WO2008098159A2

WO2008098159A2 - Genetic marker for risk of cardiovascular disorder

Info

Publication number: WO2008098159A2
Application number: PCT/US2008/053404
Authority: WO
Inventors: Ramtin Agah; Gurunathan Murugesan; Kandice Kottke-Marchant; Stephen Ellis; Raymond Tubbs
Original assignee: The Cleveland Clinic Foundation
Priority date: 2007-02-09
Filing date: 2008-02-08
Publication date: 2008-08-14
Also published as: WO2008098159A3

Abstract

A method of identifying a subject at increased risk of developing cardiovascular disorder includes obtaining a biological sample containing a nucleic acid from the subject and determining the presence of at least one polymorphism in at least one of a TGFB 1 gene, TGFB R2 gene, or TLR2 gene. The presence of the at least one polymorphism is indicative of an increased risk of a cardiovascular disorder.

Description

GENETIC MARKER FOR RISK OF CARDIOVASCULAR DISORDER

RELATED APPLICATION

[0001] This application claims priority from U.S. provisional patent application Serial No. 60/888,957, filed on February 9, 2007, the subject matter of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to the genetic markers including single nucleotide polymorphisms and haplotypes for diagnosing cardiovascular disorders in a subject.

BACKGROUND OF THE INVENTION

[0003] The genomes of all organisms undergo spontaneous mutation in the course of their continuing evolution, generating variant forms of progenitor nucleic acid sequences (Gusella, Ann. Rev. Biochem. 55, 831-854 (1986)). The variant form may confer an evolutionary advantage or disadvantage relative to a progenitor form, or may be neutral. In some instances, a variant form confers a lethal disadvantage and is not transmitted to subsequent generations of the organism. In other instances, a variant form confers an evolutionary advantage to the species and is eventually incorporated into the DNA of many or most members of the species and effectively becomes the progenitor form. In many instances, both progenitor and variant form(s) survive and co-exist in a species population. The coexistence of multiple forms of a sequence gives rise to polymorphisms. [0004] Several different types of polymorphism have been reported. A restriction fragment length polymorphism (RFLP) is a variation in DNA sequence that alters the length of a restriction fragment (Botstein et al., Am. J. Hum. Genet. 32, 314-331 (1980)). The restriction fragment length polymorphism may create or delete a restriction site, thus changing the length of the restriction fragment. RFLPs have been widely used in human and animal genetic analyses (see WO 90/13668; WO90/11369; Donis-Keller, Cell 51, 319-337 (1987); Lander et al., Genetics 121, 85-99 (1989)). When a heritable trait can be linked to a particular RFLP, the presence of the RFLP in an individual can be used to predict the likelihood that the animal will also exhibit the trait. [0005] Other polymorphisms take the form of short tandem repeats (STRs) that include tandem di-, tri- and tetra-nucleotide repeated motifs. These tandem repeats are also referred to as variable number tandem repeat (VNTR) polymorphisms. VNTRs have been used in identity and paternity analysis (U.S. Pat. No. 5,075,217; Armour et al., FEBS Lett. 307, 113-115 (1992); Horn et al., WO 91/14003; Jeffreys, EP 370,719), and in a large number of genetic mapping studies. [0006] Other polymorphisms take the form of single nucleotide variations between individuals of the same species. Such polymorphisms are far more frequent than RFLPs, STRs and VNTRs. Some single nucleotide polymorphisms (SNP) occur in protein-coding nucleic acid sequences (coding sequence SNP (cSNP)), in which case, one of the polymorphic forms may give rise to the expression of a defective or otherwise variant protein and, potentially, a genetic disease. cSNPs can alter the codon sequence of the gene and therefore specify an alternative amino acid. Such changes are called "missense" when another amino acid is substituted, and "nonsense" when the alternative codon specifies a stop signal in protein translation. When the cSNP does not alter the amino acid specified the cSNP is called "silent".

[0007] Other single nucleotide polymorphisms occur in noncoding regions. Some of these polymorphisms may also result in defective protein expression (e.g., as a result of defective splicing). Other single nucleotide polymorphisms have no phenotypic effects.

[0008] Single nucleotide polymorphisms can be used in the same manner as RFLPs and VNTRs, but offer several advantages. Single nucleotide polymorphisms occur with greater frequency and are spaced more uniformly throughout the genome than other forms of polymorphism. The greater frequency and uniformity of single nucleotide polymorphisms means that there is a greater probability that such a polymorphism will be found in close proximity to a genetic locus of interest than would be the case for other polymorphisms. The different forms of characterized single nucleotide polymorphisms are often easier to distinguish than other types of polymorphism (e.g., by use of assays employing allele- specific hybridization probes or primers). [0009] Only a small percentage of the total repository of polymorphisms in humans and other organisms has been identified. The limited number of polymorphisms identified to date is due to the large amount of work required for their detection by conventional methods. For example, a conventional approach to identifying polymorphisms might be to sequence the same stretch of DNA in a population of individuals by dideoxy sequencing. In this type of approach, the amount of work increases in proportion to both the length of sequence and the number of individuals in a population and becomes impractical for large stretches of DNA or large numbers of persons.

SUMMARY OF THE INVENTION

[0010] The present invention relates to single nucleotide polymorphism (SNP) markers, combinations of such markers and haplotypes associated with risk of cardiovascular disorders and genes associated with cardiovascular disorders to which the markers or haplotypes formed by some of these markers are located. The SNP markers and haplotypes in accordance with the present invention are located on the Transforming Growth Factor beta 1 gene (TGFBl) gene, Transforming Growth Factor beta receptor 2 (TGFB R2) gene, or Toll-like receptor 2 (TLR2) gene and can include at least one nucleotide at one or more nucleotide positions selected from the group consisting of nucleotide position 18831 of SEQ ID NO: 1, nucleotide position 24086 of SEQ ID NO: 1, nucleotide position 29505 of SEQ ID NO: 2, nucleotide position 54620 of SEQ ID NO: 2, nucleotide position 60569 of SEQ ID NO: 2, nucleotide position 67305 of SEQ ID NO: 2, nucleotide position 87000 of nucleotide NO: 2, and nucleotide position 540 of SEQ ID NO: 3.

