fashioned such that the 3′ end of the contiguous span of identity with the sequences of the Sequence Listing is present at the 3′ end of the primer. Such a configuration allows the 3′ end of the primer to hybridize to a selected nucleic acid sequence and dramatically increases the efficiency of the primer for amplification or sequencing reactions. In a preferred set of primers the contiguous span is found in one of the sequences described in Table 11. Allele specific primers may be designed such that a biallelic marker is at the 3′ end of the contiguous span and the contiguous span is present at the 3′ end of the primer. Such allele specific primers tend to selectively prime an amplification or sequencing reaction so long as they are used with a nucleic acid sample that contains one of the two alleles present at a biallelic marker. The 3′ end of primers of the invention may be located within or at least 2, 4, 6, 8, 10, 12, 15, 18, 20, 25, 50, 100, 250, 500, or 1000, to the extent that this distance is consistent with the particular Sequence ID, nucleotides upstream of an eicosanoid-related biallelic marker in said sequence or at any other location which is appropriate for their intended use in sequencing, amplification or the location of novel sequences or markers. A list of preferred amplification primers is disclosed in Table 13. A more preferred set of amplification primers is described in Table 13 in SEQ ID Nos. 26-70, 72-418, 425-489, 491-530, 532-539, 541-646, and 651-652. Primers with their 3′ ends located 1 nucleotide upstream of an eicosanoid-related biallelic marker have a special utility as microsequencing assays. Preferred microsequencing primers are described in Table 12. A more preferred set of microsequencing primers is described in Table 12 in SEQ ID Nos. 26-70, 72-418, 425-489, 491-530, 532-539, 541-646, and 651-652.
The probes of the present invention may be designed from the disclosed sequences for any method known in the art, particularly methods which allow for testing if a particular sequence or marker disclosed herein is present. A preferred set of probes may be designed for use in the hybridization assays of the invention in any manner known in the art such that they selectively bind to one allele of a biallelic marker, but not the other under any particular set of assay conditions. Preferred hybridization probes may consists of, consist essentially of, or comprise a contiguous span which ranges in length from 8, 10, 12, 15, 18 or 20 to 25, 35, 40, 50, 60, 70, or 80 nucleotides, or be specified as being 12, 15, 18, 20, 25, 35, 40, or 50 nucleotides in length and including an eicosanoid-related biallelic marker of said sequence. Optionally the original allele or alternative allele disclosed in Tables 9 and 10 may be specified as being present at the biallelic marker site. Optionally, said biallelic marker may be within 6, 5, 4, 3, 2, or 1 nucleotides of the center of the hybridization probe or at the center of said probe. A particularly preferred set of hybridization probes is disclosed in Table 14 or a sequence complementary thereto.
Any of the polynucleotides of the present invention can be labeled, if desired, by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive substances, fluorescent dyes or biotin. Preferably, polynucleotides are labeled at their 3′ and 5′ ends. A label can also be used to capture the primer, so as to facilitate the immobilization of either the primer or a primer extension product, such as amplified DNA, on a solid support. A capture label is attached to the primers or probes and can be a specific binding member which forms a binding pair with the solid's phase reagent's specific binding member (e.g. biotin and streptavidin). Therefore depending upon the type of label carried by a polynucleotide or a probe, it may be employed to capture or to detect the target DNA. Further, it will be understood that the polynucleotides, primers or probes provided herein, may, themselves, serve as the capture label. For example, in the case where a solid phase reagent's binding member is a nucleic acid sequence, it may be selected such that it binds a complementary portion of a primer or probe to thereby immobilize the primer or probe to the solid phase. In cases where a polynucleotide probe itself serves as the binding member, those skilled in the art will recognize that the probe will contain a sequence or “tail” that is not complementary to the target. In the case where a polynucleotide primer itself serves as the capture label, at least a portion of the primer will be free to hybridize with a nucleic acid on a solid phase. DNA Labeling techniques are well known to the skilled technician.
Any of the polynucleotides, primers and probes of the present invention can be conveniently immobilized on a solid support. Solid supports are known to those skilled in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, duracytes® and others. The solid support is not critical and can be selected by one skilled in the art. Thus, latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips, sheep (or other suitable animal's) red blood cells and duracytes are all suitable examples. Suitable methods for immobilizing nucleic acids on solid phases include ionic, hydrophobic, covalent interactions and the like. A solid support, as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The solid support can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent. The additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent. As yet another alternative, the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid support and which has the ability to immobilize the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding of the capture reagent to a solid support material before the performance of the assay or during the performance of the assay. The solid phase thus can be a plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cells, duracytes® and other configurations known to those of ordinary skill in the art. The polynucleotides of the invention can be attached to or immobilized on a solid support individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the inventions to a single solid support. In addition, polynucleotides other than those of the invention may be attached to the same solid support as one or more polynucleotides of the invention.
Any polynucleotide provided herein may be attached in overlapping areas or at random locations on the solid support. Alternatively the polynucleotides of the invention may be attached in an ordered array wherein each polynucleotide is attached to a distinct region of the solid support which does not overlap with the attachment site of any other polynucleotide. Preferably, such an ordered array of polynucleotides is designed to be “addressable” where the distinct locations are recorded and can be accessed as part of an assay procedure. Addressable polynucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. The knowledge of the precise location of each polynucleotides location makes these “addressable” arrays particularly useful in hybridization assays. Any addressable array technology known in the art can be employed with the polynucleotides of the invention. One particular embodiment of these polynucleotide arrays is known as the GenechipsTM, and has been generally described in U.S. Pat. No. 5,143,854; PCT publications WO 90/15070 and 92/10092. These arrays may generally be produced using mechanical synthesis methods or light directed synthesis methods, which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis (Fodor et al., Science, 251:767-777, 1991). The immobilization of arrays of oligonucleotides on solid supports has been rendered possible by the development of a technology generally identified as “Very Large Scale Immobilized Polymer Synthesis” (VLSIPSTM) in which, typically, probes are immobilized in a high density array on a solid surface of a chip. Examples of VLSIPSTM technologies are provided in U.S. Pat. Nos. 5,143,854 and 5,412,087 and in PCT Publications WO 90/15070, WO 92/10092 and WO 95/11995, which describe methods for forming oligonucleotide arrays through techniques such as light-directed synthesis techniques. In designing strategies aimed at providing arrays of nucleotides immobilized on solid supports, further presentation strategies were developed to order and display the oligonucleotide arrays on the chips in an attempt to maximize hybridization patterns and sequence information. Examples of such presentation strategies are disclosed in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212 and WO 97/31256.
Oligonucleotide arrays may comprise at least one of the sequences selected from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof or a fragment thereof of at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 consecutive nucleotides, to the extent that fragments of these lengths is consistent with the lengths of the particular Sequence ID, for determining whether a sample contains one or more alleles of the biallelic markers of the present invention. Oligonucleotide arrays may also comprise at least one of the sequences selected from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof or a fragment thereof of at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 consecutive nucleotides, to the extent that fragments of these lengths is consistent with the lengths of the particular Sequence ID, for amplifying one or more alleles of the biallelic markers of Table 7(A-B). In other embodiments, arrays may also comprise at least one of the sequences selected from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof or a fragment thereof of at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 consecutive nucleotides, to the extent that fragments of these lengths is consistent with the lengths of the particular Sequence ID, for conducting microsequencing analyses to determine whether a sample contains one or more alleles of the biallelic markers of the invention. In still further embodiments, the oligonucleotide array may comprise at least one of the sequences selecting from the group consisting of SEQ ID Nos. 26-70, 72-418, 425-489, 491-530, 532-539, 541-646, and 651-652, or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300; and the sequences complementary thereto or a fragment thereof of at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 nucleotides in length, to the extent that fragments of these lengths is consistent with the lengths of the particular Sequence ID, for determining whether a sample contains one or more alleles of the biallelic markers of the present invention. In still further embodiments, the oligonucleotide array may comprise at least one of the novel sequences listed in the fifth column of Table 8 or the sequences complementary thereto or a fragment comprising at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 consecutive nucleotides thereof to the extent that fragments of these lengths are consistent with the lengths of the particular novel sequences.
The present invention also encompasses diagnostic kits comprising one or more polynucleotides of the invention, optionally with a portion or all of the necessary reagents and instructions for genotyping a test subject by determining the identity of a nucleotide at an eicosanoid-related biallelic marker. The polynucleotides of a kit may optionally be attached to a solid support, or be part of an array or addressable array of polynucleotides. The kit may provide for the determination of the identity of the nucleotide at a marker position by any method known in the art including, but not limited to, a sequencing assay method, a microsequencing assay method, a hybridization assay method, an allele specific amplification method, or a mismatch detection assay based on polymerases and/or ligases. Optionally such a kit may include instructions for scoring the results of the determination with respect to the test subjects' risk of contracting a diseases involving arachidonic acid metabolism, or likely response to an agent acting on arachidonic acid metabolism, or chances of suffering from side effects to an agent acting on arachidonic acid metabolism. Preferably such a kit may include instructions for scoring the results of the determination with respect to the subjects risk of developing hepatotoxicity upon treatment with the anti-asthmatic drug zileuton.
B. Genomic Sequences of the 12-LO Gene and Biallelic Markers
The present invention encompasses the genomic sequence of the 12-LO gene of SEQ ID No. 651. The 12-LO genomic sequences comprise exons and introns. Particularly preferred genomic sequences of the 12-LO gene include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No. 651, wherein said contiguous span comprises at least 1 one of the following nucleotide positions of SEQ ID No. 651: 1 to 2584, 4425 to 5551, 5634 to 5757, 5881 to 5995, 6100 to 6348, 6510 to 7378, 7523 to 8644, 8855 to 12253, 12341 to 12853, 13024 to 13307, 13430 to 16566, 16668 to 16774, 16946 to 17062, 17555 to 20674; and the complements thereof. The nucleic acids defining the 12-LO intronic polynucleotides may be used as oligonucleotide primers or probes in order to detect the presence of a copy of the 12-LO gene in a test sample, or alternatively in order to amplify a target nucleotide sequence within the 12-LO sequences. Other particularly preferred genomic sequences of the invention include isolated, purified or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides from a sequence of SEQ ID No. 651 and the complements thereof; wherein said contiguous span comprises at least one nucleotide positions selected from the group consisting of: a C at position 3355, a G at position 3488, a G at position 3489, and a G at position 3708 of SEQ ID No. 651.
The present invention further provides 12-lipoxygenase intron and exon polynucleotide sequences including biallelic markers. Particularly preferred polynucleotides of the present invention include purified, isolated or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of a sequence of SEQ ID No. 651 or the complements thereof, wherein said span includes a 12-lipoxygenase-related biallelic marker. Optionally said biallelic marker is selected from the biallelic markers described in Table 2(a-c) and even more preferably said biallelic marker is selected from biallelic markers: 12-197-244, 12-208-35, 12-226-167, 12-206-366, 10-346-141, 10-347-111, 10-347-165, 10-347-203, 10-347-220, 10-349-97, 10-349-224, 10-341-116, 12-196-119, 12-214-129, 12-216-421, 12-219-230, and 12-223-207. Particularly preferred genomic sequences of the present invention include purified, isolated or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of a sequence of SEQ ID No. 651 and the complements thereof; wherein said contiguous span comprises a least one nucleotide positions selected from the group consisting of: a T at position 2323, a C at position 2341, an A at position 2623, an A at position 2832, a C at position 2844, an A at position 2934, an A at position 2947, a G at position 3802, a G at position 4062, a C at position 4088, a T at position 4109, a T at position 4170, an A at position 6019, a C at position 6375, a C at position 6429, an A at position 6467, a G at position 6484, an A at position 8658, a G at position 8703, an A at position 8777, a G at position 8785, a G at position 13341, an A at position 16836, an A at position 16854, and a T at position 17355 of SEQ ID No. 651.
The genomic sequence of the 12-LO gene contains regulatory sequences both in the non-coding 5′-flanking region and in the non-coding 3′-flanking region that border the 12-LO transcribed region containing the 14 exons of this gene. 5′-regulatory sequences of the 12-LO gene comprise the polynucleotide sequences located between the nucleotide in position 1 and the nucleotide in position 3124 of the nucleotide sequence of SEQ ID No. 651, more preferably between positions 1 and 2195 of SEQ ID No. 651. 3′-regulatory sequences of the 12-LO gene comprise the polynucleotide sequences located between the nucleotide in position 17555 and the nucleotide in position 20674 of the nucleotide sequence of SEQ ID No. 651.
The promoter activity of the regulatory regions contained in the 12-LO gene of polynucleotide sequence of SEQ ID No. 651 can be assessed by any known method. Methods for identifying the polynucleotide fragments of SEQ ID No. 651 involved in the regulation of the expression of the 12-LO gene are well-known to those skilled in the art (see Sambrook et al., Molecular Cloning A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). An example of a typical method, that can be used, involves a recombinant vector carrying a reporter gene and genomic sequences from the 12-LO genomic sequence of SEQ ID No. 651. Briefly, the expression of the reporter gene (for example beta galactosidase or chloramphenicol acetyl transferase) is detected when placed under the control of a biologically active polynucleotide fragment. Genomic sequences located upstream of the first exon of the 12-LO gene may be cloned into any suitable promoter reporter vector, such as the pSEAP-Basic, pSEAP-Enhancer, pβgal-Basic, pβgal-Enhancer, or pEGFP-1 Promoter Reporter vectors available from Clontech, or pGL2-basic or pGL3-basic promoterless luciferase reporter gene vector from Promega. Each of these promoter reporter vectors include multiple cloning sites positioned upstream of a reporter gene encoding a readily assayable protein such as secreted alkaline phosphatase, luciferase, beta galactosidase, or green fluorescent protein. The sequences upstream the first 12-LO exon are inserted into the cloning sites upstream of the reporter gene in both orientations and introduced into an appropriate host cell. The level of reporter protein is assayed and compared to the level obtained with a vector lacking an insert in the cloning site. The presence of an elevated expression level in the vector containing the insert with respect to the control vector indicates the presence of a promoter in the insert.
Promoter sequences within the 5′ non-coding regions of the 12-LO gene may be further defined by constructing nested 5′ and/or 3′ deletions using conventional techniques such as Exonuclease III or appropriate restriction endonuclease digestion. The resulting deletion fragments can be inserted into the promoter reporter vector to determine whether the deletion has reduced or obliterated promoter activity, such as described, for example, by Coles et al. (Hum. Mol. Genet., 7:791-800, 1998, the disclosure of which is incorporated herein by reference in its entirety). In this way, the boundaries of the promoters may be defined. If desired, potential individual regulatory sites within the promoter may be identified using site directed mutagenesis or linker scanning to obliterate potential transcription factor binding sites within the promoter individually or in combination. The effects of these mutations on transcription levels may be determined by inserting the mutations into cloning sites in promoter reporter vectors. This type of assays are well known to those skilled in the art and are further described in WO 97/17359, U.S. Pat. No. 5,374,544, EP 582 796, U.S. Pat. No. 5,698,389, U.S. Pat. No. 5,643,746, U.S. Pat. No. 5,502,176, and U.S. Pat. No. 5,266,488, the disclosures of which are incorporated herein by reference in their entireties.
The activity and the specificity of the promoter of the 12-LO gene can further be assessed by monitoring the expression level of a detectable polynucleotide operably linked to the 12-LO promoter in different types of cells and tissues. The detectable polynucleotide may be either a polynucleotide that specifically hybridizes with a predefined oligonucleotide probe, or a polynucleotide encoding a detectable protein, including a 12-LO polypeptide or a fragment or a variant thereof. This type of assay is well known to those skilled in the art and is described in U.S. Pat. No. 5,502,176, and U.S. Pat. No. 5,266,488, the disclosures of which are incorporated herein by reference in their entireties.
Polynucleotides carrying the regulatory elements located both at the 5′ end and at the 3′ end of the 12-LO coding region may be advantageously used to control the transcriptional and translational activity of a heterologous polynucleotide of interest, said polynucleotide being heterologous as regards to the 12-LO regulatory region.
Thus, the present invention also concerns a purified, isolated, and recombinant nucleic acid comprising a polynucleotide which, is selected from the group consisting of, the polynucleotide sequences located between the nucleotide in position 1 and the nucleotide in position 3124 of the nucleotide sequence of SEQ ID No. 651, more preferably between positions 1 and 2195 of SEQ ID No. 651 and the polynucleotide sequences located between the nucleotide in position 17555 and the nucleotide in position 20674 of SEQ ID No. 651; or a sequence complementary thereto or a biologically active fragment thereof.
A “biologically active” fragment of SEQ ID No. 651 according to the present invention is a polynucleotide comprising or alternatively consisting of a fragment of said polynucleotide which is functional as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide in a recombinant cell host.
For the purpose of the invention, a nucleic acid or polynucleotide is “functional” as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide if said regulatory polynucleotide contains nucleotide sequences which contain transcriptional and translational regulatory information, and such sequences are “operably linked” to nucleotide sequences which encode the desired polypeptide or the desired polynucleotide.
The regulatory polynucleotides according to the invention may be advantageously part of a recombinant expression vector that may be used to express a coding sequence in a desired host cell or host organism.
A further object of the invention consists of an isolated polynucleotide comprising:
a) a nucleic acid comprising a regulatory nucleotide sequence selected from the group consisting of a nucleotide sequence comprising a polynucleotide of SEQ ID No. 651;
b) a polynucleotide encoding a desired polypeptide or a nucleic acid of interest, operably linked to the nucleic acid defined in (a) above.
The polypeptide encoded by the nucleic acid described above may be of various nature or origin, encompassing proteins of prokaryotic or eukaryotic origin. Among the polypeptides expressed under the control of a 12-LO regulatory region, there may be cited bacterial, fungal or viral antigens. Also encompassed are eukaryotic proteins such as intracellular proteins, for example “house keeping” proteins, membrane-bound proteins, for example receptors, and secreted proteins, for example cytokines. In a specific embodiment, the desired polypeptide may be the 12-LO protein, especially the protein of the amino acid sequence of SEQ ID No. 653 and 654.
The desired nucleic acids encoded by the above described polynucleotide, usually a RNA molecule, may be complementary to a desired coding polynucleotide, for example to the 12-LO coding sequence, and thus useful as an antisense polynucleotide.
Such a polynucleotide may be included in a recombinant expression vector in order to express the desired polypeptide or the desired nucleic acid in host cell or in a host organism.
C. cDNA Sequences of the 12-LO Gene and Biallelic Markers
The present invention provides a 12-lipoxygenase cDNA of SEQ ID No. 652. The Open Reading Frame encoding the 12-LO protein spans from the nucleotide in position 40 to the nucleotide in position 2028 of the polynucleotide sequence of SEQ ID No. 652. The cDNA of SEQ ID No. 652 also includes a 5′-UTR region (1-40) and a 3′-UTR (2028-2343) region.
Additional preferred cDNA polynucleotides of the invention include isolated, purified or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides from a sequence of SEQ ID No. 652 and the complements thereof. Additional preferred polynucleotides include isolated, purified or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides from a sequence of SEQ ID No. 652, wherein said contiguous span comprises a T at position 1205 of SEQ ID No. 652 or nucleotide positions 2151 to 2157 of SEQ ID No. 652; and the complements thereof.
Preferred cDNA fragments comprise a biallelic marker selected from the group consisting of 10-343-231, 10-346-141, 10-347-111, 10-347-165, 10-347-203, 10-347-220, 10-349-97, 10-349-142, 10-349-216, 10-349-224, 10-507-170, 10-340-112, 10-340-130, 10-341-116 and 10-341-319. Some biallelic polymorphisms represent silent nucleotide substitutions but biallelic markers 10-346-141, 10-347-111, 10-347-165, 10-347-220, 10-349-97, 10-349-142, 10-349-216, 10-340-112, 10-340-130 are associated with amino acid changes in the corresponding 12-lipoxygenase polypeptide. One allele of biallelic marker 10-343-231 (polymorphic deletion of a C nucleotide at position 366 of SEQ ID No. 652) causes a frame shift in the open reading frame of the 12-LO cDNA of SEQ ID No. 652 resulting in the novel polypeptide of SEQ ID No. 653. 12-LO polypeptides of SEQ ID Nos. 653 and 654 of the present invention are further described below.
Other preferred cDNA fragments comprise a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides from a sequence of SEQ ID No. 652, wherein said contiguous span comprises a T at position 1205 of SEQ ID No. 652; and the complements thereof. 12-LO cDNA fragments comprise a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides from a sequence of SEQ ID No. 652, wherein said contiguous span comprises a T at position 1205 of SEQ ID No. 652 encode novel 12-LO polypeptides of SEQ ID No. 653 comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No. 653, wherein said contiguous span comprises a Leu residue at amino acid position 389 of SEQ ID No. 653.
The polynucleotide disclosed above that contains the coding sequence of the 12-LO gene of the invention may be expressed in a desired host cell or a desired host organism, when this polynucleotide is placed under the control of suitable expression signals. The expression signals may be either the expression signals contained in the regulatory regions in the 12-LO gene of the invention or may be exogenous regulatory nucleic sequences. Such a polynucleotide, when placed under the suitable expression signals, may also be inserted in a vector for its expression.
Another preferred cDNA fragment comprises the 5′-UTR (5′regulatory sequence) region beginning at position 1 and ending at position 39 of SEQ ID No. 652. Another preferred cDNA fragment comprises the 3′-UTR (3′ regulatory sequence) region beginning at position 2029 and ending at position 2343 of SEQ ID No. 652. Preferably said 3′-UTR region comprises biallelic marker 10-341-319 or nucleotide positions 2151 to 2157 of SEQ ID No. 652.
D. Polynucleotide Constructs. Recombinant Vectors, Host Cells and Transgenic Animals
The terms “polynucleotide construct” and “recombinant polynucleotide” are used interchangeably herein to refer to linear or circular, purified or isolated polynucleotides that have been artificially designed and which comprise at least two nucleotide sequences that are not found as contiguous nucleotide sequences in their initial natural environment.
Polynucleotide Constructs
1. DNA Constructs for Expressing the 12-LO Gene in Recombinant Host Cells and in Transgenic Animals.
In order to study the physiological and phenotype consequences of a lack of synthesis of the 12-LO protein, both at the cellular level and at the multicellular organism level, in particular as regards to disorders related to abnormal cell proliferation, notably cancers, the invention also encompasses DNA constructs and recombinant vectors enabling a conditional expression of a specific allele of the 12-LO genomic sequence or cDNA
A first preferred DNA construct is based on the tetracycline resistance operon tet from E. coli transposon Tn110 for controlling the 12-LO gene expression, such as described by Gossen et al. (Science, 268:1766-1769, 1995, the disclosure of which is incorporated herein by reference in its entirety). Such a DNA construct contains seven tet operator sequences from Tn10 (tetop) that are fused to either a minimal promoter or a 5′-regulatory sequence of the 12-LO gene, said minimal promoter or said 12-LO regulatory sequence being operably linked to a polynucleotide of interest that codes either for a sense or an antisense oligonucleotide or for a polypeptide, including a 12-LO polypeptide or a peptide fragment thereof. This DNA construct is functional as a conditional expression system for the nucleotide sequence of interest when the same cell also comprises a nucleotide sequence coding for either the wild type (tTA) or the mutant (rTA) repressor fused to the activating domain of viral protein VP16 of herpes simplex virus, placed under the control of a promoter, such as the HCMVIE1 enhancer/promoter or the MMTV-LTR. Indeed, a preferred DNA construct of the invention will comprise both the polynucleotide containing the tet operator sequences and the polynucleotide containing a sequence coding for the tTA or the rTA repressor. In the specific embodiment wherein the conditional expression DNA construct contains the sequence encoding the mutant tetracycline repressor rTA, the expression of the polynucleotide of interest is silent in the absence of tetracycline and induced in its presence.
2. DNA Constructs Allowing Homologous Recombination: Replacement Vectors.
A second preferred DNA construct will comprise, from 5′-end to 3′-end: (a) a first nucleotide sequence that is comprised in the 12-LO genomic sequence; (b) a nucleotide sequence comprising a positive selection marker, such as the marker for neomycine resistance (neo); and (c) a second nucleotide sequence that is comprised in the 12-LO genomic sequence, and is located on the genome downstream the first 12-LO nucleotide sequence (a).
In a preferred embodiment, this DNA construct also comprises a negative selection marker located upstream the nucleotide sequence (a) or downstream the nucleotide sequence (c). Preferably, the negative selection marker consists of the thymidine kinase (tk) gene (Thomas et al., Cell, 44:419-428, 1986, the disclosure of which is incorporated herein by reference in its entirety), the hygromycine beta gene (Te Riele et al., Nature, 348:649-651, 1990, the disclosure of which is incorporated herein by reference in its entirety), the hprt gene (Van der Lugt et al., Gene, 105:263-267, 1991; Reid et al., Proc. Natl. Acad. Sci. USA, 87:4299-4303, 1990, the disclosures of which are incorporated herein by reference in their entireties) or the Diphteria toxin A fragment (Dt-A) gene (Nada et al., Cell, 73:1125-1135, 1993; Yagi et al., Proc. Natl; Acad. Sci. USA, 87:9918-9922, 1990, the disclosures of which are incorporated herein by reference in their entireties). Preferably, the positive selection marker is located within a 12-LO exon sequence so as to interrupt the sequence encoding a 12-LO protein.
These replacement vectors are further described by Mansour et al. (Nature, 336:348-352, 1988, the disclosure of which is incorporated herein by reference in its entirety) and Koller et al. (Ann. Rev. Immunol., 10:705-730, 1992, the disclosure of which is incorporated herein by reference in its entirety).
The first and second nucleotide sequences (a) and (c) may be indifferently located within a 12-LO regulatory sequence, an intronic sequence, an exon sequence or a sequence containing both regulatory and/or intronic and/or exon sequences. The size of the nucleotide sequences (a) and (c) is ranging from 1 to 50 kb, preferably from 1 to 10 kb, more preferably from 2 to 6 kb and most preferably from 2 to 4 kb.
3. DNA Constructs Allowing Homologous Recombination: Cre-loxP System.
These new DNA constructs make use of the site specific recombination system of the P1 phage. The P1 phage possesses a recombinase called Cre which, interacts specifically with a 34 base pairs loxP site. The loxP site is composed of two palindromic sequences of 13 bp separated by a 8 bp conserved sequence (Hoess et al., Nucleic Acids Res., 14:2287-2300, 1986, the disclosure of which is incorporated herein by reference in its entirety). The recombination by the Cre enzyme between two loxP sites having an identical orientation leads to the deletion of the DNA fragment.
The Cre-loxP system used in combination with a homologous recombination technique was first described by Gu et al. (Cell, 73:1155-1164, 1993, the disclosure of which is incorporated herein by reference in its entirety). Briefly, a nucleotide sequence of interest to be inserted in a targeted location of the genome harbors at least two loxP sites in the same orientation and located at the respective ends of a nucleotide sequence to be excised from the recombinant genome. The excision event requires the presence of the recombinase (Cre) enzyme within the nucleus of the recombinant cell host. The recombinase enzyme may be brought at the desired time either by (a) incubating the recombinant cell hosts in a culture medium containing this enzyme, by injecting the Cre enzyme directly into the desired cell, such as described by Araki et al. (Proc. Natl; Acad. Sci. USA, 92: 160-164, 1995, the disclosure of which is incorporated herein by reference in its entirety), or by lipofection of the enzyme into the cells, such as described by Baubonis et al. (Nucleic Acids Res., 21:2025-2029, 1993, the disclosure of which is incorporated herein by reference in its entirety); (b) transfecting the cell host with a vector comprising the Cre coding sequence operably linked to a promoter functional in the recombinant cell host, which promoter being optionally inducible, said vector being introduced in the recombinant cell host, such as described by Gu et al. (Cell, 73:1155-1164, 1993, the disclosure of which is incorporated herein by reference in its entirety) and Sauer et al. (Proc. Natl; Acad. Sci. USA, 85:5166-5170, 1988, the disclosure of which is incorporated herein by reference in its entirety); (c) introducing in the genome of the cell host a polynucleotide comprising the Cre coding sequence operably linked to a promoter functional in the recombinant cell host, which promoter is optionally inducible, and said polynucleotide being inserted in the genome of the cell host either by a random insertion event or an homologous recombination event, such as described by Gu et al. (Science, 265:103-106, 1994, the disclosure of which is incorporated herein by reference in its entirety).
In the specific embodiment wherein the vector containing the sequence to be inserted in the 12-LO gene by homologous recombination is constructed in such a way that selectable markers are flanked by loxP sites of the same orientation, it is possible, by treatment by the Cre enzyme, to eliminate the selectable markers while leaving the 12-LO sequences of interest that have been inserted by an homologous recombination event. Again, two selectable markers are needed: a positive selection marker to select for the recombination event and a negative selection marker to select for the homologous recombination event. Vectors and methods using the Cre-loxP system are further described by Zou et al. (Curr. Biol., 4:1099-1103, 1994), the disclosure of which is incorporated herein by reference in its entirety.
Thus, a third preferred DNA construct of the invention comprises, from 5′-end to 3′-end: (a) a first nucleotide sequence that is comprised in the 12-LO genomic sequence; (b) a nucleotide sequence comprising a polynucleotide encoding a positive selection marker, said nucleotide sequence comprising additionally two sequences defining a site recognized by a recombinase, such as a loxP site, the two sites being placed in the same orientation; and (c) a second nucleotide sequence that is comprised in the 12-LO genomic sequence, and is located on the genome downstream of the first 12-LO nucleotide sequence (a).
The sequences defining a site recognized by a recombinase, such as a loxP site, are preferably located within the nucleotide sequence (b) at suitable locations bordering the nucleotide sequence for which the conditional excision is sought. In one specific embodiment, two loxP sites are located at each side of the positive selection marker sequence, in order to allow its excision at a desired time after the occurrence of the homologous recombination event.
In a preferred embodiment of a method using the third DNA construct described above, the excision of the polynucleotide fragment bordered by the two sites recognized by a recombinase, preferably two loxP sites, is performed at a desired time, due to the presence within the genome of the recombinant cell host of a sequence encoding the Cre enzyme operably linked to a promoter sequence, preferably an inducible promoter, more preferably a tissue-specific promoter sequence and most preferably a promoter sequence which is both inducible and tissue-specific, such as described by Gu et al. (Science, 265:103-106, 1994), the disclosure of which is incorporated herein by reference in its entirety.
The presence of the Cre enzyme within the genome of the recombinant cell host may result of the breeding of two transgenic animals, the first transgenic animal bearing the 12-LO-derived sequence of interest containing the loxP sites as described above and the second transgenic animal bearing the Cre coding sequence operably linked to a suitable promoter sequence, such as described by Gu et al. (Science, 265:103-106, 1994), the disclosure of which is incorporated herein by reference in its entirety.
Spatio-temporal control of the Cre enzyme expression may also be achieved with an adenovirus based vector that contains the Cre gene thus allowing infection of cells, or in vivo infection of organs, for delivery of the Cre enzyme, such as described by Anton et al. (J. Virol., 69:4600-4606, 1995) and Kanegae et al. (Nucleic Acids Res., 23:3816-3821, 1995), the disclosures of which are incorporated herein by reference in their entireties.
The DNA constructs described above may be used to introduce a desired nucleotide sequence of the invention, preferably a 12-LO genomic sequence or a 12-LO cDNA sequence, and most preferably an altered copy of a 12-LO genomic or cDNA sequence, within a predetermined location of the targeted genome, leading either to the generation of an altered copy of a targeted gene (knock-out homologous recombination) or to the replacement of a copy of the targeted gene by another copy sufficiently homologous to allow an homologous recombination event to occur (knock-in homologous recombination).
Recombinant Vectors
The term “vector” is used herein to designate either a circular or a linear DNA or RNA molecule, which is either double-stranded or single-stranded, and which comprise at least one polynucleotide of interest that is sought to be transferred in a cell host or in a unicellular or multicellular host organism.
The present invention encompasses a family of recombinant vectors that comprise a regulatory polynucleotide derived from the 12-LO genomic sequence, or a coding polynucleotide from the 12-LO genomic sequence. Consequently, the present invention further deals with a recombinant vector comprising either a regulatory polynucleotide comprised in the nucleic acid of SEQ ID No. 651 or a polynucleotide comprising the 12-LO coding sequence or both.
In a first preferred embodiment, a recombinant vector of the invention is used to amplify the inserted polynucleotide derived from a 12-LO genomic sequence selected from the group consisting of the nucleic acids of SEQ ID No. 651 or a 12-LO cDNA, for example the cDNA of SEQ ID No. 652 in a suitable host cell, this polynucleotide being amplified each time the recombinant vector replicates. Generally, a recombinant vector of the invention may comprise any of the polynucleotides described herein, including regulatory sequences and coding sequences, as well as any 12-LO primer or probe as defined above.
In a second preferred embodiment, recombinant vectors of the invention consist of expression vectors comprising either a regulatory polynucleotide or a coding nucleic acid of the invention, or both. Within certain embodiments, expression vectors are employed to express the 12-LO polypeptide which can be then purified and, for example be used in ligand screening assays or as an immunogen in order to raise specific antibodies directed against the 12-LO protein. In other embodiments, the expression vectors are used for constructing transgenic animals and also for gene therapy. Expression requires that appropriate signals are provided in the vectors, said signals including various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Dominant drug selection markers for establishing permanent, stable cell clones expressing the products are generally included in the expression vectors of the invention, as they are elements that link expression of the drug selection markers to expression of the polypeptide.
More particularly, the present invention relates to expression vectors which include nucleic acids encoding a 12-LO protein, preferably the 12-LO protein of the amino acid sequence of SEQ ID No. 653, under the control of a regulatory sequence selected among the 12-LO regulatory polynucleotides of SEQ ID Nos. 651 and 652, or alternatively under the control of an exogenous regulatory sequence.
Consequently, preferred expression vectors of the invention are selected from the group consisting of: (a) the 12-LO regulatory sequence comprised therein drives the expression of a coding polynucleotide operably linked thereto; (b) the 12-LO coding sequence is operably linked to regulation sequences allowing its expression in a suitable cell host and/or host organism.
Additionally, the recombinant expression vector described above may also comprise a nucleic acid comprising a 5′-regulatory polynucleotide, preferably a 5′-regulatory polynucleotide of the 12-LO gene. Additionally, the recombinant expression vector described above may also comprise a nucleic acid comprising a 3′-regulatory polynucleotide, preferably a 3′-regulatory polynucleotide of the 12-LO gene. The 12-LO 3′-regulatory polynucleotide may also comprise the 3′-UTR sequence contained in the nucleotide sequence of SEQ ID No. 652. The 5′-regulatory polynucleotide may also include the 5′-UTR sequence of the 12-LO cDNA, or a biologically active fragment or variant thereof. The invention also pertains to a recombinant expression vector useful for the expression of the 12-LO coding sequence, wherein said vector comprises a nucleic acid of SEQ ID No. 652.
The invention also relates to a recombinant expression vector comprising a nucleic acid comprising the nucleotide sequence beginning at the nucleotide in position 40 and ending in position 2028 of the polynucleotide of SEQ ID No. 652.
Some of the elements which can be found in the vectors of the present invention are described in further detail in the following sections.
1. General Features of the Expression Vectors of the Invention.
A recombinant vector according to the invention comprises, but is not limited to, a YAC (Yeast Artificial Chromosome), a BAC (Bacterial Artificial Chromosome), a phage, a phagemid, a cosmid, a plasmid or even a linear DNA molecule which may consist of a chromosomal, non-chromosomal, semi-synthetic and synthetic DNA. Such a recombinant vector can comprise a transcriptional unit comprising an assembly of:
(1) a genetic element or elements having a regulatory role in gene expression, for example promoters or enhancers. Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp in length that act on the promoter to increase the transcription.
(2) a structural or coding sequence which is transcribed into mRNA and eventually translated into a polypeptide, said structural or coding sequence being operably linked to the regulatory elements described in (1); and
(3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, when a recombinant protein is expressed without a leader or transport sequence, it may include a N-terminal residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.
Generally, recombinant expression vectors will include origins of replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein into the periplasmic space or the extracellular medium. In a specific embodiment wherein the vector is adapted for transfecting and expressing desired sequences in mammalian host cells, preferred vectors will comprise an origin of replication in the desired host, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′-flanking non-transcribed sequences. DNA sequences derived from the SV40 viral genome, for example SV40 origin, early promoter, enhancer, splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
The in vivo expression of a 12-LO polypeptide of SEQ ID Nos. 653 and 654 may be useful in order to correct a genetic defect related to the expression of the native gene in a host organism or to the production of a biologically inactive 12-LO protein.
Consequently, the present invention also deals with recombinant expression vectors mainly designed for the in vivo production of the 12-LO polypeptide of SEQ ID Nos. 653-654 or fragments or variants thereof by the introduction of the appropriate genetic material in the organism of the patient to be treated. This genetic material may be introduced in vitro in a cell that has been previously extracted from the organism, the modified cell being subsequently reintroduced in the said organism, directly in vivo into the appropriate tissue.
2. Regulatory Elements.
The suitable promoter regions used in the expression vectors according to the present invention are chosen taking into account the cell host in which the heterologous gene has to be expressed. The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell, such as, for example, a human or a viral promoter.
A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed. Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.
Promoter regions can be selected from any desired gene using, for example, CAT (chloramphenicol transferase) vectors and more preferably pKK232-8 and pCM7 vectors.
Preferred bacterial promoters are the LacI, LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the gpt, lambda PR, PL and trp promoters (EP 0036776, the disclosure of which is incorporated herein by reference in its entirety), the polyhedrin promoter, or the p10 protein promoter from baculovirus (Kit Novagen) (Smith et al., Mol. Cell. Biol. 3:2156-2165, 1983; O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual., W.H. Freeman and Co., New York, 1992, the disclosures of which are incorporated herein by reference in their entireties), the lambda PR promoter or also the trc promoter.
Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-L. Selection of a convenient vector and promoter is well within the level of ordinary skill in the art.
The choice of a promoter is well within the ability of a person skilled in the field of genetic egineering. For example, one may refer to the book of Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989), the disclosure of which is incorporated herein by reference in its entirety.
Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
The vector containing the appropriate DNA sequence as described above, more preferably 12-LO gene regulatory polynucleotide, a polynucleotide encoding the 12-LO polypeptide of SEQ ID Nos. 653 and 654 or both of them, can be utilized to transform an appropriate host to allow the expression of the desired polypeptide or polynucleotide.
3. Selectable Markers.
Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct. The selectable marker genes for selection of transformed host cells are preferably dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, TRP1 for S. cerevisiae or tetracycline, rifampicin or ampicillin resistance in E. coli, or levan saccharase for mycobacteria, this latter marker being a negative selection marker.
4. Preferred Vectors.
As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and a bacterial origin of replication derived from commercially available plasmids comprising genetic elements of pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), and GEM1 (Promega Biotec, Madison, Wis., USA). Large numbers of other suitable vectors are known to those of skill in the art, and commercially available, such as the following bacterial vectors: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIAexpress).
The P1 bacteriophage vector may contain large inserts ranging from about 80 to about 100 kb. The construction of P1 bacteriophage vectors such as p158 or p158/neo8 are described by Sternberg (Mamm. Genome, 5:397-404, 1994), the disclosure of which is incorporated herein by reference in its entirety. Recombinant P1 clones comprising 12-LO nucleotide sequences may be designed for inserting large polynucleotides of more than 40 kb (Linton et al., J. Clin. Invest., 92:3029-3037, 1993), the disclosure of which is incorporated herein by reference in its entirety. To generate P1 DNA for transgenic experiments, a preferred protocol is the protocol described by McCormick et al. (Genet. Anal. Tech. Appl., 11:158-164, 1994). Briefly, E. coli (preferably strain NS3529) harboring the P1 plasmid are grown overnight in a suitable broth medium containing 25 μg/ml of kanamycin. The P1 DNA is prepared from the E. coli by alkaline lysis using the Qiagen Plasmid Maxi kit (Qiagen, Chatsworth, Calif., USA), according to the manufacturer's instructions. The P1 DNA is purified from the bacterial lysate on two Qiagen-tip 500 columns, using the washing and elution buffers contained in the kit. A phenol/chloroform extraction is then performed before precipitating the DNA with 70% ethanol. After solubilizing the DNA in TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA), the concentration of the DNA is assessed by spectrophotometry.
When the goal is to express a P1 clone comprising 12-LO nucleotide sequences in a transgenic animal, typically in transgenic mice, it is desirable to remove vector sequences from the P1 DNA fragment, for example by cleaving the P1 DNA at rare-cutting sites within the P1 polylinker (SfiI, NotI or SalI). The P1 insert is then purified from vector sequences on a pulsed-field agarose gel, using methods similar using methods similar to those originally reported for the isolation of DNA from YACs (Schedl et al., Nature 362:258-261 1993; Peterson et al., Proc. Natl. Acad. Sci. USA 90:7593-7597, 1993, the disclosures of which are incorporated herein by reference in their entireties). At this stage, the resulting purified insert DNA can be concentrated, if necessary, on a Millipore Ultrafree-MC Filter Unit (Millipore, Bedford, Mass., USA—30,000 molecular weight limit) and then dialyzed against microinjection buffer (10 mM Tris-HCl, pH 7.4; 250 μM EDTA) containing 100 mM NaCl, 30 μM spermidine, 70 μM spermidine on a microdyalisis membrane (type VS, 0.025 μM from Millipore). The intactness of the purified P1 DNA insert is assessed by electrophoresis on 1% agarose (Sea Kem GTG; FMC Bio-products) pulse-field gel and staining with ethidium bromide.
A suitable vector for the expression of the 12-LO polypeptide of SEQ ID Nos. 653 and 654 is a baculovirus vector that can be propagated in insect cells and in insect cell lines. A specific suitable host vector system is the pVL1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC NoCRL 1711) which is derived from Spodoptera frugiperda. 
Other suitable vectors for the expression of the 12-LO polypeptide of SEQ ID Nos. 653 and 654 in a baculovirus expression system include those described by Chai et al. (Biotech. Appl. Biochem., 18:259-273, 1993), Vlasak et al. (Eur. J. Biochem., 135: 123-126, 1983) and Lenhard et al. (Gene, 169: 187-190, 1996), the disclosures of which are incorporated herein by reference in their entireties.
Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery systems of choice for the transfer of exogenous polynucleotides in vivo, particularly to mammals, including humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.
Particularly preferred retroviruses for the preparation or construction of retroviral in vitro or in vitro gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Mink-Cell Focus Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma virus. Particularly preferred Murine Leukemia Viruses include the 4070A and the 1504A viruses, Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Gross (ATCC No. VR-590), Rauscher (ATCC No. VR-998) and Moloney Murine Leukemia Virus (ATCC No. VR-190; PCT Application No. WO 94/24298, the disclosure of which is incorporated herein by reference in its entirety). Particularly preferred Rous Sarcoma Viruses include Bryan high titer (ATCC Nos. VR-334, VR-657, VR-726, VR-659 and VR-728). Other preferred retroviral vectors are those described in Roth et al. (Nature Medicine, 2:985-991, 1996), PCT Application No. WO 93/25234 and PCT Application No. WO 94/06920, the disclosures of which are incorporated herein by reference in their entireties.
Yet another viral vector system that is contemplated by the invention consists in the adeno-associated virus (AAV). The adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al., Current Topics in Microbiol. Immunol., 158:97-129, 1992, the disclosure of which is incorporated herein by reference in its entirety). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (McLaughlin et al., Am. J. Hum. Genet., 59: 561-569, 1989, the disclosure of which is incorporated herein by reference in its entirety). One advantageous feature of AAV derives from its reduced efficacy for transducing primary cells relative to transformed cells.
The bacterial artificial chromosome (BAC) cloning system (Shizuya et al., Proc. Natl. Acad. Sci. U.S.A. 89:8794-8797, 1992, the disclosure of which is incorporated herein by reference in its entirety) has been developed to stably maintain large fragments of genomic DNA (100-300 kb) in E. coli. A preferred BAC vector consists of pBeloBAC11 vector that has been described by Kim et al. (Genomics, 34:213-218,1996), the disclosure of which is incorporated herein by reference in its entirety. BAC libraries are prepared with this vector using size-selected genomic DNA that has been partially digested using enzymes that permit ligation into either the Bam HI or HindIII sites in the vector. Flanking these cloning sites are T7 and SP6 RNA polymerase transcription initiation sites that can be used to generate end probes by either RNA transcription or PCR methods. After the construction of a BAC library in E. coli, BAC DNA is purified from the host cell as a supercoiled circle. Converting these circular molecules into a linear form precedes both size determination and introduction of the BACs into recipient cells. The cloning site is flanked by two Not I sites, permitting cloned segments to be excised from the vector by Not I digestion. Alternatively, the DNA insert contained in the pBeloBAC11 vector may be linearized by treatment of the BAC vector with the commercially available enzyme lambda terminase that leads to the cleavage at the unique cosN site, but this cleavage method results in a full length BAC clone containing both the insert DNA and the BAC sequences.
5. Delivery of the Recombinant Vectors.
In order to effect expression of the polynucleotides and polynucleotide constructs of the invention, these constructs must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cell lines, or in vivo or ex vivo, as in the treatment of certain diseases states. One mechanism is viral infection where the expression construct is encapsidated in an infectious viral particle.
Several non-viral methods for the transfer of polynucleotides into cultured mammalian cells are also contemplated by the present invention, and include, without being limited to, calcium phosphate precipitation (Chen et al., Proc. Natl. Acad. Sci. USA, 94:10756-10761, 1987, the disclosure of which is incorporated herein by reference in its entirety), DEAE-dextran (Gopal, Mol. Cell. Biol., 5:1188-1190, 1985, the disclosure of which is incorporated herein by reference in its entirety), electroporation (Tur-Kaspa et al., Mol. Cell. Biol., 6:716-718, 1986, the disclosure of which is incorporated herein by reference in its entirety), direct microinjection (Harland et al., J. Cell. Biol. 101:1094-1095, 1985), DNA-loaded liposomes (Nicolau et al., Biochim. Biophys. Acta. 721:185-190,1982; Fraley et al., Natl. Acad. Sci. USA 76:3348-3352, 1979, the disclosures of which are incorporated herein by reference in their entireties), and receptor-mediate transfection (Wu and Wu, J. Biol. Chem. 262:44294432, 1987; Wu and Wu Biochemistry 27:887-892, 1988, the disclosures of which are incorporated herein by reference in their entireties). Some of these techniques may be successfully adapted for in vivo or ex vivo use.
Once the expression polynucleotide has been delivered into the cell, it may be stably integrated into the genome of the recipient cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle.
One specific embodiment for a method for delivering a protein or peptide to the interior of a cell of a vertebrate in vivo comprises the step of introducing a preparation comprising a physiologically acceptable carrier and a naked polynucleotide operatively coding for the polypeptide of interest into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior of the cell and has a physiological effect. This is particularly applicable for transfer in vitro but it may be applied to in vivo as well.
Compositions for use in vitro and in vivo comprising a “naked” polynucleotide are described in PCT application No. WO 90/11092 (Vical Inc.) and in PCT application No. WO 95/11307, the disclosures of which are incorporated herein by reference in their entireties.
In still another embodiment of the invention, the transfer of a naked polynucleotide of the invention, including a polynucleotide construct of the invention, into cells may be proceeded with a particle bombardment (biolistic), said particles being DNA-coated microprojectiles accelerated to a high velocity allowing them to pierce cell membranes and enter cells without killing them, such as described by Klein et al. (Nature 327:70-73, 1987), the disclosure of which is incorporated herein by reference in its entirety.
In a further embodiment, the polynucleotide of the invention may be entrapped in a liposome (Ghosh and Bacchawat, Targeting of liposomes to hepatocytes, In: Liver Diseases, Targeted diagnosis and therapy using specific rceptors and ligands, Marcel Dekeker, New York, 87-104, 1991; Wong et al., Gene 10:87-94, 1980; Nicolau et al., Biochim. Biophys. Acta. 721:185-190, 1982, the disclosures of which are incorporated herein by reference in their entireties)
In a specific embodiment, the invention provides a composition for the in vivo production of the 12-LO protein or polypeptide described herein. It comprises a naked polynucleotide operatively coding for this polypeptide, in solution in a physiologically acceptable carrier, and suitable for introduction into a tissue to cause cells of the tissue to express the said protein or polypeptide.
The amount of vector to be injected to the desired host organism varies according to the site of injection. As an indicative dose, it will be injected between 0.1 and 100 μg of the vector in an animal body, preferably a mammal body, for example a mouse body.
In another embodiment of the vector according to the invention, it may be introduced in vitro in a host cell, preferably in a host cell previously harvested from the animal to be treated and more preferably a somatic cell such as a muscle cell. In a subsequent step, the cell that has been transformed with the vector coding for the desired 12-LO polypeptide or the desired fragment thereof is reintroduced into the animal body in order to deliver the recombinant protein within the body either locally or systemically.
Host Cells
Another object of the invention consists of a host cell that have been transformed or transfected with one of the polynucleotides described therein, and more precisely a polynucleotide either comprising a 12-LO regulatory polynucleotide or the coding sequence of the 12-LO polypeptide having the amino acid sequence of SEQ ID Nos. 653 or 654. Are included host cells that are transformed (prokaryotic cells) or that are transfected (eukaryotic cells) with a recombinant vector such as one of those described above.
Generally, a recombinant host cell of the invention comprises any one of the polynucleotides or the recombinant vectors described therein.
A preferred recombinant host cell according to the invention comprises a polynucleotide selected from the following group of polynucleotides:

    
    
        a) a purified or isolated nucleic acid encoding a 12-LO polypeptide, or a polypeptide fragment or variant thereof.
        b) a purified or isolated nucleic comprising at least 8, preferably at least 15, more preferably at least 25, consecutive nucleotides of the nucleotide sequence SEQ ID No. 651, a nucleotide sequence complementary thereto, or a variant thereof.
        c) a purified or isolated nucleic acid comprising at least 8 consecutive nucleotides, preferably at least 15, more preferably at least 25 of the nucleotide sequence SEQ ID No. 652, a nucleotide sequence complementary thereto or a variant thereof.
        d) a purified or isolated nucleic acid comprising an exon of the 12-LO gene, a sequence complementary thereto or a fragment or a variant thereof.
        e) a purified or isolated nucleic acid comprising a combination of at least two exons of the 12-LO gene, or the sequences complementary thereto wherein the polynucleotides are arranged within the nucleic acid, from the 5′ end to the 3′ end of said nucleic acid, in the same order than in SEQ ID No. 651.
        f) a purified or isolated nucleic acid comprising the nucleotide sequence SEQ ID No. 651 or the sequences complementary thereto or a biologically active fragment thereof.
        g) a polynucleotide consisting of:
        
            (1) a nucleic acid comprising a regulatory polynucleotide of SEQ ID No. 651 or the sequences complementary thereto or a biologically active fragment thereof
            (2) a polynucleotide encoding a desired polypeptide or nucleic acid.
        
        
        i) a DNA construct as described previously in the present specification.
    