[0011] In one aspect of the invention, the SNPs can be used in an assay or method to detect an increased risk of a cardiovascular disorder in a subject. The method can include obtaining a biological sample containing a nucleic acid from the subject and then determining the presence of at least one polymorphism in at least one of a Transforming Growth Factor beta 1 gene (TGFBl) gene, Transforming Growth Factor beta receptor 2 (TGFB R2) gene, or Toll-like receptor 2 (TLR2) gene. The polymorphism can include at least one nucleotide at one or more nucleotide positions selected from the group consisting of nucleotide position 18831 of SEQ ID NO: 1, nucleotide position 24086 of SEQ ID NO: 1, nucleotide position 29505 of SEQ ID NO: 2, nucleotide position 54620 of SEQ ID NO: 2, nucleotide position 60569 of SEQ ID NO: 2, nucleotide position 67305 of SEQ ID NO: 2, nucleotide position 87000 of nucleotide NO: 2, and nucleotide position 540 of SEQ ID NO: 3. The presence of the at least one polymorphism is indicative of an increased risk of a cardiovascular disorder. [0012] Another aspect of the invention relates to a method of detecting an increased risk of myocardial infarction in a subject with established atherosclerosis. The increased risk of a myocardial infarction can be determined in the subject by obtaining a biological sample containing a nucleic acid from the subject. The presence of at least one polymorphism in at least one of a TGFBl gene or TGFB R2 gene is then determined from the nucleic acid sample. The polymorphism can include at least one nucleotide at one or more nucleotide positions selected from the group consisting of nucleotide position 18831 of SEQ ID NO: 1, nucleotide position 24086 of SEQ ID NO: 1, nucleotide position 29505 of SEQ ID NO: 2, nucleotide position 54620 of SEQ ID NO: 2, nucleotide position 60569 of SEQ ID NO: 2, nucleotide position 67305 of SEQ ID NO: 2, and nucleotide position 87000 of nucleotide NO: 2. The presence of the at least one polymorphism is indicative of an increased risk of a myocardial. [0013] In a further aspect of the invention the method of detecting an increased risk of myocardial infarction can comprise determining the presence of one or more of the following nucleotides selected from the group consisting of: a G at a position corresponding to nucleotide position 18831 of SEQ ID NO: 1, or a C at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 24086 of SEQ ID NO: 1, or an A at a position corresponding to a complementary position thereof; a G at a position corresponding to nucleotide position 29505 of SEQ ID NO: 2, or a C at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 54620 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 60569 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof; an A at a position corresponding to nucleotide position 67305 of SEQ ID NO: 2, or a T at a position corresponding to a complementary position thereof; and an A at a position corresponding to nucleotide position 87000 of nucleotide NO: 2 or a T at a position corresponding to a complementary position thereof.

[0014] In yet a further aspect of the invention, the one or more of the single nucleotide polymorphisms that can be used to detect increased risk of myocardial infarction can define a haplotype indicative of the increased risk of myocardial infarction. The haplotype comprises nucleotides that include an A at a position corresponding to nucleotide position 18831 of SEQ ID NO: 1, or a T at a position corresponding to a complementary position thereof and a T at a position corresponding to nucleotide position 24086 of SEQ ID NO: 1, or an A at a position corresponding to a complementary position thereof.

[0015] In a still further aspect of the invention, the haplotype can comprise an A at a position corresponding to nucleotide position 29505 of SEQ ID NO: 2, or a T at a position corresponding to a complementary position thereof; a C at a position corresponding to nucleotide position 54620 of SEQ ID NO: 2, or a G at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 60569 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof; an T at a position corresponding to nucleotide position 67305 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof; and an A at a position corresponding to nucleotide position 87000 of nucleotide or a T at a position corresponding to a complementary position thereof.

[0016] In yet another aspect of the invention, the method can be used to identify a subject with an increased risk of coronary artery disease lesion progression following bypass surgery using saphenous vein grafts. In the method, a biological sample containing a nucleic acid is obtained from the subject and the presence of at least one polymorphism in at least one of a TGFBl gene, TGFB R2 gene or TLR2 gene is determined. The polymorphism includes at least one nucleotide at one or more nucleotide positions selected from the group consisting of, nucleotide position 24086 of SEQ ID NO: 1, nucleotide position 67305 of SEQ ID NO: 2, and nucleotide position 540 of SEQ ID NO: 3. The presence of the at least one polymorphism is indicative of an increased risk of coronary artery disease lesion progression in the subject following bypass surgery using saphenous vein grafts. [0017] In another aspect of the invention, the method can include determining the presence of one or more of the following nucleotides selected from the group consisting of: a T at a position corresponding to nucleotide position 24086 of SEQ ID NO: 1, or an A at a position corresponding to a complementary position thereof; an A at a position corresponding to nucleotide position 67305 of SEQ ID NO: 2, or a T at a position corresponding to a complementary position thereof; and a T at a position corresponding to nucleotide position 540 of SEQ ID NO: 3 or an A at a position corresponding to a complementary position thereof. [0018] The information obtained from these methods can be combined with other information concerning the subject, e.g. results from blood measurements, clinical examination and questionnaires. The blood measurements include but are not restricted to the determination of plasma or serum cholesterol and high-density lipoprotein cholesterol. The information to be collected by questionnaire includes information concerning gender, age, family and medical history such as the family history of cardiovascular disorders. Clinical information collected by examination includes e.g. information concerning height, weight, hip and waist circumference, systolic and diastolic blood pressure, and heart rate. [0019] The methods of the invention allow the accurate evaluation of cardiovascular disorders before cardiovascular disorders onset, thus reducing or minimizing the debilitating effects of cardiovascular disorders. The method can be applied in persons who are free of clinical symptoms and signs of cardiovascular disorders, in those who already have clinical cardiovascular disorders, in those who have family history of cardiovascular disorders or in those who have elevated level or levels of risk factors of cardiovascular disorders.

[0020] Those skilled in the art will readily recognize that the analysis of the nucleotides present in one or several of the SNP markers of this invention in an individual's nucleic acid can be done by any method or technique capable of determining nucleotides present in a polymorphic site. As it is obvious in the art the nucleotides present in SNP markers can be determined from either nucleic acid strand or from both strands.

[0021] The major application of the current invention involves prediction of those at higher risk of developing cardiovascular disorders. Diagnostic tests that define genetic factors contributing to cardiovascular disorders might be used together with or independent of the known clinical risk factors to define an individual's risk relative to the general population. Better means for identifying those individuals at risk for cardiovascular disorders should lead to better preventive and treatment regimens, including more aggressive management of the current clinical risk factors such as cigarette smoking, hypercholesterolemia, elevated LDL cholesterol, low HDL cholesterol, hypertension and elevated blood pressure, diabetes mellitus, glucose intolerance, insulin resistance and the metabolic syndrome, obesity, lack of physical activity, and inflammatory components as reflected by increased C-reactive protein levels or other inflammatory markers. Information on genetic risk may be used by physicians to help convince particular patients to adjust life style (e.g., to stop smoking, reduce caloric intake, to increase exercise).

DETAILED DESCRIPTION

Definitions:

[0022] For convenience, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided below. [0023] The term "allele" refers to the different sequence variants found at different polymorphic regions. The sequence variants may be single or multiple base changes, including without limitation insertions, deletions, or substitutions, or may be a variable number of sequence repeats.

[0024] The term "allelic pattern" refers to the identity of an allele or alleles at one or more polymorphic regions. Alternatively, an allelic pattern may consist of either a homozygous or heterozygous state at a single polymorphic site. Alternatively, an allelic pattern may consist of the identity of alleles at more than one polymorphic site. [0025] The terms "control" or "control sample" refer to any sample appropriate to the detection technique employed. The control sample may contain the products of the allele detection technique employed or the material to be tested. Further, the controls may be positive or negative controls. By way of example, where the allele detection technique is PCR amplification, followed by size fractionation, the control sample may comprise DNA fragments of an appropriate size. [0026] A "cardiovascular disease" is a cardiovascular disorder, as defined herein, characterized by clinical events including clinical symptoms and clinical signs. Clinical symptoms are those experiences reported by a patient that indicate to the clinician the presence of pathology. Clinical signs are those objective findings on physical or laboratory examination that indicate to the clinician the presence of pathology. "Cardiovascular disease" includes both "coronary artery disease" and "peripheral vascular disease," both terms being defined below. Clinical symptoms in cardiovascular disease include chest pain, shortness of breath, weakness, fainting spells, alterations in consciousness, extremity pain, paroxysmal nocturnal dyspnea, transient ischemic attacks and other such phenomena experienced by the patient. Clinical signs in cardiovascular disease include such findings as EKG abnormalities, altered peripheral pulses, arterial bruits, abnormal heart sounds, and wheezes, jugular venous distention, neurological alterations and other such findings discerned by the clinician. Clinical symptoms and clinical signs can combine in a cardiovascular disease such as a myocardial infarction (MI) or a stroke (also termed a "cerebrovascular accident" or "CVA"), where the patient will report certain phenomena (symptoms) and the clinician will perceive other phenomena (signs) all indicative of an underlying pathology. "Cardiovascular disease" includes those diseases related to the cardiovascular disorders, such as fragile plaque disorder, occlusive disorder and stenosis. For example, a cardiovascular disease resulting from an occlusive disorder can be termed an "occlusive disease." Clinical events associated with occlusive disease include those signs and symptoms where the progressive occlusion of an artery affects the amount of circulation that reaches a target tissue. Progressive arterial occlusion may result in progressive ischemia that may ultimately progress to tissue death if the amount of circulation is insufficient to maintain the tissues. Signs and symptoms of occlusive disease include claudication, rest pain, angina, and gangrene, as well as physical and laboratory findings indicative of vessel stenosis and decreased distal perfusion.

[0027] A "cardiovascular disorder" refers broadly to both to coronary artery disorders and peripheral arterial disorders. The term "cardiovascular disorder" can apply to any abnormality of an artery, whether structural, histological, biochemical or any other abnormality. This term includes those disorders characterized by fragile plaque (termed herein "fragile plaque disorders"), those disorders characterized by vaso-occlusion (termed herein "occlusive disorders"), and those disorders characterized by restenosis. A "cardiovascular disorder" can occur in an artery primarily, that is, prior to any medical or surgical intervention. Primary cardiovascular disorders include, among others, arterial occlusion, aneurysm formation and thrombosis. A "cardiovascular disorder" can occur in an artery secondarily, that is, following a medical or surgical intervention. Secondary cardiovascular disorders include, among others, post-traumatic aneurysm formation, restenosis, and post-operative graft occlusion. [0028] A "clinical event" is an occurrence of clinically discernible signs of a disease or of clinically reportable symptoms of a disease. "Clinically discernible" indicates that the sign can be appreciated by a health care provider. "Clinically reportable" indicates that the symptom is the type of phenomenon that can be described to a health care provider. A clinical event may comprise clinically reportable symptoms even if the particular patient cannot himself or herself report them, as long as these are the types of phenomena that are generally capable of description by a patient to a health care provider.