    


Another preferred recombinant cell host according to the present invention is characterized in that its genome or genetic background (including chromosome, plasmids) is modified by the nucleic acid coding for the 12-LO polypeptide of SEQ ID Nos. 653 and 654 or fragments or variants thereof.
Preferred host cells used as recipients for the expression vectors of the invention are the following:

    
    
        a) Prokaryotic host cells: Escherichia coli strains (I.E. DH5-α strain), Bacillus subtilis, Salmonella typhimurium, and strains from species like Pseudomonas, Streptomyces and Staphylococcus. 
        b) Eukaryotic host cells: HeLa cells (ATCC NoCCL2; NoCCL2.1; NoCCL2.2), Cv 1 cells (ATCC NoCCL70), COS cells (ATCC NoCRL1650; NoCRL1651), Sf-9 cells (ATCC NoCRL1711), C127 cells (ATCC NoCRL-1804), 3T3 (ATCC NoCRL-6361), CHO (ATCC NoCCL-61), human kidney 293. (ATCC No 45504; NoCRL-1573) and BHK (ECACC No 84100501; No 84111301)
        c) Other mammalian host cells:
    
    


The 12-LO gene expression in mammalian, and typically human, cells may be rendered defective, or alternatively it may be proceeded with the insertion of a 12-LO genomic or cDNA sequence with the replacement of the 12-LO gene counterpart in the genome of an animal cell by a 12-LO polynucleotide according to the invention. These genetic alterations may be generated by homologous recombination events using specific DNA constructs that have been previously described.
One kind of host cell that may be used is mammalian zygotes, such as murine zygotes. For example, murine zygotes may undergo microinjection with a purified DNA molecule of interest, such as a purified DNA molecule that has previously been adjusted to a concentration range from 1 ng/ml (for BAC inserts) 3 ng/μl (for P1 bacteriophage inserts) in 10 mM Tris-HCl, pH 7.4, 250 μM EDTA containing 100 mM NaCl, 30 μM spermine, and 70 μM spermidine. When the DNA to be microinjected is relatively large, polyamines and high salt concentrations can be used to avoid mechanical breakage of this DNA, as described by Schedl et al. (Nucleic Acids Res. 21:4783-4787, 1993), the disclosure of which is incorporated herein by reference in its entirety.
Anyone of the polynucleotides of the invention, including the DNA constructs described herein, may be introduced in an embryonic stem (ES) cell line, preferably a mouse ES cell line. ES cell lines are derived from pluripotent, uncommitted cells of the inner cell mass of pre-implantation blastocysts. Preferred ES cell lines are the following: ES-E14TG2a (ATCC no CRL-1821), ES-D3 (ATCC no CRL1934 and no CRL-11632), YS0001 (ATCC no CRL-11776), 36.5 (ATCC no CRL-11116). To maintain ES cells in an uncommitted state, they are cultured in the presence of growth inhibited feeder cells, which provide the appropriate signals to preserve this embryonic phenotype and serve as a matrix for ES cell adherence. Preferred feeder cells consist of primary embryonic fibroblasts that are established from tissue of day 13-day 14 embryos of virtually any mouse strain, that are maintained in culture, such as described by Abbondanzo et al. (Methods in Enzymology, Academic Press, New York, 803-823, 1993), the disclosure of which is incorporated herein by reference in its entirety, and are inhibited in growth by irradiation, such as described by Robertson (“Embryo-Derived Stem Cell Lines,” E. J Robertson Ed. Teratocarcinomas and Embrionic Stem Cells: A Practical Approach. IRL Press, Oxford, 71, 1987), the disclosure of which is incorporated herein by reference in its entirety, or by the presence of an inhibitory concentration of LIF, such as described by Pease and Williams (Exp. Cell. Res. 190:09-211, 1990), the disclosure of which is incorporated herein by reference in its entirety.
The constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
Following transformation of a suitable host and growth of the host to an appropriate cell density, the selected promoter is induced by appropriate means, such as temperature shift or chemical induction, and cells are cultivated for an additional period.
Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
Microbial cells employed in the expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known by the skill artisan.
Transgenic Animals
The terms “transgenic animals” or “host animals” used herein designate animals that have their genome genetically and artificially manipulated so as to include one of the nucleic acids according to the invention. Preferred animals are non-human mammals and include those belonging to a genus selected from Mus (e.g. mice), Rattus (e.g. rats) and Oryctogalus (e.g. rabbits) which have their genome artificially and genetically altered by the insertion of a nucleic acid according to the invention.
The transgenic animals of the invention all include within a plurality of their cells a cloned recombinant or synthetic DNA sequence, more specifically one of the purified or isolated nucleic acids comprising a 12-LO coding sequence, a 12-LO regulatory polynucleotide or a DNA sequence encoding an antisense polynucleotide such as described in the present specification.
Preferred transgenic animals according to the invention contain in their somatic cells and/or in their germ line cells a polynucleotide selected from the following group of polynucleotides:

    
    
        a) a purified or isolated nucleic acid encoding a 12-LO polypeptide, or a polypeptide fragment or variant thereof.
        b) a purified or isolated nucleic comprising at least 8, preferably at least 15, more preferably at least 25, consecutive nucleotides of the nucleotide sequence SEQ ID No. 651, a nucleotide sequence complementary thereto.
        c) a purified or isolated nucleic acid comprising at least 8 consecutive nucleotides, preferably at least 15, more preferably at least 25 of the nucleotide sequence SEQ ID No. 652, a nucleotide sequence complementary thereto.
        d) a purified or isolated nucleic acid comprising an exon of the 12-LO gene, a sequence complementary thereto or a fragment or a variant thereof.
        e) a purified or isolated nucleic acid comprising a combination of at least two exons of the 12-LO gene, or the sequences complementary thereto wherein the polynucleotides are arranged within the nucleic acid, from the 5′ end to the 3′ end of said nucleic acid, in the same order than in SEQ ID No. 651.
        f) a purified or isolated nucleic acid comprising the nucleotide sequence SEQ ID No. 651 or the sequences complementary thereto or a biologically active fragment thereof.
        g) a polynucleotide consisting of:
        
            (1) a nucleic acid comprising a regulatory polynucleotide of SEQ ID No. 651 or the sequences complementary thereto or a biologically active fragment thereof
            (2) a polynucleotide encoding a desired polypeptide or nucleic acid.
        
        
        i) a DNA construct as described previously in the present specification.
    
    


The transgenic animals of the invention thus contain specific sequences of exogenous genetic material such as the nucleotide sequences described above in detail.
In a first preferred embodiment, these transgenic animals may be good experimental models in order to study the diverse pathologies related to cell differentiation, in particular concerning the transgenic animals within the genome of which has been inserted one or several copies of a polynucleotide encoding a native 12-LO protein, or alternatively a mutant 12-LO protein.
In a second preferred embodiment, these transgenic animals may express a desired polypeptide of interest under the control of the regulatory polynucleotides of the 12-LO gene, leading to good yields in the synthesis of this protein of interest, and eventually a tissue specific expression of this protein of interest.
The design of the transgenic animals of the invention may be made according to the conventional techniques well known for one skilled in the art. For more details regarding the production of transgenic animals, and specifically transgenic mice, one may refer to U.S. Pat. No. 4,873,191, issued Oct. 10, 1989, U.S. Pat. No. 5,464,764 issued Nov. 7, 1995 and U.S. Pat. No. 5,789,215, issued Aug. 4, 1998, these documents being herein incorporated by reference in their entireties to disclose methods producing transgenic mice.
Transgenic animals of the present invention are produced by the application of procedures which result in an animal with a genome that has incorporated exogenous genetic material. The procedure involves obtaining the genetic material, or a portion thereof, which encodes either a 12-LO coding sequence, a 12-LO regulatory polynucleotide or a DNA sequence encoding a 12-LO antisense polynucleotide such as described in the present specification.
A recombinant polynucleotide of the invention is inserted into an embryonic or ES stem cell line. The insertion is preferably made using electroporation, such as described by Thomas et al. (Cell 51:503-512, 1987), the disclosure of which is incorporated herein by reference in its entirety. The cells subjected to electroporation are screened (e.g. by selection via selectable markers, by PCR or by Southern blot analysis) to find positive cells which have integrated the exogenous recombinant polynucleotide into their genome, preferably via an homologous recombination event. An illustrative positive-negative selection procedure that may be used according to the invention is described by Mansour et al. (Nature 336:348-352, 1988), the disclosure of which is incorporated herein by reference in its entirety.
Then, the positive cells are isolated, cloned and injected into 3.5 days old blastocysts from mice, such as described by Bradley (“Production and Analysis of Chimaeric Mice,” E. J Robertson (Ed.), Teratocarcinomas and embryonic stem cells: A practical approach IRL Press, Oxford, 113, 1987), the disclosure of which is incorporated herein by reference in its entirety. The blastocysts are then inserted into a female host animal and allowed to grow to term.
Alternatively, the positive ES cells are brought into contact with embryos at the 2.5 days old 8-16 cell stage (morulae) such as described by Wood et al. (Proc. Natl. Acad. Sci. U.S.A. 90:4582-4585, 1993) or by Nagy et al. (Proc. Natl. Acad. Sci. USA. 90: 8424-8428, 1993), the disclosures of which are incorporated herein by reference in their entireties, the ES cells being internalized to colonize extensively the blastocyst including the cells which will give rise to the germ line.
The offspring of the female host are tested to determine which animals are transgenic e.g. include the inserted exogenous DNA sequence and which are wild-type. Thus, the present invention also concerns a transgenic animal containing a nucleic acid, a recombinant expression vector or a recombinant host cell according to the invention.
A further object of the invention consists of recombinant host cells obtained from a transgenic animal described herein.
Recombinant cell lines may be established in vitro from cells obtained from any tissue of a transgenic animal according to the invention, for example by transfection of primary cell cultures with vectors expressing onc-genes such as SV40 large T antigen, as described by Chou (Mol. Endocrinol. 3:1511-1514, 1989) and Shay et al. (Biochem. Biophys. Acta. 1072:1-7, 1991), the disclosures of which are incorporated herein by reference in their entireties.
E. 12-Lipoxygenase Polypeptides
The term “12-LO polypeptides” is used herein to embrace all of the proteins and polypeptides of the present invention. Also forming part of the invention are polypeptides encoded by the polynucleotides of the invention, as well as fusion polypeptides comprising such polypeptides. The invention embodies 12-LO proteins from humans, including isolated or purified 12-LO proteins consisting, consisting essentially, or comprising the sequence of SEQ ID Nos. 653 and 654.
Biallelic markers are associated with amino acid substitutions in the polypeptide sequence of 12-LO. It should be noted the 12-LO proteins of the invention are based on the naturally-occurring variants of the amino acid sequence of human 12-LO; wherein the Arg residue of amino acid position 189 has been replaced with a His residue (biallelic marker 10-346-141), the Asp residue of amino acid position 225 has been replaced with a His residue (biallelic marker 10-347-111), the Arg residue of amino acid position 243 has been replaced with a Cys residue (biallelic marker 10-347-165), the Gln residue of amino acid position 261 has been replaced with an Arg residue (biallelic marker 10-347-220), the Ser residue of amino acid position 322 has been replaced with a Asn residue (biallelic marker 10-349-97), the Pro residue of amino acid position 337 has been replaced with an Arg residue (biallelic marker 10-349-142), the Thr residue of amino acid position 568 has been replaced with an Asn residue (biallelic marker 10-340-112) and wherein the Met residue of amino acid position 574 has been replaced with a Lys residue (biallelic marker 10-340-112). Variant proteins and the fragments thereof which contain amino acid position 189 are collectively referred to herein as “189-His variants.” Variant proteins and the fragments thereof which contain amino acid position 225 are collectively referred to herein as “225-His variants.” Variant proteins and the fragments thereof which, contain amino acid position 243, are collectively referred to herein as “243-Cys variants.” Variant proteins and the fragments thereof which contain amino acid position 261 are collectively referred to herein as “261-Arg variants.” Variant proteins and the fragments thereof which contain amino acid position 322 are collectively referred to herein as “322-Asn variants.” Variant proteins and the fragments thereof which contain amino acid position 337 are collectively referred to herein as “337-Arg variants.” Variant proteins and the fragments thereof which contain amino acid position 568 are collectively referred to herein as “568-Asn variants.” Variant proteins and the fragments thereof which contain amino acid position 574 are collectively referred to herein as “574-Lys variants.” In each of these amino acid substitutions the original residue is replaced by a non-equivalent amino acid presenting different chemical properties. Therefore, these substitutions cause alterations in the activity, specificity and function of the 12-LO enzyme.
One allele of biallelic marker 10-349-216 is associated with the deletion of a Leu residue at amino acid position 362 of SEQ ID No. 653. 12-LO polypeptides of the present invention also include 12-LO polypeptides wherein the Leu residue at amino acid position 362 of SEQ ID No. 653 has been deleted.
One allele of biallelic marker 10-343-231 is associated with a frameshift in the open reading frame of the 12-LO gene leading to the expression of the variant 12-LO polypeptide of SEQ ID No. 654.
The present invention embodies isolated, purified, and recombinant polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No. 653, wherein said contiguous span comprises at least one amino acid position selected from the group consisting of: an His residue et amino acid position 189, an His residue at amino acid position 225, a Cys residue at amino acid position 243, an Arg residue at amino acid position 261, an Asn residue at amino acid position 322, an Arg residue at amino acid position 337, a Asn residue at amino acid position 362, an Asn at amino acid position 568 and a Lys residue at amino acid position 574.
The present invention further provides isolated, purified, and recombinant polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No. 654, wherein said contiguous span comprises at least one of amino acid positions 110-131 of SEQ ID No. 654.
The present invention further embodies isolated, purified, and recombinant polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No. 653, wherein said contiguous span comprises a Leu residue at amino acid position 389 of SEQ ID No. 653.
In other preferred embodiments the contiguous stretch of amino acids comprises the site of a mutation or functional mutation, including a deletion, addition, swap or truncation of the amino acids in the 12-LO protein sequence.
12-LO proteins are preferably isolated from human or mammalian tissue samples or expressed from human or mammalian genes. The 12-LO polypeptides of the invention can be made using routine expression methods known in the art. The polynucleotide encoding the desired polypeptide is ligated into an expression vector suitable for any convenient host. Both eukaryotic and prokaryotic host systems are used in forming recombinant polypeptides. The polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use. Purification is by any technique known in the art, for example, differential extraction, salt fractionation, chromatography, centrifugation, and the like. See, for example, Methods in Enzymology for a variety of methods for purifying proteins.
In addition, shorter protein fragments are produced by chemical synthesis. Alternatively the proteins of the invention are extracted from cells or tissues of humans or non-human animals. Methods for purifying proteins are known in the art, and include the use of detergents or chaotropic agents to disrupt particles followed by differential extraction and separation of the polypeptides by ion exchange chromatography, affinity chromatography, sedimentation according to density, and gel electrophoresis.
Any 12-LO cDNA, including SEQ ID No. 652, is used to express 12-LO proteins and polypeptides. The nucleic acid encoding the 12-LO protein or polypeptide to be expressed is operably linked to a promoter in an expression vector using conventional cloning technology. The 12-LO insert in the expression vector may comprise the full coding sequence for the 12-LO protein or a portion thereof.
The expression vector is any of the mammalian, yeast, insect or bacterial expression systems known in the art. Commercially available vectors and expression systems are available from a variety of suppliers including Genetics Institute (Cambridge, Mass.), Stratagene (La Jolla, Calif.), Promega (Madison, Wis.), and Invitrogen (San Diego, Calif.). If desired, to enhance expression and facilitate proper protein folding, the codon context and codon pairing of the sequence is optimized for the particular expression organism in which the expression vector is introduced, as explained by Hatfield, et al., U.S. Pat. No. 5,082,767, the disclosure of which is incorporated herein by reference in its entirety.
In one embodiment, the entire coding sequence of the 12-LO cDNA through the poly A signal of the cDNA is operably linked to a promoter in the expression vector. Alternatively, if the nucleic acid encoding a portion of the 12-LO protein lacks a methionine to serve as the initiation site, an initiating methionine can be introduced next to the first codon of the nucleic acid using conventional techniques. Similarly, if the insert from the 12-LO cDNA lacks a poly A signal, this sequence can be added to the construct by, for example, splicing out the Poly A signal from pSG5 (Stratagene) using BglI and SalI restriction endonuclease enzymes and incorporating it into the mammalian expression vector pXT1 (Stratagene). pXT1 contains the LTRs and a portion of the gag gene from Moloney Murine Leukemia Virus. The position of the LTRs in the construct allow efficient stable transfection. The vector includes the Herpes Simplex Thymidine Kinase promoter and the selectable neomycin gene. The nucleic acid encoding the 12-LO protein or a portion thereof is obtained by PCR from a bacterial vector containing the 12-LO cDNA of SEQ ID No. 652 using oligonucleotide primers complementary to the 12-LO cDNA or portion thereof and containing restriction endonuclease sequences for Pst I incorporated into the 5′ primer and BglII at the 5′ end of the corresponding cDNA 3′ primer, taking care to ensure that the sequence encoding the 12-LO protein or a portion thereof is positioned properly with respect to the poly A signal. The purified fragment obtained from the resulting PCR reaction is digested with PstI, blunt ended with an exonuclease, digested with Bgl II, purified and ligated to pXT1, now containing a poly A signal and digested with BglII.
The ligated product is transfected into mouse NIH 3T3 cells using Lipofectin (Life Technologies, Inc., Grand Island, N.Y.) under conditions outlined in the product specification. Positive transfectants are selected after growing the transfected cells in 600 ug/ml G418 (Sigma, St. Louis, Mo.).
Alternatively, the nucleic acids encoding the 12-LO protein or a portion thereof is cloned into pED6dpc2 (Genetics Institute, Cambridge, Mass.). The resulting pED6dpc2 constructs is transfected into a suitable host cell, such as COS I cells. Methotrexate resistant cells are selected and expanded.
The above procedures may also be used to express a mutant 12-LO protein responsible for a detectable phenotype or a portion thereof.
The expressed proteins are purified using conventional purification techniques such as ammonium sulfate precipitation or chromatographic separation based on size or charge. The protein encoded by the nucleic acid insert may also be purified using standard immunochromatography techniques. In such procedures, a solution containing the expressed 12-LO protein or portion thereof, such as a cell extract, is applied to a column having antibodies against the 12-LO protein or portion thereof is attached to the chromatography matrix. The expressed protein is allowed to bind the immunochromatography column. Thereafter, the column is washed to remove non-specifically bound proteins. The specifically bound expressed protein is then released from the column and recovered using standard techniques.
To confirm expression of the 12-LO protein or a portion thereof, the proteins expressed from host cells containing an expression vector containing an insert encoding the 12-LO protein or a portion thereof can be compared to the proteins expressed in host cells containing the expression vector without an insert. The presence of a band in samples from cells containing the expression vector with an insert which is absent in samples from cells containing the expression vector without an insert indicates that the 12-LO protein or a portion thereof is being expressed. Generally, the band will have the mobility expected for the 12-LO protein or portion thereof. However, the band may have a mobility different than that expected as a result of modifications such as glycosylation, ubiquitination, or enzymatic cleavage.
Antibodies capable of specifically recognizing the expressed 12-LO protein or a portion thereof, are described below.
If antibody production is not possible, the nucleic acids encoding the 12-LO protein or a portion thereof is incorporated into expression vectors designed for use in purification schemes employing chimeric polypeptides. In such strategies the nucleic acid encoding the 12-LO protein or a portion thereof is inserted in frame with the gene encoding the other half of the chimera. The other half of the chimera is β-globin or a nickel binding polypeptide encoding sequence. A chromatography matrix having antibody to β-globin or nickel attached thereto is then used to purify the chimeric protein. Protease cleavage sites is engineered between the β-globin gene or the nickel binding polypeptide and the 12-LO protein or portion thereof. Thus, the two polypeptides of the chimera are separated from one another by protease digestion.
One useful expression vector for generating β-globin chimerics is pSG5 (Stratagene), which encodes rabbit β-globin. Intron II of the rabbit β-globin gene facilitates splicing of the expressed transcript, and the polyadenylation signal incorporated into the construct increases the level of expression. These techniques are well known to those skilled in the art of molecular biology. Standard methods are published in methods texts such as Davis et al., (Basic Methods in Molecular Biology, L. G. Davis, M. D. Dibner, and J. F. Battey, ed., Elsevier Press, NY, 1986, the disclosure of which is incorporated herein by reference in its entirety) and many of the methods are available from Stratagene, Life Technologies, Inc., or Promega. Polypeptide may additionally be produced from the construct using in vitro translation systems such as the In vitro ExpressTM Translation Kit (Stratagene).
F. Production of Antibodies Against 12-lipoxygenase Polypeptides
Any 12-LO polypeptide or whole protein may be used to generate antibodies capable of specifically binding to expressed 12-LO protein or fragments thereof as described. The antibody compositions of the invention are capable of specifically binding to the 189-His variant of the 12-LO protein or, to the 225-His variant of the 12-LO protein or, to the 243-Cys variant of the 12-LO protein or, to the 261-Arg variant of the 12-LO protein or, to the 322-Asn variant of the 12-LO or, to the 337-Arg variant of the 12-LO protein or to the 574-Lys variant of the 12-LO protein. A preferred embodiment of the invention encompasses isolated or purified antibody compositions capable of selectively binding, or which are capable of binding to an epitope-containing fragment of a polypeptide of the invention, wherein said epitope comprises at least one amino acid position selected from the group consisting of an His residue et amino acid position 189, an His residue at amino acid position 225, a Cys residue at amino acid position 243, an Arg residue at amino acid position 261, an Asn residue at amino acid position 322, an Arg residue at amino acid position 337, a Asn residue at amino acid position 362, an Asn at amino acid position 568 and a Lys residue at amino acid position 574. For an antibody composition to specifically bind to these 12-LO variants it must demonstrate at least a 5%, 10%, 15%, 20%, 25%, 50%, or 100% greater binding affinity for full length 189-His, 225-His, 243-Cys, 261-Arg, 322-Asn, 337-Arg or 574-Lys variants in an ELISA, RIA, or other antibody-based binding assay than to full length 12-LO proteins which have the alternative amino acid specified in SEQ ID No. 653. Affinity of the antibody composition for the epitope can further be determined by preparing competitive binding curves, as described, for example, by Fisher, D., (Manual of Clinical Immunology, 2nd Ed. (Rose and Friedman, Eds.) Amer. Soc. For Microbiol., Washington, D.C., Ch. 42, 1980), the disclosure of which is incorporated herein by reference in its entirety.
Other preferred antibody compositions of the invention are capable of specifically binding to amino acid positions 110-131 of SEQ ID No. 654.
The present invention also contemplates the use of polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 50, or 100 amino acids of a 12-LO polypeptide of SEQ ID No. 653 in the manufacture of antibodies, wherein said contiguous span comprises at least one amino acid position selected from the group consisting of: an His residue et amino acid position 189, an His residue at amino acid position 225, a Cys residue at amino acid position 243, an Arg residue at amino acid position 261, an Asn residue at amino acid position 322, an Arg residue at amino acid position 337, a Asn residue at amino acid position 362, an Asn at amino acid position 568 and a Lys residue at amino acid position 574.
In a preferred embodiment such polypeptides are useful in the manufacture of antibodies to detect the presence and absence of the 189-His, 225-His, 243-Cys, 261-Arg, 322-Asn, 337-Arg, 568-Asn, or 574-Lys variant.
The present invention further encompasses the use of isolated, purified, and recombinant polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No. 654, wherein said contiguous span comprises at least one of amino acid positions 110-131 of SEQ ID No. 654.
In a preferred embodiment such polypeptides are useful in the manufacture of antibodies to detect the presence and absence of amino acid positions 110-131 of SEQ ID No. 654.
Non-human animals or mammals, whether wild-type or transgenic, which express a different species of 12-LO than the one to which antibody binding is desired, and animals which do not express 12-LO (i.e. an 12-LO knock out animal as described in herein) are particularly useful for preparing antibodies. 12-LO knock out animals will recognize all or most of the exposed regions of 12-LO as foreign antigens, and therefore produce antibodies with a wider array of 12-LO epitopes. Moreover, smaller polypeptides with only 10 to 30 amino acids may be useful in obtaining specific binding to the 189-His, 225-His, 243-Cys, 261-Arg, 322-Asn, 337-Arg, 568-Asn, or 574-Lys variants. In addition, the humoral immune system of animals which produce a species of 12-LO that resembles the antigenic sequence will preferentially recognize the differences between the animal's native 12-LO species and the antigen sequence, and produce antibodies to these unique sites in the antigen sequence. Such a technique will be particularly useful in obtaining antibodies that specifically bind to the 189-His, 225-His, 243-Cys, 261-Arg, 322-Asn, 337-Arg, 568-Asn, or 574-Lys variants. The preparation of antibody compositions is further described in Example 6.
Antibody preparations prepared according to the present invention are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample. The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body. The antibodies of the invention may be labeled, either by a radioactive, a fluorescent or an enzymatic label. Consequently, the invention is also directed to a method for detecting specifically the presence of a variant 12-LO polypeptide according to the invention in a biological sample, said method comprising the following steps: a) bringing into contact the biological sample with a polyclonal or monoclonal antibody that specifically binds a variant 12-LO polypeptide or to a peptide fragment or variant thereof; and b) detecting the antigen-antibody complex formed. The invention also concerns a diagnostic kit for detecting in vitro the presence of a variant 12-LO polypeptide according to the present invention in a biological sample, wherein said kit comprises:

    
    
        a) a polyclonal or monoclonal antibody that specifically binds a variant 12-LO polypeptide or to a peptide fragment or variant thereof, optionally labeled;
        b) a reagent allowing the detection of the antigen-antibody complexes formed, said reagent carrying optionally a label, or being able to be recognized itself by a labeled reagent, more particularly in the case when the above-mentioned monoclonal or polyclonal antibody is not labeled by itself.

II. Methods for De Novo Identification of Biallelic Markers

    
    


Large fragments of human DNA, carrying genes of interest involved in arachidonic acid metabolism; were cloned, sequenced and screened for biallelic markers. Biallelic markers within the candidate genes themselves as well as markers located on the same genomic fragment were identified. It will be clear to one of skill in the art that large fragments of human genomic DNA may be obtained from any appropriate source and may be cloned into a number of suitable vectors.
In a preferred embodiment of the invention, BAC (Bacterial Artificial Chromosomes) vectors were used to construct DNA libraries covering the entire human genome. Specific amplification primers were designed for each candidate gene and the BAC library was screened by PCR until there was at least one positive BAC clone per candidate gene. Genomic sequence, screened for biallelic markers, was generated by sequencing ends of BAC subclones. Details of a preferred embodiment are provided in Example 1. As a preferred alternative to sequencing the ends of an adequate number of BAC subclones, high throughput deletion-based sequencing vectors, which allow the generation of a high quality sequence information covering fragments of about 6 kb, may be used. Having sequence fragments longer than 2.5 or 3 kb enhances the chances of identifying biallelic markers therein. Methods of constructing and sequencing a nested set of deletions are disclosed in the related U.S. patent application entitled “High Throughput DNA Sequencing Vector” (Ser. No. 09/058,746).
In another embodiment of the invention, genomic sequences of candidate genes were available in public databases allowing direct screening for biallelic markers.
Any of a variety of methods can be used to screen a genomic fragment for single nucleotide polymorphisms such as differential hybridization with oligonucleotide probes, detection of changes in the mobility measured by gel electrophoresis or direct sequencing of the amplified nucleic acid. A preferred method for identifying biallelic markers involves comparative sequencing of genomic DNA fragments from an appropriate number of unrelated individuals.
In a first embodiment, DNA samples from unrelated individuals are pooled together, following which the genomic DNA of interest is amplified and sequenced. The nucleotide sequences thus obtained are then analyzed to identify significant polymorphisms. One of the major advantages of this method resides in the fact that the pooling of the DNA samples substantially reduces the number of DNA amplification reactions and sequencing reactions, which must be carried out. Moreover, this method is sufficiently sensitive so that a biallelic marker obtained thereby usually demonstrates a sufficient frequency of its less common allele to be useful in conducting association studies. Usually, the frequency of the least common allele of a biallelic marker identified by this method is at least 10%.
In a second embodiment, the DNA samples are not pooled and are therefore amplified and sequenced individually. This method is usually preferred when biallelic markers need to be identified in order to perform association studies within candidate genes. Preferably, highly relevant gene regions such as promoter regions or exon regions may be screened for biallelic markers. A biallelic marker obtained using this method may show a lower degree of informativeness for conducting association studies, e.g. if the frequency of its less frequent allele may be less than about 10%. Such a biallelic marker will however be sufficiently informative to conduct association studies and it will further be appreciated that including less informative biallelic markers in the genetic analysis studies of the present invention, may allow in some cases the direct identification of causal mutations, which may, depending on their penetrance, be rare mutations.
The following is a description of the various parameters of a preferred method used by the inventors for the identification of the biallelic markers of the present invention.
A. Genomic DNA Samples
The genomic DNA samples from which the biallelic markers of the present invention are generated are preferably obtained from unrelated individuals corresponding to a heterogeneous population of known ethnic background. The number of individuals from whom DNA samples are obtained can vary substantially, preferably from about 10 to about 1000, more preferably from about 50 to about 200 individuals. Usually, DNA samples are collected from at least about 100 individuals in order to have sufficient polymorphic diversity in a given population to identify as many markers as possible and to generate statistically significant results.
As for the source of the genomic DNA to be subjected to analysis, any test sample can be foreseen without any particular limitation. These test samples include biological samples, which can be tested by the methods of the present invention described herein, and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supernatants; fixed tissue specimens including tumor and non-tumor tissue and lymph node tissues; bone marrow aspirates and fixed cell specimens. The preferred source of genomic DNA used in the present invention is from peripheral venous blood of each donor. Techniques to prepare genomic DNA from biological samples are well known to the skilled technician. Details of a preferred embodiment are provided in Example 1. A person skilled in the art can choose to amplify pooled or unpooled DNA samples.
B. DNA Amplification
The identification of biallelic markers in a sample of genomic DNA may be facilitated through the use of DNA amplification methods. DNA samples can be pooled or unpooled for the amplification step. DNA amplification techniques are well known to those skilled in the art. Various methods to amplify DNA fragments carrying biallelic markers are further described hereinafter in III.B. The PCR technology is the preferred amplification technique used to identify new biallelic markers.
In a first embodiment, biallelic markers are identified using genomic sequence information generated by the inventors. Genomic DNA fragments, such as the inserts of the BAC clones described above, are sequenced and used to design primers for the amplification of 500 bp fragments. These 500 bp fragments are amplified from genomic DNA and are scanned for biallelic markers. Primers may be designed using the OSP software (Hillier L. and Green P., Methods Appl. 1: 124-8, 1991). All primers may contain, upstream of the specific target bases, a common oligonucleotide tail that serves as a sequencing primer. Those skilled in the art are familiar with primer extensions, which can be used for these purposes.
In another embodiment of the invention, genomic sequences of candidate genes are available in public databases allowing direct screening for biallelic markers. Preferred primers, useful for the amplification of genomic sequences encoding the candidate genes, focus on promoters, exons and splice sites of the genes. A biallelic marker present in these functional regions of the gene has a higher probability to be a causal mutation.
Preferred primers include those disclosed in Table 13.
C. Sequencing of Amplified Genomic DNA and Identification of Single Nucleotide Polymorphisms
The amplification products generated as described above, are then sequenced using any method known and available to the skilled technician. Methods for sequencing DNA using either the dideoxy-mediated method (Sanger method) or the Maxam-Gilbert method are widely known to those of ordinary skill in the art. Such methods are for example disclosed in Maniatis et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, 2nd Edition, 1989). Alternative approaches include hybridization to high-density DNA probe arrays as described in Chee et al. (Science 274:610, 1996).
Preferably, the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol. The products of the sequencing reactions are run on sequencing gels and the sequences are determined using gel image analysis. The polymorphism search is based on the presence of superimposed peaks in the electrophoresis pattern resulting from different bases occurring at the same position. Because each dideoxy terminator is labeled with a different fluorescent molecule, the two peaks corresponding to a biallelic site present distinct colors corresponding to two different nucleotides at the same position on the sequence. However, the presence of two peaks can be an artifact due to background noise. To exclude such an artifact, the two DNA strands are sequenced and a comparison between the peaks is carried out. In order to be registered as a polymorphic sequence, the polymorphism has to be detected on both strands.
The above procedure permits those amplification products, which contain biallelic markers to be identified. The detection limit for the frequency of biallelic polymorphisms detected by sequencing pools of 100 individuals is approximately 0.1 for the minor allele, as verified by sequencing pools of known allelic frequencies. However, more than 90% of the biallelic polymorphisms detected by the pooling method have a frequency for the minor allele higher than 0.25. Therefore, the biallelic markers selected by this method have a frequency of at least 0.1 for the minor allele and less than 0.9 for the major allele. Preferably at least 0.2 for the minor allele and less than 0.8 for the major allele, more preferably at least 0.3 for the minor allele and less than 0.7 for the major allele, thus a heterozygosity rate higher than 0.18, preferably higher than 0.32, more preferably higher than 0.42.
In another embodiment, biallelic markers are detected by sequencing individual DNA samples, the frequency of the minor allele of such a biallelic marker may be less than 0.1.
The markers carried by the same fragment of genomic DNA, such as the insert in a BAC clone, need not necessarily be ordered with respect to one another within the genomic fragment to conduct association studies. However, in some embodiments of the present invention, the order of biallelic markers carried by the same fragment of genomic DNA are determined.
D. Validation of the Biallelic Markers of the Present Invention
The polymorphisms are evaluated for their usefulness as genetic markers by validating that both alleles are present in a population. Validation of the biallelic markers is accomplished by genotyping a group of individuals by a method of the invention and demonstrating that both alleles are present. Microsequencing is a preferred method of genotyping alleles. The validation by genotyping step may be performed on individual samples derived from each individual in the group or by genotyping a pooled sample derived from more than one individual. The group can be as small as one individual if that individual is heterozygous for the allele in question. Preferably the group contains at least three individuals, more preferably the group contains five or six individuals, so that a single validation test will be more likely to result in the validation of more of the biallelic markers that are being tested. It should be noted, however, that when the validation test is performed on a small group it may result in a false negative result if as a result of sampling error none of the individuals tested carries one of the two alleles. Thus, the validation process is less useful in demonstrating that a particular initial result is an artifact, than it is at demonstrating that there is a bona fide biallelic marker at a particular position in a sequence. For an indication of whether a particular biallelic marker has been validated see Table 7(A-B). All of the genotyping, haplotyping, association, and interaction study methods of the invention may optionally be performed solely with validated biallelic markers.
E. Evaluation of the Frequency of the Biallelic Markers of the Present Invention
The validated biallelic markers are further evaluated for their usefulness as genetic markers by determining the frequency of the least common allele at the biallelic marker site. The determination of the least common allele is accomplished by genotyping a group of individuals by a method of the invention and demonstrating that both alleles are present. This determination of frequency by genotyping step may be performed on individual samples derived from each individual in the group or by genotyping a pooled sample derived from more than one individual. The group must be large enough to be representative of the population as a whole. Preferably the group contains at least 20 individuals, more preferably the group contains at least 50 individuals, most preferably the group contains at least 100 individuals. Of course the larger the group the greater the accuracy of the frequency determination because of reduced sampling error. For an indication of the frequency for the less common allele of a particular biallelic marker of the invention see Table 7(A-B). A biallelic marker wherein the frequency of the less common allele is 30% or more is termed a “high quality biallelic marker.” All of the genotyping, haplotyping, association, and interaction study methods of the invention may optionally be performed solely with high quality biallelic markers.
III. Methods of Genotyping an Individual for Biallelic Markers
Methods are provided to genotype a biological sample for one or more biallelic markers of the present invention, all of which may be performed in vitro. Such methods of genotyping comprise determining the identity of a nucleotide at an eicosanoid-related biallelic marker by any method known in the art. These methods find use in genotyping case-control populations in association studies as well as individuals in the context of detection of alleles of biallelic markers which, are known to be associated with a given trait, in which case both copies of the biallelic marker present in individual's genome are determined so that an individual may be classified as homozygous or heterozygous for a particular allele.
These genotyping methods can be performed nucleic acid samples derived from a single individual or pooled DNA samples.
Genotyping can be performed using similar methods as those described above for the identification of the biallelic markers, or using other genotyping methods such as those further described below. In preferred embodiments, the comparison of sequences of amplified genomic fragments from different individuals is used to identify new biallelic markers whereas microsequencing is used for genotyping known biallelic markers in diagnostic and association study applications.
A. Source of DNA for Genotyping
Any source of nucleic acids, in purified or non-purified form, can be utilized as the starting nucleic acid, provided it contains or is suspected of containing the specific nucleic acid sequence desired. DNA or RNA may be extracted from cells, tissues, body fluids and the like as described above in II.A. While nucleic acids for use in the genotyping methods of the invention can be derived from any mammalian source, the test subjects and individuals from which nucleic acid samples are taken are generally understood to be human.
B. Amplification of DNA Fragments Comprising Biallelic Markers
Methods and polynucleotides are provided to amplify a segment of nucleotides comprising one or more biallelic marker of the present invention. It will be appreciated that amplification of DNA fragments comprising biallelic markers may be used in various methods and for various purposes and is not restricted to genotyping. Nevertheless, many genotyping methods, although not all, require the previous amplification of the DNA region carrying the biallelic marker of interest. Such methods specifically increase the concentration or total number of sequences that span the biallelic marker or include that site and sequences located either distal or proximal to it. Diagnostic assays may also rely on amplification of DNA segments carrying a biallelic marker of the present invention.
Amplification of DNA may be achieved by any method known in the art. The established PCR (polymerase chain reaction) method or by developments thereof or alternatives. Amplification methods which can be utilized herein include but are not limited to Ligase Chain Reaction (LCR) as described in EP A 320 308 and EP A 439 182, Gap LCR (Wolcott, M. J., Clin. Mcrobiol. Rev. 5:370-386), the so-called “NASBA” or “3SR” technique described in Guatelli J. C. et al. (Proc. Natl. Acad. Sci. USA 87:1874-1878, 1990) and in Compton J. (Nature 350:91-92, 1991), Q-beta amplification as described in European Patent Application no 4544610, strand displacement amplification as described in Walker et al. (Clin. Chem. 42:9-13, 1996) and EP A 684 315 and, target mediated amplification as described in PCT Publication WO 9322461.
LCR and Gap LCR are exponential amplification techniques, both depend on DNA ligase to join adjacent primers annealed to a DNA molecule. In Ligase Chain Reaction (LCR), probe pairs are used which include two primary (first and second) and two secondary (third and fourth) probes, all of which are employed in molar excess to target. The first probe hybridizes to a first segment of the target strand and the second probe hybridizes to a second segment of the target strand, the first and second segments being contiguous so that the primary probes abut one another in 5′ phosphate-3′ hydroxyl relationship, and so that a ligase can covalently fuse or ligate the two probes into a fused product. In addition, a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a portion of the second probe in a similar abutting fashion. Of course, if the target is initially double stranded, the secondary probes also will hybridize to the target complement in the first instance. Once the ligated strand of primary probes is separated from the target strand, it will hybridize with the third and fourth probes which can be ligated to form a complementary, secondary ligated product. It is important to realize that the ligated products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. A method for multiplex LCR has also been described (WO 9320227). Gap LCR (GLCR) is a version of LCR where the probes are not adjacent but are separated by 2 to 3 bases.
For amplification of mRNAs, it is within the scope of the present invention to reverse transcribe mRNA into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Pat. No. 5,322,770 or, to use Asymmetric Gap LCR (RT-AGLCR) as described by Marshall R. L. et al. (PCR Methods and Applications 4:80-84, 1994). AGLCR is a modification of GLCR that allows the amplification of RNA.
Some of these amplification methods are particularly suited for the detection of single nucleotide polymorphisms and allow the simultaneous amplification of a target sequence and the identification of the polymorphic nucleotide as it is further described in IIIC.
The PCR technology is the preferred amplification technique used in the present invention. A variety of PCR techniques are familiar to those skilled in the art. For a review of PCR technology, see Molecular Cloning to Genetic Engineering White, B. A. Ed. in Methods in Molecular Biology 67: Humana Press, Totowa (1997) and the publication entitled “PCR Methods and Applications” (1991, Cold Spring Harbor Laboratory Press). In each of these PCR procedures, PCR primers on either side of the nucleic acid sequences to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase. The nucleic acid in the sample is denatured and the PCR primers are specifically hybridized to complementary nucleic acid sequences in the sample. The hybridized primers are extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites. PCR has further been described in several patents including U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188.
The identification of biallelic markers as described above allows the design of appropriate oligonucleotides, which can be used as primers to amplify DNA fragments comprising the biallelic markers of the present invention. Amplification can be performed using the primers initially used to discover new biallelic markers which are described herein or any set of primers allowing the amplification of a DNA fragment comprising a biallelic marker of the present invention. Primers can be prepared by any suitable method. As for example, direct chemical synthesis by a method such as the phosphodiester method of Narang S. A. et al. (Methods Enzymol. 68:90-98, 1979), the phosphodiester method of Brown E. L. et al. (Methods Enzymol. 68:109-151, 1979), the diethylphosphoramidite method of Beaucage et al. (Tetrahedron Lett. 22:1859-1862, 1981) and the solid support method described in EP 0 707 592.
In some embodiments the present invention provides primers for amplifying a DNA fragment containing one or more biallelic markers of the present invention. Preferred amplification primers are listed in Table 13. It will be appreciated that the primers listed are merely exemplary and that any other set of primers which produce amplification products containing one or more biallelic markers of the present invention.
The primers are selected to be substantially complementary to the different strands of each specific sequence to be amplified. The length of the primers of the present invention can range from 8 to 100 nucleotides, preferably from 8 to 50, 8 to 30 or more preferably 8 to 25 nucleotides. Shorter primers tend to lack specificity for a target nucleic acid sequence and generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Longer primers are expensive to produce and can sometimes self-hybridize to form hairpin structures. The formation of stable hybrids depends on the melting temperature (Tm) of the DNA. The Tm depends on the length of the primer, the ionic strength of the solution and the G+C content. The higher the G+C content of the primer, the higher is the melting temperature because G:C pairs are held by three H bonds whereas A:T pairs have only two. The G+C content of the amplification primers of the present invention preferably ranges between 10 and 75%, more preferably between 35 and 60%, and most preferably between 40 and 55%. The appropriate length for primers under a particular set of assay conditions may be empirically determined by one of skill in the art.
The spacing of the primers determines the length of the segment to be amplified. In the context of the present invention amplified segments carrying biallelic markers can range in size from at least about 25 bp to 35 kbp. Amplification fragments from 25-3000 bp are typical, fragments from 50-1000 bp are preferred and fragments from 100-600 bp are highly preferred. It will be appreciated that amplification primers for the biallelic markers may be any sequence which allow the specific amplification of any DNA fragment carrying the markers. Amplification primers may be labeled or immobilized on a solid support as described in I “Biallelic Markers and Polynucleotides Comprising Biallelic Markers.”
C. Methods of Genotyping DNA Samples for Biallelic Markers
Any method known in the art can be used to identify the nucleotide present at a biallelic marker site. Since the biallelic marker allele to be detected has been identified and specified in the present invention, detection will prove simple for one of ordinary skill in the art by employing any of a number of techniques. Many genotyping methods require the previous amplification of the DNA region carrying the biallelic marker of interest. While the amplification of target or signal is often preferred at present, ultrasensitive detection methods which do not require amplification are also encompassed by the present genotyping methods. Methods well-known to those skilled in the art that can be used to detect biallelic polymorphisms include methods such as, conventional dot blot analyzes, single strand conformational polymorphism analysis (SSCP) described by Orita et al. (Proc. Natl. Acad. Sci. U.S.A 86:27776-2770, 1989), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage detection, and other conventional techniques as described in Sheffield, V. C. et al. (Proc. Natl. Acad. Sci. USA 49:699-706, 1991), White et al. (Genomics 12:301-306, 1992), Grompe, M. et al. (Proc. Natl. Acad. Sci. USA 86:5855-5892, 1989) and Grompe, M. (Nature Genetics 5:111-117, 1993). Another method for determining the identity of the nucleotide present at a particular polymorphic site employs a specialized exonuclease-resistant nucleotide derivative as described in U.S. Pat. No. 4,656,127.
Preferred methods involve directly determining the identity of the nucleotide present at a biallelic marker site by sequencing assay, enzyme-based mismatch detection assay, or hybridization assay. The following is a description of some preferred methods. A highly preferred method is the microsequencing technique. The term “sequencing assay” is used herein to refer to polymerase extension of duplex primer/template complexes and includes both traditional sequencing and microsequencing.
1. Sequencing Assays.
The nucleotide present at a polymorphic site can be determined by sequencing methods. In a preferred embodiment, DNA samples are subjected to PCR amplification before sequencing as described above. DNA sequencing methods are described in IIC.
Preferably, the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol. Sequence analysis allows the identification of the base present at the biallelic marker site.
2. Microsequencing Assays
In microsequencing methods, a nucleotide at the polymorphic site that is unique to one of the alleles in a target DNA is detected by a single nucleotide primer extension reaction. This method involves appropriate microsequencing primers which, hybridize just upstream of a polymorphic base of interest in the target nucleic acid. A polymerase is used to specifically extend the 3′ end of the primer with one single ddNTP (chain terminator) complementary to the selected nucleotide at the polymorphic site. Next the identity of the incorporated nucleotide is determined in any suitable way.
Typically, microsequencing reactions are carried out using fluorescent ddNTPs and the extended microsequencing primers are analyzed by electrophoresis on ABI 377 sequencing machines to determine the identity of the incorporated nucleotide as described in EP 412 883. Alternatively capillary electrophoresis can be used in order to process a higher number of assays simultaneously. An example of a typical microsequencing procedure that can be used in the context of the present invention is provided in Example 2.
Different approaches can be used to detect the nucleotide added to the microsequencing primer. A homogeneous phase detection method based on fluorescence resonance energy transfer has been described by Chen and Kwok (Nucleic Acids Research 25:347-353 1997) and Chen et al. (Proc. Natl. Acad. Sci. USA 94/20 10756-10761,1997). In this method amplified genomic DNA fragments containing polymorphic sites are incubated with a 5′-fluorescein-labeled primer in the presence of allelic dye-labeled dideoxyribonucleoside triphosphates and a modified Taq polymerase. The dye-labeled primer is extended one base by the dye-terminator specific for the allele present on the template. At the end of the genotyping reaction, the fluorescence intensities of the two dyes in the reaction mixture are analyzed directly without separation or purification. All these steps can be performed in the same tube and the fluorescence changes can be monitored in real time. Alternatively, the extended primer may be analyzed by MALDI-TOF Mass Spectrometry. The base at the polymorphic site is identified by the mass added onto the microsequencing primer (see Haff L. A. and Smimov I. P., Genome Research, 7:378-388, 1997).
Microsequencing may be achieved by the established microsequencing method or by developments or derivatives thereof. Alternative methods include several solid-phase microsequencing techniques. The basic microsequencing protocol is the same as described previously, except that the method is conducted as a heterogenous phase assay, in which the primer or the target molecule is immobilized or captured onto a solid support. To simplify the primer separation and the terminal nucleotide addition analysis, oligonucleotides are attached to solid supports or are modified in such ways that permit affinity separation as well as polymerase extension. The 5′ ends and internal nucleotides of synthetic oligonucleotides can be modified in a number of different ways to permit different affinity separation approaches, e.g., biotinylation. If a single affinity group is used on the oligonucleotides, the oligonucleotides can be separated from the incorporated terminator reagent. This eliminates the need of physical or size separation. More than one oligonucleotide can be separated from the terminator reagent and analyzed simultaneously if more than one affinity group is used. This permits the analysis of several nucleic acid species or more nucleic acid sequence information per extension reaction. The affinity group need not be on the priming oligonucleotide but could alternatively be present on the template. For example, immobilization can be carried out via an interaction between biotinylated DNA and streptavidin-coated microtitration wells or avidin-coated polystyrene particles. In the same manner oligonucleotides or templates may be attached to a solid support in a high-density format. In such solid phase microsequencing reactions, incorporated ddNTPs can be radiolabeled (Syvänen, Clinica Chimica Acta 226:225-236, 1994) or linked to fluorescein (Livak and Hainer, Human Mutation 3:379-385,1994). The detection of radiolabeled ddNTPs can be achieved through scintillation-based techniques. The detection of fluorescein-linked ddNTPs can be based on the binding of antifluorescein antibody conjugated with alkaline phosphatase, followed by incubation with a chromogenic substrate (such as p-nitrophenyl phosphate). Other possible reporter-detection pairs include: ddNTP linked to dinitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate (Harju et al., Clin. Chem. 39/11 2282-2287, 1993) or biotinylated ddNTP and horseradish peroxidase-conjugated streptavidin with o-phenylenediamine as a substrate (WO 92/15712). As yet another alternative solid-phase microsequencing procedure, Nyren et al. (Analytical Biochemistry 208:171-175, 1993) described a method relying on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA).
Pastinen et al. (Genome research 7:606-614, 1997) describe a method for multiplex detection of single nucleotide polymorphism in which the solid phase minisequencing principle is applied to an oligonucleotide array format. High-density arrays of DNA probes attached to a solid support (DNA chips) are further described in III.C.5.
In one aspect the present invention provides polynucleotides and methods to genotype one or more biallelic markers of the present invention by performing a microsequencing assay. Preferred microsequencing primers include those being featured Table 12. It will be appreciated that the microsequencing primers listed in Table 12 are merely exemplary and that, any primer having a 3′ end immediately adjacent to a polymorphic nucleotide may be used. Similarly, it will be appreciated that microsequencing analysis may be performed for any biallelic marker or any combination of biallelic markers of the present invention. One aspect of the present invention is a solid support which includes one or more microsequencing primers listed in Table 12, or fragments comprising at least 8, at least 12, at least 15, or at least 20 consecutive nucleotides thereof and having a 3′ terminus immediately upstream of the corresponding biallelic marker, for determining the identity of a nucleotide at biallelic marker site.
3. Mismatch Detection Assays Based on Polymerases and Ligases
In one aspect the present invention provides polynucleotides and methods to determine the allele of one or more biallelic markers of the present invention in a biological sample, by mismatch detection assays based on polymerases and/or ligases. These assays are based on the specificity of polymerases and ligases. Polymerization reactions places particularly stringent requirements on correct base pairing of the 3′ end of the amplification primer and the joining of two oligonucleotides hybridized to a target DNA sequence is quite sensitive to mismatches close to the ligation site, especially at the 3′ end. The terms “enzyme based mismatch detection assay” are used herein to refer to any method of determining the allele of a biallelic marker based on the specificity of ligases and polymerases. Preferred methods are described below. Methods, primers and various parameters to amplify DNA fragments comprising biallelic markers of the present invention are further described above in III.B.
Allele Specific Amplification
Discrimination between the two alleles of a biallelic marker can also be achieved by allele specific amplification, a selective strategy, whereby one of the alleles is amplified without amplification of the other allele. This is accomplished by placing a polymorphic base at the 3′ end of one of the amplification primers. Because the extension forms from the 3′ end of the primer, a mismatch at or near this position has an inhibitory effect on amplification. Therefore, under appropriate amplification conditions, these primers only direct amplification on their complementary allele. Designing the appropriate allele-specific primer and the corresponding assay conditions are well with the ordinary skill in the art.
Ligation/Amplification Based Methods
The “Oligonucleotide Ligation Assay” (OLA) uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target molecules. One of the oligonucleotides is biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate that can be captured and detected. OLA is capable of detecting biallelic markers and may be advantageously combined with PCR as described by Nickerson D. A. et al. (Proc. Natl. Acad. Sci. U.S.A. 87:8923-8927, 1990). In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.
Other methods which are particularly suited for the detection of biallelic markers include LCR (ligase chain reaction), Gap LCR (GLCR) which are described above in III.B. As mentioned above LCR uses two pairs of probes to exponentially amplify a specific target. The sequences of each pair of oligonucleotides, is selected to permit the pair to hybridize to abutting sequences of the same strand of the target. Such hybridization forms a substrate for a template-dependant ligase. In accordance with the present invention, LCR can be performed with oligonucleotides having the proximal and distal sequences of the same strand of a biallelic marker site. In one embodiment, either oligonucleotide will be designed to include the biallelic marker site. In such an embodiment, the reaction conditions are selected such that the oligonucleotides can be ligated together only if the target molecule either contains or lacks the specific nucleotide(s) that is complementary to the biallelic marker on the oligonucleotide. In an alternative embodiment, the oligonucleotides will not include the biallelic marker, such that when they hybridize to the target molecule, a “gap” is created as described in WO 90/01069. This gap is then “filled” with complementary dNTPs (as mediated by DNA polymerase), or by an additional pair of oligonucleotides. Thus at the end of each cycle, each single strand has a complement capable of serving as a target during the next cycle and exponential allele-specific amplification of the desired sequence is obtained.
Ligase/Polymerase-mediated Genetic Bit AnalysisTM is another method for determining the identity of a nucleotide at a preselected site in a nucleic acid molecule (WO 95/21271). This method involves the incorporation of a nucleoside triphosphate that is complementary to the nucleotide present at the preselected site onto the terminus of a primer molecule, and their subsequent ligation to a second oligonucleotide. The reaction is monitored by detecting a specific label attached to the reaction's solid phase or by detection in solution.
4. Hybridization Assay Methods
A preferred method of determining the identity of the nucleotide present at a biallelic marker site involves nucleic acid hybridization. The hybridization probes, which can be conveniently used in such reactions, preferably include the probes defined herein. Any hybridization assay may be used including Southern hybridization, Northern hybridization, dot blot hybridization and solid-phase hybridization (see Sambrook et al., Molecular Cloning—A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., 1989).
Hybridization refers to the formation of a duplex structure by two single stranded nucleic acids due to complementary base pairing. Hybridization can occur between exactly complementary nucleic acid strands or between nucleic acid strands that contain minor regions of mismatch. Specific probes can be designed that hybridize to one form of a biallelic marker and not to the other and therefore are able to discriminate between different allelic forms. Allele-specific probes are often used in pairs, one member of a pair showing perfect match to a target sequence containing the original allele and the other showing a perfect match to the target sequence containing the alternative allele. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles. Stringent, sequence specific hybridization conditions, under which a probe will hybridize only to the exactly complementary target sequence are well known in the art (Sambrook et al., Molecular Cloning—A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., 1989). Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. By way of example and not limitation, procedures using conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C., the preferred hybridization temperature, in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20×106 cpm of 32P-labeled probe. Alternatively, the hybridization step can be performed at 65° C. in the presence of SSC buffer, 1×SSC corresponding to 0.15M NaCl and 0.05 M Na citrate. Subsequently, filter washes can be done at 37° C. for 1 h in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1×SSC at 50° C. for 45 min. Alternatively, filter washes can be performed in a solution containing 2×SSC and 0.1% SDS, or 0.5×SSC and 0.1% SDS, or 0.1×SSC and 0.1% SDS at 68° C. for 15 minute intervals. Following the wash steps, the hybridized probes are detectable by autoradiography. By way of example and not limitation, procedures using conditions of intermediate stringency are as follows: Filters containing DNA are prehybridized, and then hybridized at a temperature of 60° C. in the presence of a 5×SSC buffer and labeled probe. Subsequently, filters washes are performed in a solution containing 2×SSC at 50° C. and the hybridized probes are detectable by autoradiography. Other conditions of high and intermediate stringency which may be used are well known in the art and as cited in Sambrook et al. (Molecular Cloning—A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., 1989) and Ausubel et al. (Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y., 1989).
Although such hybridizations can be performed in solution, it is preferred to employ a solid-phase hybridization assay. The target DNA comprising a biallelic marker of the present invention may be amplified prior to the hybridization reaction. The presence of a specific allele in the sample is determined by detecting the presence or the absence of stable hybrid duplexes formed between the probe and the target DNA. The detection of hybrid duplexes can be carried out by a number of methods. Various detection assay formats are well known which utilize detectable labels bound to either the target or the probe to enable detection of the hybrid duplexes. Typically, hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected. Those skilled in the art will recognize that wash steps may be employed to wash away excess target DNA or probe. Standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the primers and probes.
Two recently developed assays allow hybridization-based allele discrimination with no need for separations or washes (see Landegren U. et al., Genome Research, 8:769-776,1998). The TaqMan assay takes advantage of the 5′ nuclease activity of Taq DNA polymerase to digest a DNA probe annealed specifically to the accumulating amplification product. TaqMan probes are labeled with a donor-acceptor dye pair that interacts via fluorescence energy transfer. Cleavage of the TaqMan probe by the advancing polymerase during amplification dissociates the donor dye from the quenching acceptor dye, greatly increasing the donor fluorescence. All reagents necessary to detect two allelic variants can be assembled at the beginning of the reaction and the results are monitored in real time (see Livak et al., Nature Genetics, 9:341-342, 1995). In an alternative homogeneous hybridization-based procedure, molecular beacons are used for allele discriminations. Molecular beacons are hairpin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogeneous solutions. When they bind to their targets they undergo a conformational reorganization that restores the fluorescence of an internally quenched fluorophore (Tyagi et al., Nature Biotechnology, 16:49-53, 1998).
The polynucleotides provided herein can be used in hybridization assays for the detection of biallelic marker alleles in biological samples. These probes are characterized in that they preferably comprise between 8 and 50 nucleotides, and in that they are sufficiently complementary to a sequence comprising a biallelic marker of the present invention to hybridize thereto and preferably sufficiently specific to be able to discriminate the targeted sequence for only one nucleotide variation. The GC content in the probes of the invention usually ranges between 10 and 75%, preferably between 35 and 60%, and more preferably between 40 and 55%. The length of these probes can range from 10, 15, 20, or 30 to at least 100 nucleotides, preferably from 10 to 50, more preferably from 18 to 35 nucleotides. A particularly preferred probe is 25 nucleotides in length. Preferably the biallelic marker is within 4 nucleotides of the center of the polynucleotide probe. In particularly preferred probes the biallelic marker is at the center of said polynucleotide. Shorter probes may lack specificity for a target nucleic acid sequence and generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Longer probes are expensive to produce and can sometimes self-hybridize to form hairpin structures. Methods for the synthesis of oligonucleotide probes have been described above and can be applied to the probes of the present invention.
Preferably the probes of the present invention are labeled or immobilized on a solid support. Labels and solid supports are further described in I. Detection probes are generally nucleic acid sequences or uncharged nucleic acid analogs such as, for example peptide nucleic acids which are disclosed in International Patent Application WO 92/20702, morpholino analogs which are described in U.S. Pat. Nos. 5,185,444; 5,034,506 and 5,142,047. The probe may have to be rendered “non-extendable” in that additional dNTPs cannot be added to the probe. In and of themselves analogs usually are non-extendable and nucleic acid probes can be rendered non-extendable by modifying the 3′ end of the probe such that the hydroxyl group is no longer capable of participating in elongation. For example, the 3′ end of the probe can be functionalized with the capture or detection label to thereby consume or otherwise block the hydroxyl group. Alternatively, the 3′ hydroxyl group simply can be cleaved, replaced or modified, U.S. patent application Ser. No. 07/049,061 filed Apr. 19, 1993 describes modifications, which can be used to render a probe non-extendable.
The probes of the present invention are useful for a number of purposes. They can be used in Southern hybridization to genomic DNA or Northern hybridization to mRNA. The probes can also be used to detect PCR amplification products. By assaying the hybridization to an allele specific probe, one can detect the presence or absence of a biallelic marker allele in a given sample.
High-Throughput parallel hybridizations in array format are specifically encompassed within “hybridization assays” and are described below.
Hybridization to Addressable Arrays of Oligonucleotides
Hybridization assays based on oligonucleotide arrays rely on the differences in hybridization stability of short oligonucleotides to perfectly matched and mismatched target sequence variants. Efficient access to polymorphism information is obtained through a basic structure comprising high-density arrays of oligonucleotide probes attached to a solid support (the chip) at selected positions. Each DNA chip can contain thousands to millions of individual synthetic DNA probes arranged in a grid-like pattern and miniaturized to the size of a dime.
The chip technology has already been applied with success in numerous cases. For example, the screening of mutations has been undertaken in the BRCA1 gene, in S. cerevisiae mutant strains, and in the protease gene of HIV-1 virus (Hacia et al., Nature Genetics, 14(4):441-447, 1996; Shoemaker et al., Nature Genetics, 14(4):450-456, 1996; Kozal et al., Nature Medicine, 2:753-759, 1996). Chips of various formats for use in detecting biallelic polymorphisms can be produced on a customized basis by Affymetrix (GeneChipTM), Hyseq (HyChip and HyGnostics), and Protogene Laboratories.
In general, these methods employ arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual which, target sequences include a polymorphic marker. EP785280 describes a tiling strategy for the detection of single nucleotide polymorphisms. Briefly, arrays may generally be “tiled” for a large number of specific polymorphisms. By “tiling” is generally meant the synthesis of a defined set of oligonucleotide probes which is made up of a sequence complementary to the target sequence of interest, as well as preselected variations of that sequence, e.g., substitution of one or more given positions with one or more members of the basis set of monomers, i.e. nucleotides. Tiling strategies are further described in PCT application No. WO 95/11995. In a particular aspect, arrays are tiled for a number of specific, identified biallelic marker sequences. In particular the array is tiled to include a number of detection blocks, each detection block being specific for a specific biallelic marker or a set of biallelic markers. For example, a detection block may be tiled to include a number of probes, which span the sequence segment that includes a specific polymorphism. To ensure probes that are complementary to each allele, the probes are synthesized in pairs differing at the biallelic marker. In addition to the probes differing at the polymorphic base, monosubstituted probes are also generally tiled within the detection block. These monosubstituted probes have bases at and up to a certain number of bases in either direction from the polymorphism, substituted with the remaining nucleotides (selected from A, T, G, C and U). Typically the probes in a tiled detection block will include substitutions of the sequence positions up to and including those that are 5 bases away from the biallelic marker. The monosubstituted probes provide internal controls for the tiled array, to distinguish actual hybridization from artefactual cross-hybridization. Upon completion of hybridization with the target sequence 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 from the scanned array is then analyzed to identify which allele or alleles of the biallelic marker are present in the sample. Hybridization and scanning may be carried out as described in PCT application No. WO 92/10092 and WO 95/11995 and U.S. Pat. No. 5,424,186.
Thus, in some embodiments, the chips may comprise an array of nucleic acid sequences of fragments of about 15 nucleotides in length. In further embodiments, the chip may comprise an array including at least one of the sequences selected from the group consisting of SEQ ID Nos. 1-70, 72-654 except SEQ ID Nos. 419-424, 490, 531 and 540 and the sequences complementary thereto, or more preferably SEQ ID Nos. 655-724, 726-1304 except SEQ ID Nos. 1073-1078, 1144, 1185, 1194 and the sequences complementary thereto, or a fragment thereof at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides. In some embodiments, the chip may comprise an array of at least 2, 3, 4, 5, 6, 7, 8 or more of these polynucleotides of the invention. Solid supports and polynucleotides of the present invention attached to solid supports are further described in I. Biallelic Markers and Polynucleotides Comprising Biallelic Markers.
5. Integrated Systems
Another technique, which may be used to analyze polymorphisms, includes multicomponent integrated systems, which miniaturize and compartmentalize processes such as PCR and capillary electrophoresis reactions in a single functional device. An example of such technique is disclosed in U.S. Pat. No. 5,589,136, which describes the integration of PCR amplification and capillary electrophoresis in chips.
Integrated systems can be envisaged mainly when micro fluidic systems are used. These systems comprise a pattern of micro channels designed onto a glass, silicon, quartz, or plastic wafer included on a microchip. The movements of the samples are controlled by electric, electroosmotic or hydrostatic forces applied across different areas of the microchip. For genotyping biallelic markers, the microfluidic system may integrate nucleic acid amplification, microsequencing, capillary electrophoresis and a detection method such as laser-induced fluorescence detection.
IV. Methods of Genetic Analysis Using the Biallelic Markers of the Present Invention
Different methods are available for the genetic analysis of complex traits (see Lander and Schork, Science, 265, 2037-2048, 1994). The search for disease-susceptibility genes is conducted using two main methods: the linkage approach in which evidence is sought for cosegregation between a locus and a putative trait locus using family studies, and the association approach in which evidence is sought for a statistically significant association between an allele and a trait or a trait causing allele (Khoury J. et al., Fundamentals of Genetic Epidemiology, Oxford University Press, NY, 1993). In general, the biallelic markers of the present invention find use in any method known in the art to demonstrate a statistically significant correlation between a genotype and a phenotype. The biallelic markers may be used in parametric and non-parametric linkage analysis methods. Preferably, the biallelic markers of the present invention are used to identify genes associated with detectable traits using association studies, an approach which does not require the use of affected families and which permits the identification of genes associated with complex and sporadic traits.
The genetic analysis using the biallelic markers of the present invention may be conducted on any scale. The whole set of biallelic markers of the present invention or any subset of biallelic markers of the present invention may be used. In some embodiments a subset of biallelic markers corresponding to one or several candidate genes of the present invention may be used. In other embodiments a subset of biallelic markers corresponding to candidate genes from a given pathway of arachidonic acid metabolism may be used. Such pathways include the cycloxygenase pathway and the lipoxygenase pathway. Alternatively, a subset of biallelic markers of the present invention localised on a specific chromosome segment may be used. Further, any set of genetic markers including a biallelic marker of the present invention may be used. A set of biallelic polymorphisms that, could be used as genetic markers in combination with the biallelic markers of the present invention, has been described in WO 98/20165. As mentioned above, it should be noted that the biallelic markers of the present invention may be included in any complete or partial genetic map of the human genome. These different uses are specifically contemplated in the present invention and claims.
A. Linkage Analysis
Linkage analysis is based upon establishing a correlation between the transmission of genetic markers and that of a specific trait throughout generations within a family. Thus, the aim of linkage analysis is to detect marker loci that show cosegregation with a trait of interest in pedigrees.
Parametric Methods
When data are available from successive generations there is the opportunity to study the degree of linkage between pairs of loci. Estimates of the recombination fraction enable loci to be ordered and placed onto a genetic map. With loci that are genetic markers, a genetic map can be established, and then the strength of linkage between markers and traits can be calculated and used to indicate the relative positions of markers and genes affecting those traits (Weir, B. S., Genetic data Analysis II: Methods for Discrete population genetic Data, Sinauer Assoc., Inc., Sunderland, Mass., USA, 1996). The classical method for linkage analysis is the logarithm of odds (lod) score method (see Morton N. E., Am. J. Hum. Genet., 7:277-318, 1955; Ott J., Analysis of Human Genetic Linkage, John Hopkins University Press, Baltimore, 1991). Calculation of lod scores requires specification of the mode of inheritance for the disease (parametric method). Generally, the length of the candidate region identified using linkage analysis is between 2 and 20 Mb. Once a candidate region is identified as described above, analysis of recombinant individuals using additional markers allows further delineation of the candidate region. Linkage analysis studies have generally relied on the use of a maximum of 5,000 microsatellite markers, thus limiting the maximum theoretical attainable resolution of linkage analysis to about 600 kb on average.
Linkage analysis has been successfully applied to map simple genetic traits that show clear Mendelian inheritance patterns and which have a high penetrance (i.e., the ratio between the number of affected carriers of allele a and the total number of a carriers in the population). However, parametric linkage analysis suffers from a variety of drawbacks. First, it is limited by its reliance on the choice of a genetic model suitable for each studied trait. Furthermore, as already mentioned, the resolution attainable using linkage analysis is limited, and complementary studies are required to refine the analysis of the typical 2 Mb to 20 Mb regions initially identified through linkage analysis. In addition, parametric linkage analysis approaches have proven difficult when applied to complex genetic traits, such as those due to the combined action of multiple genes and/or environmental factors. It is very difficult to model these factors adequately in a lod score analysis. In such cases, too large an effort and cost are needed to recruit the adequate number of affected families required for applying linkage analysis to these situations, as recently discussed by Risch, N. and Merikangas, K. (Science, 273:1516-1517, 1996).
Non-parametric Methods
The advantage of the so-called non-parametric methods for linkage analysis is that they do not require specification of the mode of inheritance for the disease, they tend to be more useful for the analysis of complex traits. In non-parametric methods, one tries to prove that the inheritance pattern of a chromosomal region is not consistent with random Mendelian segregation by showing that affected relatives inherit identical copies of the region more often than expected by chance. Affected relatives should show excess “allele sharing” even in the presence of incomplete penetrance and polygenic inheritance. In non-parametric linkage analysis the degree of agreement at a marker locus in two individuals can be measured either by the number of alleles identical by state (IBS) or by the number of alleles identical by descent (IBD). Affected sib pair analysis is a well-known special case and is the simplest form of these methods.
The biallelic markers of the present invention may be used in both parametric and non-parametric linkage analysis. Preferably biallelic markers may be used in non-parametric methods which allow the mapping of genes involved in complex traits. The biallelic markers of the present invention may be used in both IBD- and IBS-methods to map genes affecting a complex trait. In such studies, taking advantage of the high density of biallelic markers, several adjacent biallelic marker loci may be pooled to achieve the efficiency attained by multi-allelic markers (Zhao et al., Am. J. Hum. Genet., 63:225-240, 1998).
However, both parametric and non-parametric linkage analysis methods analyse affected relatives, they tend to be of limited value in the genetic analysis of drug responses or in the analysis of side effects to treatments. This type of analysis is impractical in such cases due to the lack of availability of familial cases. In fact, the likelihood of having more than one individual in a family being exposed to the same drug at the same time is extremely low.
B. Population Association Studies
The present invention comprises methods for identifying one or several genes among a set of candidate genes that are associated with a detectable trait using the biallelic markers of the present invention. In one embodiment the present invention comprises methods to detect an association between a biallelic marker allele or a biallelic marker haplotype and a trait. Further, the invention comprises methods to identify a trait causing allele in linkage disequilibrium with any biallelic marker allele of the present invention.
As described above, alternative approaches can be employed to perform association studies: genome-wide association studies, candidate region association studies and candidate gene association studies. In a preferred embodiment, the biallelic markers of the present invention are used to perform candidate gene association studies. The candidate gene analysis clearly provides a short-cut approach to the identification of genes and gene polymorphisms related to a particular trait when some information concerning the biology of the trait is available. Further, the biallelic markers of the present invention may be incorporated in any map of genetic markers of the human genome in order to perform genome-wide association studies. Methods to generate a high-density map of biallelic markers have been described in WIPO Patent application serial number PCT/IB98/01193. The biallelic markers of the present invention may further be incorporated in any map of a specific candidate region of the genome (a specific chromosome or a specific chromosomal segment for example).
As mentioned above, association studies may be conducted within the general population and are not limited to studies performed on related individuals in affected families. Association studies are extremely valuable as they permit the analysis of sporadic or multifactor traits. Moreover, association studies represent a powerful method for fine-scale mapping enabling much finer mapping of trait causing alleles than linkage studies. Studies based on pedigrees often only narrow the location of the trait causing allele. Association studies using the biallelic markers of the present invention can therefore be used to refine the location of a trait causing allele in a candidate region identified by Linkage Analysis methods. Moreover, once a chromosome segment of interest has been identified, the presence of a candidate gene such as a candidate gene of the present invention, in the region of interest can provide a shortcut to the identification of the trait causing allele. Biallelic markers of the present invention can be used to demonstrate that a candidate gene is associated with a trait. Such uses are specifically contemplated in the present invention and claims.
1. Determining the Frequency of a Biallelic Marker Allele or of a Biallelic Marker Haplotype in a Population
Association studies explore the relationships among frequencies for sets of alleles between loci. In addition, the present invention provides methods of determining the frequency in a population of an allele of a 12-LO-related biallelic marker comprising: a) genotyping individuals from said population for said biallelic marker and, b) determining the proportional representation of said biallelic marker in said population. Optionally, said 12-LO-related biallelic marker is selected from the biallelic markers described in Table 2(a-c). The present invention further provides methods of estimating the frequency of a haplotype for a set of biallelic markers in a population, comprising: a) genotyping each individual in said population for at least one 12-LO-related biallelic marker; b) genotyping each individual in said population for a second biallelic marker by determining the identity of the nucleotides at said second biallelic marker for both copies of said second biallelic marker present in the genome; and c) applying a haplotype determination method to the identities of the nucleotides determined in steps a) and b) to obtain an estimate of said frequency. Optionally, said haplotype determination method is selected from asymmetric PCR amplification, double PCR amplification of specific alleles, the Clark method, or an expectation maximization algorithm. Optionally, said 12-LO-related biallelic marker is selected from the biallelic markers described in Table 2(a-c).
Determining the Frequency of an Allele in a Population
Allelic frequencies of the biallelic markers in a population can be determined using one of the methods described above under the heading “Methods for genotyping an individual for biallelic markers,” or any genotyping procedure suitable for this intended purpose. Genotyping pooled samples or individual samples can determine the frequency of a biallelic marker allele in a population. One way to reduce the number of genotypings required is to use pooled samples. A major obstacle in using pooled samples is in terms of accuracy and reproducibility for determining accurate DNA concentrations in setting up the pools. Genotyping individual samples provides higher sensitivity, reproducibility and accuracy and; is the preferred method used in the present invention. Preferably, each individual is genotyped separately and simple gene counting is applied to determine the frequency of an allele of a biallelic marker or of a genotype in a given population.
Determining the Frequency of a Haplotype in a Population
The gametic phase of haplotypes is unknown when diploid individuals are heterozygous at more than one locus. Using genealogical information in families gametic phase can sometimes be inferred (Perlin et al., Am. J. Hum. Genet., 55:777-787, 1994). When no genealogical information is available different strategies may be used. One possibility is that the multiple-site heterozygous diploids can be eliminated from the analysis, keeping only the homozygotes and the single-site heterozygote individuals, but this approach might lead to a possible bias in the sample composition and the underestimation of low-frequency haplotypes. Another possibility is that single chromosomes can be studied independently, for example, by asymmetric PCR amplification (see Newton et al., Nucleic Acids Res., 17:2503-2516, 1989; Wu et al., Proc. Natl. Acad. Sci. USA, 86:2757, 1989) or by isolation of single chromosome by limit dilution followed by PCR amplification (see Ruano et al., Proc. Natl. Acad. Sci. USA, 87:6296-6300, 1990). Further, a sample may be haplotyped for sufficiently close biallelic markers by double PCR amplification of specific alleles (Sarkar, G. and Sommer S. S., Biotechniques, 1991). These approaches are not entirely satisfying either because of their technical complexity, the additional cost they entail, their lack of generalisation at a large scale, or the possible biases they introduce. To overcome these difficulties, an algorithm to infer the phase of PCR-amplified DNA genotypes introduced by Clark A. G. (Mol. Biol. Evol., 7:111-122, 1990) may be used. Briefly, the principle is to start filling a preliminary list of haplotypes present in the sample by examining unambiguous individuals, that is, the complete homozygotes and the single-site heterozygotes. Then other individuals in the same sample are screened for the possible occurrence of previously recognised haplotypes. For each positive identification, the complementary haplotype is added to the list of recognised haplotypes, until the phase information for all individuals is either resolved or identified as unresolved. This method assigns a single haplotype to each multiheterozygous individual, whereas several haplotypes are possible when there are more than one heterozygous site. Alternatively, one can use methods estimating haplotype frequencies in a population without assigning haplotypes to each individual. Preferably, a method based on an expectation-maximization (EM) algorithm (Dempster et al., J. R. Stat. Soc., 39B: 1-38, 1977) leading to maximum-likelihood estimates of haplotype frequencies under the assumption of Hardy-Weinberg proportions (random mating) is used (see Excoffier L. and Slatkin M., Mol. Biol. Evol., 12(5): 921-927, 1995). The EM algorithm is a generalised iterative maximum-likelihood approach to estimation that is useful when data are ambiguous and/or incomplete. The EM algorithm is used to resolve heterozygotes into haplotypes. Haplotype estimations are further described below under the heading “Statistical methods”. Any other method known in the art to determine or to estimate the frequency of a haplotype in a population may also be used.
2. Linkage Disequilibrium Analysis.
Linkage disequilibrium is the non-random association of alleles at two or more loci and represents a powerful tool for mapping genes involved in disease traits (see Ajioka R. S. et al., Am. J. Hum. Genet., 60:1439-1447, 1997). Biallelic markers, because they are densely spaced in the human genome and can be genotyped in more numerous numbers than other types of genetic markers (such as RFLP or VNTR markers), are particularly useful in genetic analysis based on linkage disequilibrium. The biallelic markers of the present invention may be used in any linkage disequilibrium analysis method known in the art.
When a disease mutation is first introduced into a population (by a new mutation or the immigration of a mutation carrier), it necessarily resides on a single chromosome and thus on a single “background” or “ancestral” haplotype of linked markers. Consequently, there is complete disequilibrium between these markers and the disease mutation: one finds the disease mutation only in the presence of a specific set of marker alleles. Through subsequent generations recombinations occur between the disease mutation and these marker polymorphisms, and the disequilibrium gradually dissipates. The pace of this dissipation is a function of the recombination frequency, so the markers closest to the disease gene will manifest higher levels of disequilibrium than those that are further away. When not broken up by recombination, “ancestral” haplotypes and linkage disequilibrium between marker alleles at different loci can be tracked not only through pedigrees but also through populations. Linkage disequilibrium is usually seen as an association between one specific allele at one locus and another specific allele at a second locus.
The pattern or curve of disequilibrium between disease and marker loci is expected to exhibit a maximum that occurs at the disease locus. Consequently, the amount of linkage disequilibrium between a disease allele and closely linked genetic markers may yield valuable information regarding the location of the disease gene. For fine-scale mapping of a disease locus, it is useful to have some knowledge of the patterns of linkage disequilibrium that exist between markers in the studied region. As mentioned above the mapping resolution achieved through the analysis of linkage disequilibrium is much higher than that of linkage studies. The high density of biallelic markers combined with linkage disequilibrium analysis provides powerful tools for fine-scale mapping. Different methods to calculate linkage disequilibrium are described below under the heading “Statistical Methods”.
3. Population-based Case-control Studies of Trait-marker Associations.
As mentioned above, the occurrence of pairs of specific alleles at different loci on the same chromosome is not random and the deviation from random is called linkage disequilibrium. Association studies focus on population frequencies and rely on the phenomenon of linkage disequilibrium. If a specific allele in a given gene is directly involved in causing a particular trait, its frequency will be statistically increased in an affected (affected) population, when compared to the frequency in a trait negative population or in a random control population. As a consequence of the existence of linkage disequilibrium, the frequency of all other alleles present in the haplotype carrying the trait-causing allele will also be increased in affected (affected) individuals compared to trait negative individuals or random controls. Therefore, association between the trait and any allele (specifically a biallelic marker allele) in linkage disequilibrium with the trait-causing allele will suffice to suggest the presence of a trait-related gene in that particular region. Case-control populations can be genotyped for biallelic markers to identify associations that narrowly locate a trait causing allele. As any marker in linkage disequilibrium with one given marker associated with a trait will be associated with the trait. Linkage disequilibrium allows the relative frequencies in case-control populations of a limited number of genetic polymorphisms (specifically biallelic markers) to be analysed as an alternative to screening all possible functional polymorphisms in order to find trait-causing alleles. Association studies compare the frequency of marker alleles in unrelated case-control populations, and represent powerful tools for the dissection of complex traits.
Case-control Populations (Inclusion Criteria)
Population-based association studies do not concern familial inheritance but compare the prevalence of a particular genetic marker, or a set of markers, in case-control populations. They are case-control studies based on comparison of unrelated case (affected or affected) individuals and unrelated control (unaffected or trait negative or random) individuals. Preferably the control group is composed of unaffected or trait negative individuals. Further, the control group is ethnically matched to the case population. Moreover, the control group is preferably matched to the case-population for the main known confusion factor for the trait under study (for example age-matched for an age-dependent trait). Ideally, individuals in the two samples are paired in such a way that they are expected to differ only in their disease status. In the following “affected population”, “case population” and “affected population” are used interchangeably.
An important step in the dissection of complex traits using association studies is the choice of case-control populations (see Lander and Schork, Science, 265, 2037-2048, 1994). A major step in the choice of case-control populations is the clinical definition of a given trait or phenotype. Any genetic trait may be analysed by the association method proposed here by carefully selecting the individuals to be included in the affected and control phenotypic groups. Four criteria are often useful: clinical phenotype, age at onset, family history and severity. The selection procedure for continuous or quantitative traits (such as blood pressure for example) involves selecting individuals at opposite ends of the phenotype distribution of the trait under study, so as to include in these affected and control individuals with non-overlapping phenotypes. Preferably, case-control populations consist of phenotypically homogeneous populations. Affected and control populations consist of phenotypically uniform populations of individuals representing each between 1 and 98%, preferably between 1 and 80%, more preferably between 1 and 50%, and more preferably between 1 and 30%, most preferably between 1 and 20% of the total population under study, and selected among individuals exhibiting non-overlapping phenotypes. The clearer the difference between the two trait phenotypes, the greater the probability of detecting an association with biallelic markers. The selection of those drastically different but relatively uniform phenotypes enables efficient comparisons in association studies and the possible detection of marked differences at the genetic level, provided that the sample sizes of the populations under study are significant enough.
In preferred embodiments, a first group of between 50 and 300 affected individuals, preferably about 100 individuals, are recruited according to their phenotypes. A similar number of trait negative individuals are included in such studies.
In the present invention, typical examples of inclusion criteria include a disease involving arachidonic acid metabolism or the evaluation of the response to a drug acting on arachidonic acid metabolism or side effects to treatment with drugs acting on arachidonic acid metabolism.
Suitable examples of association studies using biallelic markers including the biallelic markers of the present invention, are studies involving the following populations:
a case population suffering from a disease involving arachidonic acid metabolism and a healthy unaffected control population, or
a case population treated with agents acting on arachidonic acid metabolism suffering from side-effects resulting from the treatment and a control population treated with the same agents showing no side-effects, or
a case population treated with agents acting on arachidonic acid metabolism showing a beneficial response and a control population treated with same agents showing no beneficial response.
In a preferred embodiment, eicosanoid related-markers may be used to identify individuals who are prone to hepatoxicity as a result of drug treatment. This includes diagnostic and prognostic assays to identify individuals who are prone to liver toxicity as a result of drug treatment, as well as clinical trials and treatment regimes which utilize these assays. Said drug treatment may include any pharmaceutical compound suspected or known in the art to result in an increased level of hepatoxicity.
In another preferred embodiment, the trait considered was a side effect upon drug treatment; the study involved two populations derived from a clinical study of the anti-asthmatic drug zileuton. The case population was composed of asthmatic individuals treated with Zileuton showing zileuton-associated hepatotoxicity monitored by the serum level of alanine aminotransferase (ALT) and the control population was composed of asthmatic individuals treated with zileuton and having no increased serum level of ALT. Inclusion criteria and association between the biallelic markers of the present invention and zileuton-associated hepatotoxicity are further described below in IV.E. Association of Biallelic Markers of the Invention with Hepatoxicity to Anti-Asthma Drug Zileuton and in Example 5, Association between Side Effects upon Treatment with the Anti-Asthmatic Drug Zileuton (ZyfloTM) and the Biallelic Markers of the 12-lipoxygenase Gene.
Association Analysis
The general strategy to perform association studies using biallelic markers derived from a region carrying a candidate gene is to scan two groups of individuals (case-control populations) in order to measure and statistically compare the allele frequencies of the biallelic markers of the present invention in both groups.
If a statistically significant association with a trait is identified for at least one or more of the analysed biallelic markers, one can assume that: either the associated allele is directly responsible for causing the trait (the associated allele is the trait causing allele), or more likely the associated allele is in linkage disequilibrium with the trait causing allele. The specific characteristics of the associated allele with respect to the candidate gene function usually gives further insight into the relationship between the associated allele and the trait (causal or in linkage disequilibrium). If the evidence indicates that the associated allele within the candidate gene is most probably not the trait causing allele but is in linkage disequilibrium with the real trait causing allele, then the trait causing allele can be found by sequencing the vicinity of the associated marker.
Association studies are usually run in two successive steps. In a first phase, the frequencies of a reduced number of biallelic markers from one or several candidate genes are determined in the affected and control populations. In a second phase of the analysis, the identity of the candidate gene and the position of the genetic loci responsible for the given trait is further refined using a higher density of markers from the relevant region. However, if the candidate gene under study is relatively small in length, as it is the case for many of the candidate genes analysed included in the present invention, a single phase may be sufficient to establish significant associations.
Haplotype Analysis
As described above, when a chromosome carrying a disease allele first appears in a population as a result of either mutation or migration, the mutant allele necessarily resides on a chromosome having a set of linked markers: the ancestral haplotype. This haplotype can be tracked through populations and its statistical association with a given trait can be analysed. Complementing single point (allelic) association studies with multi-point association studies also called haplotype studies increases the statistical power of association studies. Thus, a haplotype association study allows one to define the frequency and the type of the ancestral carrier haplotype. A haplotype analysis is important in that it increases the statistical power of an analysis involving individual markers.
In a first stage of a haplotype frequency analysis, the frequency of the possible haplotypes based on various combinations of the identified biallelic markers of the invention is determined. The haplotype frequency is then compared for distinct populations of affected and control individuals. The number of affected individuals, which should be, subjected to this analysis to obtain statistically significant results usually ranges between 30 and 300, with a preferred number of individuals ranging between 50 and 150. The same considerations apply to the number of unaffected individuals (or random control) used in the study. The results of this first analysis provide haplotype frequencies in case-control populations, for each evaluated haplotype frequency a p-value and an odd ratio are calculated. If a statistically significant association is found the relative risk for an individual carrying the given haplotype of being affected with the trait under study can be approximated.
Interaction Analysis
The biallelic markers of the present invention may also be used to identify patterns of biallelic markers associated with detectable traits resulting from polygenic interactions. The analysis of genetic interaction between alleles at unlinked loci requires individual genotyping using the techniques described herein. The analysis of allelic interaction among a selected set of biallelic markers with appropriate level of statistical significance can be considered as a haplotype analysis. Interaction analysis consists in stratifying the case-control populations with respect to a given haplotype for the first loci and performing a haplotype analysis with the second loci with each subpopulation.
Statistical methods used in association studies are further described below in IV.C “Statistical Methods.”
4. Testing for Linkage in the Presence of Association.
The biallelic markers of the present invention may further be used in TDT (transmission/disequilibrium test). TDT tests for both linkage and association and is not affected by population stratification. TDT requires data for affected individuals and their parents or data from unaffected sibs instead of from parents (see Spielmann S. et al., Am. J. Hum. Genet., 52:506-516, 1993; Schaid D. J. et al., Genet. Epidemiol., 13:423450, 1996, Spielmann S. and Ewens W. J., Am. J. Hum. Genet., 62:450-458, 1998). Such combined tests generally reduce the false-positive errors produced by separate analyses.
C. Statistical Methods
In general, any method known in the art to test whether a trait and a genotype show a statistically significant correlation may be used.
1. Methods in Linkage Analysis.
Statistical methods and computer programs useful for linkage analysis are well-known to those skilled in the art (see Terwilliger J. D. and Ott J., Handbook of Human Genetic Linkage, John Hopkins University Press, London, 1994; Ott J., Analysis of Human Genetic Linkage, John Hopkins University Press, Baltimore, 1991).
2. Methods to Estimate Haplotype Frequencies in a Population.
As described above, when genotypes are scored, it is often not possible to distinguish heterozygotes so that haplotype frequencies cannot be easily inferred. When the gametic phase is not known, haplotype frequencies can be estimated from the multilocus genotypic data. Any method known to person skilled in the art can be used to estimate haplotype frequencies (see Lange K., Mathematical and Statistical Methods for Genetic Analysis, Springer, New York, 1997; Weir, B. S., Genetic data Analysis II: Methods for Discrete population genetic Data, Sinauer Assoc., Inc., Sunderland, Mass., USA, 1996) Preferably, maximum-likelihood haplotype frequencies are computed using an Expectation-Maximization (EM) algorithm (see Dempster et al., J. R. Stat. Soc., 39B: 1-38, 1977; Excoffier L. and Slatkin M., Mol. Biol. Evol., 12(5): 921-927, 1995). This procedure is an iterative process aiming at obtaining maximum-likelihood estimates of haplotype frequencies from multi-locus genotype data when the gametic phase is unknown. Haplotype estimations are usually performed by applying the EM algorithm using for example the EM-HAPLO program (Hawley M. E. et al., Am. J. Phys. Anthropol., 18:104, 1994) or the Arlequin program (Schneider et al., Arlequin: a software for population genetics data analysis, University of Geneva, 1997). The EM algorithm is a generalised iterative maximum likelihood approach to estimation and is briefly described below.
In what follows, phenotypes will refer to multi-locus genotypes with unknown haplotypic phase. Genotypes will refer to mutli-locus genotypes with known haplotypic phase.
Suppose one has a sample of N unrelated individuals typed for K markers. The data observed are the unknown-phase K-locus phenotypes that can be categorized with F different phenotypes. Further, suppose that we have H possible haplotypes (in the case of K biallelic markers, we have for the maximum number of possible haplotypes H=2K).
For phenotype j with cj possible genotypes, we have:




  
    
      
        
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Here, Pj is the probability of the jth phenotype, and P(hk,hl) is the probability of the ith genotype composed of haplotypes hk and hl. Under random mating (i.e. Hardy-Weinberg Equilibrium), P(hkhl) is expressed as:

P(hk,hl)=P(hk)2 for hk=hl, and 

P(hk,hl)=2P(hk)P(hl) for hk≠hl.  Equation 2

The E-M algorithm is composed of the following steps: First, the genotype frequencies are estimated from a set of initial values of haplotype frequencies. These haplotype frequencies are denoted P1(0), P2(0), P3(0), . . . , PH(0). The initial values for the haplotype frequencies may be obtained from a random number generator or in some other way well known in the art. This step is referred to the Expectation step. The next step in the method, called the Maximization step, consists of using the estimates for the genotype frequencies to re-calculate the haplotype frequencies. The first iteration haplotype frequency estimates are denoted by P1(1), P2(1), P3(1), . . . , PH(1). In general, the Expectation step at the sth iteration consists of calculating the probability of placing each phenotype into the different possible genotypes based on the haplotype frequencies of the previous iteration:




  
    
      
        
          
            
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where nj is the number of individuals with the jth phenotype and Pj(hk,hl)(s) is the probability of genotype hk, hl in phenotype j. In the Maximization step, which is equivalent to the gene-counting method (Smith, Ann. Hum. Genet., 21:254-276, 1957), the haplotype frequencies are re-estimated based on the genotype estimates:





  
    
      
        
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Here, δit is an indicator variable which counts the number of occurrences that haplotype t is present in ith genotype; it takes on values 0, 1, and 2.

The E-M iterations cease when the following criterion has been reached. Using Maximum Likelihood Estimation (MLE) theory, one assumes that the phenotypes j are distributed multinomially. At each iteration s, one can compute the likelihood function L. Convergence is achieved when the difference of the log-likehood between two consecutive iterations is less than some small number, preferably 10−7.
3. Methods to Calculate Linkage Disequilibrium Between Markers.
A number of methods can be used to calculate linkage disequilibrium between any two genetic positions, in practice linkage disequilibrium is measured by applying a statistical association test to haplotype data taken from a population.
Linkage disequilibrium between any pair of biallelic markers comprising at least one of the biallelic markers of the present invention (Mi, Mj) having alleles (ai/bi) at marker Mi and alleles (aj/bj) at marker Mj can be calculated for every allele combination (ai,aj; ai,bj; bi,aj and bi,bj), according to the Piazza formula:

Δaiaj=√θ4−√(θ4+θ3)(θ4+θ2), where:

    θ4=−−=frequency of genotypes not having allele ai at Mi and not having allele aj at Mj 
    θ3=−+=frequency of genotypes not having allele ai at Mi and having allele aj at Mj 
    θ2=+−=frequency of genotypes having allele ai at Mi and not having allele aj at Mj 


Linkage disequilibrium (LD) between pairs of biallelic markers (Mi, Mj) can also be calculated for every allele combination (ai,aj; ai,bj; bi,aj and bi,bj), according to the maximum-likelihood estimate (MLE) for delta (the composite genotypic disequilibrium coefficient), as described by Weir (Weir B. S., Genetic Data Analysis, Sinauer Ass. Eds, 1996). The MLE for the composite linkage disequilibrium is:

Daiaj=(2n1+n2+n3+n4/2)/N−2(pr(ai).pr(aj))

Where n1=Σ phenotype (ai/ai, aj/aj), n2=Σ phenotype (ai/ai, aj/bj), n3=Σ phenotype (ai/bi, aj/aj), n4=Σ phenotype (ai/bi, aj/bj) and N is the number of individuals in the sample.

This formula allows linkage disequilibrium between alleles to be estimated when only genotype, and not haplotype, data are available.
Another means of calculating the linkage disequilibrium between markers is as follows. For a couple of biallelic markers, Mi (ai/bi) and Mj (aj/bj), fitting the Hardy-Weinberg equilibrium, one can estimate the four possible haplotype frequencies in a given population according to the approach described above.
The estimation of gametic disequilibrium between ai and aj is simply:

Daiaj=pr(haplotype(ai,aj))−pr(ai).pr(aj).

Where pr(ai) is the probability of allele ai and pr(aj) is the probability of allele aj and where pr(haplotype (ai, aj)) is estimated as in Equation 3 above.
For a couple of biallelic marker only one measure of disequilibrium is necessary to describe the association between Mi and Mj.
Then a normalised value of the above is calculated as follows:

D′aiaj=Daiaj/max(−pr(ai).pr(aj),−pr(bi).pr(bj)) with Daiaj<0

D′aiaj=Daiaj/max(pr(bi).pr(aj),pr(ai).pr(bj)) with Dajij>0

The skilled person will readily appreciate that other LD calculation methods can be used without undue experimentation.
Linkage disequilibrium among a set of biallelic markers having an adequate heterozygosity rate can be determined by genotyping between 50 and 1000 unrelated individuals, preferably between 75 and 200, more preferably around 100.
4. Testing for Association.
Methods for determining the statistical significance of a correlation between a phenotype and a genotype, in this case an allele at a biallelic marker or a haplotype made up of such alleles, may be determined by any statistical test known in the art and with any accepted threshold of statistical significance being required. The application of particular methods and thresholds of significance are well with in the skill of the ordinary practitioner of the art.
Testing for association is performed by determining the frequency of a biallelic marker allele in case and control populations and comparing these frequencies with a statistical test to determine if their is a statistically significant difference in frequency which would indicate a correlation between the trait and the biallelic marker allele under study. Similarly, a haplotype analysis is performed by estimating the frequencies of all possible haplotypes for a given set of biallelic markers in case and control populations, and comparing these frequencies with a statistical test to determine if their is a statistically significant correlation between the haplotype and the phenotype (trait) under study. Any statistical tool useful to test for a statistically significant association between a genotype and a phenotype may be used. Preferably the statistical test employed is a chi-square test with one degree of freedom. A P-value is calculated (the P-value is the probability that a statistic as large or larger than the observed one would occur by chance).
Statistical Significance
In preferred embodiments, significance for diagnosis purposes, either as a positive basis for further diagnostic tests or as a preliminary starting point for early preventive therapy, the p value related to a biallelic marker association is preferably about 1×10-2 or less, more preferably about 1×10-4 or less, for a single biallelic marker analysis and about 1×10-3 or less, still more preferably 1×10-6 or less and most preferably of about 1×10-8 or less, for a haplotype analysis involving several markers. These values are believed to be applicable to any association studies involving single or multiple marker combinations.
The skilled person can use the range of values set forth above as a starting point in order to carry out association studies with biallelic markers of the present invention. In doing so, significant associations between the biallelic markers of the present invention and diseases involving arachidonic acid metabolism can be revealed and used for diagnosis and drug screening purposes.
Phenotypic Permutation
In order to confirm the statistical significance of the first stage haplotype analysis described above, it might be suitable to perform further analyses in which genotyping data from case-control individuals are pooled and randomised with respect to the trait phenotype. Each individual genotyping data is randomly allocated to two groups, which contain the same number of individuals as the case-control populations used to compile the data obtained in the first stage. A second stage haplotype analysis is preferably run on these artificial groups, preferably for the markers included in the haplotype of the first stage analysis showing the highest relative risk coefficient. This experiment is reiterated preferably at least between 100 and 10000 times. The repeated iterations allow the determination of the percentage of obtained haplotypes with a significant p-value level.
Assessment of Statistical Association
To address the problem of false positives similar analysis may be performed with the same case-control populations in random genomic regions. Results in random regions and the candidate region are compared as described in US Provisional Patent Application entitled “Methods, software and apparati for identifying genomic regions harbouring a gene associated with a detectable trait”.
5. Evaluation of Risk Factors.
The association between a risk factor (in genetic epidemiology the risk factor is the presence or the absence of a certain allele or haplotype at marker loci) and a disease is measured by the odds ratio (OR) and by the relative risk (RR). If P(R+) is the probability of developing the disease for individuals with R and P(R−) is the probability for individuals without the risk factor, then the relative risk is simply the ratio of the two probabilities, that is:

RR=P(R+)/P(R−)

In case-control studies, direct measures of the relative risk cannot be obtained because of the sampling design. However, the odds ratio allows a good approximation of the relative risk for low-incidence diseases and can be calculated:




  OR
  =
  
    
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          F
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F+ is the frequency of the exposure to the risk factor in cases and F− is the frequency of the exposure to the risk factor in controls. F+ and F− are calculated using the allelic or haplotype frequencies of the study and further depend on the underlying genetic model (dominant, recessive, additive . . . ).

One can further estimate the attributable risk (AR) which describes the proportion of individuals in a population exhibiting a trait due to a given risk factor. This measure is important in quantitating the role of a specific factor in disease etiology and in terms of the public health impact of a risk factor. The public health relevance of this measure lies in estimating the proportion of cases of disease in the population that could be prevented if the exposure of interest were absent. AR is determined as follows:

AR=PE(RR−1)/(PE(RR−1)+1)

AR is the risk attributable to a biallelic marker allele or a biallelic marker haplotype. PE is the frequency of exposure to an allele or a haplotype within the population at large; and RR is the relative risk which, is approximated with the odds ratio when the trait under study has a relatively low incidence in the general population.
D. Association of Biallelic Markers of the Invention with Asthma
In the context of the present invention, an association between biallelic marker alleles from candidate genes of the present invention and a disease linked to arachidonic acid metabolism was demonstrated. The considered trait was asthma.
Asthma affects over 5% of the population in industrialized countries. It is increasing in prevalence and severity and has a rising mortality (Rang H. P., Ritter J. M. and Dale M. M.; Pharmacology; Churchill Livingstone, N.Y., 1995). Bronchial asthma is a multifactorial syndrome rather than a single disease, defined as airway obstruction characterized by inflammatory changes in the airways and bronchial hyper-responsiveness. In addition to the evidenced impact of environmental factors on the development of asthma, patterns of clustering and segregation in asthmatic families have suggested a genetic component to asthma. However the lack of a defined and specific asthma phenotype and of suitable markers for genetic analysis is proving to be a major hurdle for reliably identifying genes associated with asthma. The identification of genes implicated in asthma would represent a major step towards the identification of new molecular targets for the development of anti-asthma drugs. Moreover there is no straightforward physiological or biological blood test for the asthmatic state. As a result, adequate asthma treatment is often delayed, thereby allowing the inflammation process to better establish itself. Thus, there is a need for the identification of asthma susceptibility genes in order to develop an efficient and reliable asthma diagnostic test.
As mentioned above, products of arachidonic acid metabolism are important inflammatory mediators and have been involved in a number of inflammatory diseases including asthma. More specifically, prostaglandins and leukotrienes are thought to play a major role in the inflammatory process observed in asthma patients.
In order to investigate and identify a genetic origin to asthma a candidate gene scan for asthma was conducted. The rational of this approach was to: 1) select candidate genes potentially involved in the pathological pathway of interest, in this case arachidonic acid metabolism, 2) to identify biallelic markers in those genes and finally 3) to measure the frequency of biallelic marker alleles in order to determine if some alleles are more frequent in asthmatic populations than in non-affected populations. Results were further validated by haplotype studies. Significant associations between biallelic marker alleles from the FLAP and 12-LO genes and asthma were demonstrated in the context of the present invention. Association studies are further described in Examples 3 and 4.
This information is extremely valuable. The knowledge of a potential genetic predisposition, even if this predisposition is not absolute, might contribute in a very significant manner to treatment efficacy of asthma patients and to the development of diagnostic tools.
E. Association of Biallelic Markers of the Invention with Hepatotoxicity to Anti-Asthma Drug Zileuton (ZyfloTM)
In the context of the present invention, an association between the 12-LO gene and side effects related to treatment with the anti-asthmatic drug zileuton was discovered.
As mentioned above, bronchial asthma is a multifactorial syndrome rather than a single disease, defined as airway obstruction characterized by inflammatory changes in the airways and bronchial hyper-responsiveness. Although initially reversible with bronchiodilators, airway obstruction becomes increasingly irreversible if treated poorly. Asthma management therefore relies on early and regular use of drugs that control the disease. As a consequence, there is a strong need for efficient and safe therapeutic opportunities for patients with asthma. There are two main categories of anti-asthmatic drugs—bronchodilators and anti-inflammatory agents. There is now general agreement on the need to implement early anti-inflammatory treatment rather than relying on symptomatic treatment with bronchiodilators alone. The leukotrienes, a family of proinflammatory mediators arising via arachidonic acid metabolism, have been implicated in the inflammatory cascade that occurs in asthmatic airways. Of great relevance to the pathogenesis of asthma is the 5-lipoxygenase, which catalyzes the initial step in the biosynthesis of leukotrienes from arachidonic acid. Given the significant role of the inflammatory process in asthma, pharmacological agents, such as leukotriene antagonists and 5-lipoxygenase inhibitors have been developed.
Zileuton (ZyfloTM) is an active inhibitor of 5-lipoxygenase, the enzyme that catalyzes the formation of leukotrienes from arachidonic acid, indicated for prophylaxis and chronic treatment of asthma. A minority of zileuton-treated patients develop liver function abnormalities. Close monitoring revealed that elevations of liver function tests may occur during treatment with zileuton. The ALT test (serum level of alanine aminotransferase) was used, which is considered the most sensitive indicator of liver injury.
In order to investigate and identify a genetic origin to zileuton-associated hepatotoxicity, a candidate gene scan was conducted. This approach comprised:

    selecting candidate genes potentially involved in the pathological pathway of interest or in the metabolism of zileuton, and
    identifying biallelic markers in those genes, and finally
    conducting association studies to identify biallelic marker alleles or haplotypes associated with elevations of liver function tests upon treatment with zileuton.


An association between elevated ALT levels upon treatment with zileuton and biallelic marker alleles from the 12-LO gene was demonstrated. Further details concerning this association study are provided in Example 5.
F. Identification of Biallelic Markers in Linkage Disequilibrium with the Biallelic Markers of the Invention
Once a first biallelic marker has been identified in a genomic region of interest, the practitioner of ordinary skill in the art, using the teachings of the present invention, can easily identify additional biallelic markers in linkage disequilibrium with this first marker. As mentioned before any marker in linkage disequilibrium with a first marker associated with a trait will be associated with the trait. Therefore, once an association has been demonstrated between a given biallelic marker and a trait, the discovery of additional biallelic markers associated with this trait is of great interest in order to increase the density of biallelic markers in this particular region. The causal gene or mutation will be found in the vicinity of the marker or set of markers showing the highest correlation with the trait.
Identification of additional markers in linkage disequilibrium with a given marker involves: (a) amplifying a genomic fragment comprising a first biallelic marker from a plurality of individuals; (b) identifying of second biallelic markers in the genomic region harboring said first biallelic marker; (c) conducting a linkage disequilibrium analysis between said first biallelic marker and second biallelic markers; and (d) selecting said second biallelic markers as being in linkage disequilibrium with said first marker. Subcombinations comprising steps (b) and (c) are also contemplated.
Methods to identify biallelic markers and to conduct linkage disequilibrium analysis are described herein and can be carried out by the skilled person without undue experimentation. The present invention then also concerns biallelic markers which are in linkage disequilibrium with the specific biallelic markers shown in Table 7(A-B) and which are expected to present similar characteristics in terms of their respective association with a given trait.
G. Identification of Functional Mutations
Once a positive association is confirmed with a biallelic marker of the present invention, the associated candidate gene can be scanned for mutations by comparing the sequences of a selected number of affected individuals and control individuals. In a preferred embodiment, functional regions such as exons and splice sites, promoters and other regulatory regions of the candidate gene are scanned for mutations. Preferably, affected individuals carry the haplotype shown to be associated with the trait and trait negative or control individuals do not carry the haplotype or allele associated with the trait. The mutation detection procedure is essentially similar to that used for biallelic site identification.

    
    
        The method used to detect such mutations generally comprises the following steps: (a) amplification of a region of the candidate gene comprising a biallelic marker or a group of biallelic markers associated with the trait from DNA samples of affected patients and trait negative controls;
        (b) sequencing of the amplified region;
        (c) comparison of DNA sequences from affected trait-positive patients and trait-negative controls; and
        (d) determination of mutations specific to affected trait-positive patients. Subcombinations which comprise steps (b) and (c) are specifically contemplated.
    