[0029] A "coronary artery disease" ("CAD") refers to a vascular disorder relating to the blockage of arteries serving the heart. Blockage can occur suddenly, by mechanisms such as plaque rupture or embolization. Blockage can occur progressively, with narrowing of the artery via myointimal hyperplasia and plaque formation. Those clinical signs and symptoms resulting from the blockage of arteries serving the heart are manifestations of coronary artery disease. Manifestations of coronary artery disease include angina, ischemia, myocardial infarction, cardiomyopathy, congestive heart failure, arrhythmias and aneurysm formation. It is understood that occlusive disease in the coronary circulation is associated with arterial stenosis accompanied by anginal symptoms, a condition commonly treated with pharmacological interventions and with angioplasty. [0030] A "disease" is a disorder characterized by clinical events including clinical signs and clinical symptoms. The diseases discussed herein include cardiovascular disease, peripheral vascular disease, CAD, cerebrovascular disease, and those diseases in any anatomic location associated with fragile plaque disorder, with occlusive disorder or with restenosis.

[0031] A "disorder associated allele" or "an allele associated with a disorder" refers to an allele whose presence in a subject indicates that the subject has or is susceptible to developing a particular disorder. One type of disorder associated allele is a "cardiovascular disorder associated allele," the presence of which in a subject indicates that the subject has or is susceptible to developing a cardiovascular disorder. These include broadly within their scope alleles which are associated with "myocardial infarction" alleles associated with "occlusive disorders," and alleles associated with restenosis.

[0032] The term "haplotype" as used herein is intended to refer to a set of alleles that are inherited together as a group (are in linkage disequilibrium) at statistically significant levels.

[0033] "Increased risk" refers to a statistically higher frequency of occurrence of the disease or condition in an individual carrying a particular polymorphic allele in comparison to the frequency of occurrence of the disease or condition in a member of a population that does not carry the particular polymorphic allele. [0034] The term "isolated" as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule. The term isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an "isolated nucleic acid" is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.

[0035] The term "marker" refers to a sequence in the genome that is known to vary among individuals.

[0036] As used herein, the term "nucleic acid" refers to polynucleotides or oligonucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs

(e.g. peptide nucleic acids) and as applicable to the embodiment being described, single (sense or antisense) and double- stranded polynucleotides.

[0037] The term "propensity to disease," also "predisposition" or "susceptibility" to disease or any similar phrase, means that certain alleles are hereby discovered to be associated with or predictive of a cardiovascular disorder. The alleles are thus over-represented in frequency in individuals with disease as compared to healthy individuals. Thus, these alleles can be used to predict disease even in pre- symptomatic or pre-diseased individuals.

[0038] A "risk factor" is a factor identified to be associated with an increased risk. A risk factor for a cardiovascular disorder or a cardiovascular disease is any factor identified to be associated with an increased risk of developing those conditions or of worsening those conditions. A risk factor can also be associated with an increased risk of an adverse clinical event or an adverse clinical outcome in a patient with a cardiovascular disorder. Risk factors for cardiovascular disease include smoking, adverse lipid profiles, elevated lipids or cholesterol, diabetes, hypertension, hypercoagulable states, elevated homocysteine levels, and lack of exercise. Carrying a particular polymorphic allele is a risk factor for a particular cardiovascular disorder, and is associated with an increased risk of the particular disorder.

[0039] As used herein, the term "specifically hybridizes" or "specifically detects" refers to the ability of a nucleic acid molecule to hybridize to at least about 6 consecutive nucleotides of a sample nucleic acid.

[0040] The term "wild-type allele" refers to an allele of a gene which, when present in two copies in a subject results in a wild-type phenotype. There can be several different wild-type alleles of a specific gene, since certain nucleotide changes in a gene may not affect the phenotype of a subject having two copies of the gene with the nucleotide changes.

[0041] The present invention is based at least in part, on the identification of alleles, single nucleotide polymorphisms, and haplotypes that are associated (to a statistically significant extent) with the development of a cardiovascular disorder in a subject. Detection of these alleles, alone or in conjunction with another means in a subject indicates that the subject has or is predisposed to the development of the cardiovascular disorders.

[0042] The alleles, SNPs, and haplotypes described herein are found more frequently in individuals with a high risk of cardiovascular disorders, such as myocardial infarction or coronary artery disease, than in a subject with low risk of cardiovascular disorders. Therefore, the single nucleotide polymorphisms and haplotypes have predictive value for detecting cardiovascular disorders or the susceptibility of the subject to the cardiovascular disorder. Detecting the SNPs and/or haplotypes can be accomplished by methods known in the art for detecting sequences at polymorphic sites.

[0043] In certain methods described herein, an individual who is at risk for the cardiovascular disorder is an individual in whom an at-risk allele, at-risk polymorphism, or an at-risk haplotype is identified. In one embodiment, the at-risk allele, at-risk polymorphism, or the at-risk haplotype is one that confers a significant risk of cardiovascular disease. In one embodiment, significance associated with an allele, SNP, or a haplotype is measured by an odds ratio. In a further embodiment, the significance is measured by a percentage. In one embodiment, a significant risk is measured as odds ratio of at least about 1.2, including by not limited to: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0 and 40.0. In a further embodiment, a significant increase or reduction in risk is at least about 20%, including but not limited to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 98%. In a further embodiment, a significant increase in risk is at least about 50%. It is understood however, that identifying whether a risk is medically significant may also depend on a variety of factors, including the specific disease, the allele or the haplotype, and often, environmental factors.

[0044] In one aspect, the method comprises assessing in an individual the presence or frequency of SNPs in, comprising portions of, a cardiovascular disorder risk gene, wherein an excess or higher frequency of the SNPs compared to a healthy control individual is indicative that the individual has a cardiovascular disease risk, or is susceptible to a cardiovascular disorder risk death. These SNP markers can correspond to an at-risk haplotypes.