    


It is preferred that candidate polymorphisms be then verified by screening a larger population of cases and controls by means of any genotyping procedure such as those described herein, preferably using a microsequencing technique in an individual test format. Polymorphisms are considered as candidate mutations when present in cases and controls at frequencies compatible with the expected association results.
Identification of mutations and low frequency polymorphisms in the 5′ flanking region of the 12-LO gene, in the exons and introns of the 12-LO gene and in the 3′ flanking region of the 12-LO gene is further described in Example 5. Forty-nine low frequency polymorphisms and mutations were identified in the region of the 12-LO gene that was scanned. Low frequency polymorphisms and mutations identified in exons 5, 6, 8, and 13 are associated with amino acid substitutions at the polypeptide level. In each of these amino acid substitutions the original residue is replaced by a non-equivalent amino acid presenting different chemical properties. As a consequence, specificity, activity and function of the 12-LO enzyme are modified. Biallelic marker 10-343-231 is associated with a frame shift in the open reading frame of the 12-LO gene leading to the expression of a variant 12-LO polypeptide comprising only 131 amino acids. This mutant 12-LO enzyme is probably inactive or shows differences in specificity, activity and function. Biallelic marker 10-343-231 is associated with the deletion of a Leu residue in the 12-LO polypeptide.
Candidate polymorphisms and mutations of the 12-LO gene suspected of being responsible for the detectable phenotype, such as hepatoxicity to zileuton or asthma, can be confirmed by screening a larger population of affected and unaffected individuals using any of the genotyping procedures described herein. Preferably the microsequencing technique is used. Such polymorphisms are considered as candidate “trait-causing” mutations when they exhibit a statistically significant correlation with the detectable phenotype.
V. Biallelic Markers of the Invention in Methods of Genetic Diagnostics
The biallelic markers of the present invention can also be used to develop diagnostics tests capable of identifying individuals who express a detectable trait as the result of a specific genotype or individuals whose genotype places them at risk of developing a detectable trait at a subsequent time. The trait analyzed using the present diagnostics may be any detectable trait, including a disease involving arachidonic acid metabolism, a response to an agent acting on arachidonic acid metabolism or side effects to an agent acting on arachidonic acid metabolism.
The diagnostic techniques of the present invention may employ a variety of methodologies to determine whether a test subject has a biallelic marker pattern associated with an increased risk of developing a detectable trait or whether the individual suffers from a detectable trait as a result of a particular mutation, including methods which enable the analysis of individual chromosomes for haplotyping, such as family studies, single sperm DNA analysis or somatic hybrids.
The present invention provides diagnostic methods to determine whether an individual is at risk of developing a disease or suffers from a disease resulting from a mutation or a polymorphism in a candidate gene of the present invention. The present invention also provides methods to determine whether an individual is likely to respond positively to an agent acting on arachidonic acid metabolism or whether an individual is at risk of developing an adverse side effect to an agent acting on arachidonic acid metabolism.
These methods involve obtaining a nucleic acid sample from the individual and, determining, whether the nucleic acid sample contains at least one allele or at least one biallelic marker haplotype, indicative of a risk of developing the trait or indicative that the individual expresses the trait as a result of possessing a particular candidate gene polymorphism or mutation (trait-causing allele).
Preferably, in such diagnostic methods, a nucleic acid sample is obtained from the individual and this sample is genotyped using methods described above in Im. Methods of Genotyping an Individual for Biallelic Markers. The diagnostics may be based on a single biallelic marker or a on group of biallelic markers.
In each of these methods, a nucleic acid sample is obtained from the test subject and the biallelic marker pattern of one or more of the biallelic markers listed in Table 7(A-B) is determined.
In one embodiment, a PCR amplification is conducted on the nucleic acid sample to amplify regions in which polymorphisms associated with a detectable phenotype have been identified. The amplification products are sequenced to determine whether the individual possesses one or more polymorphisms associated with a detectable phenotype. The primers used to generate amplification products may comprise the primers listed in Table 13, or a preferred set of primers includes those described in SEQ ID Nos. 26-70, 72-418, 425-489, 491-530, 532-539, 541-646, and 651-652. Alternatively, the nucleic acid sample is subjected to microsequencing reactions as described above to determine whether the individual possesses one or more polymorphisms associated with a detectable phenotype resulting from a mutation or a polymorphism in a candidate gene. The primers used in the microsequencing reactions may include the primers listed in Table 12, or a preferred set of primers includes those described in SEQ ID Nos. 26-70, 72-418, 425-489, 491-530, 532-539, 541-646, and 651-652. In another embodiment, the nucleic acid sample is contacted with one or more allele specific oligonucleotide probes which, specifically hybridize to one or more candidate gene alleles associated with a detectable phenotype. The probes used in the hybridization assay may include the probes listed in Table 14, or a preferred set of probes includes those described in SEQ ID Nos. 26-70, 72-418, 425-489, 491-530, 532-539, 541-646, and 651-652.
The present invention provides methods of determining whether an individual is at risk of developing asthma, or whether said individual suffers from asthma, comprising: a) genotyping said individual for at least one 12-LO-related biallelic marker; and b) correlating the result of step a) with a risk of developing asthma. In a preferred embodiment, said 12-LO-related biallelic marker is selected from the group consisting of biallelic markers: 12-197-244, 12-208-35, 12-226-167, 12-206-366, 10-346-141, 10-347-111, 10-347-165, 10-347-203, 10-347-220, 10-349-97, 10-349-224, 10-341-116, 12-196-119, 12-214-129, 12-216-421, 12-219-230, and 12-223-207. Preferably, said 12-LO-related biallelic marker is selected from the biallelic markers described in Example 5. The present invention also provides methods of determining whether an individual is at risk of developing hepatoxicity upon treatment with zileuton, comprising: a) genotyping said individual for at least one 12-LO-related biallelic marker; and b) correlating the result of step a) with a risk of developing hepatotoxicity upon treatment with zileuton. In a preferred embodiment, said 12-LO-related biallelic marker is selected from the group consisting of biallelic markers: 12-197-244, 12-208-35, 12-226-167, 12-206-366, 10-346-141, 10-347-111, 10-347-165, 10-347-203, 10-347-220, 10-349-97, 10-349-224, 10-341-116, 12-196-119, 12-214-129, 12-216-421, 12-219-230, and 12-223-207. Preferably, said 12-LO-related biallelic marker is selected from the biallelic markers described in Example 5, Association between Side Effects upon Treatment with the Anti-Asthmatic Drug Zileuton (ZyfloTM) and the Biallelic Markers of the 12-lipoxygenase Gene.
These diagnostic methods are extremely valuable as they can, in certain circumstances, be used to initiate preventive treatments or to allow an individual carrying a significant haplotype to foresee warning signs such as minor symptoms. In diseases in which attacks may be extremely violent and sometimes fatal if not treated on time, such as asthma, the knowledge of a potential predisposition, even if this predisposition is not absolute, might contribute in a very significant manner to treatment efficacy. Similarly, a diagnosed predisposition to a potential side effect could immediately direct the physician toward a treatment for which such side effects have not been observed during clinical trials.
Diagnostics, which analyze and predict response to a drug or side effects to a drug, may be used to determine whether an individual should be treated with a particular drug. For example, if the diagnostic indicates a likelihood that an individual will respond positively to treatment with a particular drug, the drug may be administered to the individual. Conversely, if the diagnostic indicates that an individual is likely to respond negatively to treatment with a particular drug, an alternative course of treatment may be prescribed. A negative response may be defined as either the absence of an efficacious response or the presence of toxic side effects.
Clinical drug trials represent another application for the markers of the present invention. One or more markers indicative of response to an agent acting on arachidonic acid metabolism or to side effects to an agent acting on arachidonic acid metabolism may be identified using the methods described above. Thereafter, potential participants in clinical trials of such an agent may be screened to identify those individuals most likely to respond favorably to the drug and exclude those likely to experience side effects. In that way, the effectiveness of drug treatment may be measured in individuals who respond positively to the drug, without lowering the measurement as a result of the inclusion of individuals who are unlikely to respond positively in the study and without risking undesirable safety problems.
VI. Computer-Related Embodiments
As used herein the term “nucleic acid codes of the invention” encompass the nucleotide sequences comprising, consisting essentially of, or consisting of any one of the following: a) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500 or 1000 nucleotides, to the extent that a polynucleotide of these lengths is consistent with the lengths of the particular Sequence ID, of a sequence selected from the group consisting of the sequences described in Table 8, and the complements thereof, excluding Sequence ID Nos. 1-10, 19, 23-25, and 647-650; b) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500 or 1000 nucleotides, to the extent that a polynucleotide of these lengths is consistent with the lengths of the particular Sequence ID, of a sequence selected from the group consisting of the sequences described in Table 9, and the complements thereof, excluding Sequence ID Nos. 11-18 and 20-21; c) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 500 nucleotides, to the extent that a polynucleotide of these lengths is consistent with the lengths of the particular Sequence ID, of a sequence selected from the group consisting of the sequences described in Table 12, more preferably a set of markers or sequences consisting of those markers or sequences found in SEQ ID Nos. 26-70, 72-418, 425-489, 491-530, 532-539, 541-646, and 651-652, and the complements thereof, wherein said span includes an eicosanoid-related biallelic marker, preferably an eicosanoid-related biallelic marker described in Table 7(A-B), preferably the biallelic markers found in SEQ ID Nos. 26-70, 72-418, 425-489, 491-530, 532-539, 541-646, and 651-652, or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, in said sequence with the alternative allele present at said biallelic marker.
The “nucleic acid codes of the invention” further encompass nucleotide sequences homologous to a contiguous span of at least 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500 or 1000 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of a sequence selected from the group consisting of the sequences described in Tables 8, 9, and 12, and the complements thereof. Homologous sequences refer to a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% homology to these contiguous spans. Homology may be determined using any method described herein, including BLAST2N with the default parameters or with any modified parameters. Homologous sequences also may include RNA sequences in which uridines replace the thymines in the nucleic acid codes of the invention. It will be appreciated that the nucleic acid codes of the invention can be represented in the traditional single character format (See the inside back cover of Stryer, Lubert. Biochemistry, 3rd edition. W. H Freeman & Co., New York.) or in any other format or code which records the identity of the nucleotides in a sequence.
It will be appreciated by those skilled in the art that the nucleic acid codes of the invention, one or more of the polypeptide codes of SEQ ID Nos. 653 and 654 can be stored, recorded, and manipulated on any medium which can be read and accessed by a computer. As used herein, the words “recorded” and “stored” refer to a process for storing information on a computer medium. A skilled artisan can readily adopt any of the presently known methods for recording information on a computer readable medium to generate manufactures comprising one or more of the nucleic acid codes of the invention and one or more of the polypeptide codes of SEQ ID Nos. 653-654. Another aspect of the present invention is a computer readable medium having recorded thereon at least 2, 5, 10, 15, 20, 25, 30, or 50 nucleic acid codes of the invention, and the complements thereof. Another aspect of the present invention is a computer readable medium having recorded thereon at least 2, 5, 10, 15, 20, 25, 30, or 50 polypeptide codes of SEQ ID Nos. 653-654.
Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media. For example, the computer readable media may be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) as well as other types of other media known to those skilled in the art.
Embodiments of the present invention include systems, particularly computer systems which store and manipulate the sequence information described herein. As used herein, “a computer system” refers to the hardware components, software components, and data storage components used to analyze the nucleotide sequences of the nucleic acid codes of the invention, or the amino acid sequences of the polypeptide codes of SEQ ID Nos. 653-654. In one embodiment, the computer system is a Sun Enterprise 1000 server (Sun Microsystems, Palo Alto, Calif.). The computer system preferably includes a processor for processing, accessing and manipulating the sequence data. The processor can be any well-known type of central processing unit, such as the Pentium 1 ml from Intel Corporation, or similar processor from Sun, Motorola, Compaq or International Business Machines. Preferably, the computer system is a general purpose system that comprises the processor and one or more internal data storage components for storing data, and one or more data retrieving devices for retrieving the data stored on the data storage components. A skilled artisan can readily appreciate that any one of the currently available computer systems are suitable. In one particular embodiment, the computer system includes a processor connected to a bus which is connected to a main memory (preferably implemented as RAM) and one or more internal data storage devices, such as a hard drive and/or other computer readable media having data recorded thereon. In some embodiments, the computer system further includes one or more data retrieving device for reading the data stored on the internal data storage devices. The data retrieving device may represent, for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, etc. In some embodiments, the internal data storage device is a removable computer readable medium such as a floppy disk, a compact disk, a magnetic tape, etc. containing control logic and/or data recorded thereon. The computer system may advantageously include or be programmed by appropriate software for reading the control logic and/or the data from the data storage component once inserted in the data retrieving device. The computer system includes a display which is used to display output to a computer user. It should also be noted that the computer system can be linked to other computer systems in a network or wide area network to provide centralized access to the computer system. Software for accessing and processing the nucleotide sequences of the nucleic acid codes of the invention, or the amino acid sequences of the polypeptide codes of SEQ ID Nos. 653-654 (such as search tools, compare tools, and modeling tools etc.) may reside in main memory during execution. In some embodiments, the computer system may further comprise a sequence comparer for comparing the above-described nucleic acid codes of the invention or polypeptide codes of SEQ ID Nos. 653-654 stored on a computer readable medium to reference nucleotide or polypeptide sequences stored on a computer readable medium. A “sequence comparer” refers to one or more programs which are implemented on the computer system to compare a nucleotide or polypeptide sequence with other nucleotide or polypeptide sequences and/or compounds including but not limited to peptides, peptidomimetics, and chemicals stored within the data storage means. For example, the sequence comparer may compare the nucleotide sequences of the nucleic acid codes of the invention, or the amino acid sequences of the polypeptide codes of SEQ ID Nos. 653-654 stored on a computer readable medium to reference sequences stored on a computer readable medium to identify homologies, motifs implicated in biological function, or structural motifs. The various sequence comparer programs identified elsewhere in this patent specification are particularly contemplated for use in this aspect of the invention.
One embodiment is a process for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database. The database of sequences can be a private database stored within the computer system, or a public database such as GENBANK, PIR OR SWISSPROT that is available through the Internet.
The process begins at a start state and then moves to a state wherein the new sequence to be compared is stored to a memory in a computer system. As discussed above, the memory could be any type of memory, including RAM or an internal storage device. The process then moves to a state wherein a database of sequences is opened for analysis and comparison. The process then moves to a state wherein the first sequence stored in the database is read into a memory on the computer. A comparison is then performed at a state to determine if the first sequence is the same as the second sequence. It is important to note that this step is not limited to performing an exact comparison between the new sequence and the first sequence in the database. Well-known methods are known to those of skill in the art for comparing two nucleotide or protein sequences, even if they are not identical. For example, gaps can be introduced into one sequence in order to raise the homology level between the two tested sequences. The parameters that control whether gaps or other features are introduced into a sequence during comparison are normally entered by the user of the computer system. Once a comparison of the two sequences has been performed at the state, a determination is made at a decision state whether the two sequences are the same. Of course, the term “same” is not limited to sequences that are absolutely identical. Sequences that are within the homology parameters entered by the user will be marked as “same” in the process. If a determination is made that the two sequences are the same, the process moves to a state wherein the name of the sequence from the database is displayed to the user. This state notifies the user that the sequence with the displayed name fulfills the homology constraints that were entered. Once the name of the stored sequence is displayed to the user, the process moves to a decision state wherein a determination is made whether more sequences exist in the database. If no more sequences exist in the database, then the process terminates at an end state. However, if more sequences do exist in the database, then the process moves to a state wherein a pointer is moved to the next sequence in the database so that it can be compared to the new sequence. In this manner, the new sequence is aligned and compared with every sequence in the database. It should be noted that if a determination had been made at the decision state that the sequences were not homologous, then the process would move immediately to the decision state in order to determine if any other sequences were available in the database for comparison. Accordingly, one aspect of the present invention is a computer system comprising a processor, a data storage device having stored thereon a nucleic acid code of the invention or a polypeptide code of SEQ ID Nos. 653-654, a data storage device having retrievably stored thereon reference nucleotide sequences or polypeptide sequences to be compared to the nucleic acid code of the invention or polypeptide code of SEQ ID Nos. 653-654 and a sequence comparer for conducting the comparison. The sequence comparer may indicate a homology level between the sequences compared or identify structural motifs in the above described nucleic acid code of the invention and polypeptide codes of SEQ ID Nos. 653-654 or it may identify structural motifs in sequences which are compared to these nucleic acid codes and polypeptide codes. In some embodiments, the data storage device may have stored thereon the sequences of at least 2, 5, 10, 15, 20, 25, 30, or 50 of the nucleic acid codes of the invention or polypeptide codes of SEQ ID Nos. 653-654.
Another aspect of the present invention is a method for determining the level of homology between a nucleic acid code of the invention and a reference nucleotide sequence, comprising the steps of reading the nucleic acid code and the reference nucleotide sequence through the use of a computer program which determines homology levels and determining homology between the nucleic acid code and the reference nucleotide sequence with the computer program. The computer program may be any of a number of computer programs for determining homology levels, including those specifically enumerated herein, including BLAST2N with the default parameters or with any modified parameters. The method may be implemented using the computer systems described above. The method may also be performed by reading 2, 5, 10, 15, 20, 25, 30, or 50 of the above described nucleic acid codes of the invention through use of the computer program and determining homology between the nucleic acid codes and reference nucleotide sequences.
One embodiment is a process in a computer for determining whether two sequences are homologous. The process begins at a start state and then moves to a state wherein a first sequence to be compared is stored to a memory. The second sequence to be compared is then stored to a memory at a state. The process then moves to a state wherein the first character in the first sequence is read and then to a state wherein the first character of the second sequence is read. It should be understood that if the sequence is a nucleotide sequence, then the character would normally be either A, T, C, G or U. If the sequence is a protein sequence, then it should be in the single letter amino acid code so that the first and sequence sequences can be easily compared.
A determination is then made at a decision state whether the two characters are the same. If they are the same, then the process moves to a state wherein the next characters in the first and second sequences are read. A determination is then made whether the next characters are the same. If they are, then the process continues this loop until two characters are not the same. If a determination is made that the next two characters are not the same, the process moves to a decision state to determine whether there are any more characters either sequence to read. If there aren't any more characters to read, then the process moves to a state wherein the level of homology between the first and second sequences is displayed to the user. The level of homology is determined by calculating the proportion of characters between the sequences that were the same out of the total number of sequences in the first sequence. Thus, if every character in a first 100 nucleotide sequence aligned with a every character in a second sequence, the homology level would be 100%. Alternatively, the computer program may be a computer program which compares the nucleotide sequences of the nucleic acid codes of the present invention, to reference nucleotide sequences in order to determine whether the nucleic acid code of the invention differs from a reference nucleic acid sequence at one or more positions. Optionally such a program records the length and identity of inserted, deleted or substituted nucleotides with respect to the sequence of either the reference polynucleotide or the nucleic acid code of the invention. In one embodiment, the computer program may be a program which determines whether the nucleotide sequences of the nucleic acid codes of the invention contain a biallelic marker or single nucleotide polymorphism (SNP) with respect to a reference nucleotide sequence. This single nucleotide polymorphism may comprise a single base substitution, insertion, or deletion, while this biallelic marker may comprise about one to ten consecutive bases substituted, inserted or deleted.
Another aspect of the present invention is a method for determining the level of homology between a polypeptide code of SEQ ID Nos. 653-654 and a reference polypeptide sequence, comprising the steps of reading the polypeptide code of SEQ ID Nos. 653-654 and the reference polypeptide sequence through use of a computer program which determines homology levels and determining homology between the polypeptide code and the reference polypeptide sequence using the computer program.
Accordingly, another aspect of the present invention is a method for determining whether a nucleic acid code of the invention differs at one or more nucleotides from a reference nucleotide sequence comprising the steps of reading the nucleic acid code and the reference nucleotide sequence through use of a computer program which identifies differences between nucleic acid sequences and identifying differences between the nucleic acid code and the reference nucleotide sequence with the computer program. In some embodiments, the computer program is a program which identifies single nucleotide polymorphisms. The method may be implemented by the computer systems described above. The method may also be performed by reading at least 2, 5, 10, 15, 20, 25, 30, or 50 of the nucleic acid codes of the invention and the reference nucleotide sequences through the use of the computer program and identifying differences between the nucleic acid codes and the reference nucleotide sequences with the computer program. In other embodiments the computer based system may further comprise an identifier for identifying features within the nucleotide sequences of the nucleic acid codes of the invention or the amino acid sequences of the polypeptide codes of SEQ ID Nos. 653-654. An “identifier” refers to one or more programs which identifies certain features within the above-described nucleotide sequences of the nucleic acid codes of the invention or the amino acid sequences of the polypeptide codes of SEQ ID Nos. 653-654. In one embodiment, the identifier may comprise a program which identifies an open reading frame in the cDNAs codes of SEQ ID No. 652.
One embodiment is an identifier process for detecting the presence of a feature in a sequence. The process begins at a start state and then moves to a state wherein a first sequence that is to be checked for features is stored to a memory in the computer system. The process then moves to a state wherein a database of sequence features is opened. Such a database would include a list of each feature's attributes along with the name of the feature. For example, a feature name could be “Initiation Codon” and the attribute would be “ATG.” Another example would be the feature name “TAATAA Box” and the feature attribute would be “TAATAA”. An example of such a database is produced by the University of Wisconsin Genetics Computer Group (www.gcg.com). Once the database of features is opened at the state, the process moves to a state wherein the first feature is read from the database. A comparison of the attribute of the first feature with the first sequence is then made at a state. A determination is then made at a decision state whether the attribute of the feature was found in the first sequence. If the attribute was found, then the process moves to a state 318 wherein the name of the found feature is displayed to the user. The process then moves to a decision state wherein a determination is made whether move features exist in the database. If no more features do exist, then the process terminates at an end state. However, if more features do exist in the database, then the process reads the next sequence feature at a state and loops back to the state wherein the attribute of the next feature is compared against the first sequence. It should be noted, that if the feature attribute is not found in the first sequence at the decision state, the process moves directly to the decision state in order to determine if any more features exist in the database. In another embodiment, the identifier may comprise a molecular modeling program which determines the 3-dimensional structure of the polypeptides codes of SEQ ID Nos. 653-654. In some embodiments, the molecular modeling program identifies target sequences that are most compatible with profiles representing the structural environments of the residues in known three-dimensional protein structures. (See, e.g., Eisenberg et al., U.S. Pat. No. 5,436,850 issued Jul. 25, 1995, the disclosure of which is incorporated herein by reference in its entirety). In another technique, the known three-dimensional structures of proteins in a given family are superimposed to define the structurally conserved regions in that family. This protein modeling technique also uses the known three-dimensional structure of a homologous protein to approximate the structure of the polypeptide codes of SEQ ID Nos. 653-654. (See e.g., Srinivasan, et al., U.S. Pat. No. 5,557,535 issued Sep. 17, 1996, the disclosure of which is incorporated herein by reference in its entirety). Conventional homology modeling techniques have been used routinely to build models of proteases and antibodies. (Sowdhamini et al., Protein Engineering 10:207, 215 (1997), the disclosure of which is incorporated herein by reference in its entirety). Comparative approaches can also be used to develop three-dimensional protein models when the protein of interest has poor sequence identity to template proteins. In some cases, proteins fold into similar three-dimensional structures despite having very weak sequence identities. For example, the three-dimensional structures of a number of helical cytokines fold in similar three-dimensional topology in spite of weak sequence homology. The recent development of threading methods now enables the identification of likely folding patterns in a number of situations where the structural relatedness between target and template(s) is not detectable at the sequence level. Hybrid methods, in which fold recognition is performed using Multiple Sequence Threading (MST), structural equivalencies are deduced from the threading output using a distance geometry program DRAGON to construct a low resolution model, and a full-atom representation is constructed using a molecular modeling package such as QUANTA.
According to this 3-step approach, candidate templates are first identified by using the novel fold recognition algorithm MST, which is capable of performing simultaneous threading of multiple aligned sequences onto one or more 3-D structures. In a second step, the structural equivalencies obtained from the MST output are converted into interresidue distance restraints and fed into the distance geometry program DRAGON, together with auxiliary information obtained from secondary structure predictions. The program combines the restraints in an unbiased manner and rapidly generates a large number of low resolution model confirmations. In a third step, these low resolution model confirmations are converted into full-atom models and subjected to energy minimization using the molecular modeling package QUANTA. (See e.g., Aszódi et al., Proteins: Structure, Function, and Genetics, Supplement 1:38-42 (1997), the disclosure of which is incorporated herein by reference in its entirety).
The results of the molecular modeling analysis may then be used in rational drug design techniques to identify agents which modulate the activity of the polypeptide codes of SEQ ID Nos. 653-654. Accordingly, another aspect of the present invention is a method of identifying a feature within the nucleic acid codes of the invention or the polypeptide codes of SEQ ID Nos. 653-654 comprising reading the nucleic acid code(s) or the polypeptide code(s) through the use of a computer program which identifies features therein and identifying features within the nucleic acid code(s) or polypeptide code(s) with the computer program. In one embodiment, computer program comprises a computer program which identifies open reading frames. In a further embodiment, the computer program identifies structural motifs in a polypeptide sequence. In another embodiment, the computer program comprises a molecular modeling program. The method may be performed by reading a single sequence or at least 2, 5, 10, 15, 20, 25, 30, or 50 of the nucleic acid codes of the invention or the polypeptide codes of SEQ ID Nos. 653-654 through the use of the computer program and identifying features within the nucleic acid codes or polypeptide codes with the computer program. The nucleic acid codes of the invention or the polypeptide codes of SEQ ID Nos. 653-654 may be stored and manipulated in a variety of data processor programs in a variety of formats. For example, the nucleic acid codes of the invention or the polypeptide codes of SEQ ID Nos. 653-654 may be stored as text in a word processing file, such as MicrosoftWORD or WORDPERFECT or as an ASCII file in a variety of database programs familiar to those of skill in the art, such as DB2, SYBASE, or ORACLE. In addition, many computer programs and databases may be used as sequence comparers, identifiers, or sources of reference nucleotide or polypeptide sequences to be compared to the nucleic acid codes of the invention or the polypeptide codes of SEQ ID Nos. 653-654. The following list is intended not to limit the invention but to provide guidance to programs and databases which are useful with the nucleic acid codes of the invention or the polypeptide codes of SEQ ID No. 653-654. The programs and databases which may be used include, but are not limited to: MacPattern (EMBL), DiscoveryBase (Molecular Applications Group), GeneMine (Molecular Applications Group), Look (Molecular Applications Group), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215: 403 (1990), the disclosure of which is incorporated herein by reference in its entirety), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444 (1988), the disclosure of which is incorporated herein by reference in its entirety), FASTDB (Brutlag et al. Comp. App. Biosci. 6:237-245, 1990, the disclosure of which is incorporated herein by reference in its entirety), Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (Molecular Simulations Inc.), Cerius2.DBAccess (Molecular Simulations Inc.), HypoGen (Molecular Simulations Inc.), Insight II, (Molecular Simulations Inc.), Discover (Molecular Simulations Inc.), CHARMm (Molecular Simulations Inc.), Felix (Molecular Simulations Inc.), DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular Simulations Inc.), Homology (Molecular Simulations Inc.), Modeler (Molecular Simulations Inc.), ISIS (Molecular Simulations Inc.), Quanta/Protein Design (Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.), WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer (Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.), the EMBL/Swissprotein database, the MDL Available Chemicals Directory database, the MDL Drug Data Report data base, the Comprehensive Medicinal Chemistry database, Derwents's World Drug Index database, the BioByteMasterFile database, the Genbank database, and the Genseqn database. Many other programs and data bases would be apparent to one of skill in the art given the present disclosure. Motifs which may be detected using the above programs include sequences encoding leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta sheets, signal sequences encoding signal peptides which direct the secretion of the encoded proteins, sequences implicated in transcription regulation such as homeoboxes, acidic stretches, enzymatic active sites, substrate binding sites, and enzymatic cleavage sites.
It should be noted that the nucleic acid codes of the invention further encompass all of the polynucleotides disclosed, described or claimed in the present invention. Also, it should be noted that the polypeptide codes of SEQ ID Nos. 653-654 further encompass all of the polypeptides disclosed, described or claimed in the present invention. Moreover, the present invention specifically contemplates the storage of such codes on computer readable media and computer systems individually or in combination, as well as the use of such codes and combinations in the methods of section “VI. Computer-Related Embodiments.”
Throughout this application, various publications, patents, and published patent applications are cited. The disclosures of the publications, patents, and published patent specifications referenced in this application are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.
VII. DNA Typing Methods and Systems
The present invention also encompasses a DNA typing system having a much higher discriminatory power than currently available typing systems. The systems and associated methods are particularly applicable in the identification of individuals for forensic science and paternity determinations. These applications have become increasingly important; in forensic science, for example, the identification of individuals by polymorphism analysis has become widely accepted by courts as evidence.
While forensic geneticists have developed many techniques to compare homologous segments of DNA to determine if the segments are identical or if they differ in one or more nucleotides, each technique still has certain disadvantages. In particular, the techniques vary widely in terms of expense of analysis, time required to carry out an analysis and statistical power.
RFLP Analysis Methods
The best known and most widespread method in forensic DNA typing is the restriction fragment length polymorphism (RFLP) analysis. In RFLP testing, a repetitive DNA sequence referred to as a variable number tandem repeat (VNTR) which varies between individuals is analyzed. The core repeat is typically a sequence of about 15 base pairs in length, and highly polymorphic VNTR loci can have an average of about 20 alleles. DNA restriction sites located on either site of the VNTR are exploited to create DNA fragments from about 0.5 Kb to less than 10 Kb which are then separated by electrophoresis, indicating the number of repeats found in the individual at the particular loci. RFLP methods generally consist of (1) extraction and isolation of DNA, (2) restriction endonuclease digestion; (3) separation of DNA fragments by electrophoresis; (4) capillary transfer; (5) hybridization with radiolabelled probes; (6) autoradiography; and (7) interpretation of results (Lee, H. C. et al., Am. J. Forensic. Med. Pathol. 15(4): 269-282 (1994)). RFLP methods generally combine analysis at about 5 loci and have much higher discriminate potential than other available test due the highly polymorphic nature of the VNTRs. However, autoradiography is costly and time consuming and an analysis generally takes weeks or months for turnaround. Additionally, a large amount of sample DNA is required, which is often not available at a crime scene. Furthermore, the reliability of the system and its credibility as evidence is decreased because the analysis of tightly spaced bands on electrophoresis results in a high rate of error.
PCR Methods
PCR based methods offer an alternative to RFLP methods. In a first method called AmpFLP, DNA fragments containing VNTRs are amplified and then separated electrophoretically, without the restriction step of RFLP method. While this method allows small quantities of sample DNA to be used, decreases analysis time by avoiding autoradiography, and retains high discriminatory potential, it nevertheless requires electrophoretic separation which takes substantial time and introduces an significant error rate. In another AmpFLP method, short tandem repeats (STRs) of 2 to 8 base pairs are analyzed. STRs are more suitable to analysis of degraded DNA samples since they require smaller amplified fragments but have the disadvantage of requiring separation of the amplified fragments. While STRs are far less informative than longer repeats, similar discriminatory potential can be achieved if enough STRs are used in a single analysis.
Other methods include sequencing of mitochondrial DNA, which is especially suitable for situations where sample DNA is very degraded or in small quantities. However, only a small region of 1 Kb of the mitochondrial DNA referred to as the D-Loop locus has been found useful for typing because of its polymorphic nature, resulting in lower discriminatory potential than with RFLP or AmpFLP methods. Furthermore, DNA sequencing is expensive to carry out on a large number of samples.
Further available methods include dot-blot methods, which involve using allele specific oligonucleotide probes which hybridize sequence specifically to one allele of a polymorphic site. Systems include the HLA DQ-alpha kit developed by Cetus Corp. which has a discriminatory value of about 1 in 20, and a dot-blot strip referred to as the Polymarker strip combining five genetic loci for a discriminatory value of about one in a few thousand. (Weedn, V., Clinics in Lab. Med. 16(1): 187-196 (1996)).
In addition to difficulties in analysis and time consuming laboratory procedures, it remains desirable for all DNA typing systems to have a higher discriminatory power. Several applications exist in which even the most discriminating tests need improvement in order to remove the considerable remaining doubt resulting from such analyses. Table 3 below lists characteristics of currently available forensic testing systems (Weedn, (1996)) and compares them with the method of the invention.












TABLE 3









Sensitivity





Turnaround
Discriminatory
(amount


Test type
Technology
time
potential
DNA)
Sample





















RFLP
VNTR
Weeks or
106 to 109
10
ng
Highly intact



(autoradiography)
months



DNA


AmpFLP
VNTR
Days
103 to 106
100
pg
Moderate



(PCR based)




degradation


Dot blot (ex.
Sequence specific
Days
101 to 103
1
ng
Moderate


HLADQA1)
oligonucleotide




degradation



probes


Mitochondrial
D-loop sequence
Days
102
1
pg
Severe


DNA
(PCR based)




degradation


Present marker
Biallelic Markers
Hours to
106, 1047, 10650
100
pg
Moderate


set of the
(set of 13, set of
Days



degradation


invention
100, set of 500, set
(throughput



of 650)
dependent)









Applications

As described above, an important application of DNA typing tests is to determine whether a DNA sample (e.g. from a crime scene) originated from an individual suspected of leaving said DNA sample.
There are several applications for DNA typing which require a particularly powerful genotyping system. In a first application, a high powered typing system is advantageous when for example a suspect is identified by searching a DNA profile database such as that maintained by the U.S. Federal Bureau of Investigation. Since databases may contain large numbers of data entries that are expected to increase consistently, currently used forensic systems can be expected to identify several matching DNA profiles due to their relative lack of power. While database searches generally reinforce the evidence by excluding other possible suspects, low powered typing systems resulting in the identification of several individuals may often tend to diminish the overall case against a defendant.
In another application, a target population is systematically tested to identify an individual having the same DNA profile as that of a DNA sample. In such a situation, a defendant is chosen at random based on DNA profile from a large population of innocent individuals. Since the population tested can often be large enough that at least one positive match is identified, and it is usually not possible to exhaustively test a population, the usefulness of the evidence will depend on the level of significance of the forensic test. In order to render such an application useful as a sole or primary source of evidence, DNA typing systems of extremely high discriminatory potential are required.
In yet another application, it is desirable to be able to discriminate between related individuals. Because related individuals will be expected to share a large portion of alleles at polymorphic sites, a very high powered DNA typing assay would be required to discriminate between them. This can have important effects if a sample is found to match the defendant's DNA profile and no evidence that the perpetrator is a relative can be found.
Accordingly, there a need in this art for a rapid, simple, inexpensive and accurate technique having a very high resolution value to determine relationships between individuals and differences in degree of relationships. Also, there is a need in the art for a very accurate genetic relationship test procedure which uses very small amounts of an original DNA sample, yet produces very accurate results.
The present invention thus involves methods for the identification of individuals comprising determining the identity of the nucleotides at set of genetic markers in a biological sample, wherein said set of genetic markers comprises at least one eicosanoid-related biallelic marker. The present invention provides an extensive set of biallelic markers allowing a higher discriminatory potential than the genetic markers used in current forensic typing systems. Also, biallelic markers can be genotyped in individuals with much higher efficiency and accuracy than the genetic markers used in current forensic typing systems. In preferred embodiments, the invention comprises determining the identity of a nucleotide at an eicosanoid-related biallelic marker by single nucleotide primer extension, which does not require electrophoresis as in techniques described above and results in lower rate of experimental error. As shown in Table 3, herein, in comparison with PCR based VNTR based methods which allow discriminatory potential of thousands to millions, and RFLP based methods which allow discriminatory potential of merely millions to billions under optimal assumptions, the biallelic marker based method of the present invention provides a radical increase in discriminatory potential.
Any suitable set of genetic markers and biallelic markers of the invention may be used, and may be selected according to the discriminatory power desired. Biallelic markers, sets of biallelic markers, probes, primers, and methods for determining the identity of said biallelic markers are further described herein.
Discriminatory Potential of Biallelic Marker Typing
Calculating Discriminatory Potential
The discriminatory potential of the forensic test can be determined in terms of the profile frequency, also referred to as the random match probability, by applying the product rule. The product rule involves multiplying the allelic frequencies of all the individual alleles tested, and multiplying by an additional factor of 2 for each heterozygous locus.
In one example discussed below, the discriminatory potential of biallelic marker typing can be considered in the context of forensic science. In order to determine the discriminatory potential with respect to the numbers of biallelic markers to be used in a genetic typing system, the formulas and calculations below assume that (1) the population under study is sufficiently large (so that we can assume no consanguinity); (2) all markers chosen are not correlated, so that the product rule (Lander and Budlowle (1992)) can be applied; and (3) the ceiling rule can be applied or that the allelic frequencies of markers in the population under study are known with sufficient accuracy.
As noted in Weir, B. S., Genetic data Analysis II: Methods for Discrete population genetic Data, Sinauer Assoc., Inc., Sunderland, Mass., USA, 1996, the example assumes a crime has been committed and a sample of DNA from the perpetrator (P) is available for analysis. The genotype of this DNA sample can be determined for several genetic markers, and the profile A of the perpetrator can thereby be determined.
In this example, one suspect (S) is available for typing. The same set of genetic markers, such as the biallelic markers of the invention, are typed and the same profile A is obtained for (S) and (P). Two hypotheses are thus presented as follows:
(1) either S is P (event C)
(2) either S is not P (event C).
The ratio L of both probabilities can then be calculated using the following equation:




  L
  =
  
    
      pr
      ⁡
      
        (
        
          
            S
            =
            A
          
          ,
          
            P
            =
            
              A
              /
              C
            
          
        
        )
      
    
    
      pr
      ⁡
      
        (
        
          
            S
            =
            A
          
          ,
          
            P
            =
            
              A
              /
              
                C
                _
              
            
          
        
        )
      
    
  




L can then further be calculated by the following equation:




  
    
      
        L
        =
        
          
            1
            
              pr
              ⁡
              
                (
                
                  
                    P
                    =
                    
                      
                        A
                        /
                        S
                      
                      =
                      A
                    
                  
                  ,
                  
                    C
                    _
                  
                
                )
              
            
          
          ⁢
          
            (
            1
            )
          
        
      
    
    
      
        Equation
        ⁢
        
          
        
        ⁢
        1
      
    
  




These probabilities as well as L can be calculated in several settings, notably for different kinship coefficients between P and S for a genetic marker (see Weir, (1996)).
Assuming that all genetic markers chosen are independent of each other, the global ratio L for a set of genetic markers will be the product over each genetic marker of all L.
It is further possible to estimate the mean number of biallelic markers or VNTRs required to have a ratio L equal to 108 or 106 by calculating the expectancy of the random variable L using the following equation:




  
    E
    ⁡
    
      (
      L
      )
    
  
  =
  
    
      ∏
      
        i
        =
        1
      
      N
    
    ⁢
    
      E
      ⁡
      
        (
        
          L
          i
        
        )
      
    
  




where N is the number of loci





  
    
      E
      ⁡
      
        (
        
          L
          i
        
        )
      
    
    =
    
      
        ∑
        
          j
          =
          1
        
        
          G
          i
        
      
      ⁢
      
        
          pr
          ⁡
          
            (
            
              
                P
                =
                
                  
                    
                      A
                      ij
                    
                    /
                    S
                  
                  =
                  
                    A
                    ij
                  
                
              
              ,
              
                C
                _
              
            
            )
          
        
        ·
        
          L
          ij
        
      
    
  
  ,




where Aij is the genotype j at the ith marker, Li the ratio associated with such genotype, Gi being the number of genotypes at locus i. From equation 1, it can easily be derived that the expectancy of Li is Gi, the number of possible genotypes of this marker.

The general expectancy for a set of genetic markers can then be expressed by the following equation:




  
    
      
        
          E
          ⁡
          
            (
            L
            )
          
        
        =
        
          
            ∏
            
              i
              =
              1
            
            N
          
          ⁢
          
            
              G
              i
            
            ⁡
            
              (
              2
              )
            
          
        
      
    
    
      
        Equation
        ⁢
        
          
        
        ⁢
        2
      
    
  




A. Biallelic Marker-based DNA Typing Systems

Using the equations described above, it is possible to select biallelic marker-based DNA typing systems having a desired discriminatory potential.
Using biallelic markers, E(L) can thus be expressed as 3N When using VNTR-based DNA typing systems, assuming the VNTRs have 10 alleles, E(L) can be expressed as 55N. Based on these results, the number of biallelic markers or VNTRs needed to obtain, in mean, a ratio of at least 106 or 108 can calculated, and are set forth below in Table 4.











TABLE 4







Marker sets
L = 106
L = 108




















Biallelic
13
17



5-allele markers (e.g. VNTR)
5
7



10-allele markers (e.g. VNTR)
4
5










Thus, in a first embodiment, DNA typing systems and methods of the invention may comprise genotyping a set of at least 13 or at least 17 biallelic markers to obtain a ratio of at least 106 or 108, assuming a flat distribution of L across the biallelic markers. In preferred embodiments, a greater number of biallelic markers is genotyped to obtain a higher L value. Preferably at least 1, 2, 3, 4, 5, 10, 13, 15, 17, 20, 25, 30, 40, 50, 70, 85, 100, 150, 200, 300, 400, 500, 600 or all of the eicosanoid-related biallelic markers are genotyped. Said DNA typing systems of the invention would result in L values as listed in Table 5 below as an indication of the discriminate potential of the systems of the invention.








TABLE 5





Number of biallelic



markers
L
















50
7.2 * 1023


100
5 * 1047


650
3{circumflex over ( )}650









In situations where the distribution of L is not flat, such as in the worst case when the perpetrator is homozygous for the major allele at each genetic locus and L thus takes the lowest value, a larger number of biallelic markers is required for the same discriminatory potential. Therefore, in preferred embodiments, DNA typing systems and methods of the invention using a larger number of biallelic markers allow for uneven distributions of L across the biallelic markers. For example, assuming unrelated individuals, a set of independent markers having an allelic frequency of 0.1/0.9, and the genetic profile of a homozygote at each genetic loci for the major allele, 66 biallelic markers are required to obtain a ratio of 106, and 88 biallelic markers are required to obtain a ratio of 108. Thus, in preferred embodiments based on the use of markers having a major allele of sufficiently high frequency, this is a first estimation of the upper bound of markers required in a DNA typing system.
In further embodiments, it is also desirable to have the ability to discriminate between relatives. Although unrelated individuals have a low probability of sharing genetic profiles, the probability is greatly increased for relatives. For example, the DNA profile of a suspect matches the DNA profile of a sample at a crime scene, and the probability of obtaining the same DNA profile if left by an untyped relative is required. Table 6 below (Weir (1996)) lists probabilities for several different types of relationships, assuming alleles Ai and Aj, and population frequencies pi and pj, and lists likelihood ratios assuming genetic loci having allele frequencies of 0.1.










TABLE 6





Genotype
Relationship
Pr(p = A|S = A)
L


















Ai Aj
Full brothers
(1 + pi + pj + 2pi pj)/4
3.3



Father and son
(pi + pj)/2
10.0



Half brothers
(pi + pj + 4pi pj)/4
16.7



Uncle and nephew
(1 + pi + pj + 2pi pj)/4
16.7



First cousins
(1 + pi + pj + 12pi pj)/8
25.0



Unrelated
2pi pj
50.0


Aj Aj
Full brothers
(1 + pi)2/4
3.3



Father and son
pi
10.0



Half brothers
pi (1 + pi)/2
18.2



Uncle and nephew
pi (1 + pi)/2
18.2



First cousins
pi (1 + 3pi)/4
30.8



Unrelated
pi2
100.0









In one example, where the suspect is the full brother of the perpetrator, the number of required biallelic markers will be 187 assuming the profile is that of a homozygote for the major allele at each biallelic marker.
In yet further embodiments, the DNA typing systems and methods of the present invention may further take into account effects of subpopulations on the discriminatory potential. In embodiments described above for example, DNA typing systems consider close familial relationships, but do not take into account membership in the same population. While population membership is expected to have little effect, the invention may further comprise genotyping a larger set of biallelic markers to achieve higher discriminatory potential. Alternatively, a larger set of biallelic markers may be optimized for typing selected populations; alternatively, the ceiling principle may be used to study allele frequencies from individuals in various populations of interest, taking for any particular genotype the maximum allele frequency found among the populations.
The invention thus encompasses methods for genotyping comprising determining the identity of a nucleotide at least 13, 15, 17, 20, 25, 30, 40, 50, 66, 70, 85, 88, 100, 187, 200, 300, 500, 700, 1000 or 2000 biallelic markers in a biological sample, wherein at least 1, 2, 3, 4, 5, 10, 13, 17, 20, 25, 30, 40, 50, 70, 85, 100, 150, 200, 300, 400, 500, 600 or all of said biallelic markers are eicosanoid-related biallelic markers selected from the group consisting of the markers provided in Table 7(A-B).
Any markers known in the art may be used with the eicosanoid-related biallelic markers of the present invention in the DNA typing methods and systems described herein, for example in anyone of the following web sites offering collections of SNPs and information about those SNPs:
The Genetic Annotation Initiative (http://cgap.nci.nih.gov/GAI/). An NIH run site which contains information on candidate SNPs thought to be related to cancer and tumorigenesis generally.
dbSNP Polymorphism Repository (http://www.ncbi.nlm.nih.gov/SNP/). A more comprehensive NIH-run database containing information on SNPs with broad applicability in biomedical research.
HUGO Mutation Database Initiative http://ariel.ucs.unimelb.edu.au:80/ ̃cotton/mdi.htm). A database meant to provide systematic access to information about human mutations including SNPs. This site is maintained by the Human Genome Organization (HUGO).
Human SNP Database (http://www-genome.wi.mit.edu/SNP/human/index.html). Managed by the Whitehead Institute for Biomedical Research Genome Institute, this site contains information about SNPs resulting from the many Whitehead research projects on mapping and sequencing.
SNPs in the Human-Genome SNP database (http://www.ibc.wustl.edu/SNP. This website provides access to SNPs that have been organized by chromosomes and cytogenetic location. The site is run by Washington University.
HGBase (http://hgbase.cgr.ki.se/). HGBASE is an attempt to summarize all known sequence variations in the human genome, to facilitate research into how genotypes affect common diseases, drug responses, and other complex phenotypes, and is run by the Karolinska Institute of Sweden.
The SNP Consortium Database (http://snp.cshl.org/db/snp/map). A collection of SNPs and related information resulting from the collaborative effort of a number of large pharmaceutical and information processing companies.
GeneSNPs (http://www.genome.utah.edu/genesnps/). Run by the University of Utah, this site contains information about SNPs resulting from the U.S. National Institute of Environmental Health's initiative to understand the relationship between genetic variation and response to environmental stimuli and xenobiotics.
In addition, biallelic markers provided in the following patents and patent applications may also be used with the eicosanoid-related biallelic markers of the invention in the DNA typing methods and systems described above: U.S. Ser. No. 60/206,615, filed 24 Mar. 2000; U.S. Ser. No. 60/216,745, filed 30 Jun. 2000; WIPO Serial No. PCT/IB00/00184, filed 11 Feb. 2000; WIPO Serial No. PCT/IB98/01193, filed 17 Jul. 1998; PCT Publication No. WO 99/54500, filed 21 Apr. 1999; and WIPO Serial No. PCT/IB00/00403, filed 24 Mar. 2000.
Biallelic markers, sets of biallelic markers, probes, primers, and methods for determining the identity of a nucleotide at said biallelic markers are also encompassed and are further described herein, and may encompass any further limitation described in this disclosure, alone or in any combination.
Forensic matching by microsequencing is further described in Example 8 below.
EXAMPLES
Several of the methods of the present invention are described in the following examples, which are offered by way of illustration and not by way of limitation. Many other modifications and variations of the invention as herein set forth can be made without departing from the spirit and scope thereof and therefore only such limitations should be imposed as are indicated by the appended claims.
Example 1
De Novo Identification of Biallelic Markers
The biallelic markers set forth in this application were isolated from human genomic sequences. To identify biallelic markers, genomic fragments were amplified, sequenced and compared in a plurality of individuals.
DNA Samples
Donors were unrelated and healthy. They represented a sufficient diversity for being representative of a French heterogeneous population. The DNA from 100 individuals was extracted and tested for the de novo identification of biallelic markers.
DNA samples were prepared from peripheral venous blood as follows. Thirty ml of peripheral venous blood were taken from each donor in the presence of EDTA. Cells (pellet) were collected after centrifugation for 10 minutes at 2000 rpm. Red cells were lysed in a lysis solution (50 ml final volume: 10 mM Tris pH7.6; 5 mM MgCl2; 10 mM NaCl). The solution was centrifuged (10 minutes, 2000 rpm) as many times as necessary to eliminate the residual red cells present in the supernatant, after resuspension of the pellet in the lysis solution. The pellet of white cells was lysed overnight at 42° C. with 3.7 ml of lysis solution composed of: (a) 3 ml TE 10-2 (Tris-HCl 10 mM, EDTA 2 mM)/NaCl 0.4 M; (b) 200 μl SDS 10%; and (c) 500 μl proteinase K (2 mg proteinase K in TE 10-2/NaCl 0.4 M).
For the extraction of proteins, 1 ml saturated NaCl (6M) (1/3.5 v/v) was added. After vigorous agitation, the solution was centrifuged for 20 minutes at 10000 rpm. For the precipitation of DNA, 2 to 3 volumes of 100% ethanol were added to the previous supernatant, and the solution was centrifuged for 30 minutes at 2000 rpm. The DNA solution was rinsed three times with 70% ethanol to eliminate salts, and centrifuged for 20 minutes at 2000 rpm. The pellet was dried at 37° C., and resuspended in 1 ml TE 10-1 or 1 ml water. The DNA concentration was evaluated by measuring the OD at 260 nm (1 unit OD=50 μg/ml DNA). To determine the presence of proteins in the DNA solution, the OD 260/OD 280 ratio was determined. Only DNA preparations having a OD 260/OD 280 ratio between 1.8 and 2 were used in the subsequent examples described below. DNA pools were constituted by mixing equivalent quantities of DNA from each individual.
Amplification of Genomic DNA by PCR
Amplification of specific genomic sequences was carried out on pooled DNA samples obtained as described above.
Amplification Primers
The primers used for the amplification of human genomic DNA fragments were defined with the OSP software (Hillier & Green, 1991). Preferably, primers included, upstream of the specific bases targeted for amplification, a common oligonucleotide tail useful for sequencing. Primers PU contain the following additional PU 5′ sequence: TGTAAAACGACGGCCAGT; primers RP contain the following RP 5′ sequence: CAGGAAACAGCTATGACC. Primers are listed in Table 13.