[0045] The method can include obtaining a biological sample containing a nucleic acid from the subject and determining the presence of at least one polymorphism in at least one of a TGFB 1 gene, TGFB R2 gene, or TLR2 gene. The polymorphism can at least one nucleotide at one or more nucleotide positions selected from the group consisting of nucleotide position 18831 of SEQ ID NO: 1, nucleotide position 24086 of SEQ ID NO: 1, nucleotide position 29505 of SEQ ID NO: 2, nucleotide position 54620 of SEQ ID NO: 2, nucleotide position 60569 of SEQ ID NO: 2, nucleotide position 67305 of SEQ ID NO: 2, nucleotide position 87000 of nucleotide NO: 2, and nucleotide position 540 of SEQ ID NO: 3. The presence of the at least one polymorphism is indicative of an increased risk of a cardiovascular disorder.

[0046] In an aspect of the invention, the method can be used to detect the inherited risk of myocardial infarction in a subject with established atherosclerosis. The method can use markers (e.g., SNPs and haplotypes) in the TGFBl and TGFB R2 genes to stratify patients with atherosclerosis for an increased risk of myocardial infarction.

[0047] In this aspect of the invention, a biological sample containing a nucleic acid can be obtained from a subject with atherosclerosis. The presence of at least one polymorphism or haplotype in at least one of a TGFBl gene or TGFB R2 gene can then be determined. The polymorphism can include at least one nucleotide at one or more nucleotide positions selected from the group consisting of nucleotide position 18831 of SEQ ID NO: 1, nucleotide position 24086 of SEQ ID NO: 1, nucleotide position 29505 of SEQ ID NO: 2, nucleotide position 54620 of SEQ ID NO: 2, nucleotide position 60569 of SEQ ID NO: 2, nucleotide position 67305 of SEQ ID NO: 2, and nucleotide position 87000 of nucleotide NO: 2. The presence of the at least one polymorphism is indicative of an increased risk of a myocardial. [0048] The minor allele at each of the respective nucleotide positions can include a G at a position corresponding to nucleotide position 18831 of SEQ ID NO: 1, or a C at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 24086 of SEQ ID NO: 1, or a A at a position corresponding to a complementary position thereof; a G at a position corresponding to nucleotide position 29505 of SEQ ID NO: 2, or a C at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 54620 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 60569 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof; an A at a position corresponding to nucleotide position 67305 of SEQ ID NO: 2, or a T at a position corresponding to a complementary position thereof; and an A at a position corresponding to nucleotide position 87000 of nucleotide NO: 2 or a T at a position corresponding to a complementary position thereof.

[0049] In a further aspect of the invention, the one or more SNPs can define a first haplotype of the TGFB 1 gene indicative of the inherited risk of myocardial infarction. The haplotype can comprise nucleotides that include an A at a position corresponding to nucleotide position 18831 of SEQ ID NO: 1, or a T at a position corresponding to a complementary position thereof and a T at a position corresponding to nucleotide position 24086 of SEQ ID NO: 1, or an A at a position corresponding to a complementary position thereof.

[0050] In a still further aspect of the invention, the one or more SNPs can define a second haplotype of the TGFB R2 gene indicative of the inherited risk of myocardial infarction. The second haplotype can comprise an A at a position corresponding to nucleotide position 29505 of SEQ ID NO: 2, or a T at a position corresponding to a complementary position thereof; a C at a position corresponding to nucleotide position 54620 of SEQ ID NO: 2, or a G at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 60569 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof; an T at a position corresponding to nucleotide position 67305 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof; and an A at a position corresponding to nucleotide position 87000 of nucleotide or a T at a position corresponding to a complementary position thereof.

[0051] Detection of the first haplotype of the TGFBl gene and/or the second haplotype of the TGFB R2 gene in the subject with established atherosclerosis is indicative of increased risk of myocardial infarction in the subject. These markers unlike protein markers, which assess the risk only at the time of measurement, can be used to determine lifelong risk in the subject.

[0052] In yet another aspect of the invention, the method can be used to identify a subject at increased risk of coronary artery disease lesion progression following bypass surgery using saphenous vein grafts. It was found that the TGFBl gene, the TGFB2 gene, and the TLR2 gene are partially responsible for the pathogenesis of coronary artery disease lesion progression in reversed saphenous vein grafts placed as conduits during coronary bypass graft surgery.

[0053] In this aspect a biological sample containing nucleic acid can be obtained from a subject that is a candidate for coronary revascularization using a saphenous vein graft. The presence of at least one polymorphism in at least one of a TGFB 1, TGFB R2 gene, or TLR2 gene can then be determined. The polymorphism includes at least one nucleotide at one or more nucleotide positions selected from the group consisting of, nucleotide position 24086 of SEQ ID NO: 1, nucleotide position 67305 of SEQ ID NO: 2, and nucleotide position 540 of SEQ ID NO: 3. [0054] The minor allele at each of the respective nucleotide positions can include a T at a position corresponding to nucleotide position 24086 of SEQ ID NO: 1, or an A at a position corresponding to a complementary position thereof; an A at a position corresponding to nucleotide position 67305 of SEQ ID NO: 2, or a T at a position corresponding to a complementary position thereof; and a T at a position corresponding to nucleotide position 540 of SEQ ID NO: 3 or an A at a position corresponding to a complementary position thereof. [0055] The presence of the at least one polymorphism is indicative of an increased risk of coronary artery disease lesion progression in the subject following bypass surgery using saphenous vein grafts. Other methods of revascularization, such as with coronary stents, can then be selected if the polymorphisms are detected.

[0056] Many methods are available for detecting specific alleles at human polymorphic loci. The preferred method for detecting a specific polymorphic allele will depend, in part, upon the molecular nature of the polymorphism. For example, the various allelic forms of the polymorphic locus may differ by a single base-pair of the DNA. Such single nucleotide polymorphisms (or SNPs) are major contributors to genetic variation, comprising some 80% of all known polymorphisms, and their density in the human genome is estimated to be on average 1 per 1,000 base pairs. SNPs are most frequently biallelic-occurring in only two different forms (although up to four different forms of an SNP, corresponding to the four different nucleotide bases occurring in DNA, are theoretically possible). Nevertheless, SNPs are mutationally more stable than other polymorphisms, making them suitable for association studies in which linkage disequilibrium between markers and an unknown variant is used to map disease- causing mutations. In addition, because SNPs typically have only two alleles, they can be genotyped by a simple plus/minus assay rather than a length measurement, making them more amenable to automation.