Amplification


PCR assays were performed using the following protocol:

















Final volume
25
μl


DNA
2
ng/μl


MgCl2
2
mM


dNTP (each)
200
μM


primer (each)
2.9
ng/μl


Ampli Taq Gold DNA polymerase
0.05
unit/μl


PCR buffer (10× = 0.1 M TrisHCl pH 8.3 0.5 M KCl)
1×









DNA amplification was performed on a Genius II thermocycler. After heating at 94° C. for 10 min, 40 cycles were performed. Cycling times and temperatures were: 30 sec at 94° C., 55° C. for 1 min and 30 sec at 72° C. Holding for 7 min at 72° C. allowed final elongation. The quantities of the amplification products obtained were determined on 96-well microtiter plates, using a fluorometer and Picogreen as intercalant agent (Molecular Probes).
Sequencing of Amplified Genomic DNA and Identification of Biallelic Polymorphisms
Sequencing of the amplified DNA was carried out on ABI 377 sequencers. The sequences of the amplification products were determined using automated dideoxy terminator sequencing reactions with a dye terminator cycle sequencing protocol. The products of the sequencing reactions were run on sequencing gels and the sequences were determined using gel image analysis (ABI Prism DNA Sequencing Analysis software 2.1.2 version).
The sequence data were further evaluated to detect the presence of biallelic markers within the amplified fragments. The polymorphism search was based on the presence of superimposed peaks in the electrophoresis pattern resulting from different bases occurring at the same position. However, the presence of two peaks can be an artifact due to background noise. To exclude such an artifact, the two DNA strands were sequenced and a comparison between the two strands was carried out. In order to be registered as a polymorphic sequence, the polymorphism had to be detected on both strands. Further, some biallelic single nucleotide polymorphisms were confirmed by microsequencing as described below.
Biallelic markers were identified in the analyzed fragments and are shown in Table 7. Also, the genomic structure of the FLAP gene and 12-LO gene including the relative location of some biallelic markers is shown in FIG. 1 and FIG. 3, respectively.
Example 2
Genotyping of Biallelic Markers
The biallelic markers identified as described above were further confirmed and their respective frequencies were determined through microsequencing. Microsequencing was carried out on individual DNA samples obtained as described herein.
Microsequencing Primers
Amplification of genomic DNA fragments from individual DNA samples was performed as described in Example 1 using the same set of PCR primers. Microsequencing was carried out on the amplified fragments using specific primers. See Table 12. The preferred primers used in microsequencing had about 19 nucleotides in length and hybridized just upstream of the considered polymorphic base.
The microsequencing reactions were performed as follows: 5 μl of PCR products were added to 5 μl purification mix (2U SAP (Shrimp alkaline phosphate) (Amersham E70092X)); 2U Exonuclease I (Amersham E70073Z); and 1 μl SAP buffer (200 mM Tris-HCl pH8, 100 mM MgCl2) in a microtiter plate. The reaction mixture was incubated 30 minutes at 37° C., and denatured 10 minutes at 94° C. afterwards. To each well was then added 20 μl of microsequencing reaction mixture containing: 10 pmol microsequencing oligonucleotide (19mers, GENSET, crude synthesis, 5 OD), 1 U Thermosequenase (Amersham E79000G), 1.25 μl Thermosequenase buffer (260 mM Tris HCl pH 9.5, 65 mM MgCl2), and the two appropriate fluorescent ddNTPs complementary to the nucleotides at the polymorphic site corresponding to both polymorphic bases (11.25 nM TAMRA-ddTTP; 16.25 nM ROX-ddCTP; 1.675 nM REG-ddATP; 1.25 nM RHO-ddGTP; Perkin Elmer, Dye Terminator Set 401095). After 4 minutes at 94° C., 20 PCR cycles of 15 sec at 55° C., 5 sec at 72° C., and 10 sec at 94° C. were carried out in a Tetrad PTC-225 thermocycler (MJ Research). The microtiter plate was centrifuged 10 sec at 1500 rpm. The unincorporated dye terminators were removed by precipitation with 19 μl MgCl2 2 mM and 55 μl 100% ethanol. After 15 minute incubation at room temperature, the microtiter plate was centrifuged at 3300 rpm 15 minutes at 4° C. After discarding the supernatants, the microplate was evaporated to dryness under reduced pressure (Speed Vac). Samples were resuspended in 2.5 μl formamide EDTA loading buffer and heated for 2 min at 95° C. 0.8 μl microsequencing reaction were loaded on a 10% (19:1) polyacrylamide sequencing gel. The data were collected by an ABI PRISM 377 DNA sequencer and processed using the GENESCAN software (Perkin Elmer).
Frequency of Biallelic Markers
Frequencies are reported for the less common allele only and are shown in Table 7.
Example 3
Association Study Between Asthma and the Biallelic Markers of the FLAP Gene
Collection of DNA Samples from Case and Control Individuals
The disease trait followed in this association study was asthma, a disease involving the leukotriene pathway. The asthmatic population corresponded to 298 individuals that took part in a clinical study for the evaluation of the anti-asthmatic drug Zileuton. More than 90% of these 298 asthmatic individuals had a Caucasian ethnic background. The control population was composed of 286 individuals from a random US Caucasian population.
Genotyping of Case and Control Individuals
The general strategy to perform the association studies was to individually scan the DNA samples from all individuals in each of the populations described above in order to establish the allele frequencies of the above described biallelic markers in each of these populations.
Allelic frequencies of the above-described biallelic marker alleles in each population were determined by performing microsequencing reactions on amplified fragments obtained by genomic PCR performed on the DNA samples from each individual. Genomic PCR and microsequencing were performed as detailed above in Examples 1 and 2 using the described PCR and microsequencing primers.
Frequency of the Biallelic Marker Alleles of the FLAP Gene and Association with Asthma
Frequencies of biallelic marker alleles were compared in the case-control populations described above. The association curve in FIG. 2 shows the p-value obtained for each marker and the localization of the markers in the genomic region harboring the FLAP gene. As shown in FIG. 2, the biallelic marker 10-35-390 presented a strong association with asthma, this association being highly significant (pvalue=2.29×10−3). The two markers 10-32-357 and 10-33-234 showed weak association when tested independently. The biallelic marker 10-35/390 is located in the FLAP gene. Therefore, the association studies results show that a polymorphism of the FLAP gene seems to be related to asthma. The biallelic marker 10-35-390 can be then used in diagnostics with a test based on this marker or on a combination of biallelic markers comprising this marker.
Haplotype Frequency Analysis
The results of the haplotype analysis using 9 biallelic markers (10-253-298, 10-32-357, 10-33-175, 10-33-234, 10-33-327, 10-35-358, 10-35-390, 12-628-306, and 12-629-241) are shown in Table 15. Haplotype analysis for association of FLAP markers and asthma was performed by estimating the frequencies of all possible 2, 3 and 4 marker haplotypes in the asthmatic and Caucasian US control populations. Haplotype estimations were performed by applying the Expectation-Maximization (EM) algorithm (Excoffier and Slatkin, 1995), using the EM-HAPLO program (Hawley et al., 1994). Estimated haplotype frequencies in the asthmatic and control populations were compared by means of a chi-square statistical test.
The most significant haplotypes obtained are shown in Table 15.
Preferred haplotypes comprise either the marker 10-33-234 (allele A) or the marker 10-35-390 (allele T). Preferred haplotype No. 1 (A at 10-33-234 and T at 10-35-390) presented a p-value of 8.2×10−4 and an odd-ratio of 1.61. Estimated haplotype frequencies were 28.3% in the cases and 19.7% in the US controls. Also preferred are haplotypes No. 2 (A at 10-33-234 and G at 12-629-241) and haplotype No. 3 (T at 10-33/327 and T at 10-33/390) which presented respectively a p-value of 1.6×10−3 and 1.8×10−3, an odd-ratio of 1.65 and 1.53 and haplotypes frequencies of 0.305 and 0.307 for the asthmatic population and of 0.210 and 0.224 for the US control population.
Preferred haplotypes consisting of three markers (haplotype nos. 37, 38, 39 and 41) comprise the marker 10-33-234 (allele A) and the marker 10-35-390 (allele T). Preferred haplotype No. 37 (A at 10-33-234, T at 10-33-390 and C at 12-628-306) presented a p-value of 8.6×10−4 and an odd-ratio of 1.76. Estimated haplotype frequencies were 26.5% in the cases and 17.1% in the US controls. Haplotype No. 40 (A at 10-33-234, C at 12-628-306 and G at 12-629-241) is also very significantly associated with asthma.
Four-marker haplotypes (haplotype Nos. 121 to 125), five-marker haplotypes (haplotype Nos. 247 and 248) and a six-marker haplotype (haplotype No. 373) also showed significant p-values. They all comprise markers 10-33-234 (allele A) and 10-35/390 (allele T), except haplotype no. 124. Other markers in these haplotypes are chosen from the group consisting of 10-235-298 (allele C), 10-35-358 (allele G), 12-628-306 (allele C) and 12-629-241 (allele G).
Haplotype No. 1 is the preferred haplotype of the invention. It can be used in diagnosis of asthma. Moreover, most of the haplotypes significantly associated with asthma comprise the biallelic marker 10-35-390 (allele A) and could also be used in diagnosis.
The statistical significance of the results obtained for the haplotype analysis was evaluated by a phenotypic permutation test reiterated 1000 or 10,000 times on a computer. For this computer simulation, data from the asthmatic and control individuals were pooled and randomly allocated to two groups which contained the same number of individuals as the case-control populations used to produce the data summarized in Table 15. A haplotype analysis was then run on these artificial groups for the 2 markers included in the haplotype No. 1, which showed the strongest association with asthma. This experiment was reiterated 1000 and 10,000 times and the results are shown in Table 16. These results demonstrate that among 1000 iterations none and among 10,000 iterations only 1 of the obtained haplotypes had a p-value comparable to the one obtained for the haplotype No. 1. These results clearly validate the statistical significance of the association between this haplotype and asthma.
Example 4
Association Between Asthma and the Biallelic Markers of the 12-lipoxygenase Gene
Collection of DNA Samples from Case and Control Individuals
The disease trait followed in this association study was asthma, a disease involving the leukotriene pathway. The asthmatic population corresponded to 297 individuals that took part in a clinical study for the evaluation of the anti-asthmatic drug zileuton. More than 90% of these 297 asthmatic individuals had a Caucasian ethnic background. The control population corresponded to 186 individuals from a random US Caucasian population.
Genotyping of Case and Control Individuals
The general strategy to perform the association studies was to individually scan the DNA samples from all individuals in each of the populations described above in order to establish the allele frequencies of the above described biallelic markers in each of these populations.
Allelic frequencies of the above-described biallelic marker alleles in each population were determined by performing microsequencing reactions on amplified fragments obtained by genomic PCR performed on the DNA samples from each individual. Genomic PCR and microsequencing were performed as detailed above in Examples 1 and 2 using the described PCR and microsequencing primers.
Haplotype Frequency Analysis
None of the single marker alleles showed a significant association with asthma however, significant results were obtained in haplotype studies. Allelic frequencies were useful to check that the markers used in the haplotype studies meet the Hardy-Weinberg proportions (random mating).
Haplotype analysis was performed using 12 biallelic markers and 17 biallelic markers. The results of the haplotype analysis using 12 biallelic markers (12-208-35, 12-226-167, 12-206-366, 10-347-203, 10-347-220, 10-349-97, 10-349-224, 12-196-119, 12-214-129, 12-216-421, 12-219-230 and 12-223-207) are shown in Table 17. The results of the haplotype analysis using 17 biallelic markers (12-197-244, 12-208-35, 12-226-167, 12-206-366, 10-346-141, 10-347-111, 10-347-165, 10-347-203, 10-347-220, 10-349-97, 10-349-224, 10-341-116, 12-196-119, 12-214-129, 12-216-421 and 12-219-230) are shown in Table 18. Haplotype analysis for association of 12-LO biallelic markers and asthma was performed by estimating the frequencies of all possible 2, 3 and 4 marker haplotypes in the asthmatic and control populations described above. Haplotype estimations were performed by applying the Expectation-Maximization (EM) algorithm (Excoffier and Slatkin, Mol. Biol. Evol., 12:921-927, 1995), using the EM-HAPLO program (Hawley et al., Am. J. Phys. Anthropol., 18:104, 1994) as described above. Estimated haplotype frequencies in the asthmatic and control population were compared by means of a chi-square statistical test (one degree of freedom).
Table 17 shows the most significant haplotypes obtained from the 12 biallelic marker analysis. Haplotype No.1 consisting of three biallelic markers (10-347-220, 12-214-129 and 12-219-230) presented a p-value of 2.10−5 and an odd-ratio of 3.38. Estimated haplotype frequencies were 12.3% in the cases and 4% in the controls. Haplotype No.14 consisting of four biallelic markers (10-347-203, 12-196-119, 12-216-421 and 12-219-230) had a p-value of 4.10−6 and an odd ratio of 4.18. Estimated haplotype frequencies were 11.8% in the cases and 3.1% in the controls. Haplotype No.1 and haplotype No.14, are both strongly associated with asthma. Haplotypes Nos. 2-13 and 15-24 also showed very significant Association (see Table 17).
Table 18 shows the most significant haplotypes obtained from the 17 biallelic marker analysis. Haplotype No. 1 consisting of two biallelic markers (12-206-366 and 10-349-224) presented a p-value of 1.8 10−4 and an odd-ratio of 2.05. Estimated haplotype frequencies were 42.4% in the cases and 26.5% in the controls. Haplotype No. 7 consisting of three biallelic markers (10-349-97, 12-214-129, 12-219-230) had a p-value of 2.3 10−5 and an odd ratio of 3.32. Estimated haplotype frequencies were 12.5% in the cases and 4.1% in the controls. Haplotype No. 27 consisting of four biallelic markers (10-349-97, 12-196-119, 12-216-421 and 12-219-230) had a p-value of 5.4 10−6 and an odd ratio of 3.90. Estimated haplotype frequencies were 12.4% in the cases and 3.5% in the controls. Haplotypes Nos. 1, 7 and 27 are strongly associated with asthma. Other haplotypes also showed very significant association (see Table 18).
The statistical significance of the results obtained for the haplotype analysis was evaluated by a phenotypic permutation test reiterated 1000 or 10,000 times on a computer. For this computer simulation, data from the asthmatic and control individuals were pooled and randomly allocated to two groups which contained the same number of individuals as the case-control populations used to produce the data summarized in Tables 17 and 18. A haplotype analysis was then run on these artificial groups for the markers included in haplotype No. 14 from Table 17 and for the markers included in haplotypes Nos. 7 and 27 from Table 18, which showed the strongest association with asthma. This experiment was reiterated 1000 and 10,000 times and the results are shown in Table 21 and Table 22, respectively. These results demonstrate that among 1000 iterations only 7 and among 10,000 iterations only 39 of the obtained haplotypes from the 12 biallelic marker set had a p-value comparable to the one obtained for haplotype No.14 from Table 17. Also, among 1000 iterations only 2 of the obtained haplotypes from the 17 biallelic marker set had a p-value comparable to the one obtained for haplotype No. 7 from Table 18. These results further demonstrate that among 1000 iterations none of the obtained haplotypes had a p-value comparable to the one obtained for haplotype No. 27 from Table 18. These results clearly validate the statistical significance of the association between the haplotypes shown in Tables 17 and 18 and asthma.
Example 5
Association Between Side Effects Upon Treatment with the Anti-Asthmatic Drug Zileuton (ZyfloTM) and the Biallelic Markers of the 12-lipoxygenase Gene
Collection of DNA Samples from Case and Control Individuals
The side effect examined in this study was the hepatotoxicity experienced by asthmatic individuals as a result of their treatment with Zileuton as part of a clinical study. Asthmatic individuals were unrelated and more than 90% of the individuals had a Caucasian ethnic background. Hepatotoxicity was monitored by measuring the serum levels of alanine aminotransferase (ALT), which is a sensitive indicator of liver cell damage.
More than 90% of the asthmatic individuals participating in this study did not experience Zileuton-associated ALT increase compared to their ALT levels prior to zileuton intake. As mentioned above, an association study is more informative if the case-control populations present extreme phenotypes. Therefore, the asthmatic individuals, which were selected for the side effect positive trait (ALT+), corresponded to 89 individuals that presented at least 3 times the upper limit of normal (ULN) level of ALT. On the other side, the asthmatic individuals that were selected for the side effect negative trait (ALT−) corresponded to 208 individuals that presented less than 1×ULN of ALT. ALT+ and ALT− populations corresponded to 4% and 35% respectively of the total asthmatic individuals that participated in this study.
Genotyping of Case and Control Individuals
The general strategy to perform the association studies was to individually scan the DNA samples from all individuals in each of the populations described above in order to establish the allele frequencies of the above described biallelic markers in each of these populations.
Allelic frequencies of the above-described biallelic marker alleles in each population were determined by performing microsequencing reactions on amplified fragments obtained by genomic PCR performed on the DNA samples from each individual. Genomic PCR and microsequencing were performed as detailed above in Examples 1 and 2 using the described PCR and microsequencing primers.
Haplotype Frequency Analysis
None of the single marker alleles showed a significant association with hepatoxicity to zileuton, however, significant results were obtained in haplotype studies.
Haplotype analysis was performed using 12 biallelic markers and 17 biallelic markers. The results of the haplotype analysis using 12 biallelic markers (12-208-35, 12-226-167, 12-206-366, 10-347-203, 10-347-220, 10-349-97, 10-349-224, 12-196-119, 12-214-129, 12-216-421, 12-219-230 and 12-223-207) are shown in Table 19. The results of the haplotype analysis using 17 biallelic markers (12-197-244, 12-208-35, 12-226-167, 12-206-366, 10-346-141, 10-347-111, 10-347-165, 10-347-203, 10-347-220, 10-349-97, 10-349-224, 10-341-116, 12-196-119, 12-214-129, 12-216-421 and 12-219-230) are shown in Table 20. Haplotype analysis for association of 12-LO biallelic markers and asthma was performed by estimating the frequencies of all possible 2, 3, 4 and 5 marker haplotypes in the ALT+ and ALT− populations described above. Haplotype estimations were performed by applying the Expectation-Maximization (EM) algorithm (Excoffier and Slatkin, Mol. Biol. Evol., 12:921-927, 1995), using the EM-HAPLO program (Hawley et al., Am. J. Phys. Anthropol., 18:104, 1994) as described above. Estimated haplotype frequencies in the ALT+ and ALT− populations were compared by means of a chi-square statistical test (one degree of freedom).
Table 19 shows the most significant haplotypes obtained from the 12 biallelic marker analysis. Haplotype No.3 consisting of three biallelic markers (10-349-224, 12-216-421 and 12-223-207) presented a p-value of 4.10−5 and an odd-ratio of 3.53. Estimated haplotype frequencies were 15.1% in the cases and 4.8% in the controls. Haplotype No. 8 consisting of four biallelic markers (12-206-366, 10-349-224, 12-216-421 and 12-223-207) had a p-value of 2.9.10−6 and an odd ratio of 4.56. Estimated haplotype frequencies were 15.8% in the cases and 4% in the controls. Both haplotypes showed strong association with elevated serum ALT level upon treatment with zileuton. Both haplotypes are related as three out of four biallelic marker alleles (T at 10-349-224, A at 12-216-421 and T at 12-223-207) are common to both haplotypes. Haplotypes Nos. 4-7 and 9-25 also showed very significant association.
Table 20 shows the most significant haplotypes obtained from the 17 biallelic marker analysis. Haplotype No. 11 consisting of three biallelic markers (12-197/244, 10-349-224 and 12-216-421) presented a p-value of 1.7.10-3 and an odd-ratio of 2.66, for alleles CTA respectively. Estimated haplotype frequencies were 13.7% in the cases and 5.6% in the controls. The p-value obtained by a chi-square distribution with 7 df for this combination of markers is 2.310−2 by Omnibus test suggesting that result is highly significant. Another haplotype consisting of four biallelic markers (12-208-35, 10-512/36, 12-196-119 and 12-219/230) presented a p-value of 3.7.10−5 and an odd-ratio of 3.74. Estimated haplotype frequencies were 14.7% in the cases and 4.4% in the controls. The p-value obtained by a chi-square distribution with 15 df for this combination of markers is 5.410−4 by Omnibus test. Both haplotypes showed strong association with elevated serum ALT level upon treatment with zileuton. Both haplotypes are related as three out of four biallelic marker alleles (C at 12-197/244, T at 10-349-224 and A at 12-216-421) are common to both haplotypes. Other haplotypes also showed very significant association.
The statistical significance of the results obtained for the haplotype analysis was evaluated by a phenotypic permutation test reiterated 100, 1000 or 10,000 times on a computer. For this computer simulation, data from the ALT+ and ALT− populations were pooled and randomly allocated to two groups which contained the same number of individuals as the ALT+ and ALT− populations used to produce the data summarized in Tables 19 and 20. A haplotype analysis was then run on the artificial groups for the 4 markers included in haplotype No. 8 from Table 17 and on the artificial groups for the 4 markers included in haplotype No. 13 from Table 18, which showed the strongest association with secondary effects to zileuton. This experiment was reiterated 1000 and 10,000 times and the results are shown in Table 21 and Table 22, resepectively. These results demonstrate that among 1000 iterations only 5 and among 10,000 iterations only 77 of the obtained haplotypes from the 12 biallelic markers had a p-value comparable to the one obtained for haplotype No. 8. These results demonstrate that among 100 iterations only 3 of the obtained haplotypes from the 17 biallelic markers had a p-value comparable to the one obtained for haplotype No. 11. The p-value obtained by permutating affected status for the omnibus LR test is 2.2.10−2. These results clearly validate the statistical significance of the association between hepatotoxicity to ZyfloTM and the haplotypes Nos. 3-25 and Nos. 6-30 shown in Table 19 and Table 20, respectively.
Allele Frequency Analysis
Allele frequencies were determined in a random US caucasian population, in an asthmatic population showing no side effects upon treatment with ZyfloTM (ALT−) and in an asthmatic population showing elevated alanine aminotransferase levels upon treatment with ZyfloTM (ALT+). Table 23 is a chart containing a list of preferred 12-LO-related biallelic markers with an indication of the frequency of the least common allele determined by genotyping as described in Example 2.
Example 6
Identification of Mutations and of Low Frequency Alleles of the 12-LO Gene
Exons 6, 8 and 14 of the 12-lipoxygenase gene were screened for mutations by comparing their sequence in individuals exhibiting elevated ALT levels upon treatment with zileuton (ALT+) and in individuals showing normal ALT levels upon treatment with zileuton (ALT−). ALT+ and ALT− individuals are further described in Example 5. Intron sequences immediately flanking these exons were also screened.
To identify mutations, fragments of the 12-LO gene were amplified, sequenced and compared in ALT+ and ALT− individuals. DNA samples from each individual were processed separately.
DNA Samples
Individual DNA samples were obtained as described in Example 1.
Amplification of the 12-LO Gene
Amplification primers are described in Table 13. PCR assays were performed as described in Example 1.
Sequencing of Amplified Genomic DNA: Identification of Mutations and of Low Frequency Polymorphisms
Sequencing of the amplified DNA was carried out on ABI 377 sequencers. The sequences of the amplification products were determined using automated dideoxy terminator sequencing reactions with a dye terminator cycle sequencing protocol. The products of the sequencing reactions were run on sequencing gels and the sequences were determined using gel image analysis (ABI Prism DNA Sequencing Analysis software 2.1.2 version).
The sequence data was further analyzed to detect the presence of mutations and of low frequency alleles. The sequences of exon 6, exon 8, exon 14 and flanking intronic sequences in 79 ALT+ individuals and 105 ALT− individuals were compared. New polymorphisms/mutations were detected and the genotype of each individual for these markers was determined. Results are shown below:

















Position in 12-LO
Least Common
Original



Marker ID
Gene
Allele/Mutation
Allele









10-508-191
5′ flanking region
C
T



10-508-245
5′ flanking region
T
C



10-511-62
5′ flanking region
T
C



10-511-337
5′ flanking region
Insertion T
—



10-512-36
5′ flanking region
C
G



10-512-318
5′ flanking region
A
G



10-513-250
5′ flanking region
A
G



10-513-262
5′ flanking region
T
C



10-513-352
5′ flanking region
A
G



10-513-365
5′ flanking region
A
G



10-343-231
Exon 2
Deletion C
—



10-343-366
Intron 2
C
T



10-343-278
Intron 2
T
C



10-343-339
Intron 4
T
G



10-346-23
Intron 4
G
A



10-346-141
Exon 5
A
G



10-346-263
Intron 5
G
C



10-346-305
Intron 5
C
T



10-347-74
Intron 5
A
G



10-347-111
Exon 6
G
C



10-347-165
Exon 6
T
C



10-347-203
Exon 6
G
A



10-347-220
Exon 6
A
G



10-347-271
Intron 6
T
A



10-347-348
Intron 6
A
G



10-348-391
Intron 7
A
G



10-349-47
Intron 7
C
T



10-349-97
Exon 8
G
A



10-349-142
Exon 8
G
C



10-349-216
Exon 8
Deletion CTG
—



10-349-224
Exon 8
T
G



10-349-368
Intron 8
C
T



10-350-72
Intron 8
T
C



10-350-332
Intron 9
C
T



10-507-170
Exon 11
G
A



10-507-321
Intron 11
A
C



10-507-353
Intron 11
T
C



10-507-364
Intron 11
T
C



10-507-405
Intron 11
T
C



10-339-32
Intron 11
T
C



10-339-124
Intron 11
T
C



10-340-112
Exon 13
A
C



10-340-130
Exon 13
A
T



10-340-238
Intron 13
A
G



10-341-116
Exon 14
A
G



10-341-319
Exon 14
T
C




(5′UTR)



10-342-301
3′ flanking region
Insertion A
—



10-342-373
3′ flanking region
T
C










Low frequency polymorphisms and mutations identified in exons 5, 6, 8, and 13 are associated with amino acid substitutions at the polypeptide level. In each of these amino acid substitutions the original residue is replaced by a non-equivalent amino acid presenting different chemical properties. As a consequence, specificity, activity and function of the 12-LO enzyme are modified. Biallelic marker 10-343-231 is associated with a frame shift in the open reading frame of the 12-LO gene leading to the expression of a variant 12-LO polypeptide comprising only 131 amino acids. This mutant 12-LO enzyme is probably inactive or shows differences in specificity, activity and function. Biallelic marker 10-343-231 is associated with the deletion of a Leu residue in the 12-LO polypeptide.
The mutations and low frequency polymorphisms listed above represent potential functional mutations of the 12-LO gene.
Example 7
15 Preparation of Antibody Compositions to 12-lipoxygenase Variants
Preferably antibody compositions, specifically binding the 189-His variant of the 12-LO protein or, to the 225-His variant of the 12-LO protein or, to the 243-Cys variant of the 12-LO protein or, to the 261-Arg variant of the 12-LO protein or, to the 322-Asn variant of the 12-LO or, to the 337-Arg variant of the 12-LO protein or to the 574-Lys variant of 12-LO, are prepared. Other preferred antibody compositions of the invention are capable of specifically binding to amino acid positions 110-131 of SEQ ID No. 654.
Substantially pure protein or polypeptide is isolated from transfected or transformed cells containing an expression vector encoding the 12-LO protein or a portion thereof. The concentration of protein in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms per ml. Monoclonal or polyclonal antibodies to the protein can then be prepared as follows:
Monoclonal Antibody Production by Hybridoma Fusion
Monoclonal antibody to epitopes in the 12-LO protein or a portion thereof can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (Nature, 256:495, 1975, the disclosure of which is incorporated herein by reference in its entirety) or derivative methods thereof (see Harlow and Lane, Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, pp. 53-242, 1988, the disclosure of which is incorporated herein by reference in its entirety).
Briefly, a mouse is repetitively inoculated with a few micrograms of the 12-LO protein or a portion thereof over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall, E., Meth. Enzymol. 70:419 (1980), , the disclosure of which is incorporated herein by reference in its entirety, and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis, L. et al. Basic Methods in Molecular Biology Elsevier, New York. Section 21-2, the disclosure of which is incorporated herein by reference in its entirety.
Polyclonal Antibody Production by Immunization
Polyclonal antiserum containing antibodies to heterogeneous epitopes in the 12-LO protein or a portion thereof can be prepared by immunizing suitable non-human animal with the 12-LO protein or a portion thereof, which can be unmodified or modified to enhance immunogenicity. A suitable non-human animal is preferably a non-human mammal is selected, usually a mouse, rat, rabbit, goat, or horse. Alternatively, a crude preparation which, has been enriched for 12-LO concentration can be used to generate antibodies. Such proteins, fragments or preparations are introduced into the non-human mammal in the presence of an appropriate adjuvant (e.g. aluminum hydroxide, RIBI, etc.) which is known in the art. In addition the protein, fragment or preparation can be pretreated with an agent which will increase antigenicity, such agents are known in the art and include, for example, methylated bovine serum albumin (mBSA), bovine serum albumin (BSA), Hepatitis B surface antigen, and keyhole limpet hemocyanin (KLH). Serum from the immunized animal is collected, treated and tested according to known procedures. If the serum contains polyclonal antibodies to undesired epitopes, the polyclonal antibodies can be purified by immunoaffinity chromatography.
Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. Also, host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable. Techniques for producing and processing polyclonal antisera are known in the art, see for example, Mayer and Walker (1987), the disclosure of which is incorporated herein by reference in its entirety. An effective immunization protocol for rabbits can be found in Vaitukaitis, J. et al. J. Clin. Endocrinol. Metab. 33:988-991 (1971), the disclosure of which is incorporated herein by reference in its entirety. Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony, O. et al., Chap. 19 in: Handbook of Experimental Immunology D. Wier (ed) Blackwell (11973), the disclosure of which is incorporated herein by reference in its entirety. Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 μM). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, D., Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980), the disclosure of which is incorporated herein by reference in its entirety.
Antibody preparations prepared according to either the monoclonal or the polyclonal protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample. The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.
Example 8
Forensic Matching by Microsequencing
DNA samples are isolated from forensic specimens of, for example, hair, semen, blood or skin cells by conventional methods. A panel of PCR primers based on a number of the sequences of of the invention is then utilized according to the methods described herein to amplify DNA of approximately 500 bases in length from the forensic specimen. The alleles present at each of the selected biallelic markers site according to biallelic markers of the invention are then identified according Examples discussed herein. A simple database comparison of the analysis results determines the differences, if any, between the sequences from a subject individual or from a database and those from the forensic sample. In a preferred method, statistically significant differences between the suspect's DNA sequences and those from the sample conclusively prove a lack of identity. This lack of identity can be proven, for example, with only one sequence. Identity, on the other hand, should be demonstrated with a large number of sequences, all matching. Preferably, a minimum of 13, 17, 20, 25, 30, 40, 50, 66, 70, 85, 88, 100, 187, 200 or 500 biallelic markers are used to test identity between the suspect and the sample.
The disclosures of all issued patents, published PCT applications, scientific references or other publications cited herein are incorporated herein by reference in their entireties.
Although this invention has been described in terms of certain preferred embodiments, other embodiments which will be apparent to those of ordinary skill in the art of view of the disclosure herein are also within the scope of this invention. Accordingly, the scope of the invention is intended to be defined only by reference to the appended claims.
In accordance with the regulations relating to Sequence Listings, the following codes have been used in the Sequence Listing to indicate the locations of biallelic markers within the sequences and to identify each of the alleles present at the polymorphic base. The code “r” in the sequences indicates that one allele of the polymorphic base is a guanine, while the other allele is an adenine. The code “y” in the sequences indicates that one allele of the polymorphic base is a thymine, while the other allele is a cytosine. The code “m” in the sequences indicates that one allele of the polymorphic base is an adenine, while the other allele is a cytosine. The code “k” in the sequences indicates that one allele of the polymorphic base is a guanine, while the other allele is a thymine. The code “s” in the sequences indicates that one allele of the polymorphic base is a guanine, while the other allele is a cytosine. The code “w” in the sequences indicates that one allele of the polymorphic base is an adenine, while the other allele is a thymine.
In some instances, the polymorphic bases of the biallelic markers alter the identity of amino acids in the encoded polypeptide. This is indicated in the accompanying Sequence Listing by use of the feature VARIANT, placement of a Xaa at the position of the polymorphic amino acid, and definition of Xaa as the two alternative amino acids. For example, if one allele of a biallelic marker is the codon CAC, which encodes histidine, while the other allele of the biallelic marker is CAA, which encodes glutamine, the Sequence Listing for the encoded polypeptide will contain an Xaa at the location of the polymorphic amino acid. In this instance, Xaa would be defined as being histidine or glutamine.
In other instances, Xaa may indicate an amino acid whose identity is unknown because of nucleotide sequence ambiguity. In this instance, the feature UNSURE is used, Xaa is placed at the position of the unknown amino acid, and Xaa is defined as being any of the 20 amino acids or a limited number of amino acids suggested by the genetic code.







TABLE 7A







List of all of the eicosanoid-related biallelic markers.















BIALLELIC

GENOTYPING





MARKER
VALIDATION
LEAST COMMON ALLELE



BIALLELIC
SEQ ID
POSITION IN
MICRO-
FREQUENCY


GENE
MARKER ID
NO.
SEQ ID NO.
SEQUENCING
%
















FLAP
10-253-118
1
478
N




FLAP
10-253-298
2
478
Y
G
4.57


FLAP
10-253-315
3
478
N


FLAP
10-499-155
4
478
N


FLAP
10-520-256
5
478
N
T
40.8


FLAP
10-500-258
6
478
N


FLAP
10-500-410
7
478
N


FLAP
10-503-159
8
478
N


FLAP
10-504-172
9
478
N


FLAP
10-504-243
10
478
N


FLAP
10-204-326
11
478
Y
A
6.63


FLAP
10-32-357
12
478
Y
A
33.5


FLAP
10-33-175
13
478
Y
T
2.30


FLAP
10-33-211
14
478
N


FLAP
10-33-234
15
478
Y
A
44.0


FLAP
10-33-270
16
478
Y
G/G


FLAP
10-33-327
17
478
Y
C
24.5


FLAP
10-34-290
18
478
N


FLAP
10-35-358
19
478
Y
C
31.3


FLAP
10-35-390
20
478
Y
T
23.0


FLAP
10-36-164
21
478
Y
G/G


FLAP
10-498-192
22
478
N


FLAP
12-628-306
23
478
Y
T
10.3


FLAP
12-628-311
24
478
N


FLAP
12-629-241
25
478
Y
C
28.3


12-LO
12-206-366
26
478
Y
C
38.2


12-LO
10-343-339
27
478
N


12-LO
10-347-74
28
478
N


12-LO
10-347-111
29
478
N
G/G


12-LO
10-347-165
30
478
N
C/C


12-LO
10-347-203
31
478
Y
G
41.6


12-LO
10-347-220
32
478
Y
A
40.5


12-LO
10-347-271
33
478
N


12-LO
10-347-348
34
478
N


12-LO
10-348-391
35
478
N


12-LO
10-349-47
36
478
N


12-LO
10-349-97
37
478
Y
G
39.6


12-LO
10-349-142
38
478
N
C/C


12-LO
10-349-224
39
478
Y
T
39.6


12-LO
10-349-368
40
478
N


12-LO
10-339-32
41
478
N


12-LO
10-341-116
42
478
Y
A
10.8


12-LO
10-341-319
43
478
N


12-LO
12-196-119
44
119
Y
C
29.1


12-LO
12-197-244
45
243
Y
C
32.8


12-LO
12-198-128
46
128
N


12-LO
12-206-81
47
478
N


12-LO
12-208-35
48
35
Y
A
42.3


12-LO
12-214-129
49
129
Y
C
38.7


12-LO
12-214-151
50
151
N


12-LO
12-214-360
51
358
N


12-LO
12-215-467
52
466
N


12-LO
12-216-421
53
418
Y
A
36.0


12-LO
12-219-230
54
229
Y
G
32.1


12-LO
12-219-256
55
255
N


12-LO
12-220-48
56
478
N


12-LO
12-221-302
57
302
N


12-LO
12-223-179
58
179
N


12-LO
12-223-207
59
207
Y
C
38.4


12-LO
12-225-541
60
540
Y
C
37.4


12-LO
12-226-167
61
166
Y
G
41.2


12-LO
12-226-458
62
455
N


12-LO
12-229-332
63
332
N


12-LO
12-229-351
64
351
N


12-LO
12-230-364
65
364
N


12-LO
12-231-100
66
99
N


12-LO
12-231-148
67
147
N


12-LO
12-231-266
68
265
N


cPLA2
10-231-23
69
500
Y
A
8.79


cPLA2
10-233-386
70
501
Y
G
28.3


cPLA2
10-239-368
72
501
N


cPLA2
10-223-30
73
501
Y
G
22.5


cPLA2
10-223-72
74
501
N


cPLA2
10-223-130
75
501
N


cPLA2
10-223-262
76
501
N


cPLA2
10-223-392
77
501
N


cPLA2
10-224-341
78
501
N


cPLA2
10-227-282
79
501
Y
G
3.93


ANX1
10-240-241
80
501
N


ANX1
10-249-185
81
501
N


ANX1
10-251-128
82
501
N


ANX1
10-252-209
83
501
N


ANX1
12-387-32
84
501
Y
G
33.9


ANX1
10-242-316
85
500
N


ANX1
10-245-412
86
501
N


ANX1
12-378-171
87
501
N


ANX1
12-378-228
88
501
N


ANX1
12-378-450
89
501
N


ANX1
12-379-65
90
501
N


ANX1
12-382-204
91
501
Y
G
50.0


ANX1
12-383-117
92
501
N


ANX1
12-383-170
93
501
N


ANX1
12-383-268
94
501
N


ANX1
12-384-336
95
501
N


ANX1
12-384-451
96
501
N


ANX1
12-385-123
97
258
N


ANX1
12-385-427
98
501
N


ANX1
12-386-155
99
443
Y
G
8.15


ANX1
12-386-24
100
313
N


ANX1
12-387-177
101
501
Y
T
33.5


ANX1
12-389-431
102
501
N


ANX1
12-391-366
103
501
N


ANX1
12-394-85
104
501
N


ANX1
12-395-382
105
385
N


ANX1
12-400-217
106
501
Y
G
27.2


ANX1
12-400-280
107
501
N


ANX1
12-401-378
108
380
N


ANX1
12-402-126
109
323
N


ANX1
12-404-265
110
317
N


ANX1
12-406-52
111
501
N


ANX1
12-406-409
112
501
N


ANX1
12-407-217
113
501
N


ANX1
12-407-399
114
501
N


ANX1
12-408-355
115
501
Y
G
2.69


ANX1
12-409-221
116
229
N


ANX1
12-410-301
117
486
N


ANX2
10-395-101
118
501
N


ANX2
10-395-124
119
501
N


ANX2
10-395-155
120
501
N


ANX2
10-395-294
121
501
N


ANX2
10-396-100
122
501
N


ANX2
10-397-201
123
501
N


ANX2
10-399-178
124
501
N


ANX2
10-400-369
125
501
N


ANX2
10-392-20
126
497
N


ANX2
10-392-103
127
501
N


ANX2
10-392-324
128
501
N


ANX2
10-393-27
129
501
N


ANX2
10-393-324
130
501
N


ANX2
12-727-237
131
501
N


ANX2
12-728-224
132
501
N


ANX2
12-730-142
133
501
N


ANX2
12-730-193
134
501
N


ANX2
12-731-60
135
501
N


ANX2
12-731-119
136
501
N


ANX2
12-731-137
137
501
N


ANX2
12-731-146
138
501
N


ANX2
12-731-398
139
501
N


ANX2
12-732-113
140
501
N


ANX2
12-732-164
141
501
N


ANX2
12-732-165
142
501
Y
C
27.4


ANX2
12-732-445
143
501
N


ANX2
12-734-201
144
501
N


ANX2
12-735-42
145
501
N


ANX2
12-736-363
146
501
N


ANX2
12-737-69
147
501
Y
A
36.8


ANX2
12-737-296
148
501
N


ANX2
12-738-429
149
501
Y
T
35.5


ANX2
12-740-112
150
501
Y
G
37.6


ANX2
12-740-118
151
501
N


ANX2
12-741-265
152
501
N


ANX2
12-741-327
153
501
N


ANX2
12-741-376
154
501
N


ANX2
12-745-30
155
501
N


ANX2
12-745-75
156
501
N


ANX2
12-745-343
157
501
N


ANX2
12-745-350
158
501
N


ANX2
12-746-320
159
501
N


ANX2
12-747-181
160
501
N


ANX2
12-747-302
161
501
N


ANX2
12-749-240
162
501
N


ANX2
12-749-255
163
501
N


ANX2
12-752-37
164
508
N


ANX2
12-752-85
165
501
N


ANX2
12-752-196
166
501
N


ANX2
12-752-484
167
501
N


ANX2
12-753-139
168
501
N


ANX2
12-753-376
169
501
N


ANX2
12-754-172
170
501
N


ANX2
12-754-218
171
501
N


ANX2
12-754-328
172
501
N


ANX2
12-754-396
173
501
N


ANX2
12-755-280
174
501
N


ANX2
12-757-384
175
501
N


ANX2
12-758-257
176
501
N


ANX2
12-758-374
177
501
N


ANX2
12-758-424
178
501
N


ANX2
12-761-23
179
541
N


ANX2
12-761-178
180
501
N


ANX2
12-764-329
181
501
N


ANX2
12-764-377
182
501
N


ANX2
12-765-168
183
501
N


ANX2
12-765-504
184
501
N


ANX3
10-372-279
185
501
N


ANX3
10-375-136
186
501
N


ANX3
10-376-281
187
501
N


ANX3
10-369-392
188
501
N


ANX3
10-371-257
189
501
N


ANX3
12-513-389
190
501
N


ANX3
12-513-494
191
501
N


ANX3
12-515-394
192
501
N


ANX3
12-516-97
193
501
Y
T
37.2


ANX3
12-520-287
194
501
N


ANX3
12-520-323
195
501
Y
A
21.5


ANX3
12-523-179
196
501
Y
A
29.9


ANX3
12-523-270
197
501
N


ANX3
12-527-367
198
501
Y
T
18.9


ANX3
12-529-376
199
501
N


ANX3
12-529-489
200
501
N


ANX3
12-530-134
201
501
Y
T
39.3


ANX3
12-530-393
202
501
N


ANX3
12-531-173
203
501
Y
C
37.6


ANX3
12-539-441
204
501
N


ANX3
12-543-78
205
501
N


ANX3
12-543-79
206
501
N


ANX3
12-546-235
207
501
N


ANX3
12-549-287
208
501
N


ANX3
12-550-287
209
501
N


ANX3
12-552-175
210
501
N


ANX3
12-554-330
211
501
N


ANX3
12-556-312
212
501
N


ANX3
12-556-443
213
501
N


ANX3
12-558-205
214
501
N


ANX3
12-558-238
215
501
N


ANX3
12-558-305
216
501
N


ANX3
12-769-39
217
501
N


ANX3
12-769-430
218
501
N


ANX3
12-770-73
219
501
N


ANX3
12-772-200
220
501
N


ANX3
12-772-254
221
501
N


CAL1
10-87-73
222
72
N


CAL1
10-87-74
223
73
N


CAL1
10-87-80
224
79
N


CAL1
10-87-140
225
138
N


CAL1
10-88-81
226
81
Y
C
44.7


CAL1
10-89-41
227
41
N


CAL1
10-90-35
228
35
Y
A
1.14


CAL1
10-91-274
229
274
N


CAL1
10-93-133
230
133
N


CAL1
10-94-197
231
197
Y
G/G


CAL1
10-94-198
232
198
N


CAL1
10-166-362
233
362
N


CAL2
10-207-386
234
387
Y
C/C


CAL2
10-207-409
235
409
Y
G
9.04


CAL2
10-118-307
236
307
Y
A
0.27


CAL2
10-173-247
237
247
N


CAL2
10-173-294
238
294
Y
G
2.87


CAL2
10-173-347
239
347
Y
C/C


CAL3
10-103-104
240
104
N


CAL3
10-103-323
241
323
Y
T
22.3


CAL3
10-103-402
242
403
N


CAL3
10-106-98
243
98
N


CAL3
10-106-288
244
288
Y


CAL3
10-106-378
245
380
Y


CAL3
10-168-160
246
160
Y
T
42.1


CAL3
10-168-206
247
206
Y


CAL3
10-168-284
248
283
N


CAL3
10-169-318
249
317
N


CALPA1
12-86-79
250
501
Y
C
37.4


CALPA1
12-88-393
251
501
N


CALPA1
12-89-369
252
501
Y
G
36.3


CALPA1
12-89-91
253
501
N


CALPA1
12-94-210
254
501
N


CALPA1
12-94-516
255
521
N


CALPA1
12-96-64
256
501
Y
T
8.52


CALPA1
12-97-83
257
501
N


CALPA1
12-99-296
258
501
Y
T
6.45


CALPA1
12-100-266
259
501
Y
G
32.2


CALPA1
12-811-174
260
501
N


CALPA1
12-815-94
261
501
N


CALPA1
12-815-383
262
501
N


CALPA1
12-815-384
263
500
N


CALPA1
12-815-391
264
501
N


CALPA1
12-817-214
265
501
N


CALPA1
12-817-355
266
501
N


CALPA1
12-819-437
267
501
N


CALPA1
12-821-62
268
501
N


CALPA1
12-821-483
269
501
N


CALPA1
12-825-173
270
501
N


CALPA1
12-826-312
271
501
N


CALPA1
12-831-59
272
501
N


CALPA1
12-833-264
273
501
N


CALPA1
12-833-279
274
501
N


CALPA1
12-833-280
275
502
N


CALPA1
12-833-373
276
501
N


CALPA1
12-834-183
277
483
N


CALPA1
12-835-54
278
501
N


CALPA1
12-836-134
279
501
N


CALPA1
12-836-237
280
500
N


CALPA1
12-836-238
281
476
N


CALPA1
12-836-257
282
498
N


CALPA1
12-836-275
283
501
N


CALPA1
12-838-179
284
501
N


CALPA1
12-839-397
285
501
N


CALPA1
12-840-47
286
501
N


CALPA1
12-840-77
287
501
N


CALPA1
12-841-445
288
445
N


CALPA1
12-842-215
289
501
N


CALPA1
12-842-447
290
499
N


CALPA1
12-844-167
291
501
N


CALPA1
12-845-364
292
501
N


CALPA1
12-846-209
293
501
N


CALPA1
12-847-123
294
501
N


CALPA1
12-849-242
295
501
N


CYP2J2
10-336-58
296
501
N


CYP2J2
10-336-137
297
501
N


CYP2J2
10-336-232
298
501
N


CYP2J2
12-102-104
299
379
N


CYP2J2
12-102-111
300
386
N


CYP2J2
12-102-275
301
501
N


CYP2J2
12-103-202
302
501
Y
C
14.3


CYP2J2
12-103-214
303
501
N


CYP2J2
12-104-351
304
501
Y
T
27.4


CYP2J2
12-105-435
305
439
N


CYP2J2
12-109-149
306
278
Y
A
8.51


CYP2J2
12-109-197
307
326
N


CYP2J2
12-109-209
308
338
N


CYP2J2
12-109-284
309
413
N


CYP2J2
12-113-276
310
501
Y
G
31.2


CYP2J2
12-115-57
311
501
Y
G
8.87


CYP2J2
12-119-26
312
501
Y
G
29.8


COX1
12-347-308
313
501
N


COX1
12-354-334
314
501
Y
C/C


COX1
12-357-140
315
501
Y
C
7.14


COX1
12-361-320
316
501
Y
G
18.3


COX1
12-361-388
317
501
Y
A
18.5


COX1
12-365-251
318
501
Y
C
18.8


COX1
12-374-261
319
501
Y
T
21.3


COX1
10-308-116
320
501
N


COX1
10-311-274
321
501
N


COX1
10-314-76
322
501
N


COX1
10-306-265
323
501
N


COX2
10-52-386
324
386
N


COX2
10-62-240
325
240
Y
C
12.23


COX2
10-65-276
326
276
Y


COX2
10-67-42
327
42
N


COX2
10-67-340
328
341
Y


COX2
10-55-265
329
264
Y
C
40.9


COX2
10-57-278
330
278
Y


COX2
10-59-176
331
176
Y


COX2
10-60-114
332
114
N


PGDS
10-27-176
333
176
Y
A
5.32


PGDS
10-28-242
334
242
Y


PGDS
10-30-349
335
350
Y
A/A


PGDS
10-181-42
336
42
Y
C
30.2


PGDS
10-181-372
337
374
Y
C
26.3


PGDS
10-183-260
338
259
N


PG15OH
10-475-163
339
501
N


PG15OH
12-884-203
340
501
Y
T
29.7


PG15OH
10-479-266
341
501
N


PG15OH
10-479-350
342
501
N


PG15OH
10-479-394
343
501
N


PG15OH
10-482-145
344
501
N


PG15OH
12-854-64
345
501
N


PG15OH
12-854-472
346
501
N


PG15OH
12-855-194
347
501
N


PG15OH
12-855-288
348
501
N


PG15OH
12-855-423
349
501
N


PG15OH
12-857-25
350
476
N


PG15OH
12-858-346
351
501
Y
T
37.2


PG15OH
12-858-443
352
501
N


PG15OH
12-860-388
353
501
N


PG15OH
12-861-270
354
501
N


PG15OH
12-862-349
355
501
N


PG15OH
12-862-365
356
501
N


PG15OH
12-862-452
357
501
N


PG15OH
12-866-423
358
501
Y
C
46.2


PG15OH
12-867-47
359
501
N


PG15OH
12-868-181
360
501
N


PG15OH
12-868-198
361
501
N


PG15OH
12-868-282
362
501
N


PG15OH
12-869-128
363
501
N


PG15OH
12-870-491
364
501
N


PG15OH
12-872-52
365
501
N


PG15OH
12-872-293
366
501
N


PG15OH
12-873-185
367
501
N


PG15OH
12-873-319
368
501
N


PG15OH
12-875-248
369
501
Y
G
28.8


PG15OH
12-876-265
370
501
N


PG15OH
12-876-280
371
501
N


PG15OH
12-876-454
372
501
N


PG15OH
12-877-59
373
501
N


PG15OH
12-877-69
374
501
N


PG15OH
12-877-79
375
501
N


PG15OH
12-878-153
376
501
N


PG15OH
12-878-419
377
501
N


PG15OH
12-879-67
378
501
N


PG15OH
12-879-439
379
501
N


PG15OH
12-881-210
380
501
N


PG15OH
12-881-389
381
501
N


PG15OH
12-883-273
382
501
N


PG15OH
12-885-196
383
501
N


PG15OH
12-885-333
384
501
N


PG15OH
12-885-407
385
501
N


PG15OH
12-885-410
386
501
N


PG15OH
12-886-195
387
501
Y
A
21.1


PG15OH
12-886-348
388
501
N


PG15OH
12-887-201
389
501
N


PG15OH
12-887-467
390
501
N


PG15OH
12-888-98
391
501
N


PG15OH
12-888-203
392
501
Y
G
38.3


PG15OH
12-888-315
393
501
N


PG15OH
12-889-518
394
479
N


PG15OH
12-894-266
395
501
N


PG15OH
12-895-391
396
501
Y
C
34.6


PG15OH
12-896-140
397
501
N


PG15OH
12-897-115
398
501
N


PG15OH
12-897-225
399
501
N


PG15OH
12-898-49
400
528
N


CYP8
12-164-119
401
501
Y
C
11.8


CYP8
12-168-84
402
501
Y
T
20.1


CYP8
12-168-365
403
501
N


CYP8
12-170-299
404
501
Y
T
6.52


CYP8
12-171-360
405
501
Y
T
8.70


CYP8
12-173-59
406
501
Y
G
26.0


CYP8
12-175-214
407
501
Y
A
10.1


CYP8
12-177-183
408
501
Y
G
25.4


CYP8
12-177-366
409
501
N


TAX2
10-128-45
410
45
Y
T/T


TAX2
10-128-63
411
63
N


TAX2
10-123-177
412
177
N


TAX2
10-123-402
413
402
N


TAX2
10-120-137
414
136
Y
A
1.60


TAX2
10-120-141
415
140
Y
A
3.09


TAX2
10-179-39
416
39
N


TAX2
10-180-65
417
65
Y
C
44.7


TAX2
10-179-257
418
257
Y


15-LOA
10-43-124
419
123
N


15-LOA
10-43-134
420
133
N


15-LOA
10-43-193
421
192
N


15-LOA
10-43-195
422
194
N


15-LOA
10-43-233
423
232
N


15-LOA
10-43-138
424
137
Y


15-LOA
10-46-372
425
369
Y
T
2.43


15-LOA
10-46-36
426
35
N


15-LOA
10-47-103
427
102
Y


15-LOA
10-47-125
428
124
Y
T
5.68


15-LOA
10-48-184
429
183
Y
T
28.0


15-LOA
10-48-381
430
382
Y
T
31.4


15-LOA
10-49-33
431
33
Y
T
14.3


15-LOA
10-39-148
432
150
Y
G
14.5


15-LOA
10-40-222
433
222
Y
A
47.6


15-LOA
10-40-252
434
250
N


15-LOA
10-42-354
435
354
Y


15-LOA
10-154-42
436
42
N


15-LOA
10-154-156
437
156
Y
T
24.2


15-LOA
10-154-226
438
226
N


15-LOB
12-776-259
439
501
N


5-LO
10-384-109
440
501
N


5-LO
12-296-388
441
501
Y
G
37.6


5-LO
10-388-379
442
501
N


5-LO
10-389-116
443
501
N


5-LO
10-389-349
444
501
N


5-LO
10-391-94
445
501
N


5-LO
12-277-147
446
501
Y
T
44.9


5-LO
12-278-413
447
501
Y
A
33.9


5-LO
12-288-190
448
501
N


5-LO
12-289-35
449
501
N


5-LO
12-296-119
450
501
N


5-LO
12-297-291
451
501
N


5-LO
12-298-105
452
501
N


5-LO
12-300-126
453
501
N


5-LO
12-300-410
454
501
N


5-LO
12-301-379
455
501
N


5-LO
12-302-264
456
501
N


5-LO
12-309-405
457
501
N


5-LO
12-310-105
458
501
N


5-LO
12-314-453
459
501
Y
A
18.8


5-LO
12-316-292
460
501
Y
C
40.8


LTA4H
10-281-314
461
501
N


LTA4H
10-268-381
462
501
N


LTA4H
12-54-297
463
501
Y
C
9.34


LTA4H
10-276-407
464
501
N


LTA4H
12-44-50
465
501
Y
A
25.9


LTA4H
12-44-67
466
501
N


LTA4H
12-45-145
467
501
N


LTA4H
12-45-166
468
501
N


LTA4H
12-45-305
469
501
N


LTA4H
12-46-92
470
501
Y
G
31.9


LTA4H
12-47-132
471
501
Y
C
4.84


LTA4H
12-47-61
472
501
N


LTA4H
12-48-100
473
501
N


LTA4H
12-48-323
474
501
N


LTA4H
12-48-369
475
501
N


LTA4H
12-48-37
476
501
N


LTA4H
12-49-131
477
501
Y
A
40.1


LTA4H
12-49-53
478
501
N


LTA4H
12-49-64
479
501
N


LTA4H
12-51-234
480
501
Y
A
43.3


LTA4H
12-51-253
481
501
N


LTA4H
12-51-370
482
501
N


LTA4H
12-52-400
483
501
N


LTA4H
12-57-192
484
501
Y
T
41.2


LTA4H
12-57-221
485
501
Y
T
4.40


LTA4H
12-57-510
486
501
N


LTB4H2
10-1-139
487
139
Y
G
36.3


LTB4H2
10-1-212
488
212
Y
T
16.3


LTB4H2
10-1-241
489
241
Y
A
5.84


LTB4H2
10-9-143
490
143
Y


LTB4H2
10-9-185
491
185
Y
T/T


LTB4H2
10-9-264
492
264
Y


LTB4H2
10-11-22
493
22
N


LTB4H2
10-13-152
494
152
Y
T
20.8


LTB4H2
10-13-256
495
256
Y


LTB4H2
10-13-282
496
282
Y
C
25.0


LTB4H2
10-15-281
497
281
N


LTB4H2
10-17-142
498
142
Y
C/C


LTB4H2
10-18-302
499
302
N


LTB4H2
10-23-331
500
331
N


LTB4H2
10-25-152
501
152
Y


LTB4H2
10-25-258
502
258
N


LTB4H2
10-3-103
503
103
Y
T
47.7


LTB4H2
10-3-144
504
144
Y


LTB4H2
10-3-275
505
275
Y


LTB4H2
10-5-227
506
227
Y
A
28.1


LTB4H2
10-7-155
507
155
Y
T
30.4


LTB4H2
10-7-383
508
381
N


LTB4H2
10-7-98
509
98
N


LTB412OH
12-561-270
510
501
Y
T
35.2


LTB412OH
12-563-87
511
501
Y
C
28.0


LTB412OH
12-564-64
512
501
Y
T
36.0


LTB412OH
12-564-214
513
501
N


LTB412OH
12-568-207
514
501
N


LTB412OH
12-568-365
515
501
N


LTB412OH
12-568-367
516
501
N


LTB412OH
12-571-337
517
501
Y
G
17.9


LTB412OH
12-573-378
518
501
Y
A
6.91


LTB412OH
10-294-256
519
501
N


LTB412OH
10-294-304
520
501
N


LTB412OH
10-295-201
521
501
N


LTB412OH
10-296-80
522
501
N


LTB412OH
10-296-373
523
501
N


LTB412OH
10-298-122
524
501
N


LTB412OH
10-298-158
525
501
N


LTB412OH
10-300-49
526
501
N


LTB412OH
10-300-185
527
501
N


LTB4H3
10-10-328
528
327
Y
A
12.5


LTB4H3
10-12-52
529
52
N


LTB4H3
10-14-46
530
46
Y
T
39.3


LTB4H3
10-19-358
531
357
Y


LTB4H3
10-20-111
532
110
Y
A
15.8


LTB4H3
10-20-274
533
273
Y
A/A


LTB4H3
10-24-90
534
90
Y
C
19.2


LTB4H3
10-24-204
535
204
Y
A
25.0


LTB4H3
10-24-221
536
221
N


LTB4H3
10-24-234
537
234
Y
A
36.1


LTB4H3
10-24-288
538
288
N


LTB4H3
10-24-311
539
311
N


LTB4H3
10-26-289
540
289
N


LTB4H3
10-8-39
541
39
Y


LTB4H3
10-8-120
542
120
N


LTB4H3
10-8-154
543
154
N


LTB4H3
10-8-101
544
101
Y


LTB4H3
10-8-86
545
86
Y


LTB4H3
10-8-92
546
92
N


LTB4H3
10-8-94
547
94
N


LTB4R
12-61-472
548
501
N


LTB4R
12-63-402
549
416
N


LTB4R
12-63-74
550
88
N


LTB4R
12-64-271
551
287
Y
C
28.6


LTB4R
12-65-98
552
439
N


LTB4R
12-70-147
553
501
Y
C
11.5


LTB4R
12-70-397
554
501
Y
T
39.7


LTB4R
12-71-320
555
501
Y
A
4.49


LTB4R
12-73-150
556
501
N


LTB4R
12-73-49
557
501
Y
A
43.3


LTB4R
12-73-56
558
501
N


LTB4R
12-74-38
559
501
Y
C
44.1


LTB4R
12-76-238
560
501
Y
T
20.6


LTB4R
12-77-217
561
501
N


LTB4R
12-77-478
562
501
Y
A
4.40


LTB4R
12-80-114
563
501
N


LTB4R
12-80-233
564
501
Y
C
4.55


LTB4R
12-82-250
565
250
N


LTC4
10-176-85
566
85
Y
T
0.54


LTC4
10-176-51
567
51
N


LTC4
10-176-207
568
207
N


LTC4
10-176-397
569
397
Y
A
1.63


LTC4
10-177-219
570
219
Y
C
29.0


12-LO
12-214-85
571
85
N


12-LO
12-215-272
572
271
N


12-LO
12-221-163
573
163
N


12-LO
12-225-82
574
82
N


cPLA2
10-234-179
575
214
Y
Deletion AA
32.6


cPLA2
10-235-272
576
491
N


ANX1
10-251-342
577
498
N


ANX2
10-395-367
578
497
N


ANX2
12-730-58
579
498
N


ANX2
12-735-208
580
412
Y
Deletion
21.5


ANX2
12-739-22
581
498
Y
Insertion G
23.4


ANX3
12-540-363
582
498
N


ANX3
12-550-206
583
497
N


CAL2
12-207-410
584
409
N


CAL3
10-171-254
585
255
N


CALPA1
12-94-110
586
498
Y
Deletion
32.5







AATT


CALPA1
12-834-290
587
498
N


COX2
10-55-115
588
114
Y
Deletion
3.01







TTATA


PG15OH
12-857-122
589
498
N


PG15OH
12-872-175
590
498
N


PG15OH
12-882-40
591
498
N


PG15OH
12-888-234
592
498
N


5-LO
12-278-353
593
499
N


5-LO
12-283-386
594
498
N


LTA4H
12-44-181
595
458
N


ANX3
10-370-132
596
501
N


ANX3
10-370-254
597
501
N


15PGDHB
10-485-256
598
501
N


15PGDHB
10-485-257
599
501
N


15PGDHB
10-474-320
600
501
N


5LO
10-387-371
601
501
N


LTB412OH
12-570-239
602
501
N


LTB412OH
12-570-344
603
501
N


LTB412OH
12-570-393
604
501
N


LTB412OH
12-570-421
605
501
N


LTB412OH
12-570-62
606
502
N


LTB4H3
10-4-144
607
141
N


LTB4H3
10-4-161
608
158
N


LTB4H3
10-4-270
609
267
N


LTB4H3
10-4-340
610
337
N


LTB4H3
10-4-369
611
366
N


LTB4H3
10-4-420
612
417
N


LTB4H2
10-13-396
613
396
N


12-LO
10-509-284
614
501
N


12-LO
10-509-295
615
501
N


12-LO
10-339-124
616
501
N


12-LO
10-340-112
617
501
N


12-LO
10-340-130
618
501
N


12-LO
10-340-238
619
501
N


12-LO
10-342-301
620
501
N


12-LO
10-342-373
621
501
N


12-LO
10-343-231
622
501
N


12-LO
10-343-278
623
501
N


12-LO
10-346-141
624
501
N
G/G


12-LO
10-346-23
625
500
N


12-LO
10-346-263
626
501
N


12-LO
10-346-305
627
501
N


12-LO
10-349-216
628
501
N


12-LO
10-350-332
629
501
N


12-LO
10-350-72
630
501
N


12-LO
10-507-170
631
501
N


12-LO
10-507-321
632
501
N


12-LO
10-507-353
633
501
N


12-LO
10-507-364
634
501
N


12-LO
10-507-405
635
501
N


12-LO
10-508-191
636
501
N


12-LO
10-508-245
637
501
N


12-LO
10-510-173
638
501
N


12-LO
10-511-337
639
501
N


12-LO
10-512-36
640
501
Y
C
39.4


12-LO
10-511-62
641
501
N


12-LO
10-512-318
642
501
N


12-LO
10-513-250
643
501
N


12-LO
10-513-262
644
501
N


12-LO
10-513-352
645
501
N


12-LO
10-513-365
646
501
N


FLAP
10-517-100
647
501
N


FLAP
10-518-125
648
501
N


FLAP
10-518-194
649
501
N


FLAP
10-522-71
650
501
N
















TABLE 7B







List of all of the eicosanoid-related biallelic markers (47mers)

















GENOTYPING





BIALLELIC

LEAST COMMON





MARKER
VALIDATION
ALLELE



BIALLELIC
SEQ ID
POSITION IN
MICRO-
FREQUENCY


GENE
MARKER ID
NO.
SEQ ID NO.
SEQUENCING
%
















FLAP
10-253-118
655
24
N




FLAP
10-253-298
656
24
Y
G
4.57


FLAP
10-253-315
657
24
N


FLAP
10-499-155
658
24
N


FLAP
10-520-256
659
24
N
T
40.8


FLAP
10-500-258
660
24
N


FLAP
10-500-410
661
24
N


FLAP
10-503-159
662
24
N


FLAP
10-504-172
663
24
N


FLAP
10-504-243
664
24
N


FLAP
10-204-326
665
24
Y
A
6.63


FLAP
10-32-357
666
24
Y
A
33.5


FLAP
10-33-175
667
24
Y
T
2.30


FLAP
10-33-211
668
24
N


FLAP
10-33-234
669
24
Y
A
44.0


FLAP
10-33-270
670
24
Y
G/G


FLAP
10-33-327
671
24
Y
C
24.5


FLAP
10-34-290
672
24
N


FLAP
10-35-358
673
24
Y
C
31.3


FLAP
10-35-390
674
24
Y
T
23.0


FLAP
10-36-164
675
24
Y
G/G


FLAP
10-498-192
676
24
N


FLAP
12-628-306
677
24
Y
T
10.3


FLAP
12-628-311
678
24
N


FLAP
12-629-241
679
24
Y
C
28.3


12-LO
12-206-366
680
24
Y
C
38.2


12-LO
10-343-339
681
24
N


12-LO
10-347-74
682
24
N


12-LO
10-347-111
683
24
N
G/G


12-LO
10-347-165
684
24
N
C/C


12-LO
10-347-203
685
24
Y
G
41.6


12-LO
10-347-220
686
24
Y
A
40.5


12-LO
10-347-271
687
24
N


12-LO
10-347-348
688
24
N


12-LO
10-348-391
689
24
N


12-LO
10-349-47
690
24
N


12-LO
10-349-97
691
24
Y
G
39.6


12-LO
10-349-142
692
24
N
C/C


12-LO
10-349-224
693
24
Y
T
39.6


12-LO
10-349-368
694
24
N


12-LO
10-339-32
695
24
N


12-LO
10-341-116
696
24
Y
A
10.8


12-LO
10-341-319
697
24
N


12-LO
12-196-119
698
24
Y
C
29.1


12-LO
12-197-244
699
24
Y
C
32.8


12-LO
12-198-128
700
24
N


12-LO
12-206-81
701
24
N


12-LO
12-208-35
702
24
Y
A
42.3


12-LO
12-214-129
703
24
Y
C
38.7


12-LO
12-214-151
704
24
N


12-LO
12-214-360
705
24
N


12-LO
12-215-467
706
24
N


12-LO
12-216-421
707
24
Y
A
36.0


12-LO
12-219-230
708
24
Y
G
32.1


12-LO
12-219-256
709
24
N


12-LO
12-220-48
710
24
N


12-LO
12-221-302
711
24
N


12-LO
12-223-179
712
24
N


12-LO
12-223-207
713
24
Y
C
38.4


12-LO
12-225-541
714
24
Y
C
37.4


12-LO
12-226-167
715
24
Y
G
41.2


12-LO
12-226-458
716
24
N


12-LO
12-229-332
717
24
N


12-LO
12-229-351
718
24
N


12-LO
12-230-364
719
24
N


12-LO
12-231-100
720
24
N


12-LO
12-231-148
721
24
N


12-LO
12-231-266
722
24
N


cPLA2
10-231-23
723
24
Y
A
8.79


cPLA2
10-233-386
724
24
Y
G
28.3


cPLA2
10-239-368
726
24
N


cPLA2
10-223-30
727
24
Y
G
22.5


cPLA2
10-223-72
728
24
N


cPLA2
10-223-130
729
24
N


cPLA2
10-223-262
730
24
N


cPLA2
10-223-392
731
24
N


cPLA2
10-224-341
732
24
N


cPLA2
10-227-282
733
24
Y
G
3.93


ANX1
10-240-241
734
24
N


ANX1
10-249-185
735
24
N


ANX1
10-251-128
736
24
N


ANX1
10-252-209
737
24
N


ANX1
12-387-32
738
24
Y
G
33.9


ANX1
10-242-316
739
24
N


ANX1
10-245-412
740
24
N


ANX1
12-378-171
741
24
N


ANX1
12-378-228
742
24
N


ANX1
12-378-450
743
24
N


ANX1
12-379-65
744
24
N


ANX1
12-382-204
745
24
Y
G
50.0


ANX1
12-383-117
746
24
N


ANX1
12-383-170
747
24
N


ANX1
12-383-268
748
24
N


ANX1
12-384-336
749
24
N


ANX1
12-384-451
750
24
N


ANX1
12-385-123
751
24
N


ANX1
12-385-427
752
24
N


ANX1
12-386-155
753
24
Y
G
8.15


ANX1
12-386-24
754
24
N


ANX1
12-387-177
755
24
Y
T
33.5


ANX1
12-389-431
756
24
N


ANX1
12-391-366
757
24
N


ANX1
12-394-85
758
24
N


ANX1
12-395-382
759
24
N


ANX1
12-400-217
760
24
Y
G
27.2


ANX1
12-400-280
761
24
N


ANX1
12-401-378
762
24
N


ANX1
12-402-126
763
24
N


ANX1
12-404-265
764
24
N


ANX1
12-406-52
765
24
N


ANX1
12-406-409
766
24
N


ANX1
12-407-217
767
24
N


ANX1
12-407-399
768
24
N


ANX1
12-408-355
769
24
Y
G
2.69


ANX1
12-409-221
770
24
N


ANX1
12-410-301
771
24
N


ANX2
10-395-101
772
24
N


ANX2
10-395-124
773
24
N


ANX2
10-395-155
774
24
N


ANX2
10-395-294
775
24
N


ANX2
10-396-100
776
24
N


ANX2
10-397-201
777
24
N


ANX2
10-399-178
778
24
N


ANX2
10-400-369
779
24
N


ANX2
10-392-20
780
24
N


ANX2
10-392-103
781
24
N


ANX2
10-392-324
782
24
N


ANX2
10-393-27
783
24
N


ANX2
10-393-324
784
24
N


ANX2
12-727-237
785
24
N


ANX2
12-728-224
786
24
N


ANX2
12-730-142
787
24
N


ANX2
12-730-193
788
24
N


ANX2
12-731-60
789
24
N


ANX2
12-731-119
790
24
N


ANX2
12-731-137
791
24
N


ANX2
12-731-146
792
24
N


ANX2
12-731-398
793
24
N


ANX2
12-732-113
794
24
N


ANX2
12-732-164
795
24
N


ANX2
12-732-165
796
24
Y
C
27.4


ANX2
12-732-445
797
24
N


ANX2
12-734-201
798
24
N


ANX2
12-735-42
799
24
N


ANX2
12-736-363
800
24
N


ANX2
12-737-69
801
24
Y
A
36.8


ANX2
12-737-296
802
24
N


ANX2
12-738-429
803
24
Y
T
35.5


ANX2
12-740-112
804
24
Y
G
37.6


ANX2
12-740-118
805
24
N


ANX2
12-741-265
806
24
N


ANX2
12-741-327
807
24
N


ANX2
12-741-376
808
24
N


ANX2
12-745-30
809
24
N


ANX2
12-745-75
810
24
N


ANX2
12-745-343
811
24
N


ANX2
12-745-350
812
24
N


ANX2
12-746-320
813
24
N


ANX2
12-747-181
814
24
N


ANX2
12-747-302
815
24
N


ANX2
12-749-240
816
24
N


ANX2
12-749-255
817
24
N


ANX2
12-752-37
818
24
N


ANX2
12-752-85
819
24
N


ANX2
12-752-196
820
24
N


ANX2
12-752-484
821
24
N


ANX2
12-753-139
822
24
N


ANX2
12-753-376
823
24
N


ANX2
12-754-172
824
24
N


ANX2
12-754-218
825
24
N


ANX2
12-754-328
826
24
N


ANX2
12-754-396
827
24
N


ANX2
12-755-280
828
24
N


ANX2
12-757-384
829
24
N


ANX2
12-758-257
830
24
N


ANX2
12-758-374
831
24
N


ANX2
12-758-424
832
24
N


ANX2
12-761-23
833
24
N


ANX2
12-761-178
834
24
N


ANX2
12-764-329
835
24
N


ANX2
12-764-377
836
24
N


ANX2
12-765-168
837
24
N


ANX2
12-765-504
838
24
N


ANX3
10-372-279
839
24
N


ANX3
10-375-136
840
24
N


ANX3
10-376-281
841
24
N


ANX3
10-369-392
842
24
N


ANX3
10-371-257
843
24
N


ANX3
12-513-389
844
24
N


ANX3
12-513-494
845
24
N


ANX3
12-515-394
846
24
N


ANX3
12-516-97
847
24
Y
T
37.2


ANX3
12-520-287
848
24
N


ANX3
12-520-323
849
24
Y
A
21.5


ANX3
12-523-179
850
24
Y
A
29.9


ANX3
12{circumflex over ( )}523-270
851
24
N


ANX3
12-527-367
852
24
Y
T
18.9


ANX3
12-529-376
853
24
N


ANX3
12-529-489
854
24
N


ANX3
12-530-134
855
24
Y
T
39.3


ANX3
12-530-393
856
24
N


ANX3
12-531-173
857
24
Y
C
37.6


ANX3
12-539-441
858
24
N


ANX3
12-543-78
859
24
N


ANX3
12-543-79
860
24
N


ANX3
12-546-235
861
24
N


ANX3
12-549-287
862
24
N


ANX3
12-550-287
863
24
N


ANX3
12-552-175
864
24
N


ANX3
12-554-330
865
24
N


ANX3
12-556-312
866
24
N


ANX3
12-556-443
867
24
N


ANX3
12-558-205
868
24
N


ANX3
12-558-238
869
24
N


ANX3
12-558-305
870
24
N


ANX3
12-5769-39
871
24
N


ANX3
12-769-430
872
24
N


ANX3
12-770-73
873
24
N


ANX3
12-772-200
874
24
N


ANX3
12-772-254
875
24
N


CAL1
10-87-73
876
24
N


CAL1
10-87-74
877
24
N


CAL1
10-87-80
878
24
N


CAL1
10-87-140
879
24
N


CAL1
10-88-81
880
24
Y
C
44.7


CAL1
10-89-41
881
24
N


CAL1
10-90-35
882
24
Y
A
1.14


CAL1
10-91-274
883
24
N


CAL1
10-93-133
884
24
N


CAL1
10-94-197
885
24
Y
G/G


CAL1
10-94-198
886
24
N


CAL1
10-166-362
887
24
N


CAL2
10-207-386
888
24
Y
C/C


CAL2
10-207-409
889
24
Y
G
9.04


CAL2
10-118-307
890
24
Y
A
0.27


CAL2
10-173-247
891
24
N


CAL2
10-173-294
892
24
Y
G
2.87


CAL2
10-173-347
893
24
Y
C/C


CAL3
10-103-104
894
24
N


CAL3
10-103-323
895
24
Y
T
22.3


CAL3
10-103-402
896
24
N


CAL3
10-106-98
897
24
N


CAL3
10-106-288
898
24
Y


CAL3
10-106-378
899
24
Y


CAL3
10-168-160
900
24
Y
T
42.1


CAL3
10-168-206
901
24
Y


CAL3
10-168-284
902
24
N


CAL3
10-169-318
903
24
N


CALPA1
12-86-79
904
24
Y
C
37.4


CALPA1
12-88-393
905
24
N


CALPA1
12-89-369
906
24
Y
G
36.3


CALPA1
12-89-91
907
24
N


CALPA1
12-94-210
908
24
N


CALPA1
12-94-516
909
24
N


CALPA1
12-96-64
910
24
Y
T
8.52


CALPA1
12-97-83
911
24
N


CALPA1
12-99-296
912
24
Y
T
6.45


CALPA1
12-100-266
913
24
Y
G
32.2


CALPA1
12-811-174
914
24
N


CALPA1
12-815-94
915
24
N


CALPA1
12-815-383
916
24
N


CALPA1
12-815-384
917
24
N


CALPA1
12-815-391
918
24
N


CALPA1
12-817-214
919
24
N


CALPA1
12-817-355
920
24
N


CALPA1
12-819-437
921
24
N


CALPA1
12-821-62
922
24
N


CALPA1
12-821-483
923
24
N


CALPA1
12-825-173
924
24
N


CALPA1
12-826-312
925
24
N


CALPA1
12-831-59
926
24
N


CALPA1
12-833-264
927
24
N


CALPA1
12-833-279
928
24
N


CALPA1
12-833-280
929
24
N


CALPA1
12-833-373
930
24
N


CALPA1
12-834-183
931
24
N


CALPA1
12-835-54
932
24
N


CALPA1
12-836-134
933
24
N


CALPA1
12-836-237
934
24
N


CALPA1
12-836-238
935
24
N


CALPA1
12-836-257
936
24
N


CALPA1
12-836-275
937
24
N


CALPA1
12-838-179
938
24
N


CALPA1
12-839-397
939
24
N


CALPA1
12-840-47
940
24
N


CALPA1
12-840-77
941
24
N


CALPA1
12-841-445
942
24
N


CALPA1
12-842-215
943
24
N


CALPA1
12-842-447
944
24
N


CALPA1
12-844-167
945
24
N


CALPA1
12-845-364
946
24
N


CALPA1
12-846-209
947
24
N


CALPA1
12-847-123
948
24
N


CALPA1
12-849-242
949
24
N


CYP2J2
10-336-58
950
24
N


CYP2J2
10-336-137
951
24
N


CYP2J2
10-336-232
952
24
N


CYP2J2
12-102-104
953
24
N


CYP2J2
12-102-111
954
24
N


CYP2J2
12-102-275
955
24
N


CYP2J2
12-103-202
956
24
Y
C
14.3


CYP2J2
12-103-214
957
24
N


CYP2J2
12-104-351
958
24
Y
T
27.4


CYP2J2
12-105-435
959
24
N


CYP2J2
12-109-149
960
24
Y
A
8.51


CYP2J2
12-109-197
961
24
N


CYP2J2
12-109-209
962
24
N


CYP2J2
12-109-284
963
24
N


CYP2J2
12-113-276
964
24
Y
G
31.2


CYP2J2
12-115-57
965
24
Y
G
8.87


CYP2J2
12-119-26
966
24
Y
G
29.8


COX1
12-347-308
967
24
N


COX1
12-354-334
968
24
Y
C/C


COX1
12-357-140
969
24
Y
C
7.14


COX1
12-361-320
970
24
Y
G
18.3


COX1
12-361-388
971
24
Y
A
18.5


COX1
12-365-251
972
24
Y
C
18.8


COX1
12-374-261
973
24
Y
T
21.3


COX1
10-308-116
974
24
N


COX1
10-311-274
975
24
N


COX1
10-314-76
976
24
N


COX1
10-306-265
977
24
N


COX2
10-52-386
978
24
N


COX2
10-62-240
979
24
Y
C
12.23


COX2
10-65-276
980
24
Y


COX2
10-67-42
981
24
N


COX2
10-67-340
982
24
Y


COX2
10-55-265
983
24
Y
C
40.9


COX2
10-57-278
984
24
Y


COX2
10-59-176
985
24
Y


COX2
10-60-114
986
24
N


PGDS
10-27-176
987
24
Y
A
5.32


PGDS
10-28-242
988
24
Y


PGDS
10-30-349
989
24
Y
A/A


PGDS
10-181-42
990
24
Y
C
30.2


PGDS
10-181-372
991
24
Y
C
26.3


PGDS
10-183-260
992
24
N


PG15OH
10-475-163
993
24
N


PG15OH
12-884-203
994
24
Y
T
29.7


PG15OH
10-479-266
995
24
N


PG15OH
10-479-350
996
24
N


PG15OH
10-479-394
997
24
N


PG15OH
10-482-145
998
24
N


PG15OH
12-854-64
999
24
N


PG15OH
12-854-472
1000
24
N


PG15OH
12-855-194
1001
24
N


PG15OH
12-855-288
1002
24
N


PG15OH
12-855-423
1003
24
N


PG15OH
12-857-25
1004
24
N


PG15OH
12-858-346
1005
24
Y
T
37.2


PG15OH
12-858-443
1006
24
N


PG15OH
12-860-388
1007
24
N


PG15OH
12-861-270
1008
24
N


PG15OH
12-862-349
1009
24
N


PG15OH
12-862-365
1010
24
N


PG15OH
12-862-452
1011
24
N


PG15OH
12-866-423
1012
24
Y
C
46.2


PG15OH
12-867-47
1013
24
N


PG15OH
12-868-181
1014
24
N


PG15OH
12-868-198
1015
24
N


PG15OH
12-868-282
1016
24
N


PG15OH
12-869-128
1017
24
N


PG15OH
12-870-491
1018
24
N


PG15OH
12-872-52
1019
24
N


PG15OH
12-872-293
1020
24
N


PG15OH
12-873-185
1021
24
N


PG15OH
12-873-319
1022
24
N


PG15OH
12-875-248
1023
24
Y
G
28.8


PG15OH
12-876-265
1024
24
N


PG15OH
12-876-280
1025
24
N


PG15OH
12-876-454
1026
24
N


PG15OH
12-877-59
1027
24
N


PG15OH
12-877-69
1028
24
N


PG15OH
12-877-79
1029
24
N


PG15OH
12-878-153
1030
24
N


PG15OH
12-878-419
1031
24
N


PG15OH
12-879-67
1032
24
N


PG15OH
12-879-439
1033
24
N


PG15OH
12-881-210
1034
24
N


PG15OH
12-881-389
1035
24
N


PG15OH
12-883-273
1036
24
N


PG15OH
12-885-196
1037
24
N


PG15OH
12-885-333
1038
24
N


PG15OH
12-885-407
1039
24
N


PG15OH
12-885-410
1040
24
N


PG15OH
12-886-195
1041
24
Y
A
21.1


PG15OH
12-886-348
1042
24
N


PG15OH
12-887-201
1043
24
N


PG15OH
12-887-467
1044
24
N


PG15OH
12-888-98
1045
24
N


PG15OH
12-888-203
1046
24
Y
G
38.3


PG15OH
12-888-315
1047
24
N


PG15OH
12-889-518
1048
24
N


PG15OH
12-894-266
1049
24
N


PG15OH
12-895-391
1050
24
Y
C
34.6


PG15OH
12-896-140
1051
24
N


PG15OH
12-897-115
1052
24
N


PG15OH
12-897-225
1053
24
N


PG15OH
12-898-49
1054
24
N


CYP8
12-164-119
1055
24
Y
C
11.8


CYP8
12-168-84
1056
24
Y
T
20.1


CYP8
12-168-365
1057
24
N


CYP8
12-170-299
1058
24
Y
T
6.52


CYP8
12-171-360
1059
24
Y
T
8.70


CYP8
12-173-59
1060
24
Y
G
26.0


CYP8
12-175-214
1061
24
Y
A
10.1


CYP8
12-177-183
1062
24
Y
G
25.4


CYP8
12-177-366
1063
24
N


TAX2
10-128-45
1064
24
Y
T/T


TAX2
10-128-63
1065
24
N


TAX2
10-123-177
1066
24
N


TAX2
10-123-402
1067
24
N


TAX2
10-120-137
1068
24
Y
A
1.60


TAX2
10-120-141
1069
24
Y
A
3.09


TAX2
10-179-39
1070
24
N


TAX2
10-180-65
1071
24
Y
C
44.7


TAX2
10-179-257
1072
24
Y


15-LOA
10-43-124
1073
24
N


15-LOA
10-43-134
1074
24
N


15-LOA
10-43-193
1075
24
N


15-LOA
10-43-195
1076
24
N


15-LOA
10-43-233
1077
24
N


15-LOA
10-43-138
1078
24
Y


15-LOA
10-46-372
1079
24
Y
T
2.43


15-LOA
10-46-36
1080
24
N


15-LOA
10-47-103
1081
24
Y


15-LOA
10-47-125
1082
24
Y
T
5.68


15-LOA
10-48-184
1083
24
Y
T
28.0


15-LOA
10-48-381
1084
24
Y
T
31.4


15-LOA
10-49-33
1085
24
Y
T
14.3


15-LOA
10-39-148
1086
24
Y
G
14.5


15-LOA
10-40-222
1087
24
Y
A
47.6


15-LOA
10-40-252
1088
24
N


15-LOA
10-42-354
1089
24
Y


15-LOA
10-154-42
1090
24
N


15-LOA
10-154-156
1091
24
Y
T
24.2


15-LOA
10-154-226
1092
24
N


15-LOB
12-776-259
1093
24
N


5-LO
10-384-109
1094
24
N


5-LO
12-296-388
1095
24
Y
G
37.6


5-LO
10-388-379
1096
24
N


5-LO
10-389-116
1097
24
N


5-LO
10-389-349
1098
24
N


5-LO
10-391-94
1099
24
N


5-LO
12-277-147
1100
24
Y
T
44.9


5-LO
12-278-413
1101
24
Y
A
33.9


5-LO
12-288-190
1102
24
N


5-LO
12-289-35
1103
24
N


5-LO
12-296-119
1104
24
N


5-LO
12-297-291
1105
24
N


5-LO
12-298-105
1106
24
N


5-LO
12-300-126
1107
24
N


5-LO
12-300-410
1108
24
N


5-LO
12-301-379
1109
24
N


5-LO
12-302-264
1110
24
N


5-LO
12-309-405
1111
24
N


5-LO
12-310-105
1112
24
N


5-LO
12-314-453
1113
24
Y
A
18.8


5-LO
12-316-292
1114
24
Y
C
40.8


LTA4H
10-281-314
1115
24
N


LTA4H
10-268-381
1116
24
N


LTA4H
12-54-297
1117
24
Y
C
9.34


LTA4H
10-276-407
1118
24
N


LTA4H
12-44-50
1119
24
Y
A
25.9


LTA4H
12-44-67
1120
24
N


LTA4H
12-45-145
1121
24
N


LTA4H
12-45-166
1122
24
N


LTA4H
12-45-305
1123
24
N


LTA4H
12-46-92
1124
24
Y
G
31.9


LTA4H
12-47-132
1125
24
Y
C
4.84


LTA4H
12-47-61
1126
24
N


LTA4H
12-48-100
1127
24
N


LTA4H
12-48-323
1128
24
N


LTA4H
12-48-369
1129
24
N


LTA4H
12-48-37
1130
24
N


LTA4H
12-49-131
1131
24
Y
A
40.1


LTA4H
12-49-53
1132
24
N


LTA4H
12-49-64
1133
24
N


LTA4H
12-51-234
1134
24
Y
A
43.3


LTA4H
12-51-253
1135
24
N


LTA4H
12-51-370
1136
24
N


LTA4H
12-52-400
1137
24
N


LTA4H
12-57-192
1138
24
Y
T
41.2


LTA4H
12-57-221
1139
24
Y
T
4.40


LTA4H
12-57-510
1140
24
N


LTB4H2
10-1-139
1141
24
Y
G
36.3


LTB4H2
10-1-212
1142
24
Y
T
16.3


LTB4H2
10-1-241
1143
24
Y
A
5.84


LTB4H2
10-9-143
1144
24
Y


LTB4H2
10-9-185
1145
24
Y
T/T


LTB4H2
10-9-264
1146
24
Y


LTB4H2
10-11-22
1147
24
N


LTB4H2
10-13-152
1148
24
Y
T
20.8


LTB4H2
10-13-256
1149
24
Y


LTB4H2
10-13-282
1150
24
Y
C
25.0


LTB4H2
10-15-281
1151
24
N


LTB4H2
10-17-142
1152
24
Y
C/C


LTB4H2
10-18-302
1153
24
N


LTB4H2
10-23-331
1154
24
N


LTB4H2
10-25-152
1155
24
Y


LTB4H2
10-25-258
1156
24
N


LTB4H2
10-3-103
1157
24
Y
T
47.7


LTB4H2
10-3-144
1158
24
Y


LTB4H2
10-3-275
1159
24
Y


LTB4H2
10-5-227
1160
24
Y
A
28.1


LTB4H2
10-7-155
1161
24
Y
T
30.4


LTB4H2
10-7-383
1162
24
N


LTB4H2
10-7-98
1163
24
N


LTB412OH
12-561-270
1164
24
Y
T
35.2


LTB412OH
12-563-87
1165
24
Y
C
28.0


LTB412OH
12-564-64
1166
24
Y
T
36.0


LTB412OH
12-564-214
1167
24
N


LTB412OH
12-568-207
1168
24
N


LTB412OH
12-568-365
1169
24
N


LTB412OH
12-568-367
1170
24
N


LTB412OH
12-571-337
1171
24
Y
G
17.9


LTB412OH
12-573-378
1172
24
Y
A
6.91


LTB412OH
10-294-256
1173
24
N


LTB412OH
10-294-304
1174
24
N


LTB412OH
10-295-201
1175
24
N


LTB412OH
10-296-80
1176
24
N


LTB412OH
10-296-373
1177
24
N


LTB412OH
10-298-122
1178
24
N


LTB412OH
10-298-158
1179
24
N


LTB412OH
10-300-49
1180
24
N


LTB412OH
10-300-185
1181
24
N


LTB4H3
10-10-328
1182
24
Y
A
12.5


LTB4H3
10-12-52
1183
24
N


LTB4H3
10-14-46
1184
24
Y
T
39.3


LTB4H3
10-19-358
1185
24
Y


LTB4H3
10-20-111
1186
24
Y
A
15.8


LTB4H3
10-20-274
1187
24
Y
A/A


LTB4H3
10-24-90
1188
24
Y
C
19.2


LTB4H3
10-24-204
1189
24
Y
A
25.0


LTB4H3
10-24-221
1190
24
N


LTB4H3
10-24-234
1191
24
Y
A
36.1


LTB4H3
10-24-288
1192
24
N


LTB4H3
10-24-311
1193
24
N


LTB4H3
10-26-289
1194
24
N


LTB4H3
10-8-39
1195
24
Y


LTB4H3
10-8-120
1196
24
N


LTB4H3
10-8-154
1197
24
N


LTB4H3
10-8-101
1198
24
Y


LTB4H3
10-8-86
1199
24
Y


LTB4H3
10-8-92
1200
24
N


LTB4H3
10-8-94
1201
24
N


LTB4R
12-61-472
1202
24
N


LTB4R
12-63-402
1203
24
N


LTB4R
12-63-74
1204
24
N


LTB4R
12-64-271
1205
24
Y
C
28.6


LTB4R
12-65-98
1206
24
N


LTB4R
12-70-147
1207
24
Y
C
11.5


LTB4R
12-70-397
1208
24
Y
T
39.7


LTB4R
12-71-320
1209
24
Y
A
4.49


LTB4R
12-73-150
1210
24
N


LTB4R
12-73-49
1211
24
Y
A
43.3


LTB4R
12-73-56
1212
24
N


LTB4R
12-74-38
1213
24
Y
C
44.1


LTB4R
12-76-238
1214
24
Y
T
20.6


LTB4R
12-77-217
1215
24
N


LTB4R
12-77-478
1216
24
Y
A
4.40


LTB4R
12-80-114
1217
24
N


LTB4R
12-80-233
1218
24
Y
C
4.55


LTB4R
12-82-250
1219
24
N


LTC4
10-176-85
1220
24
Y
T
0.54


LTC4
10-176-51
1221
24
N


LTC4
10-176-207
1222
24
N


LTC4
10-176-397
1223
24
Y
A
1.63


LTC4
10-177-219
1224
24
Y
C
29.0


12-LO
12-214-85
1225
24
N


12-LO
12-215-272
1226
24
N


12-LO
12-221-163
1227
24
N


12-LO
12-225-82
1228
24
N


cPLA2
10-234-179
1229
24
Y
Deletion AA
32.6


cPLA2
10-235-272
1230
24
N


ANX1
10-251-342
1231
24
N


ANX2
10-395-367
1232
24
N


ANX2
12-730-58
1233
24
N


ANX2
12-735-208
1234
24
Y
Deletion
21.5


ANX2
12-739-22
1235
24
Y
Insertion G
23.4


ANX3
12-540-363
1236
24
N


ANX3
12-550-206
1237
24
N


CAL2
12-207-410
1238
24
N


CAL3
10-171-254
1239
24
N


CALPA1
12-94-110
1240
24
Y
Deletion
32.5







AATT


CALPA1
12-834-290
1241
24
N


COX2
10-55-115
1242
24
Y
Deletion
3.01







TTATA


PG15OH
12-857-122
1243
24
N


PG15OH
12-872-175
1244
24
N


PG15OH
12-882-40
1245
24
N


PG15OH
12-888-234
1246
24
N


5-LO
12-278-353
1247
24
N


5-LO
12-283-386
1248
24
N


LTA4H
12-44-181
1249
24
N


ANX3
10-370-132
1250
24
N


ANX3
10-370-254
1251
24
N


15PGDHB
10-485-256
1252
24
N


15PGDHB
10-485-257
1253
24
N


15PGDHB
10-474-320
1254
24
N


5LO
10-387-371
1255
24
N


LTB412OH
12-570-239
1256
24
N


LTB412OH
12-570-344
1257
24
N


LTB412OH
12-570-393
1258
24
N


LTB412OH
12-570-421
1259
24
N


LTB412OH
12-570-62
1260
24
N


LTB4H3
10-4-144
1261
24
N


LTB4H3
10-4-161
1262
24
N


LTB4H3
10-4-270
1263
24
N


LTB4H3
10-4-340
1264
24
N


LTB4H3
10-4-369
1265
24
N


LTB4H3
10-4-420
1266
24
N


LTB4H2
10-13-396
1267
24
N


12-LO
10-509-284
1268
24
N


12-LO
10-509-295
1269
24
N


12-LO
10-339-124
1270
24
N


12-LO
10-340-112
1271
24
N


12-LO
10-340-130
1272
24
N


12-LO
10-340-238
1273
24
N


12-LO
10-342-301
1274
24
N


12-LO
10-342-373
1275
24
N


12-LO
10-343-231
1276
24
N


12-LO
10-343-278
1277
24
N


12-LO
10-346-141
1278
24
N
G/G


12-LO
10-346-23
1279
24
N


12-LO
10-346-263
1280
24
N


12-LO
10-346-305
1281
24
N


12-LO
10-349-216
1282
24
N


12-LO
10-350-332
1283
24
N


12-LO
10-350-72
1284
24
N


12-LO
10-507-170
1285
24
N


12-LO
10-507-321
1286
24
N


12-LO
10-507-353
1287
24
N


12-LO
10-507-364
1288
24
N


12-LO
10-507-405
1289
24
N


12-LO
10-508-191
1290
24
N


12-LO
10-508-245
1291
24
N


12-LO
10-510-173
1292
24
N


12-LO
10-511-337
1293
24
N


12-LO
10-512-36
1294
24
Y
C
39.4


12-LO
10-511-62
1295
24
N


12-LO
10-512-318
1296
24
N


12-LO
10-513-250
1297
24
N


12-LO
10-513-262
1298
24
N


12-LO
10-513-352
1299
24
N


12-LO
10-513-365
1300
24
N


FLAP
10-517-100
1301
24
N


FLAP
10-518-125
1302
24
N


FLAP
10-518-194
1303
24
N


FLAP
10-522-71
1304
24
N




















TABLE 8





SEQ ID
BIALLELIC
1ST
2ND
POSITION RANGE OF PREFERRED


NO.
MARKER ID
ALLELE
ALLELE
SEQUENCE



















1
10-253-118
A
G
[1-955]


2
10-253-298
G
C
[1-840]


3
10-253-315
C
T
[1-823]


4
10-499-155
A
G
[1-556], [898-955]


5
10-520-256
C
T
[1-384], [726-955]


6
10-500-258
G
T
[1-311], [653-955]


7
10-500-410
A
G
[1-160], [502-955]


8
10-503-159
G
T
[143-160], [388-408], [447-955]


9
10-504-172
A
T
[1-85], [124-792]


10
10-504-243
A
C
[1-15], [54-722]


19
10-35-358
G
C
[555-842]


23
12-628-306
G
A
[1-868], [904-955]


24
12-628-311
T
C
[1-873], [909-955]


25
12-629-241
G
C
[1-17], [247-658], [705-787], [882-955]


27
10-343-339
G
T
[487-506], [733-904]


28
10-347-74
A
G
[1-134], [240-487], [784-956]


35
10-348-391
A
G
[351-552], [682-776]


40
10-349-368
C
T
[416-525]


44
12-196-119
C
T
[1-469]


45
12-197-244
C
T
[153-206]


48
12-208-35
A
T
[1-346], [453-507]


52
12-215-467
G
T
[1-161], [254-499]


53
12-216-421
A
G
[1-486]


54
12-219-230
A
G
[1-485]


55
12-219-256
C
T
[1-485]


56
12-220-48
G
A
[1-577], [883-956]


57
12-221-302
A
C
[1-64], [265-286]


58
12-223-179
A
G
[1-468]


59
12-223-207
C
T
[1-468]


60
12-225-541
C
T
[1-60], [368-598]


61
12-226-167
G
C
[1-255], [344-508]


62
12-226-458
C
T
[1-255], [344-508]


63
12-229-332
G
C
[1-456]


64
12-229-351
G
C
[1-456]


65
12-230-364
C
T
[1-420]


66
12-231-100
C
T
[1-490]


67
12-231-148
C
T
[1-490]


68
12-231-266
C
T
[1-490]


72
10-239-368
C
T
[1-144], [373-618]


73
10-223-30
G
C
[1-653], [729-1001]


74
10-223-72
A
G
[1-612], [688-1001]


75
10-223-130
A
T
[1-555], [631-1001]


76
10-223-262
A
G
[1-424], [500-1001]


77
10-223-392
A
G
[1-294], [370-1001]


78
10-224-341
C
T
[137-176], [428-563], [920-1001]


82
10-251-128
A
G
[202-240], [373-415], [464-518], [581-777]


84
12-387-32
A
G
[1-396], [464-1001]


85
10-242-316
G
C
[1-350], [418-1000]


86
10-245-412
A
G
[367-701]


87
12-378-171
T
C
[1-731]


88
12-378-228
G
A
[1-788]


89
12-378-450
T
A
[1-1001]


90
12-379-65
A
G
[1-1001]


91
12-382-204
A
G
[1-1001]


92
12-383-117
A
G
[1-37], [246-317], [383-1001]


93
12-383-170
A
G
[193-264], [330-1001]


94
12-383-268
G
T
[95-166], [232-1001]


98
12-385-427
G
T
[257-826]


99
12-386-155
G
T
[272-682], [823-943]


100
12-386-24
C
T
[272-682]


101
12-387-177
C
T
[1-251], [319-1001]


102
12-389-431
C
T
[1-386], [470-583], [644-996]


103
12-391-366
C
T
[293-1001]


104
12-394-85
A
C
[1-103], [184-266], [345-1001]


105
12-395-382
A
G
[1-885]


108
12-401-378
A
G
[1-880]


109
12-402-126
C
T
[99-823]


110
12-404-265
A
G
[1-261], [314-501], [715-733], [782-817]


111
12-406-52
C
T
136-952], [984-1001]


112
12-406-409
A
G
[1-595], [627-1001]


113
12-407-217
G
C
[247-673]


114
12-407-399
A
T
[1-491], [955-1001]


115
12-408-355
G
C
[80-907]


116
12-409-221
A
C
[1-500]


117
12-410-301
C
T
[111-986]


118
10-395-101
A
G
[1-529], [633-1001]


119
10-395-124
A
G
[1-539], [611-1001]


120
10-395-155
A
T
[1-509], [581-1001]


121
10-395-294
C
T
[1-371], [443-858]


122
10-396-100
A
G
[1-506], [635-776], [952-1001]


124
10-399-178
A
G
[1-142], [178-514], [632-1001]


125
10-400-369
A
T
[1-285], [385-513], [555-844], [878-941]


126
10-392-20
A
G
[75-203], [245-534], [568-631], [746-849], [898-997]


127
10-392-103
A
G
[1-552], [663-770], [819-1001]


128
10-392-324
G
C
[1-331], [442-549], [598-891], [977-1001]


129
10-393-27
G
C
[1-76], [187-294], [343-636], [722-1001]


130
10-393-324
A
G
[1-340], [423-1001]


131
12-727-237
A
G
[513-1001]


132
12-728-224
A
G
[352-507], [661-772], [862-1001]


133
12-730-142
A
G
[1-1001]


134
12-730-193
A
G
[1-1001]


135
12-731-60
C
T
[97-665], [711-729], [898-1001]


136
12-731-119
C
T
[1-606], [652-670], [839-1001]


137
12-731-137
G
T
[1-588], [634-652], [821-1001]


138
12-731-146
A
C
[1-579], [625-643], [812-1001]


139
12-731-398
C
T
[1-327], [373-391], [560-743], [823-1001]


140
12-732-113
A
G
[58-1001]


141
12-732-164
A
G
[1-1001]


142
12-732-165
G
C
[1-1001]


143
12-732-445
C
T
[1-935], [975-1001]


144
12-734-201
T
C
[161-1001]


145
12-735-42
G
A
[1-343], [374-566], [656-682], [731-961]


146
12-736-363
G
A
[1-1001]


147
12-737-69
T
C
[1-739]


148
12-737-296
G
A
[1-960]


149
12-738-429
G
A
[1-205], [411-1001]


150
12-740-112
A
G
[1-26], [144-616], [743-1001]


151
12-740-118
C
T
[1-20], [138-610], [737-1001]


152
12-741-265
G
A
[1-1001]


153
12-741-327
T
A
[1-1001]


154
12-741-376
G
A
[1-1001]


155
12-745-30
G
A
[1-1001]


156
12-745-75
T
C
[1-1001]


157
12-745-343
T
G
[1-1001]


158
12-745-350
C
A
[1-1001]


159
12-746-320
C
T
[1-1001]


160
12-747-181
C
T
[1-1001]


161
12-747-302
C
T
[1-1001]


164
12-752-37
G
A
[1-1003]


165
12-752-85
C
G
[1-1001]


166
12-752-196
T
C
[1-62], [108-1001]


167
12-752-484
T
C
[396-1001]


168
12-753-139
C
T
[1-1001]


169
12-753-376
C
T
[1-778], [855-1001]


170
12-754-172
C
T
[1-1001]


171
12-754-218
C
T
[1-1001]


172
12-754-328
G
C
[1-1001]


173
12-754-396
G
T
[1-1001]


174
12-755-280
G
C
[1-1001]


176
12-758-257
A
C
[1-1001]


177
12-758-374
A
C
[1-1001]


178
12-758-424
A
G
[1-1001]


179
12-761-23
G
A
[1-177], [253-701]


180
12-761-178
G
A
[1-292], [368-1001]


181
12-764-329
G
A
[1-1001]


182
12-764-377
G
A
[1-1001]


183
12-765-168
G
A
[1-906]


184
12-765-504
T
C
[1-1002]


190
12-513-389
C
T
[1-1001]


191
12-513-494
G
C
[1-999]


192
12-515-394
A
T
[77-950]


193
12-516-97
C
T
[1-744], [798-1001]


194
12-520-287
A
T
[179-468], [506-885]


195
12-520-323
A
G
[143-432], [470-849]


196
12-523-179
G
A
[1-291], [344-1001]


197
12-523-270
G
A
[1-382], [435-1001]


198
12-527-367
T
A
[1-496], [595-1001]


199
12-529-376
T
C
[279-1001]


200
12-529-489
T
C
[1-37], [391-1001]


201
12-530-134
A
T
[1-94], [166-224], [316-803]


202
12-530-393
C
T
[57-544], [766-1001]


203
12-531-173
C
T
[1-231], [414-735], [789-1001]


204
12-539-441
C
T
[1-1001]


205
12-543-78
G
A
[1-836]


206
12-543-79
C
G
[1-837]


207
12-546-235
C
T
[1-403], [492-1001]


208
12-549-287
T
C
[149-494]


209
12-550-287
A
G
[304-1001]


210
12-552-175
G
A
[1-750], [831-883]


211
12-554-330
G
T
[1-1001]


212
12-556-312
A
C
[1-1001]


213
12-556-443
C
T
[1-1001]


214
12-558-205
C
G
[1-1001]


215
12-558-238
T
C
[1-1001]


216
12-558-305
T
A
[1-1001]


217
12-769-39
G
T
[1-292], [593-624], [690-1001]


218
12-769-430
C
T
[202-233], [299-633]


219
12-770-73
G
A
[1-716]


220
12-772-200
G
A
[1-732], [788-1001]


221
12-772-254
T
C
[1-786], [842-1001]


233
10-166-362
A
C


250
12-86-79
G
A
[70-653], [748-1001]


252
12-89-369
G
C
[1-51], [102-1001]


253
12-89-91
A
G
[1-329], [380-1000]


254
12-94-210
C
T
[573-588]


255
12-94-516
A
T
[287-302]


256
12-96-64
C
A
[1-630], [936-1001]


257
12-97-83
A
C
[1-20], [543-649], [719-916], [964-1001]


258
12-99-296
G
A
[1-210], [305-522], [904-1001]


259
12-100-266
T
C
[504-545], [927-949]


260
12-811-174
T
C
[1-945]


261
12-815-94
A
G
[1-1001]


262
12-815-383
A
G
[1-1001]


263
12-815-384
G
C
[1-1001]


264
12-815-391
C
T
[1-1001]


268
12-821-62
T
G
[1-294], [376-437], [621-887]


269
12-821-483
T
G
[1-48], [460-510], [664-715], [797-858]


270
12-825-173
A
C
[1-34], [522-1001]


273
12-833-264
T
A
[1-86], [216-446], [558-1001]


274
12-833-279
G
A
[1-101], [231-461], [573-1001]


275
12-833-280
T
C
[1-102], [232-462], [574-1001]


276
12-833-373
G
A
[1-195], [325-555], [667-1001]


277
12-834-183
A
G
[295-990]


278
12-835-54
A
G
[1-1001]


279
12-836-134
C
T
[84-249], [354-587], [633-1001]


280
12-836-237
A
G
[1-147], [252-945]


281
12-836-238
A
T
[1-123], [228-919]


282
12-836-257
A
G
[1-123], [228-919]


283
12-836-275
A
C
[1-108], [213-904]


284
12-838-179
A
G
[1-519], [718-1001]


285
12-839-397
G
A
[1-43], [110-1001]


286
12-840-47
C
G
[1-553], [659-1001]


287
12-840-77
T
C
[1-583], [689-1001]


288
12-841-445
G
C
[1-502]


291
12-844-167
T
C
[186-1001]


292
12-845-364
G
A
[1-849]


293
12-846-209
A
T
[1-817]


294
12-847-123
A
G
[1-1001]


295
12-849-242
C
A
[1-27], [490-658]


298
10-336-232
A
G
[507-1001]


299
12-102-104
A
G
[1-630], [712-790]


300
12-102-111
A
G
[1-630], [712-790]


301
12-102-275
A
G
[1-581], [663-741], [834-851], [891-1001]


302
12-103-202
C
T
[188-767]


303
12-103-214
A
G
[176-755]


304
12-104-351
T
G
[1-20], [336-402], [438-511], [911-935]


305
12-105-435
A
G
[1-147], [492-924]


306
12-109-149
A
G
[1-59], [289-607]


307
12-109-197
A
G
[1-59], [289-607]


308
12-109-209
A
G
[1-59], [289-607]


309
12-109-284
A
G
[1-59], [289-607]


310
12-113-276
T
C
[1-1001]


311
12-115-57
A
G
[507-1001]


312
12-119-26
T
C
[1-569]


314
12-354-334
G
A
[1-750]


315
12-357-140
C
T
[1-1001]


316
12-361-320
G
T
[1-201], [268-1001]


317
12-361-388
A
G
[1-133], [200-1001]


318
12-365-251
G
C
[1-41], [132-151], [232-622], [688-933]


319
12-374-261
G
A
[249-1001]


321
10-311-274
C
T
[125-305], [472-878]


322
10-314-76
C
T
[1-224], [290-535], [803-1001]


335
10-30-349
A
G


340
12-884-203
C
T
[1-349], [464-1001]


342
10-479-350
C
T
[1-280], [446-1001]


343
10-479-394
A
G
[1-236], [402-1001]


345
12-854-64
A
G
[1-1001]


346
12-854-472
G
T
[1-1001]


347
12-855-194
T
G
[1-1001]


348
12-855-288
T
C
[1-1001]


349
12-855-423
T
G
[1-1001]


350
12-857-25
C
T
[221-985]


351
12-858-346
T
C
[1-1001]


352
12-858-443
G
A
[1-1001]


353
12-860-388
G
A
[1-30], [157-628], [831-1001]


354
12-861-270
C
T
[1-780]


355
12-862-349
A
G
[78-1001]


356
12-862-365
C
T
[62-1001]


357
12-862-452
G
T
[1-1000]


358
12-866-423
C
T
[1-434], [521-1001]


359
12-867-47
C
T
[81-769]


360
12-868-181
A
G
[306-1001]


361
12-868-198
A
G
[289-1001]


362
12-868-282
C
T
[205-1001]


363
12-869-128
A
C
[1-128], [908-1001]


365
12-872-52
A
G
[436-1001]


366
12-872-293
A
G
[185-1001]


367
12-873-185
T
C
[114-257], [288-377], [572-1001]


368
12-873-319
T
A
[1-139], [248-391], [422-511], [706-1001]


369
12-875-248
T
C
[1-408], [525-1001]


370
12-876-265
T
A
[1-1001]


371
12-876-280
C
G
[1-1001]


372
12-876-454
G
A
[1-1001]


373
12-877-59
C
T
[329-1001]


374
12-877-69
G
T
[319-1001]


375
12-877-79
C
T
[309-1001]


376
12-878-153
C
T
[207-937]


377
12-878-419
G
T
[1-629], [734-929]


378
12-879-67
G
C
[1-200], [261-460], [527-1001]


379
12-879-439
A
G
[1-89], [156-796]


380
12-881-210
A
G
[1-1001]


381
12-881-389
G
T
[1-841]


382
12-883-273
G
C
[1-56], [96-1001]


383
12-885-196
T
C
[1-1001]


384
12-885-333
C
G
[1-1001]


385
12-885-407
T
C
[1-1001]


386
12-885-410
C
G
[1-1001]


387
12-886-195
T
C
[1-815], [867-1001]


388
12-886-348
T
C
[1-968]


389
12-887-201
G
A
[1-59], [181-1001]


390
12-887-467
T
C
[295-325], [447-1001]


391
12-888-98
G
A
[1-717], [916-1001]


392
12-888-203
C
A
[1-822]


393
12-888-315
T
G
[1-1001]


394
12-889-518
G
A
[1-89], [280-320], [441-1001]


395
12-894-266
T
C
[1-1001]


396
12-895-391
G
A
[148-1001]


397
12-896-140
T
A
[60-76], [126-1001]


398
12-897-115
T
C
[259-557]


399
12-897-225
G
A
[369-667]


400
12-898-49
G
A
[1-283], [372-781]


401
12-164-119
T
G
[1-646], [979-1001]


403
12-168-365
C
G
[1-600]


407
12-175-214
A
G
[1-154], [227-317], [391-660], [747-1001]


408
12-177-183
C
G
[1-837], [975-1001]


409
12-177-366
C
A
[1-1001]


427
10-47-103
A
C


428
10-47-125
A
T


433
10-40-222
A
G


434
10-40-252
C
T


442
10-388-379
C
T
[1-202], [383-1001]


443
10-389-116
A
G
[1-538], [693-1001]


444
10-389-349
C
T
[1-305], [460-1001]


445
10-391-94
A
G
[1-259], [301-575], [691-928]


446
12-277-147
A
T
[1-693]


447
12-278-413
A
G
[1-151], [365-733], [775-1001]


448
12-288-190
G
A
[1-701]


449
12-289-35
A
G
[1-791], [946-1001]


450
12-296-119
A
G
[451-550]


451
12-297-291
C
T
[1-1001]


452
12-298-105
G
A
[1-162], [348-1001]


453
12-300-126
A
G
[1-782]


454
12-300-410
A
C
[1-415], [447-498]


455
12-301-379
A
T
[1-627], [932-1001]


456
12-302-264
G
A
[1-1001]


458
12-310-105
G
C
[293-1001]


459
12-314-453
A
T
[1-392], [439-558], [643-799]


460
12-316-292
C
T
[1-460]


461
10-281-314
G
T
[1-282], [453-832], [921-1001]


462
10-268-381
C
T
[1-197], [383-895]


463
12-54-297
C
T
[97-326], [404-518], [658-1001]


464
10-276-407
C
T
[1-97], [510-615], [954-1001]


465
12-44-50
T
C
[220-534], [918-1001]


466
12-44-67
T
C
[237-551], [935-1001]


469
12-45-305
C
T
[1-63], [488-816]


470
12-46-92
A
G
[83-1001]


471
12-47-132
C
T
[1-184], [457-685], [799-871], [987-1001]


472
12-47-61
C
T
[72-255], [528-756], [870-942]


473
12-48-100
A
G
[1-1001]


474
12-48-323
A
G
[1-747]


475
12-48-369
C
T
[1-682]


476
12-48-37
C
T
[1-1001]


477
12-49-131
T
C
[1-609], [677-749], [920-1001]


478
12-49-53
G
A
[1-531], [599-671], [842-1001]


479
12-49-64
G
A
[1-542], [610-682], [853-1001]


480
12-51-234
T
C
[1-47], [182-541], [919-1001]


481
12-51-253
C
A
[1-66], [201-560], [938-1001]


482
12-51-370
G
A
[1-182], [317-676]


483
12-52-400
G
A
[1-100], [404-1001]


484
12-57-192
G
A
[1-168], [286-752]


485
12-57-221
G
A
[1-197], [315-781]


486
12-57-510
C
A
[1-163], [251-486], [604-1010]