[0057] A variety of methods are available for detecting the presence of a particular single nucleotide polymorphic allele in an individual. Advancements in this field have provided accurate, easy, and inexpensive large-scale SNP genotyping. Most recently, for example, several new techniques have been described including dynamic allele- specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMan system as well as various DNA "chip" technologies such as the Affymetrix SNP chips. These methods require amplification of the target genetic region, typically by PCR. Still other newly developed methods, based on the generation of small signal molecules by invasive cleavage followed by mass spectrometry or immobilized padlock probes and rolling-circle amplification, might eventually eliminate the need for PCR. Several of the methods known in the art for detecting specific single nucleotide polymorphisms are summarized below. The method of the present invention is understood to include all available methods. [0058] Several methods have been developed to facilitate analysis of single nucleotide polymorphisms. In one embodiment, the single base polymorphism can be detected by using a specialized exonuclease -resistant nucleotide, as disclosed, e.g., in Mundy, C. R. (U.S. Pat. No.4,656,127). According to the method, a primer complementary to the allelic sequence immediately 3' to the polymorphic site is permitted to hybridize to a target molecule obtained from a particular animal or human. If the polymorphic site on the target molecule contains a nucleotide that is complementary to the particular exonuclease-resistant nucleotide derivative present, then that derivative will be incorporated onto the end of the hybridized primer. Such incorporation renders the primer resistant to exonuclease, and thereby permits its detection. Since the identity of the exonuclease-resistant derivative of the sample is known, a finding that the primer has become resistant to exonucleases reveals that the nucleotide present in the polymorphic site of the target molecule was complementary to that of the nucleotide derivative used in the reaction. This method has the advantage that it does not require the determination of large amounts of extraneous sequence data.

[0059] In another embodiment of the invention, a solution-based method is used for determining the identity of the nucleotide of a polymorphic site. Cohen, D. et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087). As in the Mundy method of U.S. Pat. No. 4,656,127, a primer is employed that is complementary to allelic sequences immediately 3' to a polymorphic site. The method determines the identity of the nucleotide of that site using labeled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymorphic site will become incorporated onto the terminus of the primer.

[0060] An alternative method, known as Genetic Bit Analysis or GBA is described by Goelet, P. et al. (PCT Appln. No. 92/15712). The method of Goelet, P. et al. uses mixtures of labeled terminators and a primer that is complementary to the sequence 3' to a polymorphic site. The labeled terminator that is incorporated is thus determined by, and complementary to, the nucleotide present in the polymorphic site of the target molecule being evaluated. In contrast to the method of Cohen et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087) the method of Goelet, P. et al. is preferably a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase.

[0061] Recently, several primer-guided nucleotide incorporation procedures for assaying polymorphic sites in DNA have been described (Komher, J. S. et al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. Acids Res. 18:3671 (1990); Syvanen, A. -C, et al., Genomics 8:684-692 (1990); Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147 (1991); Prezant, T. R. et al., Hum. Mutat. 1: 159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P. et al., Anal. Biochem. 208:171-175 (1993)). These methods differ from GBA in that they all rely on the incorporation of labeled deoxynucleotides to discriminate between bases at a polymorphic site. In such a format, since the signal is proportional to the number of deoxynucleotides incorporated, polymorphisms that occur in runs of the same nucleotide can result in signals that are proportional to the length of the run (Syvanen, A. -C, et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

[0062] Any cell type or tissue may be utilized to obtain nucleic acid samples for use in the diagnostics described herein. In a preferred embodiment, the DNA sample is obtained from a bodily fluid, e.g., blood, obtained by known techniques (e.g. venipuncture) or saliva. Alternatively, nucleic acid tests can be performed on dry samples (e.g. hair or skin). When using RNA or protein, the cells or tissues that may be utilized must express a TGFB 1 gene, TGFB R2 gene, or TLR2 gene. [0063] Diagnostic procedures may also be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Nucleic acid reagents may be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, G. J., 1992, PCR in situ hybridization: protocols and applications, Raven Press, NY).

[0064] In addition to methods which focus primarily on the detection of one nucleic acid sequence, profiles may also be assessed in such detection schemes. Fingerprint profiles may be generated, for example, by utilizing a differential display procedure, Northern analysis and/or RT-PCR.

[0065] In one aspect of the invention, the detection method comprises an allele specific hybridization using probes overlapping a region of at least one allele of a TGFBl gene, TGFBR2 gene, or TLR2 gene and having about 5, 10, 20, 25, or 30 nucleotides around the mutation or polymorphic region. In another aspect of the invention, several probes capable of hybridizing specifically to other allelic variants involved in a cardiovascular disorder are attached to a solid phase support, e.g., a "chip" (which can hold up to about 250,000 oligonucleotides). Oligonucleotides can be bound to a solid support by a variety of processes, including lithography. Mutation detection analysis using these chips comprising oligonucleotides, also termed "DNA probe arrays" is described e.g., in Cronin et al. (1996) Human Mutation 7:244. In one embodiment, a chip comprises all the allelic variants of at least one polymorphic region of a gene. The solid phase support is then contacted with a test nucleic acid and hybridization to the specific probes is detected. Accordingly, the identity of numerous allelic variants of one or more genes can be identified in a simple hybridization experiment. [0066] These techniques may also comprise the step of amplifying the nucleic acid before analysis. Amplification techniques are known to those of skill in the art and include, but are not limited to cloning, polymerase chain reaction (PCR), polymerase chain reaction of specific alleles (ASA), ligase chain reaction (LCR), nested polymerase chain reaction, self sustained sequence replication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), and Q-Beta Replicase (Lizardi, P. M. et al., 1988, Bio/Technology 6 : 1197) .