494
10-13-152
C
T


510
12-561-270
C
T
[188-203], [496-642], [697-738]


511
12-563-87
C
T
[1-929]


512
12-564-64
G
T
[1-213], [381-1001]


513
12-564-214
C
T
[1-64], [232-1001]


514
12-568-207
G
T
[424-513], [613-1001]


515
12-568-365
G
T
[266-355], [455-1001]


516
12-568-367
G
T
[264-353], [453-1001]


517
12-571-337
G
C
[1-53], [327-897]


518
12-573-378
A
G
[1-335], [437-910]


519
10-294-256
G
C
[1-53], [327-897]


520
10-294-304
G
C
[279-849], [942-1001]


522
10-296-80
A
G
[359-397], [531-906]


523
10-296-373
A
G
[60-105], [239-623], [924-1001]


524
10-298-122
C
T
[1-565], [737-873]


525
10-298-158
A
G
[1-529], [701-837]


526
10-300-49
A
G
[285-643], [808-854]


527
10-300-185
C
T
[92-507], [672-718], [976-1001]


549
12-63-402
A
G
[1-472]


550
12-63-74
A
G
[1-472]


551
12-64-271
C
T
[1-787]


552
12-65-98
C
T
[112-272], [334-864]


553
12-70-147
A
C
[1-211], [491-1001]


554
12-70-397
C
T
[241-1001]


555
12-71-320
A
G
[1-1001]


556
12-73-150
C
T
[1-140], [275-607], [646-821]


557
12-73-49
A
G
[1-240], [375-707], [746-921]


558
12-73-56
A
T
[1-233], [368-700], [739-914]


559
12-74-38
G
A
[1-1001]


561
12-77-217
C
T
[1-822]


562
12-77-478
A
G
[1-562]


563
12-80-114
T
C
[1-1001]


564
12-80-233
G
A
[1-1001]


565
12-82-250
A
T
[404-454]


571
12-214-85
CCTAT
—
[1-101], [259-305]


572
12-215-272
T
—
[1-161], [254-499]


573
12-221-163
GTCCTCA
T
[1-64], [265-286]


574
12-225-82
T
—
[1-60], [368-598]


577
10-251-342
GG
C
[1-56], [156-301], [364-560]


578
10-395-367
A
—
[1-263], [367-717], [764-783]


579
12-730-58
ACAA
—
[162-251], [287-321], [517-767]


580
12-735-208
—
Deletion
[1-689], [779-805], [854-1002]


581
12-739-22
G
—
[1-39], [386-640], [791-1002]


582
12-540-363
T
—
[1-1002]


583
12-550-206
T
—
[380-1002]


587
12-834-290
G
—
[196-1002]


589
12-857-122
CTCT
—
[145-1002]


590
12-872-175
T
—
[1-41], [310-1102]


592
12-888-234
C
—
[1-850], [950-1002]


593
12-278-353
A
—
[1-208], [422-790], [832-1001]


595
12-44-181
C
—
[308-622], [983-1002]


602
12-570-239
T
C
[386, 671], [724, 727], [947, 1001]


603
12-570-344
T
C
[1, 51], [491, 601], [727, 776], [829, 832]


619
10-340-238
A
G
[231, 310], [487, 601]


620
10-342-301
Insertion
—
[432, 576], [605, 609], [676, 722]


621
10-342-373
C
T
[360, 504], [533, 537], [604, 650], [930, 1001]


625
10-346-23
A
G
[1, 144], [233, 274], [305, 347], [478, 592], [696, 945]


626
10-346-263
G
C
[1, 37], [68, 110], [241, 355], [459, 708]


627
10-346-305
C
T
[1, 68], [199, 313], [417, 666], [961, 1001]


629
10-350-332
C
T
[1, 913]


630
10-350-72
C
T
[1, 1001]


632
10-507-321
A
C
[1, 308], [440, 462], [552, 652], [711, 1000]


633
10-507-353
C
T
[1, 276], [408, 430], [520, 620], [679, 1000]


634
10-507-364
C
T
[1, 265], [397, 609], [668, 1000]


635
10-507-405
C
T
[1, 224], [356, 378], [468, 568], [627, 1000]


636
10-508-191
C
T
[1, 403], [442, 444], [491, 640], [942, 1000]


637
10-508-245
C
T
[1, 349], [388, 390], [463, 586], [888, 1000]


638
10-510-173
ATTTA
TTTTTT
[243, 380], [411, 546]


647
10-517-100
G
C
[1, 1000]


648
10-518-125
G
T
[1, 1000]


649
10-518-194
A
G
[1, 1000]


650
10-522-71
A
G
[1, 806], [844, 863], [911, 920], [950, 1000]



















TABLE 9





SEQ ID
BIALLELIC
ORIGINAL
ALTERNATIVE


NO.
MARKER ID
ALLELE
ALLELE


















11
10-204-326
G
A


12
10-32-357
C
A


13
10-33-175
C
T


14
10-33-211
C
T


15
10-33-234
A
C


16
10-33-270
G
A


17
10-33-327
T
C


18
10-34-290
G
T


20
10-35-390
C
T


21
10-36-164
G
A


26
12-206-366
T
C


29
10-347-111
G
C


30
10-347-165
C
T


33
10-347-271
A
T


34
10-347-348
G
A


36
10-349-47
T
C


38
10-349-142
C
G


41
10-339-32
C
T


43
10-341-319
C
T


46
12-198-128
G
A


47
12-206-81
G
A


49
12-214-129
C
T


50
12-214-151
G
C


51
12-214-360
G
C


69
10-231-23
G
A


70
10-233-386
A
G


79
10-227-282
A
G


80
10-240-241
A
G


83
10-252-209
G
A


95
12-384-336
C
T


96
12-384-451
G
C


97
12-385-123
C
T


106
12-400-217
A
G


107
12-400-280
A
G


162
12-749-240
G
A


163
12-749-255
G
T


175
12-757-384
T
C


185
10-372-279
T
C


186
10-375-136
T
C


187
10-376-281
A
T


188
10-369-392
C
T


222
10-87-73
C
T


223
10-87-74
A
T


224
10-87-80
A
G


225
10-87-140
C
T


226
10-88-81
T
C


227
10-89-41
G
A


228
10-90-35
G
A


229
10-91-274
T
G


231
10-94-197
G
A


232
10-94-198
T
G


234
10-207-386
C
G


235
10-207-409
G
C


236
10-118-307
G
A


237
10-173-247
G
A


238
10-173-294
A
G


239
10-173-347
C
T


240
10-103-104
C
T


241
10-103-323
T
C


242
10-103-402
C
T


243
10-106-98
C
A


246
10-168-160
T
C


247
10-168-206
C
A


248
10-168-284
T
A


249
10-169-318
C
A


251
12-88-393
A
C


265
12-817-214
G
A


266
12-817-355
T
C


267
12-819-437
A
G


271
12-826-312
G
A


272
12-831-59
G
C


289
12-842-215
T
C


290
12-842-447
A
G


297
10-336-137
T
A


313
12-347-308
G
A


320
10-308-116
C
T


326
10-65-276
G
A


327
10-67-42
A
T


328
10-67-340
T
C


331
10-59-176
C
T


332
10-60-114
A
G


334
10-28-242
G
A


336
10-181-42
C
T


337
10-181-372
C
T


338
10-183-260
C
G


341
10-479-266
G
A


364
12-870-491
A
G


402
12-168-84
A
C


404
12-170-299
G
A


405
12-171-360
C
T


406
12-173-59
A
G


410
10-128-45
T
C


411
10-128-63
A
G


412
10-123-177
G
A


414
10-120-137
G
A


415
10-120-141
C
A


425
10-46-372
C
T


429
10-48-184
C
T


430
10-48-381
C
T


431
10-49-33
C
T


432
10-39-148
A
G


435
10-42-354
T
C


436
10-154-42
C
T


437
10-154-156
C
T


438
10-154-226
G
A


439
12-776-259
A
G


440
10-384-109
C
T


441
12-296-388
A
G


457
12-309-405
A
G


467
12-45-145
A
G


468
12-45-166
G
A


487
10-1-139
G
T


488
10-1-212
G
T


489
10-1-241
C
A


491
10-9-185
T
C


492
10-9-264
C
G


493
10-11-22
T
C


495
10-13-256
C
T


496
10-13-282
T
C


497
10-15-281
T
G


498
10-17-142
C
T


499
10-18-302
C
T


500
10-23-331
G
A


501
10-25-152
T
C


502
10-25-258
C
T


503
10-3-103
C
T


504
10-3-144
T
C


505
10-3-275
G
T


506
10-5-227
A
C


507
10-7-155
T
C


508
10-7-383
C
T


509
10-7-98
G
C


533
10-20-274
A
G


534
10-24-90
A
C


536
10-24-221
G
T


546
10-8-92
T
C


547
10-8-94
C
T


548
12-61-472
C
T


560
12-76-238
G
T


566
10-176-85
C
T


567
10-176-51
C
T


568
10-176-207
G
T


569
10-176-397
C
A


570
10-177-219
A
C


575
10-234-179
AA
—


576
10-235-272
T
—


584
10-207-410
—
C


585
10-171-254
GG
—


586
12-94-110
—
AATT


588
10-55-115
TTATA
—


591
12-882-40
A
—


594
12-283-386
T
—


598
10-485-256
A
G


599
10-485-257
T
C


600
10-474-320
Insertion A
—


601
10-387-371
T
C


604
12-570-393
C
T


605
12-570-421
T
G


606
12-570-62
Insertion TG
—


607
10-4-144
C
A


608
10-4-161
A
C


609
10-4-270
G
C


610
10-4-340
A
G


611
10-4-369
C
T


612
10-4-420
G
T


613
10-13-396
Insertion AAT
—


614
10-509-284
C
T


616
10-339-124
C
T


617
10-340-112
C
A


618
10-340-130
T
A


622
10-343-231
Insertion C
—


623
10-343-278
C
T


624
10-346-141
G
A


628
10-349-216
Insertion CTG
—


631
10-507-170
A
G


639
10-511-337
Deletion
—


640
10-512-36
G
C


641
10-511-62
C
T


642
10-512-318
G
A


643
10-513-250
G
A


644
10-513-262
T
C


645
10-513-352
G
A


646
10-513-365
G
A



















TABLE 10






BIALLELIC
1ST
2ND


SEQ ID NO.
MARKER ID
ALLELE
ALLELE


















22
10-498-192
A
G


31
10-347-203
A
G


32
10-347-220
A
G


37
10-349-97
A
G


39
10-349-224
G
T


42
10-341-116
A
G


81
10-249-185
A
G


123
10-397-201
G
T


189
10-371-257
A
C


230
10-93-133
C
T


244
10-106-288
C
T


245
10-106-378
C
T


296
10-336-58
C
T


323
10-306-265
A
G


324
10-52-386
C
T


325
10-62-240
G
C


329
10-55-265
C
T


330
10-57-278
C
T


333
10-27-176
A
G


339
10-475-163
A
G


344
10-482-145
A
G


413
10-123-402
A
G


416
10-179-39
C
T


417
10-180-65
G
C


418
10-179-257
G
T


426
10-46-36
T
A


521
10-295-201
G
T


528
10-10-328
G
A


529
10-12-52
C
T


530
10-14-46
C
T


532
10-20-111
A
C


535
10-24-204
A
G


537
10-24-234
A
G


538
10-24-288
A
G


539
10-24-311
G
C


541
10-8-39
A
C


542
10-8-120
A
G


543
10-8-154
G
C


544
10-8-101
A
T


545
10-8-86
C
T


596
10-370-132
C
T


597
10-370-254
C
T


615
10-509-295
Insertion




and Deletion
















TABLE 11







Sequences that are useful for designing some


of the primers and probes of the invention








SEQ ID



NO.
POSITION RANGE OF NOVEL SEQUENCE











26
[569-588], [815-956]


29
[1-97], [203-450], [747-956]


30
[1-43], [149-396], [693-956]


31
[111-358], [655-956]


32
[94-341], [638-956]


33
[44-291], [588-956]


34
[1-214], [511-844]


36
[734-843]


37
[684-793]


38
[639-748]


39
[557-666]


41
[217-319], [721-781]


42
[1-96], [276-387], [881-956]


43
[72-184], [678-820]


46
[1-56], [193-400]


47
[855-874]


49
[1-101], [259-305]


50
[1-101], [259-305]


51
[1-101], [259-305]


79
[1-311], [512-1001]


80
[709-1001]


81
[1-231], [723-741]


83
[291-476]


95
[1-138], [532-662], [970-1001]


96
[59-254], [648-778], [918-1001]


97
[318-757]


106
[88-182], [309-461], [798-843]


107
[1-119], [246-398], [735-780]


123
[1-449], [568-1001]


162
[264-407], [801-833]


163
[249-392], [786-818]


175
[1-419]


185
[267-360], [549-599], [651-807], [851-1001]


186
[1-459], [691-1001]


187
[1-311], [557-1001]


188
[1-155], [662-1001]


189
[1-39], [554-1001]


251
[746-1001]


265
[315-445], [873-1001]


266
[174-304], [732-1001]


271
[1-173], [572-844], [884-917]


272
[1-75], [556-576]


289
[1-191]


290
[160-421]


296
[1-151], [681-1001]


297
[1-72], [602-1001]


313
[1-319]


320
[592-1001]


339
[1-24], [804-1001]


341
[1-364], [530-1001]


364
[1-270], [554-1001]


402
[1-319]


404
[1-319], [767-830]


405
[1-222], [639-1001]


439
[1-73], [608-900]


440
[1-40], [732-1001]


441
[182-281]


457
[1-315], [838-1001]


467
[1-222], [647-1001]


468
[1-201], [626-954]


521
[1-138], [281-412], [529-880]


548
[60-80]


560
[539-810]


588
[406-418]


591
[150-320], [777-824], [864-1002]


594
[300-450]


596
[196-237], [920-1001]


597
[74-115], [798-1001]


598
[557-1001]


599
[556-1001]


600
[256-267], [669-670], [833-835]


604
[1-100], [540-650], [776-825], [878-881], [969-985]


605
[1-128], [568-678], [804-853], [906-909], [997-1001]


606
[210-320], [446-495], [548-551], [771-1001]


607
[1-54]


608
[1-54]


609
[1-54]


610
[1-54]


611
[1-54]


612
[1-54]


613
[1-30], [138-179]


614
[725-814]


615
[714-803]


616
[1-252], [634-713], [890-1001]


617
[106-155], [357-436], [613-727]


618
[88-137], [339-418], [595-709]


622
[138-178], [592-638], [863-1001]


623
[91-131], [545-591], [816-1001]


624
[1-29], [118-159], [190-232], [363-477], [581-830]


628
[587-698]


631
[199-459], [591-613], [703-803], [862-1000]


641
[1-159], [190-325]


646
[1-20]
















TABLE 12







Microsequencing primers











COMPLEMENTARY



POSITION RANGE OF
POSITION RANGE OF


SEQ ID
MICROSEQUENCING
MICROSEQUENCING


NO.
PRIMERS
PRIMERS












1
458-477
479-498


2
 459-477*
479-498


3
458-477
479-498


4
458-477
479-498


5
458-477
479-498


6
458-477
479-498


7
458-477
479-498


8
458-477
479-498


9
458-477
479-498


10
458-477
479-498


11
458-477
 479-497*


12
 459-477*
479-498


13
 459-477*
479-498


14
458-477
479-498


15
 459-477*
479-498


16
 459-477*
479-498


17
 459-477*
479-498


18
458-477
479-498


19
 459-477*
479-498


20
 459-477*
479-498


21
458-477
 479-497*


22
458-477
479-498


23
458-477
 479-497*


24
458-477
479-498


25
 459-477*
479-498


26
 459-477*
479-498


27
458-477
479-498


28
458-477
479-498


29
458-477
479-498


30
458-477
479-498


31
 459-477*
479-498


32
458-477
 479-497*


33
458-477
479-498


34
458-477
479-498


35
458-477
479-498


36
458-477
479-498


37
458-477
 479-497*


38
458-477
479-498


39
458-477
 479-497*


40
458-477
479-498


41
458-477
479-498


42
458-477
 479-497*


43
458-477
479-498


44
 100-118*
120-139


45
 224-242*
244-263


46
108-127
129-148


47
458-477
479-498


48
 16-34*
36-55


49
 110-128*
130-149


50
131-150
152-171


51
338-357
359-378


52
446-465
467-486


53
398-417
 419-437*


54
209-228
 230-248*


55
235-254
256-275


56
458-477
479-498


57
282-301
303-322


58
159-178
180-199


59
 188-206*
208-227


60
 521-539*
541-560


61
 147-165*
 167-185*


62
435-454
456-475


63
312-331
333-352


64
331-350
352-371


65
344-363
365-384


66
79-98
100-119


67
127-146
148-167


68
245-264
266-285


69
480-499
 501-519*


70
481-500
 502-520*


72
481-500
502-521


73
 482-500*
502-521


74
481-500
502-521


75
481-500
502-521


76
481-500
502-521


77
481-500
502-521


78
481-500
502-521


79
481-500
 502-520*


80
481-500
502-521


81
481-500
502-521


82
481-500
502-521


83
481-500
502-521


84
 482-500*
502-521


85
480-499
501-520


86
481-500
502-521


87
481-500
502-521


88
481-500
502-521


89
481-500
502-521


90
481-500
502-521


91
 482-500*
502-521


92
481-500
502-521


93
481-500
502-521


94
481-500
502-521


95
481-500
502-521


96
481-500
502-521


97
238-257
259-278


98
481-500
502-521


99
423-442
 444-462*


100
293-312
314-333


101
481-500
 502-520*


102
481-500
502-521


103
481-500
502-521


104
481-500
502-521


105
365-384
386-405


106
 482-500*
502-521


107
481-500
502-521


108
360-379
381-400


109
303-322
324-343


110
297-316
318-337


111
481-500
502-521


112
481-500
502-521


113
481-500
502-521


114
481-500
502-521


115
 482-500*
502-521


116
209-228
230-249


117
466-485
487-506


118
481-500
502-521


119
481-500
502-521


120
481-500
502-521


121
481-500
502-521


122
481-500
502-521


123
481-500
502-521


124
481-500
502-521


125
481-500
502-521


126
477-496
498-517


127
481-500
502-521


128
481-500
502-521


129
481-500
502-521


130
481-500
502-521


131
481-500
502-521


132
481-500
502-521


133
481-500
502-521


134
481-500
502-521


135
481-500
502-521


136
481-500
502-521


137
481-500
502-521


138
481-500
502-521


139
481-500
502-521


140
481-500
502-521


141
481-500
502-521


142
481-500
 502-520*


143
481-500
502-521


144
481-500
502-521


145
481-500
502-521


146
481-500
502-521


147
 482-500*
502-521


148
481-500
502-521


149
481-500
 502-520*


150
 482-500*
502-521


151
481-500
502-521


152
481-500
502-521


153
481-500
502-521


154
481-500
502-521


155
481-500
502-521


156
481-500
502-521


157
481-500
502-521


158
481-500
502-521


159
481-500
502-521


160
481-500
502-521


161
481-500
502-521


162
481-500
502-521


163
481-500
502-521


164
488-507
509-528


165
481-500
502-521


166
481-500
502-521


167
481-500
502-521


168
481-500
502-521


169
481-500
502-521


170
481-500
502-521


171
481-500
502-521


172
481-500
502-521


173
481-500
502-521


174
481-500
502-521


175
481-500
502-521


176
481-500
502-521


177
481-500
502-521


178
481-500
502-521


179
521-540
542-561


180
481-500
502-521


181
481-500
502-521


182
481-500
502-521


183
481-500
502-521


184
481-500
502-521


185
481-500
502-521


186
481-500
502-521


187
481-500
502-521


188
481-500
502-521


189
481-500
502-521


190
481-500
502-521


191
481-500
502-521


192
481-500
502-521


193
481-500
 502-520*


194
481-500
502-521


195
481-500
 502-520*


196
 482-500*
502-521


197
481-500
502-521


198
481-500
 502-520*


199
481-500
502-521


200
481-500
502-521


201
 482-500*
502-521


202
481-500
502-521


203
481-500
 502-520*


204
481-500
502-521


205
481-500
502-521


206
481-500
502-521


207
481-500
502-521


208
481-500
502-521


209
481-500
502-521


210
481-500
502-521


211
481-500
502-521


212
481-500
502-521


213
481-500
502-521


214
481-500
502-521


215
481-500
502-521


216
481-500
502-521


217
481-500
502-521


218
481-500
502-521


219
481-500
502-521


220
481-500
502-521


221
481-500
502-521


222
52-71
73-92


223
53-72
74-93


224
59-78
80-99


225
118-137
139-158


226
 62-80*
 82-101


227
21-40
42-61


228
15-34
 36-54*


229
254-273
275-294


230
113-132
134-153


231
 178-196*
198-217


232
178-197
199-218


233
342-361
363-382


234
 368-386*
388-407


235
 390-408*
410-429


236
287-306
 308-326*


237
227-246
248-267


238
274-293
 295-313*


239
 328-346*
348-367


240
 84-103
105-124


241
 304-322*
324-343


242
383-402
404-423


243
78-97
 99-118


244
 269-287*
289-308


245
 361-379*
381-400


246
 141-159*
161-180


247
 187-205*
207-226


248
263-282
284-303


249
297-316
318-337


250
481-500
 502-520*


251
481-500
502-521


252
 482-500*
502-521


253
481-500
502-521


254
481-500
502-521


255
501-520
522-541


256
 482-500*
502-521


257
481-500
502-521


258
 482-500*
502-521


259
 482-500*
502-521


260
481-500
502-521


261
481-500
502-521


262
481-500
502-521


263
480-499
501-520


264
481-500
502-521


265
481-500
502-521


266
481-500
502-521


267
481-500
502-521


268
481-500
502-521


269
481-500
502-521


270
481-500
502-521


271
481-500
502-521


272
481-500
502-521


273
481-500
502-521


274
481-500
502-521


275
482-501
503-522


276
481-500
502-521


277
463-482
484-503


278
481-500
502-521


279
481-500
502-521


280
480-499
501-520


281
456-475
477-496


282
478-497
499-518


283
481-500
502-521


284
481-500
502-521


285
481-500
502-521


286
481-500
502-521


287
481-500
502-521


288
425-444
446-465


289
481-500
502-521


290
479-498
500-519


291
481-500
502-521


292
481-500
502-521


293
481-500
502-521


294
481-500
502-521


295
481-500
502-521


296
481-500
502-521


297
481-500
502-521


298
481-500
502-521


299
359-378
380-399


300
366-385
387-406


301
481-500
502-521


302
 482-500*
502-521


303
481-500
502-521


304
481-500
 502-520*


305
419-438
440-459


306
258-277
 279-297*


307
306-325
327-346


308
318-337
339-358


309
393-412
414-433


310
 482-500*
502-521


311
481-500
 502-520*


312
 482-500*
502-521


313
481-500
502-521


314
 482-500*
502-521


315
481-500
 502-520*


316
481-500
 502-520*


317
 482-500*
502-521


318
 482-500*
502-521


319
 482-500*
502-521


320
481-500
502-521


321
481-500
502-521


322
481-500
502-521


323
481-500
502-521


324
366-385
387-406


325
 221-239*
241-260


326
256-275
 277-295*


327
22-41
43-62


328
 322-340*
342-361


329
 245-263*
265-284


330
 259-277*
279-298


331
 157-175*
177-196


332
 94-113
115-134


333
156-175
 177-195*


334
222-241
 243-261*


335
330-349
 351-369*


336
 23-41*
43-62


337
 355-373*
375-394


338
239-258
260-279


339
481-500
502-521


340
 482-500*
502-521


341
481-500
502-521


342
481-500
502-521


343
481-500
502-521


344
481-500
502-521


345
481-500
502-521


346
481-500
502-521


347
481-500
502-521


348
481-500
502-521


349
481-500
502-521


350
456-475
477-496


351
 482-500*
502-521


352
481-500
502-521


353
481-500
502-521


354
481-500
502-521


355
481-500
502-521


356
481-500
502-521


357
481-500
502-521


358
 482-500*
502-521


359
481-500
502-521


360
481-500
502-521


361
481-500
502-521


362
481-500
502-521


363
481-500
502-521


364
481-500
502-521


365
481-500
502-521


366
481-500
502-521


367
481-500
502-521


368
481-500
502-521


369
 482-500*
502-521


370
481-500
502-521


371
481-500
502-521


372
481-500
502-521


373
481-500
502-521


374
481-500
502-521


375
481-500
502-521


376
481-500
502-521


377
481-500
502-521


378
481-500
502-521


379
481-500
502-521


380
481-500
502-521


381
481-500
502-521


382
481-500
502-521


383
481-500
502-521


384
481-500
502-521


385
481-500
502-521


386
481-500
502-521


387
 482-500*
502-521


388
481-500
502-521


389
481-500
502-521


390
481-500
502-521


391
481-500
502-521


392
 482-500*
502-521


393
481-500
502-521


394
459-478
480-499


395
481-500
502-521


396
481-500
 502-520*


397
481-500
502-521


398
481-500
502-521


399
481-500
502-521


400
508-527
529-548


401
481-500
 502-520*


402
 482-500*
502-521


403
481-500
502-521


404
481-500
 502-520*


405
 482-500*
502-521


406
481-500
 502-520*


407
481-500
 502-520*


408
481-500
 502-520*


409
481-500
502-521


410
 26-44*
46-65


411
43-62
64-83


412
157-176
178-197


413
382-401
403-422


414
 117-135*
137-156


415
 121-139*
141-160


416
19-38
40-59


417
 46-64*
66-85


418
237-256
 258-276*


425
 350-368*
370-389


426
15-34
36-55


427
 82-101
103-122


428
 105-123*
125-144


429
 164-182*
184-203


430
362-381
383-402


431
 14-32*
34-53


432
130-149
 151-169*


433
202-221
 223-241*


434
230-249
251-270


435
334-353
 355-373*


436
22-41
43-62


437
 137-155*
157-176


438
206-225
227-246


439
481-500
502-521


440
481-500
502-521


441
 482-500*
502-521


442
481-500
502-521


443
481-500
502-521


444
481-500
502-521


445
481-500
502-521


446
 482-500*
502-521


447
 482-500*
502-521


448
481-500
502-521


449
481-500
502-521


450
481-500
502-521


451
481-500
502-521


452
481-500
502-521


453
481-500
502-521


454
481-500
502-521


455
481-500
502-521


456
481-500
502-521


457
481-500
502-521


458
481-500
502-521


459
 482-500*
502-521


460
481-500
 502-520*


461
481-500
502-521


462
481-500
502-521


463
 482-500*
502-521


464
481-500
502-521


465
 482-500*
502-521


466
481-500
502-521


467
481-500
502-521


468
481-500
502-521


469
481-500
502-521


470
481-500
 502-520*


471
 482-500*
502-521


472
481-500
502-521


473
481-500
502-521


474
481-500
502-521


475
481-500
502-521


476
481-500
502-521


477
 482-500*
502-521


478
481-500
502-521


479
481-500
502-521


480
 483-500*
502-521


481
481-500
502-521


482
481-500
502-521


483
481-500
502-521


484
481-500
 502-520*


485
 482-500*
 502-520*


486
481-500
502-521


487
119-138
 140-158*


488
192-211
 213-231*


489
 222-240*
242-261


491
 166-184*
186-205


492
 245-263*
265-284


493
 2-21
23-42


494
 133-151*
153-172


495
 237-255*
257-276


496
 263-281*
283-302


497
261-280
282-301


498
 123-141*
143-162


499
282-301
303-322


500
311-330
332-351


501
 133-151*
153-172


502
238-257
259-278


503
 84-102*
104-123


504
 125-143*
145-164


505
255-274
 276-294*


506
 208-226*
228-247


507
 136-154*
156-175


508
361-380
382-401


509
78-97
 99-118


510
481-500
 502-520*


511
 482-500*
502-521


512
481-500
 502-520*


513
481-500
502-521


514
481-500
502-521


515
481-500
502-521


516
481-500
502-521


517
 482-500*
502-521


518
 482-500*
502-521


519
481-500
502-521


520
481-500
502-521


521
481-500
502-521


522
481-500
502-521


523
481-500
502-521


524
481-500
502-521


525
481-500
502-521


526
481-500
502-521


527
481-500
502-521


528
307-326
 328-346*


529
32-51
53-72


530
 27-45*
47-66


532
 91-109*
111-130


533
253-272
 274-292*


534
 71-89*
 91-110


535
184-203
 205-223*


536
201-220
222-241


537
214-233
 235-253*


538
268-287
289-308


539
291-310
312-331


541
 20-38*
40-59


542
100-119
121-140


543
134-153
155-174


544
 81-100
 102-119*


545
 67-85*
 87-106


546
72-91
 93-112


547
74-93
 95-114


548
481-500
502-521


549
396-415
417-436


550
68-87
 89-108


551
 268-286*
288-307


552
419-438
440-459


553
 482-500*
502-521


554
 482-500*
502-521


555
481-500
 502-520*


556
481-500
502-521


557
 482-500*
502-521


558
481-500
502-521


559
481-500
 502-520*


560
481-500
 502-520*


561
481-500
502-521


562
481-500
 502-520*


563
481-500
502-521


564
481-500
 502-520*


565
230-249
251-270


566
 66-84*
 86-105


567
31-50
52-71


568
187-206
208-227


569
 378-396*
 398-416*


570
 200-218*
220-239


571
65-84
—


572
251-270
—


573
143-162
—


574
62-81
—


575
 195-213*
—


576
471-490
—


577
478-497
—


578
477-496
—


579
478-497
—


580
—
 459-477*


581
—
 498-516*


582
478-497
—


583
477-496
—


584
389-408
—


585
235-254
—


586
 479-497*
—


587
478-497
—


588
 95-113*
—


589
478-497
—


590
478-497
—


591
478-497
—


592
478-497
—


593
479-498
—


594
478-497
—


595
438-457
—


596
481-500
502-521


597
481-500
502-521


598
481-500
502-521


599
481-500
502-521


600
481-500
—


601
481-500
502-521


602
481-500
502-521


603
481-500
502-521


604
481-500
502-521


605
481-500
502-521


606
—
503-522


607
121-140
142-161


608
138-157
159-178


609
247-266
268-287


610
317-336
338-357


611
346-365
367-386


612
397-416
418-437


613
374-395
—


614
481-500
502-521


615
481-500
—


616
481-500
502-521


617
481-500
502-521


618
481-500
502-521


619
481-500
502-521


620
481-500
—


621
481-500
502-521


622
481-500
—


623
481-500
502-521


624
481-500
 502-520*


625
480-499
501-520


626
481-500
502-521


627
481-500
502-521


628
481-500
—


629
481-500
502-521


630
481-500
502-521


631
481-500
502-521


632
481-500
502-521


633
481-500
502-521


634
481-500
502-521


635
481-500
502-521


636
481-500
502-521


637
481-500
502-521


638
481-500
—


639
481-500
—


640
481-500
502-521


641
481-500
502-521


642
481-500
502-521


643
481-500
502-521


644
481-500
502-521


645
481-500
502-521


646
481-500
502-521


647
481-500
502-521


648
481-500
502-521


649
481-500
502-521


650
481-500
502-521
















TABLE 13







Amplification primers











COMPLEMENTARY



POSITION
POSITION



RANGE OF
RANGE OF


SEQ ID
AMPLIFICATION
AMPLIFICATION


NO.
PRIMERS
PRIMERS












1
361-379
761-780


2
181-199
581-600


3
164-182
564-583


4
324-343
536-553


5
294-310
743-760


6
221-237
670-687


7
70-86
519-536


8
326-343
760-780


9
307-324
557-575


10
237-254
487-505


11
153-170
590-607


12
121-139
522-541


13
304-322
705-723


14
268-286
669-687


15
245-263
646-664


16
209-227
610-628


17
152-170
553-571


18
189-206
525-542


19
120-137
526-543


20
 88-105
494-511


21
315-334
741-760


22
287-306
621-638


23
266-286
764-782


24
271-291
769-787


25
238-257
617-637


26
222-239
635-654


27
140-157
553-572


28
405-422
826-845


29
368-385
789-808


30
314-331
735-754


31
276-293
697-716


32
259-276
680-699


33
209-226
630-649


34
132-149
553-572


35
 90-109
488-507


36
432-451
829-848


37
382-401
779-798


38
337-356
734-753


39
255-274
652-671


40
114-133
511-530


41
447-464
845-864


42
363-380
771-789


43
160-177
568-586


44
 1-20
450-469


45
 1-19
380-399


46
 1-20
380-400


47
398-415
835-854


48
 1-21
487-507


49
 1-20
429-448


50
 1-20
429-448


51
 1-20
429-448


52
 1-20
479-499


53
 1-20
467-486


54
 1-20
465-485


55
 1-20
465-485


56
76-96
505-525


57
 1-21
387-407


58
 1-20
449-468


59
 1-20
449-468


60
 1-19
581-598


61
 1-19
490-508


62
 1-19
490-508


63
 1-21
437-456


64
 1-21
437-456


65
 1-20
401-420


66
 1-19
470-490


67
 1-19
470-490


68
 1-19
470-490


69
478-495
879-898


70
119-137
540-557


72
138-157
538-556


73
472-490
900-917


74
431-449
859-876


75
374-392
802-819


76
243-261
671-688


77
113-131
541-558


78
161-179
561-580


79
220-238
620-638


80
261-279
595-614


81
317-335
720-738


82
374-393
732-751


83
293-312
701-720


84
470-488
901-921


85
185-203
590-609


86
 90-107
509-528


87
 83-103
652-671


88
140-160
709-728


89
361-381
930-949


90
437-456
885-905


91
298-318
727-747


92
385-404
832-852


93
332-351
779-799


94
234-253
681-701


95
347-367
816-836


96
463-483
932-952


97
136-155
591-611


98
75-94
530-550


99
290-310
669-688


100
290-310
669-688


101
325-343
756-776


102
71-90
503-519


103
136-154
571-590


104
417-435
930-949


105
 5-23
530-549


106
285-304
749-769


107
222-241
686-706


108
 4-22
446-466


109
198-218
634-654


110
53-70
504-523


111
450-468
881-899


112
 93-111
524-542


113
285-303
746-766


114
103-121
564-584


115
147-165
587-607


116
 9-29
435-455


117
187-206
636-656


118
406-425
797-816


119
384-403
775-794


120
354-373
745-764


121
216-235
607-626


122
402-420
735-752


123
301-319
707-724


124
324-341
618-635


125
134-153
533-552


126
478-495
906-924


127
399-416
827-845


128
178-195
606-624


129
475-493
879-896


130
179-197
583-600


131
265-284
694-714


132
277-295
720-740


133
360-378
867-887


134
309-327
816-836


135
442-462
898-918


136
383-403
839-859


137
365-385
821-841


138
356-376
812-832


139
104-124
560-580


140
389-408
879-898


141
338-357
828-847


142
337-356
827-846


143
57-76
547-566


144
301-320
682-701


145
10-30
524-542


146
386-406
844-862


147
47-67
547-566


148
268-288
768-787


149
478-498
903-922


150
391-410
828-846


151
385-404
822-840


152
316-336
745-765


153
378-398
807-827


154
427-447
856-876


155
67-86
512-530


156
112-131
557-575


157
380-399
825-843


158
387-406
832-850


159
183-201
672-692


160
321-340
767-787


161
200-219
646-666


162
262-281
761-780


163
247-266
746-765


164
 1-21
527-544


165
42-62
568-585


166
153-173
679-696


167
441-461
967-984


168
364-382
900-920


169
127-145
663-683


170
330-349
769-788


171
284-303
723-742


172
176-195
615-634


173
110-129
549-568


174
222-242
688-708


175
118-135
628-647


176
245-264
749-768


177
128-147
632-651


178
79-98
583-602


179
 1-21
545-563


180
116-136
660-678


181
355-373
811-828


182
403-421
859-876


183
74-94
649-668


184
408-428
 983-1002


185
224-242
627-646


186
366-385
765-784


187
221-239
646-665


188
111-129
512-531


189
246-263
661-679


190
114-133
610-627


191
 9-28
505-522


192
107-126
537-557


193
405-424
891-911


194
216-234
653-673


195
180-198
617-637


196
129-149
661-678


197
220-240
752-769


198
352-372
849-867


199
349-369
856-875


200
461-481
968-987


201
369-389
798-817


202
110-130
539-558


203
329-346
723-741


204
62-79
567-587


205
130-150
558-578


206
131-151
559-579


207
267-284
795-815


208
304-324
770-787


209
216-234
712-731


210
188-207
659-674


211
172-192
645-663


212
194-214
674-693


213
63-83
543-562


214
236-256
687-705


215
268-288
719-737


216
335-355
786-804


217
463-482
899-918


218
72-91
508-527


219
115-135
555-573


220
236-256
681-700


221
290-310
735-754


222
 1-18
345-362


223
 1-18
345-362


224
 1-18
345-362


225
 1-18
345-362


226
 1-18
349-368


227
 1-19
401-420


228
 1-18
409-427


229
 1-18
402-420


230
 1-19
407-426


231
 1-19
403-420


232
 1-19
403-420


233
 1-18
363-380


234
 1-12
444-464


235
 1-12
444-464


236
 1-18
343-361


237
 1-19
418-435


238
 1-19
418-435


239
 1-19
418-435


240
 1-19
420-439


241
 1-19
420-439


242
 1-19
420-439


243
 1-19
406-424


244
 1-19
406-424


245
 1-19
406-424


246
 1-18
354-371


247
 1-18
354-371


248
 1-18
354-371


249
 1-20
408-425


250
130-149
562-579


251
444-464
875-894


252
133-151
564-584


253
411-429
842-862


254
292-312
819-839


255
 6-26
533-553


256
101-121
547-564


257
419-437
866-886


258
347-366
776-796


259
253-273
749-768


260
127-146
656-675


261
408-428
849-859


262
119-139
560-570


263
118-138
559-569


264
111-131
552-562


265
288-307
791-810


266
147-166
650-669


267
65-84
527-546


268
17-37
542-562


269
438-458
963-983


270
329-347
774-794


271
337-357
792-811


272
 92-112
540-559


273
315-335
746-764


274
330-350
761-779


275
331-351
762-780


276
424-444
855-873


277
306-326
737-757


278
449-468
879-898


279
368-386
909-929


280
266-284
807-827


281
242-260
783-803


282
242-260
783-803


283
227-245
768-788


284
323-342
895-915


285
448-468
877-897


286
 98-118
527-547


287
128-148
557-577


288
 1-21
483-502


289
248-268
697-715


290
478-498
927-945


291
245-265
649-667


292
415-435
843-863


293
294-312
786-796


294
379-397
835-855


295
293-312
723-742


296
444-462
845-863


297
365-383
766-784


298
270-288
671-689


299
276-296
733-753


300
276-296
733-753


301
227-247
684-704


302
301-318
733-751


303
289-306
721-739


304
373-393
831-851


305
 5-23
442-462


306
130-148
577-597


307
130-148
577-597


308
130-148
577-597


309
130-148
577-597


310
288-307
756-775


311
445-463
901-921


312
41-61
509-526


313
195-213
636-656


314
307-327
816-834


315
362-381
794-814


316
182-200
677-696


317
114-132
609-628


318
252-270
697-717


319
262-280
741-761


320
386-403
754-771


321
228-245
632-651


322
426-443
827-846


323
238-255
660-678


324
 1-18
412-429


325
 1-20
419-438


326
 1-19
408-425


327
 2-20
403-422


328
 2-20
403-422


329
 1-17
405-418


330
 1-19
410-429


331
 1-19
401-420


332
 1-20
409-428


333
 1-18
290-307


334
 3-20
410-429


335
 1-18
368-385


336
 1-18
424-443


337
 1-18
424-443


338
 1-18
430-447


339
340-358
739-758


340
299-317
817-837


341
236-253
638-657


342
152-169
554-573


343
108-125
510-529


344
357-374
779-798


345
438-457
942-962


346
30-49
534-554


347
234-254
674-694


348
328-348
768-788


349
463-483
903-923


350
452-471
965-985


351
371-391
827-846


352
468-488
924-943


353
367-387
866-885


354
232-251
668-688


355
153-171
607-626


356
137-155
591-610


357
50-68
504-523


358
79-98
589-609


359
455-474
898-918


360
322-340
789-809


361
305-323
772-792


362
221-239
688-708


363
374-394
813-833


364
11-31
528-548


365
450-470
893-911


366
209-229
652-670


367
151-169
667-685


368
285-303
801-819


369
251-271
727-747


370
242-261
745-765


371
257-276
760-780


372
431-450
934-954


373
443-462
875-895


374
433-452
865-885


375
423-442
855-875


376
349-369
839-859


377
 83-103
573-593


378
435-453
951-971


379
64-82
580-600


380
292-311
723-743


381
113-132
544-564


382
229-248
674-694


383
235-255
677-696


384
372-392
814-833


385
446-466
888-907


386
449-469
891-910


387
246-266
674-694


388
399-419
827-847


389
184-204
683-701


390
450-470
949-967


391
148-168
578-598


392
253-273
683-703


393
365-385
795-815


394
426-445
976-995


395
309-329
745-765


396
348-368
870-889


397
138-158
622-640


398
106-126
595-615


399
216-236
705-725


400
 6-26
558-576


401
116-134
600-619


402
105-125
566-584


403
386-406
847-865


404
296-315
781-799


405
142-160
521-541


406
443-460
901-921


407
287-306
765-785


408
217-236
664-683


409
399-418
846-865


410
 1-20
349-366


411
 1-20
349-366


412
 1-18
412-429


413
 1-18
412-429


414
 1-16
333-349


415
 1-16
333-349


416
 1-18
400-420


417
 1-18
407-424


418
 1-18
400-420


425
 1-18
398-417


426
 1-18
398-417


427
 1-20
404-423


428
 1-20
404-423


429
 1-18
403-421


430
 1-18
403-421


431
 1-19
402-419


432
 1-20
405-422


433
 1-18
335-352


434
 1-18
335-352


435
 1-18
413-432


436
 1-19
283-300


437
 1-19
283-300


439
243-263
674-692


440
393-412
802-819


441
114-134
543-563


442
123-141
542-561


443
386-403
792-809


444
153-170
559-576


445
409-426
821-838


446
355-374
783-803


447
 90-109
600-620


448
199-219
670-690


449
467-486
915-935


450
383-403
812-832


451
211-229
688-707


452
33-53
586-605


453
376-395
840-860


454
 92-111
556-576


455
124-142
553-573


456
286-306
745-764


457
456-475
884-904


458
397-415
826-845


459
49-67
536-556


460
210-229
679-698


461
188-207
600-617


462
121-138
521-540


463
210-228
647-667


464
 95-113
511-530


465
147-166
530-550


466
164-183
547-567


467
357-375
804-824


468
336-354
783-803


469
198-216
645-665


470
410-429
792-811


471
370-388
756-776


472
441-459
827-847


473
403-421
904-921


474
180-198
681-698


475
134-152
635-652


476
466-484
967-984


477
180-200
613-630


478
102-122
535-552


479
113-133
546-563


480
329-347
717-734


481
348-366
736-753


482
464-482
852-869


483
451-471
881-900


484
151-168
672-692


485
180-197
701-721


486
469-486
 990-1010


487
 1-20
429-448


488
 1-20
429-448


489
 1-20
429-448


491
 1-18
332-351


492
 1-18
332-351


493
 1-18
402-421


494
 1-19
402-420


495
 1-19
402-420


496
 1-19
402-420


497
 1-20
409-428


498
 1-20
413-431


499
 1-19
403-422


500
 1-19
427-446


501
 1-18
408-427


502
 1-18
408-427


503
 1-19
411-430


504
 1-19
411-430


505
 1-19
411-430


506
 1-20
404-421


507
 1-20
399-418


508
 1-20
399-418


509
 1-20
399-418


510
232-252
703-723


511
415-433
862-882


512
438-456
920-940


513
289-307
771-791


514
295-313
812-829


515
137-155
654-671


516
135-153
652-669


517
165-183
647-667


518
124-144
590-610


519
246-264
650-669


520
198-216
602-621


521
301-320
701-720


522
423-440
833-850


523
131-148
541-558


524
384-401
806-825


525
348-365
770-789


526
453-470
802-820


527
317-334
666-684


528
 1-20
414-433


529
 1-18
330-349


530
 1-18
414-431


532
 1-19
400-419


533
 1-19
400-419


534
 1-20
416-435


535
 1-20
416-435


536
 1-20
416-435


537
 1-20
416-435


538
 1-20
416-435


539
 1-20
416-435


541
 1-20
427-446


542
 1-20
427-446


543
 1-20
427-446


544
 1-20
427-446


545
 1-20
427-446


546
 1-20
427-446


547
 1-20
427-446


548
39-57
548-568


549
15-35
445-464


550
15-35
445-464


551
19-38
407-425


552
342-360
777-797


553
355-374
784-804


554
105-124
534-554


555
187-206
703-721


556
353-370
799-818


557
453-470
899-918


558
446-463
892-911


559
 88-106
519-538


560
264-283
671-689


561
288-306
772-792


562
28-46
512-532


563
101-121
595-614


564
220-240
714-733


565
 1-21
436-454


566
 1-18
404-423


567
 1-18
404-423


568
 1-18
404-423


569
 1-18
404-423


570
 1-18
400-419


571
 1-20
429-448


572
 1-20
479-499


573
 1-21
387-407


574
 1-19
581-598


575
36-56
346-366


576
220-237
553-571


577
157-176
515-534


578
140-159
531-550


579
441-459
948-968


580
133-153
647-665


581
476-494
907-927


582
425-445
939-957


583
292-310
788-807


584
 1-18
443-463


585
 1-19
333-351


586
389-409
916-936


587
207-227
638-658


588
 1-19
401-418


589
376-395
889-909


590
324-344
767-785


591
459-478
904-924


592
281-301
711-731


593
147-166
657-677


594
114-132
547-567


595
235-254
618-638


596
371-389
772-791


597
249-267
650-669


598
246-264
652-669


599
245-263
651-668


600
182-199
590-609


601
131-149
535-552


602
255-274
719-738


603
360-379
824-843


604
409-428
873-892


605
437-456
901-920


606
79-98
543-562


607
 1-18
427-444


608
 1-18
427-444


609
 1-18
427-444


610
 1-18
427-444


611
 1-18
427-444


613
 1-19
402-420


614
218-235
618-637


615
207-224
607-626


616
379-396
776-795


617
390-408
791-809


618
372-390
773-791


619
264-282
665-683


620
201-220
606-623


621
129-148
534-551


622
271-288
684-703


623
224-241
637-656


624
363-381
763-780


625
478-496
878-895


626
241-259
641-658


627
199-217
599-616


628
286-305
683-702


629
172-189
513-532


630
430-447
771-790


631
332-350
739-758


632
181-199
588-607


633
149-167
556-575


634
138-156
545-564


635
 97-115
504-523


636
311-328
727-746


637
257-274
673-692


638
329-347
729-748


639
165-182
569-587


640
147-164
551-569


641
440-457
844-862


642
184-203
607-626


643
252-269
660-677


644
240-257
648-665


645
150-167
558-575


646
137-154
545-562


647
402-420
722-740


648
378-396
630-648


649
309-327
561-579


650
431-449
885-903
















TABLE 14







Preferred probes useful in genotyping eicosanoid-related


biallelic markers by hybridization assays.