[0067] Amplification products may be assayed in a variety of ways, including size analysis, restriction digestion followed by size analysis, detecting specific tagged oligonucleotide primers in the reaction products, allele-specific oligonucleotide (ASO) hybridization, allele specific 5' exonuclease detection, sequencing, hybridization, and the like. [0068] PCR based detection means can include multiplex amplification of a plurality of markers simultaneously. For example, it is well known in the art to select PCR primers to generate PCR products that do not overlap in size and can be analyzed simultaneously. Alternatively, it is possible to amplify different markers with primers that are differentially labeled and thus can each be differentially detected. Of course, hybridization based detection means allow the differential detection of multiple PCR products in a sample. Other techniques are known in the art to allow multiplex analyses of a plurality of markers. [0069] In a merely illustrative embodiment, the method includes the steps of (i) collecting a sample of cells from a patient, (ii) isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, (iii) contacting the nucleic acid sample with one or more primers which specifically hybridize 5' and 3' to at least one allele of an cardiovascular disease polymorphisms or haplotype under conditions such that hybridization and amplification of the allele occurs, and (iv) detecting the amplification product. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

[0070] In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the allele. Exemplary sequencing reactions include those based on techniques developed by Maxim and Gilbert ((1977) Proc. Natl Acad Sci USA 74:560) or Sanger (Sanger et al (1977) Proc. Nat. Acad. Sci USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures may be utilized when performing the subject assays (see, for example Biotechniques (1995) 19:448), including sequencing by mass spectrometry (see, for example PCT publication WO 94/16101; Cohen et al. (1996) Adv Chromatogr 36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol 38:147-159). It will be evident to one of skill in the art that, for certain embodiments, the occurrence of only one, two or three of the nucleic acid bases need be determined in the sequencing reaction. For instance, A-track or the like, e.g., where only one nucleic acid is detected, can be carried out. [0071] In a further embodiment, protection from cleavage agents (such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine) can be used to detect mismatched bases in RNA/RNA or RNA/DNA or DNA/DNA heteroduplexes (Myers, et al. (1985) Science 230:1242). In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type allele with the sample. The double- stranded duplexes are treated with an agent which cleaves single- stranded regions of the duplex such as which will exist due to base pair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S 1 nuclease to enzymatically digest the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al (1988) Proc. Natl Acad Sci USA 85:4397; and Saleeba et al (1992) Methods Enzymol. 217:286-295. In one embodiment, the control DNA or RNA can be labeled for detection.

[0072] Examples of other techniques for detecting alleles include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation or nucleotide difference (e.g., in allelic variants) is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al (1989) Proc. Natl Acad. Sci USA 86:6230). Such allele specific oligonucleotide hybridization techniques may be used to test one mutation or polymorphic region per reaction when oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations or polymorphic regions when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.

[0073] Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation or polymorphic region of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al (1989), Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238). In addition, it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al (1992) MoI. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

[0074] Another embodiment of the invention is directed to kits for detecting a predisposition of a subject for developing a cardiovascular disorder. This kit may contain one or more oligonucleotides, including 5' and 3' oligonucleotides that hybridize 5' and 3' to at least one cardiovascular disorder allele of a TGFBl gene, TGFB R2 gene, or TLR2 gene. PCR amplification oligonucleotides should hybridize between 25 and 2500 base pairs apart, preferably between about 100 and about 500 bases apart, in order to produce a PCR product of convenient size for subsequent analysis.

[0075] Particularly preferred primers for use in the diagnostic method of the invention include those that amplify a portion of the nucleic acids containing the polymorphisms of interest and the flanking sequences. For example, the primers can be designed to amplify all or a portion of SEQ ID NO: 4 of the a TGFB 1 gene, SEQ ID NO: 5 of the a TGFBl gene, SEQ ID NO: 6 of the TGFBR2 gene, SEQ ID NO: 7 of the TGFB R2 gene, SEQ ID NO: 8 of the TGFBR2 gene, SEQ ID NO: 9 of the TGFBR2 gene, SEQ ID NO: 10 of the TGFB R2 gene, or SEQ ID NO: 11 of the TLR2 gene.

[0076] Primers for the detection of a human polymorphism in these genes can be readily designed using this sequence information and standard techniques known in the art for the design and optimization of primers sequences. Optimal design of such primer sequences can be achieved, for example, by the use of commercially available primer selection programs. [0077] For use in a kit, oligonucleotides may be any of a variety of natural and/or synthetic compositions such as synthetic oligonucleotides, restriction fragments, cDNAs, synthetic peptide nucleic acids (PNAs), and the like. The assay kit and method may also employ labeled oligonucleotides to allow ease of identification in the assays. Examples of labels which may be employed include radio-labels, enzymes, fluorescent compounds, streptavidin, avidin, biotin, magnetic moieties, metal binding moieties, antigen or antibody moieties, and the like. [0078] The kit may, optionally, also include DNA sampling means. DNA sampling means are well known to one of skill in the art and can include, but not be limited to substrates, such as filter papers, the AmpliCard (University of Sheffield, Sheffield, England SlO 2JF; Tarlow, JW, et al., J. of Invest. Dermatol. 103:387-389 (1994)) and the like; DNA purification reagents, such as Nucleon kits, lysis buffers, proteinase solutions and the like; PCR reagents, such as 10x reaction buffers, thermostable polymerase, dNTPs, and the like; and allele detection means such as the Hinfl restriction enzyme, allele specific oligonucleotides, degenerate oligonucleotide primers for nested PCR from dried blood.