SEQ ID
POSITION RANGE OF



NO.
PROBES














1
466-490



2
466-490



3
466-490



4
466-490



5
466-490



6
466-490



7
466-490



8
466-490



9
466-490



10
466-490



11
466-490



12
466-490



13
466-490



14
466-490



15
466-490



16
466-490



17
466-490



18
466-490



19
466-490



20
466-490



21
466-490



22
466-490



23
466-490



24
466-490



25
466-490



26
466-490



27
466-490



28
466-490



29
466-490



30
466-490



31
466-490



32
466-490



33
466-490



34
466-490



35
466-490



36
466-490



37
466-490



38
466-490



39
466-490



40
466-490



41
466-490



42
466-490



43
466-490



44
107-131



45
231-255



46
116-140



47
466-490



48
23-47



49
117-141



50
139-163



51
346-370



52
454-478



53
406-430



54
217-241



55
243-267



56
466-490



57
290-314



58
167-191



59
195-219



60
528-552



61
154-178



62
443-467



63
320-344



64
339-363



65
352-376



66
 87-111



67
135-159



68
253-277



69
488-512



70
489-513



72
489-513



73
489-513



74
489-513



75
489-513



76
489-513



77
489-513



78
489-513



79
489-513



80
489-513



81
489-513



82
489-513



83
489-513



84
489-513



85
488-512



86
489-513



87
489-513



88
489-513



89
489-513



90
489-513



91
489-513



92
489-513



93
489-513



94
489-513



95
489-513



96
489-513



97
246-270



98
489-513



99
431-455



100
301-325



101
489-513



102
489-513



103
489-513



104
489-513



105
373-397



106
489-513



107
489-513



108
368-392



109
311-335



110
305-329



111
489-513



112
489-513



113
489-513



114
489-513



115
489-513



116
217-241



117
474-498



118
489-513



119
489-513



120
489-513



121
489-513



122
489-513



123
489-513



124
489-513



125
489-513



126
485-509



127
489-513



128
489-513



129
489-513



130
489-513



131
489-513



132
489-513



133
489-513



134
489-513



135
489-513



136
489-513



137
489-513



138
489-513



139
489-513



140
489-513



141
489-513



142
489-513



143
489-513



144
489-513



145
489-513



146
489-513



147
489-513



148
489-513



149
489-513



150
489-513



151
489-513



152
489-513



153
489-513



154
489-513



155
489-513



156
489-513



157
489-513



158
489-513



159
489-513



160
489-513



161
489-513



162
489-513



163
489-513



164
496-520



165
489-513



166
489-513



167
489-513



168
489-513



169
489-513



170
489-513



171
489-513



172
489-513



173
489-513



174
489-513



175
489-513



176
489-513



177
489-513



178
489-513



179
529-553



180
489-513



181
489-513



182
489-513



183
489-513



184
489-513



185
489-513



186
489-513



187
489-513



188
489-513



189
489-513



190
489-513



191
489-513



192
489-513



193
489-513



194
489-513



195
489-513



196
489-513



197
489-513



198
489-513



199
489-513



200
489-513



201
489-513



202
489-513



203
489-513



204
489-513



205
489-513



206
489-513



207
489-513



208
489-513



209
489-513



210
489-513



211
489-513



212
489-513



213
489-513



214
489-513



215
489-513



216
489-513



217
489-513



218
489-513



219
489-513



220
489-513



221
489-513



222
60-84



223
61-85



224
67-91



225
126-150



226
69-93



227
29-53



228
23-47



229
262-286



230
121-145



231
185-209



232
186-210



233
350-374



234
375-399



235
397-421



236
295-319



237
235-259



238
282-306



239
335-359



240
 92-116



241
311-335



242
391-415



243
 86-110



244
276-300



245
368-392



246
148-172



247
194-218



248
271-295



249
305-329



250
489-513



251
489-513



252
489-513



253
489-513



254
489-513



255
509-533



256
489-513



257
489-513



258
489-513



259
489-513



260
489-513



261
489-513



262
489-513



263
488-512



264
489-513



265
489-513



266
489-513



267
489-513



268
489-513



269
489-513



270
489-513



271
489-513



272
489-513



273
489-513



274
489-513



275
490-514



276
489-513



277
471-495



278
489-513



279
489-513



280
488-512



281
464-488



282
486-510



283
489-513



284
489-513



285
489-513



286
489-513



287
489-513



288
433-457



289
489-513



290
487-511



291
489-513



292
489-513



293
489-513



294
489-513



295
489-513



296
489-513



297
489-513



298
489-513



299
367-391



300
374-398



301
489-513



302
489-513



303
489-513



304
489-513



305
427-451



306
266-290



307
314-338



308
326-350



309
401-425



310
489-513



311
489-513



312
489-513



313
489-513



314
489-513



315
489-513



316
489-513



317
489-513



318
489-513



319
489-513



320
489-513



321
489-513



322
489-513



323
489-513



324
374-398



325
228-252



326
264-288



327
30-54



328
329-353



329
252-276



330
266-290



331
164-188



332
102-126



333
164-188



334
230-254



335
338-362



336
30-54



337
362-386



338
247-271



339
489-513



340
489-513



341
489-513



342
489-513



343
489-513



344
489-513



345
489-513



346
489-513



347
489-513



348
489-513



349
489-513



350
464-488



351
489-513



352
489-513



353
489-513



354
489-513



355
489-513



356
489-513



357
489-513



358
489-513



359
489-513



360
489-513



361
489-513



362
489-513



363
489-513



364
489-513



365
489-513



366
489-513



367
489-513



368
489-513



369
489-513



370
489-513



371
489-513



372
489-513



373
489-513



374
489-513



375
489-513



376
489-513



377
489-513



378
489-513



379
489-513



380
489-513



381
489-513



382
489-513



383
489-513



384
489-513



385
489-513



386
489-513



387
489-513



388
489-513



389
489-513



390
489-513



391
489-513



392
489-513



393
489-513



394
467-491



395
489-513



396
489-513



397
489-513



398
489-513



399
489-513



400
516-540



401
489-513



402
489-513



403
489-513



404
489-513



405
489-513



406
489-513



407
489-513



408
489-513



409
489-513



410
33-57



411
51-75



412
165-189



413
390-414



414
124-148



415
128-152



416
27-51



417
53-77



418
245-269



425
357-381



426
23-47



427
 90-114



428
112-136



429
171-195



430
370-394



431
21-45



432
138-162



433
210-234



434
238-262



435
342-366



436
30-54



437
144-168



438
214-238



439
489-513



440
489-513



441
489-513



442
489-513



443
489-513



444
489-513



445
489-513



446
489-513



447
489-513



448
489-513



449
489-513



450
489-513



451
489-513



452
489-513



453
489-513



454
489-513



455
489-513



456
489-513



457
489-513



458
489-513



459
489-513



460
489-513



461
489-513



462
489-513



463
489-513



464
489-513



465
489-513



466
489-513



467
489-513



468
489-513



469
489-513



470
489-513



471
489-513



472
489-513



473
489-513



474
489-513



475
489-513



476
489-513



477
489-513



478
489-513



479
489-513



480
489-513



481
489-513



482
489-513



483
489-513



484
489-513



485
489-513



486
489-513



487
127-151



488
200-224



489
229-253



491
173-197



492
252-276



493
10-34



494
140-164



495
244-268



496
270-294



497
269-293



498
130-154



499
290-314



500
319-343



501
140-164



502
246-270



503
 91-115



504
132-156



505
263-287



506
215-239



507
143-167



508
369-393



509
 86-110



510
489-513



511
489-513



512
489-513



513
489-513



514
489-513



515
489-513



516
489-513



517
489-513



518
489-513



519
489-513



520
489-513



521
489-513



522
489-513



523
489-513



524
489-513



525
489-513



526
489-513



527
489-513



528
315-339



529
40-64



530
34-58



532
 98-122



533
261-285



534
 78-102



535
192-216



536
209-233



537
222-246



538
276-300



539
299-323



541
27-51



542
108-132



543
142-166



544
 89-113



545
74-98



546
 80-104



547
 82-106



548
489-513



549
404-428



550
 76-100



551
275-299



552
427-451



553
489-513



554
489-513



555
489-513



556
489-513



557
489-513



558
489-513



559
489-513



560
489-513



561
489-513



562
489-513



563
489-513



564
489-513



565
238-262



566
73-97



567
39-63



568
195-219



569
385-409



570
207-231



596
489-513



597
489-513



598
489-513



599
489-513



601
489-513



602
489-513



603
489-513



604
489-513



605
489-513



607
129-153



608
146-170



609
255-279



610
325-349



611
354-378



612
405-429



614
489-513



616
489-513



617
489-513



618
489-513



619
489-513



621
489-513



623
489-513



624
489-513



625
488-512



626
489-513



627
489-513



629
489-513



630
489-513



631
489-513



632
489-513



633
489-513



634
489-513



635
489-513



636
489-513



637
489-513



640
489-513



641
489-513



642
489-513



643
489-513



644
489-513



645
489-513



646
489-513



647
489-513



648
489-513



649
489-513



650
489-513

















TABLE 15





HAPLOTYPE FREQUENCY ANALYSIS
















MARKERS



FLAP















cases/controls
10-253-298
10-33-175
10-33-234
10-33-327
10-35-358
10-35-390
12-628-306
12-629-241












freq %
5′ gene
exon 2
intron 2
intron 4
3′ gene















case/controls
287/186
295/174
295/274
295/270
291/280
295/272
284/185
283/182


diff. freq. all.
95/95 (C)
99/98 (C)
49/44 (A)
78/76 (T)
72/69 (G)
31/23 (C)
88/90 (C)
76/72 (G)


(cases - controls)
0.5
1.8
5.3
2.6
3.4
9
2.1
4.6


pvalue
6.55E−01
1.35E−02
6.93E−02
2.94E−01
2.06E−01
2.29E−03
3.17E−01
1.14E−01



















1
293 vs 265


A


T




2
281 vs 177


A




G


3
293 vs 261



T

T


4
289 vs 271




G
T


5
293 vs 168

C



T


6
293 vs 265


A
T


7
282 vs 178





T

G


37
281 vs 176


A


T
C


38
280 vs 173


A


T

G


39
289 vs 264


A

G
T


40
278 vs 175


A



C
G


41
284 vs 176
C

A


T


121
277 vs 171


A


T
C
G


122
278 vs 173


A

G
T

G


123
279 vs 176


A

G
T
C


124
276 vs 175


A

G

C
G


125
280 vs 174
C

A


T
C


247
275 vs 171


A

G
T
C
G


248
276 vs 169
C

A


T
C
G


373
274 vs 169
C

A

G
T
C
G


457
273 vs 163
C

A
T
G
T
C
G














MARKERS





FLAP



cases/controls










freq %












case/controls
ESTIMATED FREQUENCIES














diff. freq. all.
Frequencies






(cases - controls)
haplotype
Odds

Pvalue














pvalue
cases
controls
ratio
Chi-S
(1 df)



















1
293 vs 265
0.283
0.197
1.61
11.18
(8.2e−04)



2
281 vs 177
0.305
0.210
1.65
9.97
(1.6e−03)



3
293 vs 261
0.307
0.224
1.53
9.62
(1.8e−03)



4
289 vs 271
0.304
0.231
1.46
7.77
(5.2e−03)



5
293 vs 168
0.309
0.226
1.53
7.26
(6.9e−03)



6
293 vs 265
0.276
0.208
1.46
7.17
(7.3e−03)



7
282 vs 178
0.314
0.233
1.50
7.01
(7.7e−03)



37
281 vs 176
0.265
0.171
1.76
11.04
(8.6e−04)



38
280 vs 173
0.292
0.194
1.71
10.71
(1.0e−03)



39
289 vs 264
0.283
0.199
1.59
10.56
(1.1e−03)



40
278 vs 175
0.271
0.180
1.70
9.94
(1.6e−03)



41
284 vs 176
0.287
0.195
1.66
9.77
(1.7e−03)



121
277 vs 171
0.265
0.169
1.77
11.07
(8.6e−04)



122
278 vs 173
0.290
0.195
1.69
10.29
(1.3e−03)



123
279 vs 176
0.264
0.175
1.70
9.80
(1.7e−03)



124
276 vs 175
0.271
0.181
1.69
9.72
(1.7e−03)



125
280 vs 174
0.265
0.176
1.69
9.68
(1.8e−03)



247
275 vs 171
0.265
0.170
1.77
10.91
(9.1e−04)



248
276 vs 169
0.265
0.172
1.74
10.30
(1.3e−03)



373
274 vs 169
0.265
0.172
1.73
10.13
(1.4e−03)



457
273 vs 163
0.247
0.167
1.64
7.74
(5.2e−03)

















TABLE 16





HAPLOTYPE FREQUENCY ANALYSIS PERMUTATIONS TEST RESULTS (>1000 Iterations)


















10-33-234
10-35-390


Markers
intron 2
intron 4





ALT vs US
A
T











cases vs US controls
5.3 (51 vs 56)
6.93E−02
9 (31 vs 23)
2.29E−03


ASSOCIATION
diff all. Freq
pvalue
diff all. Freq
pvalue















sample
haplotype

PERMUTATIONS



sizes
frequencies

TEST RESULTS

















HAPLOTYPE
cases vs

con-

odds-


Av.
Max
>Iter/


(AT)
controls
cases
trols
p-excess
ratio
chi-S
P value
Chi-S
Chi-S
nb of Iter.




















Asthmatics vs
293 vs 265
0.283
0.197
10.7
1.61
11.18
8.20E−04
1.2
7.4
0/1000


US controls







1.2
12.9
1/10 000
















TABLE 17





HAPLOTYPE FREQUENCY ANALYSIS (Asthma)


297 Asthmatics vs 186 US controls randoms
















MARKERS















12-lipoxygenase
12-208-35
12-226-167
12-206-366
10-347-203
10-347-220
10-349-97
10-349-224













cases/controls
5′ gene
intron 2
exon 6
exon 8
12-196-119
12-214-129
















frequency %
284/182
288/188
272/89
285/184
274/184
282/182
271/177
281/184
282/181


(case/controls)
59/58 (T)
62/59 (C)
57/62 (T)
57/58 (A)
58/60 (G)
59/60 (A)
57/60 (G)
70/71 (T)
61/61 (T)


diff freq. all. (cases
0.9
3.4
−4.6
−1.2
−1.7
−1.9
−3.1
−1.2
−0.7


controls)
7.52e−01
2.94e−01
2.73e−01
6.55e−01
5.84e−01
5.27e−01
3.43e−01
6.55e−01
7.52e−01


pvalue
*
*
*
*
*
*
*
*
*




















1
268 vs 176




G



C


2
277 vs 174





A


C


3
274 vs 179




G


4
282 vs 176





A


5
280 vs 176



A




C


6
285 vs 178



A


7
270 vs 176

C




T


8
247 vs 86 


C

A

T


9
255 vs 85 


C
G


T


10
253 vs 84 


C


G
T


11
267 vs 172
A





T


12
281 vs 181

C



G


13
274 vs 182

C


A


14
278 vs 174



A



T


15
267 vs 175




G


T


16
276 vs 173





A

T


17
273 vs 172



A



T
C


18
268 vs 172




G



C


19
261 vs 172




G


T
C


20
271 vs 171





A

T
C


21
277 vs 169





A


C


22
280 vs 171



A




C


23
264 vs 170




G
A


C


24
264 vs 81 
T

C


G













MARKERS













12-lipoxygenase






cases/controls
12-216-421
12-219-230
12-223-207



frequency %
288/182
288/187
287/186













(case/controls)
61/64 (G)
64/68 (A)
62/62 (T)
ESTIMATED FREQUENCIES

















diff freq. all. (cases
−2.9
−4.2
0.8
haplotype







controls)
3.71e−01
1.80e−01
7.52e−01
frequencies
p-
Odds


















pvalue
*
*
*
cases
controls
excess
ratio
Chi-S
Pvalue (1 df)























1
268 vs 176

G

0.123
0.040
8.63
3.38
17.85
(2.3e−05)



2
277 vs 174

G

1.125
0.041
8.71
3.31
17.75
(2.5e−05)



3
274 vs 179
A
G

0.123
0.041
8.49
3.26
17.47
(2.9e−05)



4
282 vs 176
A
G

0.125
0.043
8.57
3.20
17.29
(3.2e−05)



5
280 vs 176

G

0.115
0.037
8.08
3.36
16.81
(3.9e−05)



6
285 vs 178
A
G

0.113
0.039
7.73
3.16
15.62
(7.4e−05)



7
270 vs 176


T
0.130
0.055
7.98
2.58
13.40
(2.5e−04)



8
247 vs 86 



0.405
0.256
19.96
1.97
12.10
(5.0e−04)



9
255 vs 83 



0.406
0.259
19.81
1.95
11.80
(5.6e−04)



10
253 vs 84 



0.399
0.253
19.62
1.97
11.73
(5.9e−04)



11
267 vs 172


T
0.088
0.030
5.97
3.09
11.45
(7.0e−04)



12
281 vs 181


T
0.136
0.066
7.50
2.22
11.10
(8.6e−04)



13
274 vs 182


T
0.137
0.067
7.52
2.21
11.05
(8.6e−04)



14
278 vs 174
A
G

0.118
0.031
9.01
4.18
21.01
(4.4e−06)



15
267 vs 175
A
G

0.124
0.035
9.27
3.92
20.87
(4.8e−06)



16
276 vs 173
A
G

0.124
0.035
9.23
3.91
20.65
(5.4e−06)



17
273 vs 172

G

0.121
0.034
9.01
3.90
20.02
(7.3e−06)



18
268 vs 172
A
G

0.124
0.036
9.14
3.76
19.84
(8.2e−06)



19
261 vs 172

G

0.126
0.037
9.20
3.74
19.81
(8.2e−06)



20
271 vs 171

G

0.125
0.037
9.11
3.69
19.49
(1.0e−05)



21
277 vs 169
A
G

0.125
0.038
9.06
3.64
19.10
(1.2e−05)



22
280 vs 171
A
G

0.116
0.033
8.56
3.81
18.76
(1.5e−05)



23
264 vs 170

G

0.125
0.040
8.91
3.45
18.15
(2.0e−05)



24
264 vs 81 
G


0.197
0.056
14.96
4.13
18.01
(2.1e−05)

















TABLE 18A





ALLELE FREQUENCY ANALYSIS (Asthma)


CASES (297 ALT) vs CONTROLS (186 US CAUCASIAN)





















MARKERS
12-197/244
12-205/35
12-226/167
12-206/366
10-346/141
10-347/111











PROTEIN 12-LO
5′ gene
In2
ex5
ex6
















cases/controls
277/180
284/182
288/188
272/89
285/185
284/180


frequency % (case/controls)
66/67 (T)
58/57 (T)
62/58 (C)
57/61 (T)
99/100 (G)
99/100 (G)


diff freq. all. (cases - controls)
−1.0
0.9
3.4
−4.6
−0.4
−0.2


pvalue
7.52e−01
7.52e−01
2.94e−01
2.73e−01
HOM
HOM



*
*
*
*














Test
cases vs
0.034 (HWD)
−0.002 (HWE)
−0.001 (HWE)
−0.014 (HWE)
0.000 (HWD)
0.000 (HWD)


Hardy Weinberg
controls
0.054 (HWD)
−0.020 (HWE)
0.022 (HWE)
0.000 (HWE)
0.000 (HWD)
0.000 (HWD)
















MARKERS
10-347/165
10-347/203
10-347/220
10-349/97
10-349/224
10-341/116










PROTEIN 12-LO
ex6
ex8
ex14
















cases/controls
268/185
280/184
283/184
287/182
277/177
286/176


frequency % (case/controls)
99/100 (C)
57/58 (A)
57/59 (G)
59/60 (A)
56/60 (G)
89/89 (G)


diff freq. all. (cases - controls)
−0.2
−1.1
−2.1
−1.4
−4.1
0.1


pvalue
5.92e−01#
6.55e−01
4.80e−01
6.55e−01
2.06e−01
7.52e−01



*
*
*
*
*
*














Test
cases vs
0.000 (HWD)
−0.011 (HWE)
−0.005 (HWE)
0.003 (HWE)
−0.010 (HWE)
−0.008 (HWE)


Hardy Weinberg
controls
0.000 (HWD)
0.012 (HWE)
0.021 (HWE)
0.008 (HWE)
−0.004 (HWE)
−0.000 (HWE)

















MARKERS
12-196/119
12-214/129
12-216/421
12-219/230
12-223/207











PROTEIN 12-LO
markers in bac



















cases/controls
281/184
282/181
288/182
288/187
287/186



frequency % (case/controls)
69/70 (T)
60/61 (T)
61/64 (G)
63/67 (A)
62/61 (T)



diff freq. all. (cases - controls)
−1.2
−0.7
−2.9
−4.2
0.8



pvalue
6.55e−01
7.52e−01
3.71e−01
1.80e−01
7.52e−01




*
*
*
*
*















Test
cases vs
0.012 (HWE)
−0.013 (HWE)
−0.012 (HWE)
−0.010 (HWE)
0.012 (HWD)



Hardy Weinberg
controls
0.030 (HWE)
0.016 (HWE)
0.024 (HWE)
−0.001 (HWE)
−0.019 (HWD)

















TABLE 18B





HAPLOTYPE FREQUENCY ANALYSIS (Asthma)


CASES (297 ALT) vs CONTROLS (186 US CAUCASIAN)

















ESTIMATED FREQUENCIES










Haplotype




frequencies
p-
















Marker 1
Marker 2
Marker 3
Marker 4
Haplotype
cases
controls
excess





















haplotype 1
PT2
265 vs 86
12-206/366
10-349/224


CT
0.424
0.265
21.72


haplotype 2
PT2
267 vs 89
12-206/366
10-347/220


CA
0.423
0.274
20.56


haplotype 3
PT2
266 vs 88
12-206/366
10-347/203


CG
0.421
0.277
19.96


haplotype 4
PT2
271 vs 87
12-206/366
10-349/97


CG
0.408
0.270
18.88


haplotype 5
PT2
271 vs 174
12-197/244
12-214/129


CC
0.148
0.077
7.66


haplotype 6
PT2
285 vs 175
10-341/116
12-223/207


AT
0.042
0.008
3.45


haplotype 7
PT3
282 vs 174
10-349/97
12-214/129
12-219/230

ACG
0.125
0.041
8.73


haplotype 8
PT3
287 vs 176
10-349/97
12-216/421
12-219/230

AAG
0.126
0.043
8.67


haplotype 9
PT3
277 vs 176
10-347/220
12-214/129
12-219/230

GCG
0.119
0.040
8.28


haplotype 10
PT3
275 vs 176
10-347/203
12-214/129
12-219/230

ACG
0.115
0.037
8.05


haplotype 11
PT3
283 vs 179
10-347/220
12-216/421
12-219/230

GAG
0.119
0.041
8.11


haplotype 12
PT3
266 vs 171
12-197/244
10-347/203
12-214/129

CAC
0.070
0.012
5.96


haplotype 13
PT3
248 vs 85
12-206/366
10-347/165
10-349/224

CCT
0.427
0.255
23.11


haplotype 14
PT3
271 vs 168
12-197/244
10-349/97
12-214/129

CAC
0.069
0.012
5.83


haplotype 15
PT3
261 vs 86
12-206/366
10-347/220
10-349/224

CAT
0.423
0.256
22.45


haplotype 16
PT3
276 vs 176
12-226/167
10-349/224
12-223/207

CTT
0.137
0.055
8.64


haplotype 17
PT3
280 vs 178
10-347/203
12-216/421
12-219/230

AAG
0.112
0.039
7.59


haplotype 18
PT3
268 vs 170
12-197/244
10-347/220
12-214/129

CGC
0.067
0.012
5.60


haplotype 19
PT3
249 vs 88
12-206/366
10-347/165
10-347/220

CCA
0.428
0.265
22.14


haplotype 20
PT3
264 vs 86
12-206/366
10-346/141
10-349/224

CGT
0.426
0.265
21.94


haplotype 21
PT3
261 vs 85
12-206/366
10-347/203
10-349/224

CGT
0.418
0.259
21.40


haplotype 22
PT3
264 vs 84
12-206/366
10-349/97
10-349/224

CGT
0.411
0.253
21.19


haplotype 23
PT3
248 vs 87
12-206/366
10-347/165
10-347/203

CCG
0.425
0.268
21.54


haplotype 24
PT3
261 vs 86
12-206/366
10-347/111
10-349/224

CGT
0.421
0.265
21.30


haplotype 25
PT3
266 vs 164
12-197/244
10-347/111
12-214/129

CGC
0.151
0.068
8.89


haplotype 26
PT3
265 vs 89
12-206/366
10-346/141
10-347/220

CGA
0.426
0.274
21.00


haplotype 27
PT4
280 vs 173
10-349/97
12-196/119
12-216/421
12-219/230
ATAG
0.124
0.035
9.20


haplotype 28
PT4
274 vs 174
10-347/203
12-196/119
12-216/421
12-219/230
ATAG
0.117
0.031
8.89


haplotype 29
PT4
275 vs 171
10-349/97
12-196/119
12-214/129
12-219/230
ATCG
0.126
0.037
9.21


haplotype 30
PT4
276 vs 175
10-347/220
12-196/119
12-216/421
12-219/230
GTAG
0.121
0.035
8.92


haplotype 31
PT4
269 vs 172
10-347/203
12-196/119
12-214/129
12-219/230
ATCG
0.120
0.034
8.90


haplotype 32
PT4
280 vs 165
10-349/97
10-341/116
12-214/129
12-219/230
AGCG
0.127
0.038
9.30


haplotype 33
PT4
270 vs 172
10-347/220
12-196/119
12-214/129
12-219/230
GTCG
0.124
0.037
9.00


haplotype 34
PT4
282 vs 169
10-349/97
12-214/129
12-216/421
12-219/230
ACAG
0.124
0.038
9.00


haplotype 35
PT4
267 vs 167
12-197/244
12-208/35
12-214/129
12-223/207
CTCC
0.055
0.000
5.49


haplotype 36
PT4
285 vs 167
10-349/97
10-341/116
12-216/421
12-219/230
AGAG
0.127
0.039
9.12


haplotype 37
PT4
277 vs 172
10-347/220
12-214/129
12-216/421
12 219/230
GCAG
0.120
0.036
8.71


haplotype 38
PT4
275 vs 171
10-347/203
12-214/129
12-216/421
12-219/230
ACAG
0.116
0.033
8.52


haplotype 39
PT4
276 vs 162
12-208/35
10-341/116
12-214/129
12-219/230
AGCG
0.054
0.000
5.40


haplotype 40
PT4
245 vs 85
12-206/366
10-347/165
10-347/220
10-349/224
CCAT
0.429
0.246
24.18


haplotype 41
PT4
268 vs 81
12-208/35
12-206/366
10-349/97
12-216/421
TCGG
0.196
0.056
14.80














PERMUTATIONS



ESTIMATED FREQUENCIES
TEST RESULTS














Odds


Av.
Max
>Iter/



ratio
Chi-S
Pvalue (1 df)
Chi-S
Chi-s
nb of Iter






















haplotype 1
PT2
265 vs 86
2.05
13.97
(1.8e−04)
****
2.2
8.3
[0/100]



haplotype 2
PT2
267 vs 89
1.94
12.55
(3.9e−04)
***
2.3
9.3
[0/100]



haplotype 3
PT2
266 vs 88
1.90
11.64
(6.3e−04)
***
2.1
7.1
[0/100]



haplotype 4
PT2
271 vs 87
1.86
10.69
(1.1e−03)
***
1.7
5.9
[0/100]



haplotype 5
PT2
271 vs 174
2.08
10.03
(1.5e−03)
***
1.8
9.7
[0/100]



haplotype 6
PT2
285 vs 175
5.48
8.95
(2.7e−03)
***
1.4
9.9
[2/100]



haplotype 7
PT3
282 vs 174
3.32
17.87
(2.3e−05)
*****
1.8
8.5
[0/100]



haplotype 8
PT3
287 vs 176
3.23
17.65
(2.6e−05)
*****
1.5
13.6
[0/100]



haplotype 9
PT3
277 vs 176
3.27
16.90
(3.7e−05)
****
3.0
15.4
[0/100]



haplotype 10
PT3
275 vs 176
3.35
16.64
(4.4e−05)
****
2.4
19.2
[1/100]



haplotype 11
PT3
283 vs 179
3.15
16.40
(5.1e−05)
****
2.6
14.4
[0/100]



haplotype 12
PT3
266 vs 171
6.46
15.97
(6.3e−05)
****
1.9
11.3
[0/100]



haplotype 13
PT3
248 vs 85
2.18
15.86
(6.7e−05)
****
2.7
18.8
[1/100]



haplotype 14
PT3
271 vs 168
6.37
15.34
(8.7e−05)
****
1.8
10.4
[0/100]



haplotype 15
PT3
261 vs 86
2.13
15.27
(9.2e−05)
****
2.4
7.8
[0/100]



haplotype 16
PT3
276 vs 176
2.72
15.27
(9.2e−05)
****
1.4
7.1
[0/100]



haplotype 17
PT3
280 vs 178
3.12
15.15
(9.7e−05)
****
2.2
21.1
[2/100]



haplotype 18
PT3
268 vs 170
6.10
14.68
(1.3e−04)
****
2.0
12.7
[0/100]



haplotype 19
PT3
249 vs 88
2.07
14.54
(1.3e−04)
****
2.8
9.4
[0/100]



haplotype 20
PT3
264 vs 86
2.06
14.23
(1.6e−04)
****
2.6
11.2
[0/100]



haplotype 21
PT3
261 vs 85
2.05
13.71
(2.0e−04)
****
2.2
8.3
[0/100]



haplotype 22
PT3
264 vs 84
2.06
13.68
(2.1e−04)
****
2.4
7.2
[0/100]



haplotype 23
PT3
248 vs 87
2.03
13.55
(2.3e−04)
****
2.2
8.5
[0/100]



haplotype 24
PT3
261 vs 86
2.02
13.43
(2.4e−04)
****
2.1
6.1
[0/100]



haplotype 25
PT3
266 vs 164
2.44
13.33
(2.5e−04)
****
1.9
25.3
[1/100]



haplotype 26
PT3
265 vs 89
1.97
13.04
(3.0e−04)
****
2.3
7.0
[0/100]



haplotype 27
PT4
280 vs 173
3.90
20.63
(5.4e−06)
*****
1.5
11.6
[0/100]



haplotype 28
PT4
274 vs 174
4.14
20.59
(5.7e−06)
*****
2.7
19.0
[0/100]



haplotype 29
PT4
275 vs 171
3.72
19.86
(8.2e−06)
*****
2.0
11.8
[0/100]



haplotype 30
PT4
276 vs 175
3.80
19.84
(8.2e−06)
*****
2.4
14.7
[0/100]



haplotype 31
PT4
269 vs 172
3.86
19.61
(9.1e−06)
*****
2.4
12.2
[0/100]



haplotype 32
PT4
280 vs 165
3.73
19.55
(9.5e−06)
*****
1.9
16.0
[0/100]



haplotype 33
PT4
270 vs 172
3.68
19.31
(1.1e−05)
*****
2.8
19.3
[0/100]



haplotype 34
PT4
282 vs 169
3.62
18.98
(1.3e−05)
*****
1.8
10.5
[0/100]



haplotype 35
PT4
267 vs 167
100.00
18.96
(1.3e−05)
*****
2.6
21.7
[2/100]



haplotype 36
PT4
285 vs 167
3.55
18.89
(1.4e−05)
*****
2.0
12.3
[0/100]



haplotype 37
PT4
277 vs 172
3.62
18.59
(1.6e−05)
*****
2.3
25.8
[1/100]



haplotype 38
PT4
275 vs 171
3.80
18.57
(1.6e−05)
*****
2.7
21.6
[1/100]



haplotype 39
PT4
276 vs 162
0.00
18.10
(2.0e−05)
*****
2.7
18.6
[1/100]



haplotype 40
PT4
245 vs 85
2.30
17.77
(2.5e−05)
*****
3.0
7.5
[0/100]



haplotype 41
PT4
268 vs 81
4.09
17.76
(2.5e−05)
*****
1.8
15.1
[0/100]

















TABLE 19





HAPLOTYPE FREQUENCY ANALYSIS (Zyflo secondary effects)


89 ALT+ vs 208 ALT−
















MARKERS















12-lipoxygenase









Size
12-208-35
12-226-167
12-206-366
10-347-203
10-347-220
10-349-97
10-349-224













(cases/controls)
5′ gene
Intron 2
exon 6
exon 8
12-196-119
12-214-129
















frequency %
87/197
89/199
86/186
88/197
86/188
86/196
86/185
86/195
89/193


(cases/controls)
58/59 (T)
61/63 (C)
55/58 (T)
56/58 (A)
56/59 (G)
58/59 (A)
54/59 (G)
72/69 (T)
59/61 (T)


diff freq. all.
−0.8
−2.1
−3.7
−2.2
−3.0
−1.4
−4.8
3.4
−2.4


(cases - controls)
7.52e−01
5.84e−01
4.03e−01
5.84e−01
4.80e−01
7.52e−01
2.73e−01
4.03e−01
5.84e−01


p value
*
*
*
*
*
*
*
*
*




















1
87 vs 197
A
G









2
83 vs 184




A


C


3
85 vs 185






T


4
85 vs 186


C


5
85 vs 179






T

C


6
85 vs 180


C





C


7
86 vs 188




A


8
82 vs 174


C



T


9
85 vs 179






T

C


10
83 vs 177




A

T


11
82 vs 183





G
T


12
85 vs 183



G


T


13
82 vs 168


C



T

C


14
84 vs 175


C

A


15
84 vs 184


C
G


16
85 vs 180


C





C


17
82 vs 181






T
T


18
83 vs 187
A






T
T


19
83 vs 171




A

T

C


20
83 vs 174


C



T


21
82 vs 178





G
T

C


22
82 vs 168


C



T

C


23
82 vs 172


C
G


T


24
81 vs 166


C

A

T


25
80 vs 171


C



T
T













MARKERS











2-lipoxygenase




Size












(cases/controls)
12-216-421

12-223-207



frequency %
89/199
12-219230
88/199













(cases/controls)
58/63 (G)
89/199
62/63 (T)
ESTIMATED FREQUENCIES















diff freq. all.
−4.7
67/62 (A)
−0.6
haplotype





(cases - controls)
2.73e−01
4.5
7.52e−01
frequencies
Odds

















p value
*
2.94e−01
*
cases
controls
ratio
Chi-S
Pvalue(1 df)






















1
87 vs 197



0.123
0.070
1.87
4.34
(3.6e−02)



2
83 vs 184



0.209
0.140
1.63
4.09
(4.3e−02)



3
85 vs 185
A

T
0.151
0.048
3.53
16.76
(4.2e−05)



4
85 vs 186
A

T
0.148
0.059
2.77
11.62
(6.3e−04)



5
85 vs 179


T
0.151
0.066
2.52
9.85
(1.7e−03)



6
85 vs 180


T
0.149
0.070
2.34
8.42
(3.6e−03)



7
86 vs 188
A

T
0.157
0.076
2.25
8.36
(3.8e−03)



8
82 vs 174
A

T
0.158
0.040
4.56
21.85
(2.9e−06)



9
85 vs 179
A

T
0.157
0.052
3.37
16.03
(6.0e−05)



10
83 vs 177
A

T
0.162
0.057
3.22
15.30
(9.2e−05)



11
82 vs 183
A

T
0.147
0.050
3.23
14.18
(1.6e−04)



12
85 vs 183
A

T
0.143
0.049
3.21
13.97
(1.8e−04)



13
82 vs 168


T
0.156
0.058
3.02
13.08
(3.0e−04)



14
84 vs 175
A

T
0.155
0.062
2.78
11.77
(5.9e−04)



15
84 vs 184
A

T
0.147
0.059
2.74
11.22
(7.8e−04)



16
85 vs 180
A

T
0.145
0.058
2.74
11.05
(8.6e−04)



17
82 vs 181
A

T
0.132
0.050
2.85
10.62
(1.1e−03)



18
83 vs 187


T
0.148
0.066
2.48
9.50
(2.1e−03)



19
83 vs 171


T
0.162
0.074
2.41
9.29
(2.3e−03)



20
83 vs 174
A
A

0.111
0.041
2.92
9.24
(2.3e−03)



21
82 vs 178


T
0.152
0.068
2.45
9.22
(2.3e−03)



22
82 vs 168
A

T
0.161
0.043
4.27
20.43
(6.0e−06)



23
82 vs 172
A

T
0.146
0.040
4.07
18.03
(2.1e−05)



24
81 vs 166
A

T
0.160
0.047
3.82
17.77
(2.5e−05)



25
80 vs 171
A

T
0.137
0.037
4.17
17.18
(3.4e−05)

















TABLE 20A





ALLELE FREQUENCY ANALYSIS (Zyflo secondary effects)


CASES (85 ALT+) vs CONTROLS (208 ALT−)





















MARKERS
12-197/244
12-208/35
12-226/167
12-206/366
10-346/141
10-347/111











PROTEIN 12-LO
5′gene
In2
ex5
ex6
















cases/controls
81/196
87/197
89/199
86/186
88/197
88/196


frequency % (case/controls)
70/65(T)
58/59(T)
61/63(C)
55/58(T)
100/99(G)
99/100(G)


diff freq. all. (cases - controls)
5.8
−0.8
−2.1
−3.7
0.5
−0.6


pvalue
1.80e−01
7.52e−01
5.84e−01
4.03e−01
HOM
HOM



*
*
*
*














Test
cases vs
−0.001 (HWE)
0.008 (HWE)
−0.020 (HWE)
−0.031 (HWE)
0.000 (HWD)
0.000 (HWD)


Hardy Weinberg
controls
0.048 (HWD)
−0.007 (HWE)
0.007 (HWE)
−0.007 (HWE)
0.000 (HWD)
0.000 (HWD)
















MARKERS
10-347/165
10-347/203
10-347/220
10-349/97
10-349/224
10-341/116










PROTEIN 12-LO
ex6
ex8
ex14
















cases/controls
69/199
83/197
87/196
89/198
83/194
89/197


frequency % (case/controls)
100/99(C)
56/58(A)
43/57(G)
59/59(A)
54/57(G)
90/89(G)


diff freq. all. (cases - controls)
0.3
−1.8
0.1
−0.1
−3.0
1.6


pvalue
7.43e−01#
6.55e−01
7.52e−01
7.52e−01
4.80e−01
5.27e−01



*
*
*
*
*
*














Test
cases vs
0.000 (HWD)
−0.037 (HWE)
0.020 (HWE)
0.000 (HWE)
−0.029 (HWE)
0.002 (HWE)


Hardy Weinberg
controls
0.000 (HWD)
0.000 (HWE)
0.002 (HWE)
0.004 (HWE)
−0.003 (HWD)
0.764 (HWE)

















MARKERS
12-196/119
12-214/129
12-216/421
12-219/230
12-223/207











PROTEIN 12-LO
markers in bac



















cases/controls
86/195
89/193
89/199
89/199
88/199



frequency % (case/controls)
72/69(T)
59/61(T)
58/63(G)
67/62(G)
62/63(T)



diff freq. all. (cases - controls)
3.4
−2.4
−4.7
4.5
−0.6



pvalue
4.03e−01
5.84e−01
2.73e−01
2.94e−01
7.52e−01




*
*
*
*
*















Test
cases vs
0.015 (HWE)
−0.011 (HWE)
−0.031 (HWE)
0.002 (HWE)
0.037 (HWE)



Hardy Weinberg
controls
0.010 (HWE)
−0.014 (HWE)
−0.004 (HWE)
−0.016 (HWE)
0.001 (HWE)

















TABLE 20B





HAPLOTYPE FREQUENCY ANALYSIS (Zyflo secondary effects)


CASES (85 ALT+) vs CONTROLS (208 ALT−)

















ESTIMATED



FREQUENCIES



Haplotype



frequencies
















MARKER 1
MARKER 2
MARKER 3
MARKER 4
MARKER 5
HAPLOTYPE
cases
controls





















haplotype 1
PT2
79 vs 192
12-197/244
12-196/119



TT
0.542
0.436


haplotype 2
PT2
87 vs 197
12-208/35
12-226/167



AG
0.123
0.070


haplotype 3
PT2
84 vs 183
12-206/366
12-196/119



CC
0.205
0.139


haplotype 4
PT2
84 vs 192
10-347/220
12-196/119



GT
0.486
0.400


haplotype 5
PT2
81 vs 193
10-347/203
12-196/119



GC
0.207
0.144


haplotype 6
PT3
82 vs 194
10-349/224
12-216/421
12-223/207


TAT
0.158
0.064


haplotype 7
PT3
85 vs 186
12-206/366
12-216/421
12-223/207


CAT
0.148
0.059


haplotype 8
PT3
77 vs 180
12-197/244
12-206/366
12-196/119


TTT
0.434
0.286


haplotype 9
PT3
78 vs 190
12-197/244
10-347/220
12-196/119


TGT
0.433
0.291


haplotype 10
PT3
76 vs 187
12-197/244
10-349/224
12-196/119


TGT
0.435
0.293


haplotype 11
PT3
77 vs 191
12-197/244
10-349/224
12-216/421


CTA
0.137
0.056


haplotype 12
PT3
75 vs 191
12-197/244
10-347/203
12-196/119


TAT
0.431
0.294


haplotype 13
PT4
81 vs 183
12-206/366
10-349/224
12-216/421
12-223/207

CTAT
0.160
0.058


haplotype 14
PT4
84 vs 185
12-206/366
10-346/141
12-216/421
12-223/207

CGAT
0.158
0.058


haplotype 15
PT4
82 vs 188
10-349/224
12-214/129
12-216/421
12-223/207

TCAT
0.161
0.063


haplotype 16
PT4
81 vs 184
12-206/366
10-347/203
12-216/421
12-223/207

CGAT
0.153
0.059


haplotype 17
PT4
82 vs 191
10-347/111
10-349/224
12-216/421
12-223/207

GTAT
0.159
0.064


haplotype 18
PT4
82 vs 192
10-346/141
10-349/224
12-216/421
12-223/207

GTAT
0.158
0.065


haplotype 19
PT4
81 vs 192
10-347/220
10-349/224
12-216/421
12-223/207

ATAT
0.159
0.066


haplotype 20
PT4
84 vs 183
12-206/366
10-347/220
12-216/421
12-223/207

CAAT
0.150
0.059


haplotype 21
PT4
85 vs 183
12-206/366
10-347/111
12-216/421
12-223/207

CGAT
0.148
0.059


haplotype 22
PT4
76 vs 185
12-197/244
10-346/141
10-349/224
12-196/119

TGGT
0.435
0.284


haplotype 23
PT4
85 vs 180
12-206/366
12-214/129
12-216/421
12-223/207

CCAT
0.145
0.058


haplotype 24
PT4
80 vs 192
10-347/203
10-349/224
12-216/421
12-223/207

GTAT
0.156
0.065


haplotype 25
PT4
82 vs 190
10-347/203
10-341/116
12-214/129
12-223/207

GGCT
0.125
0.046


haplotype 26
PT5
77 vs 190
12-197/244
12-208/35
12-196/119
12-216/421
12-219/230
TATGA
0.138
0.050


haplotype 27
PT5
77 vs 189
12-197/244
12-208/35
10-349/97
12-196/119
12-223/207
TTATC
0.127
0.045


haplotype 28
PT5
77 vs 184
12-197/244
12-208/35
12-196/119
12-214/129
12-219/230
TATTA
0.126
0.047


haplotype 29
PT5
76 vs 188
12-197/244
12-208/35
10-347/220
12-196/119
12-223/207
TTGTC
0.121
0.048


haplotype 30
PT5
76 vs 176
12-197/244
12-208/35
12-206/366
10-341/116
12-196/119
TTTGT
0.195
0.099















PERMUTATIONS




ESTIMATED FREQUENCIES
TEST RESULTS















p-
Odds



Max
>Iter/



excess
ratio
Chi-S
Pvalue (1 df)
Av. Chi-S
Chi-s
No. of Iter























haplotype 1
PT2
79 vs 192
18.89
1.53
5.11
(2.3e−02)
**
1.7
6.7
[3/100]



haplotype 2
PT2
87 vs 197
5.73
1.87
4.34
(3.6e−02)
*
1.1
7.3
[4/100]



haplotype 3
PT2
84 vs 183
7.68
1.60
3.75
(5.1e−02)
*
1.3
8.0
[11/100] 



haplotype 4
PT2
84 vs 192
14.25
1.41
3.49
(6.1e−02)
*
1.0
5.3
[8/100]



haplotype 5
PT2
81 vs 193
7.46
1.56
3.43
(6.1e−02)
*
1.3
10.3
[9/100]



haplotype 6
PT3
82 vs 194
10.09
2.76
12.35
(4.3e−04)
***
2.1
11.0
[0/100]



haplotype 7
PT3
85 vs 186
9.44
2.77
11.62
(6.3e−04)
***
2.8
24.7
[3/100]



haplotype 8
PT3
77 vs 180
20.67
1.91
10.62
(1.1e−03)
***
1.8
10.9
[1/100]



haplotype 9
PT3
78 vs 190
19.96
1.86
9.98
(1.6e−03)
***
1.7
9.3
[0/100]



haplotype 10
PT3
76 vs 187
20.17
1.86
9.88
(1.7e−03)
***
1.8
6.9
[0/100]



haplotype 11
PT3
77 vs 191
8.56
2.66
9.76
(1.7e−03)
***
1.6
11.3
[1/100]



haplotype 12
PT3
75 vs 191
19.42
1.82
9.13
(2.4e−03)
***
1.3
6.1
[0/100]



haplotype 13
PT4
81 vs 183
10.82
3.10
14.38
(1.5e−04)
****
2.0
13.9
[0/100]



haplotype 14
PT4
84 vs 185
10.62
3.04
14.20
(1.6e−04)
****
3.3
23.7
[2/100]



haplotype 15
PT4
82 vs 188
10.50
2.86
13.12
(2.8e−04)
****
2.9
25.2
[3/100]



haplotype 16
PT4
81 vs 184
10.03
2.89
12.50
(3.9e−04)
***
2.7
13.0
[1/100]



haplotype 17
PT4
82 vs 191
10.14
2.77
12.33
(4.3e−04)
***
2.3
15.7
[2/100]



haplotype 18
PT4
82 vs 192
9.96
2.70
11.85
(5.6e−04)
***
2.6
18.1
[3/100]



haplotype 19
PT4
81 vs 192
10.02
2.69
11.78
(5.9e−04)
***
2.2
9.9
[0/100]



haplotype 20
PT4
84 vs 183
9.60
2.79
11.72
(5.9e−04)
***
2.3
17.1
[3/100]



haplotype 21
PT4
85 vs 183
9.42
2.76
11.45
(7.0e−04)
***
2.0
12.3
[1/100]



haplotype 22
PT4
76 vs 185
21.12
1.94
11.16
(8.2e−04)
***
2.0
8.7
[0/100]



haplotype 23
PT4
85 vs 180
9.20
2.74
11.03
(8.6e−04)
***
2.3
15.7
[3/100]



haplotype 24
PT4
80 vs 192
9.65
2.63
11.00
(8.6e−04)
***
1.7
14.4
[3/100]



haplotype 25
PT4
82 vs 190
8.22
2.94
10.86
(9.6e−04)
***
1.8
18.8
[2/100]



haplotype 26
PT5
77 vs 190
9.27
3.06
12.24
(4.5e−04)
***
2.6
13.2
[2/100]



haplotype 27
PT5
77 vs 189
8.56
3.08
11.42
(7.0e−04)
***
1.6
10.3
[0/100]



haplotype 28
PT5
77 vs 184
8.30
2.93
10.42
(1.2e−03)
***
2.4
14.6
[4/100]



haplotype 29
PT5
76 vs 188
7.63
2.71
8.84
(2.9e−03)
***
1.4
9.5
[1/100]



haplotype 30
PT5
76 vs 176
10.67
2.21
8.82
(2.9e−03)
***
1.3
7.3
[0/100]

















TABLE 21





Summary of Association Study Results and Permutation Tests







12-Lipoxygenase














12-206-366
10-347-203
10-349-224







intron 2
exon 6
exon 8
12-196-119
12-216-421
12-219-230
12-223-207
MARKERS

















C

T

A

T
HAPLOTYPE 8 Zyflo









secondary effects









(ALT+ vs ALT−)



A

T
A
G

HAPLOTYPE 14









Asthma (ALT vs US)















4.03E−01
5.84E−01
7.52E−01
4.03E−01
2.73E−01
2.94E−01
7.52E−01
pvalue
ALT+ vs


−3.7
−2.2
−1.4
3.4
−4.7
4.5
−0.6
diff all. Freq
ALT−


2.73E−01
6.55E−01
5.27E−01
6.55E−01
3.71E−01
1.80E−01
7.52E−01
pvalue
ALT vs


−4.6
−1.2
−1.9
−1.2
−2.9
−4.2
0.8
diff all. Freq
caucasian US















sample

PERMUTATIONS




sizes

TEST RESULTS















HAPLOTYPE 8 (ALT+ vs ALT−)
cases vs
haplotype frequencies
odds-

Av.
Max
>Iter/nb
















Zyflo secondary effects
controls
cases
controls
ratio
chi-S
P value
Chi-S
Chi-S
of Iter.



















ALT+ vs
82 vs 174
0.158
0.04
4.56
21.85
2.90E−06
3.1
29.9
5/1 000


ALT−






3.3
40.9
77/10 000 


ALT vs
256 vs 83 
0.059
0

10.12
1.40E−03
3.5
37.6
82/1 000 


caucasian US















sample

PERMUTATIONS




sizes

TEST RESULTS















HAPLOTYPE 14 (ALT vs US)
cases vs
haplotype frequencies
odds-

Av.
Max
>Iter/nb
















Asthma
controls
cases
controls
ratio
chi-S
P value
Chi-S
Chi-S
of Iter.



















ALT+ vs
85 vs 193
0.097
0.109
−1.34
0.18
6.50E−01
2.1
24.1
785/1 000


ALT−


ALT vs
278 vs 174 
0.118
0.031
4.18
21.01
4.40E−06
2.8
38.6
39/10 000


caucasian US






2.8
29.9
 7/1 000
















TABLE 22





Permutations Test Results







12-Lipoxygenase














12-206/366
10-349/97
10-349/224
12-195/119
12-214/129
12-216/421
12-219/230
12-223/207











In2
ex8
ex8
in bac (not localization in Bac: 3′ or 5′ gene)
MARKERS



















C

T


A

T
HAPLO-
(ALT+










TYPE 1
vs











ALT−)



A


C

G

HAPLO-
(ALT vs










TYPE 2
US)



A

T

A
G

HAPLO-










TYPE 3


4.03e−01
7.52e−01
4.80e−01
4.03e−01
5.84e−01
2.73e−01
2.94e−01
7.52e−01
pvalue
ALT+


−3.7
−0.1
−3.0
3.4
−2.4
−4.7
4.5
−0.6
(cases
vs


(54 vs 58)
(58 vs 59)
(54 vs 57)
(72 vs 68)
(58 vs 61)
(57 vs 62)
(66 vs 62)
(61 vs 62)
vs
ALT−










con-










trols)


2.73e−01
6.55e−01
2.06e−01
6.55e−01
7.52e−01
3.71e−01
1.80e−01
7.52e−01
pvalue
ALT vs











cauca-











sian











US


−4.6
−1.4
−4.1
−1.2
−0.7
−2.9
−4.2
0.8
(cases


(57 vs 61)
(59 vs 60)
(56 vs 60)
(69 vs 70)
(60 vs 61)
(61 vs 64)
(63 vs 67)
(62 vs 61)
vs










con-










trols)










Zyflo secondary effects







HAPLOTYPE 1


(ALT+ vs









ALT−)
PERMUTATIONS













(Zyflo
sample
haplotype

TEST RESULTS












secondary
sizes
frequencies

>Iter/
















effects)
cases vs

con-
p-
odds-

Av.
Max
nb of

















CTAT
controls
cases
trols
excess
ratio
chi-S
P value
Chi-S
Chi-S
Iter.





















ALT+ vs
 81 vs 183
0.16
0.058
10.82
3.10
14.38
1.60E−04
****
2
13.9
0/100


ALT−








2.7
33.6
18/1000


ALT+ vs
81 vs 99
0.16
0.065
10.11
2.72
8.28
4.00E−03
***
3.3
23.1
118/1000 


ALT− (1)


ALT+ vs
81 vs 84
0.16
0.044
12.12
4.15
12.23
4.50E−04
***
2.7
19.6
20/1000


ALT− (2)


ALT vs
264 vs 83 
0.071
0
302.77#
7.08
12.37
4.30E−04
***TH
2.6
23
25/1000


caucasian


US










Asthma gene
















PERMUTATIONS



HAPLOTYPE
sample
haplotype

TEST RESULTS











2 (ALT vs
sizes
frequencies

>Iter/
















US) (Asthma
cases vs

con-
p-
odds-

Av.
Max
nb of

















gene)ACG
controls
cases
trols
excess
ratio
chi-S
P value
Chi-S
Chi-S
Iter.





















ALT+ vs
89 vs 193
0.131
0.121
1.14
1.10
0.11
6.50E−01
*
1.3
18.1
760/1000


ALT−


ALT+ vs
89 vs 104
0.131
0.115
1.84
1.16
0.20
5.80E−01
*
1.5
14.6
883/1000


ALT− (1)


ALT+ vs
89 vs 89 
0.131
0.134
−0.26
0.98
0.00
7.50E−01
*
1.4
16.2
946/1000


ALT− (2)


ALT vs
282 vs 174 
0.125
0.041
8.73
3.32
17.87
2.30E−05
*****
1.8
8.5
 0/100


caucasian








2
19.9
 2/1000


US



















PERMUTATIONS



HAPLOTYPE
sample
haplotype

TEST RESULTS











3 (ALT vs
sizes
frequencies

>Iter/
















US) (Asthma
cases vs

con-
p-
odds-

Av.
Max
nb of

















gene) ATAG
controls
cases
trols
excess
ratio
chi-S
P value
Chi-S
Chi-S
Iter.





















ALT+ vs
 86 vs 194
0.123
0.114
1.04
1.09
0.10
7.50E−01
*
1.5
15.5
816/1000


ALT−


ALT+ VS
 88 vs 100
0.123
0.108
1.69
1.16
0.21
5.80E−01
*
1.6
16.6
735/1000


ALT− (1)


ALT+ vs
86 vs 94
0.123
0.11
1.54
1.14
0.16
6.50E−01
*
1.5
19.3
750/1000


ALT− (2)


ALT vs
280 vs 173
0.124
0.035
9.2
3.9
20.63
5.40E−06
******
1.5
11.6
 0/100


caucasian








2
18.7
 0/1000


US
















TABLE 23







Allele Frequency











ALT+
ALT−
US caucasian























PROTEINS
Marker
size
A
C
G
T
size
A
C
G
T
size
A
C
G
T


























12-LO
12-197/244
81

29.63

70.37
196

35.46

64.54
180

32.78

67.22


2
12-208/35
87
41.95


58.05
197
41.12


58.88
182
42.31


57.69


3
12-226/167
89

60.67
39.33

199

62.81
37.19

188

58.78
41.22


4
12-206/366
86

45.35

54.65
186

41.67

58.33
89

38.20

61.80


5
10-346/141
88


HOM

197
0.51

99.49

185


HOM


6
10-347/111
88

0.57
99.43

196


HOM

180


HOM


7
10-347/165
69

HOM


199

99.75

0.25
185

HOM


8
10-347/203
83
56.02

43.98

197
57.87

42.13

184
58.42

41.58


9
10-347/220
87
42.53

57.47

196
42.60

57.40

184
40.49

59.51


10
10-349/97
89
58.99

41.01

198
59.09

40.91

182
60.44

39.56


11
10-349/224
83


54.22
45.78
194


57.22
42.78
177


60.45
39.55


12
10-341/116
89
9.55

90.45

197
11.17

88.83

176
10.80

89.20


13
12-196/119
86

27.91

72.09
195

31.28

68.72
184

29.08

70.92


14
12-214/129
89

41.01

58.99
193

38.60

61.40
181

38.67

61.33


15
12-216/421
89
42.13

57.87

199
37.44

62.56

182
35.99

64.01


16
12-219/230
89
66.85

33.15

199
62.31

37.69

187
67.91

32.09


17
12-223/207
88

38.07

61.93
199

37.44

62.56
186

38.44

61.56

## Claims

1. A method of determining the frequency in a population of 12-LO-related biallelic marker comprising:
(a) genotyping individuals from said population for biallelic marker 10-349-224 and 12-206-366; and
(b) determining the proportional representation of said biallelic marker in said population.

2. The method according to claim 1, wherein step (a) is performed on each individual of said population.

3. The method according to clam 1, wherein step (a) is performed on a single pooled biological sample derived from said population.

4. A method of detecting an association between an allele and a phenotype, comprising the steps of:
(a) determining the frequency of biallelic markers 10-349-224 and 12-206-366 in an affected population comprising:
(i) genotyping individuals from a population for biallelic markers 10-349-224 and 12-206-366; and
(ii) determining the proportional representation of said biallelic markers in said population;

(b) determining the frequency of said biallelic markers in a control population comprising:
(i) genotyping individuals from a population for biallelic markers 10-349-224 and 12-206-366; and
(ii) determining the proportional representation of said biallelic markers in said population; and

(c) determining whether a statistically significant association exists between said biallelic markers and asthma.

5. The method according to claim 4, wherein said control population is either a trait negative population or a random population.

6. The method according to claim 4, wherein steps (a) and (b) are performed on a single pooled biological sample derived from each of said populations.

7. The method according to claim 4, wherein steps (a) and (b) are performed separately on biological samples derived from each individual in said populations.

8. The method according to claim 4, wherein the identity of the nucleotides at all of the biallelic markers described in FIG. 2(A-B) is determined in steps (a) and (b).

9. A method of estimating the frequency of a haplotype for a set of biallelic markers in a population, comprising the steps of:
(a) genotyping each individual in said population for a first biallelic marker;
(b) genotyping each individual in said population for a second biallelic marker by determining the identity of the nucleotides at said second biallelic marker for both copies of said second biallelic marker present in the genome; and
(c) applying a haplotype determination method to the identities of the nucleotides determined in steps (a) and (b) to obtain an estimate of said frequency;
wherein said biallelic markers are 10-349-224 and 12-206-366.

10. The method according to claim 9, wherein said haplotype determination method is selected from the group consisting of asymmetric PCR amplification, double PCR amplification of specific alleles, the Clark method, and an expectation maximization algorithm.

11. A method of detecting an association between a haplotype and a phenotype, comprising the steps of:
(a) estimating the frequency of at least one haplotype in an affected population comprising:
(i) genotyping each individual in said population for a first biallelic marker;
(ii) genotyping each individual in said population for a second biallelic marker by determining the identity of the nucleotides at said second biallelic marker for both copies of said second biallelic marker present in the genome; and
(iii) applying a haplotype determination method to the identities of the nucleotides determined in steps (a) and (b) to obtain an estimate of said frequency;

(b) estimating the frequency of said haplotype in a control population comprising:
(i) genotyping each individual in said population for a first biallelic marker;
(ii) genotyping each individual in said population for a second biallelic marker by determining the identity of the nucleotides at said second biallelic marker for both copies of said second biallelic marker present in the genome; and
(iii) applying a haplotype determination method to the identities of the nucleotides determined in steps (a) and (b) to obtain an estimate of said frequency; and

(c) determining whether a statistically significant association exists between said haplotype and asthma;

wherein said biallelic markers are 10-349-224 and 12-206-366.

12. The method according to claim 11, wherein said control population is either a trait negative population or a random population.