Example 1

Genes Associated with Stable Heart Disease: A Haplotype Association Study

[0079] Coronary atherosclerosis leading to coronary plaque formation is the most common underlying cause for myocardial infarction (MI). The major pathological event leading to MI is plaque rupture with superimposed thrombosis. Recent studies suggest that vulnerability to plaque rupture is dependent on plaque composition rather than size. Lipid-rich and soft plaques are more prone to rupture than collagen-rich hard plaques. Although patients with unstable angina (clinical presentation preceding MI) have a different plaque composition than patients with stable angina, the genetic predisposition to develop unstable angina is not known at the present time.

[0080] Currently, serum levels of certain protein markers such as troponin and high sensitivity C-reactive protein (hs-CRP) are used to stratify patients at risk for cardiac events. However, these markers are subject to variation; they are not sufficient to distinguish patients with stable heart disease (stable angina) from those with unstable heart disease (unstable angina).

[0081] Our study, "Genes Associated with Stable Heart disease" (GASH), seeks to identify genetic markers that can be associated with development of an unstable plaque and angina. We will investigate about 50 candidate genes that represent a multifactorial process centered on factors directly affecting plaque stability in more than 1,000 patients with distinct clinical history. We will examine about 50-100 selective single nucleotide polymorphisms (SNPs) present in these genes that define distinct haplotypes and identify those variants associated with plaque stability. Once identified, individual SNPs or set of SNPs (haplotype) can be used as genetic markers to stratify patients with propensity for MI.

Experimental Approach

[0082] Patients have been classified into 'case' (unstable angina) and 'control' (stable angina) groups. Each patient in the case group has been matched with a patient in the control group with a similar demographic profile. The patient population chosen is unique in that both groups have coronary disease but are separated by stable versus unstable angina. Genomic DNA from these patients was isolated from the white blood cells (buffy coat from whole blood drawn into an EDTA tube), and stored at -8O⁰C until use. Genotyping involves amplification of the target gene product by polymerase chain reaction (PCR) of genomic DNA followed by melt curve analysis. It is a semi-automated, high-throughput process consisting of three components. TECAN Genesis 150 (Millipore, Billerica, Mass.), a liquid handling system capable of handling four 384-well PCR plates simultaneously, is used to pipette DNA and a PCR master mix containing the SNP- specific fluorescent probe, and oil overlay. Inclusion of probe in the master mix allows for a homogenous reaction while eliminating post-PCR handling of amplicons. The PCR is done in a 384-well format using the twin-block GeneAmp PCR System 9700 (Applied Biosystems, Foster City, Calif.). Amplicons are then denatured in the same PCR machine and subjected to "melt curve" analysis in a new generation of genotyping platform, the LightTyper (recently introduced by Roche Applied Science Corporation, Indianapolis, Indiana. Genotyping Assay

[0083] The genotyping assay is based on melt curve analysis of probe that binds to the target sequence of the amplicon. The assay exploits the thermal properties of DNA, namely melting temperature (Tm). Tm is the temperature at which 50 percent of the DNA strands are unwound. When a fluorescently labeled sequence- specific oligonucleotide probe (about 20 base length) hybridizes with a target DNA sequence to form a duplex, it can generate fluorescent signal. Upon heating, the probe will melt off/separate from the target sequence of the duplex at its Tm, resulting in the loss of fluorescent signal. This change can be captured as a melt curve and can be converted into the derivative melt peak, from which the genotype can be derived.

[0084] If the probe sequence, designed to match the wild type DNA, and the target DNA sequence are perfectly complementary to each other, the probe Tm will be high. For a mismatched (variant/mutant) duplex, probe Tm will be low. This discrimination in Tm allows or assigning genotypes. The LightTyper system can use both hybridization probe (two probes: an anchor probe and a sensor probe) and simple probe (one probe).

[0085] Hybridization probes work based on the fluorescence resonance energy transfer (FRET) principle. In this method, an upstream fluorescein- labeled probe (donor) and a downstream LCR-640-labeled probe (acceptor) are designed to anneal to the complementary target on the amplicon to generate fluorescent signal. When heated, one of the annealed probes will melt off from the template, resulting in loss of fluorescent signal. The Tm of each sample is an indicator of its classification as wild type, homozygote mutant, or heterozygote. [0086] The simple probe utilizes a sequence-specific single probe to generate fluorescent signal upon annealing to the target sequence. The simple probe also gives information similar to that obtained with the hybridization probe. Results

[0087] We determined three markers (one SNP and two haplotypes; 7 SNPs altogether) in two separate genes to stratify patients with atherosclerosis for an increased risk for MI. Proposed genetic markers are: Gene: TGF-Bl; Haplotype: A-T (two SNPs); SNP; 24086 (one SNP; constituent of haplotype) Gene: TGF-BR2; Haplotype: A-C-T-T-A; (five SNPs). These markers have a "p" value of <0.05 both for initial and subsequent cohort studies.

TABLE

TGBFl Gene:

SNP #1: Reference Sequence (RS): Not Available in dbSNP

Position in Reference Sequence: 18831

Absolute Position: Chrl9:46535301

Relative Position: 15488A/G

Minor Allele (G) Frequency: 14%

Assigned Name: IIPGA-TGFB1-18831

Flanking Sequence:

TCTATATATAGTTGACCCTGAATATATATACAGAGAGAGAGAGAGATGAT TTTGTAGATTTTGAAAAAAGTCCTAAGAGA GGCCAGGTATGGTAGCTCAC R(A/G)

CCCGTAATCCCAGCACTTTGGGAGGCTGAGGCAGGTGGATCACTTGAGGTCAGGA GTTGGAGACCAGCCTGGCCAACATGGTGAAACCCTGTCTGTACTA (SEQ ID NO: 4)

SNP # 2: RS8179181 Position in Reference Sequence: 24086 Absolute Position: Chrl9:46530046 Relative Position: 20743C/T Minor Allele (T) Frequency: 16% Assigned Name: IIPGA-TGFB 1-24086 Flanking Sequence:

TCTCGTAGCCCGGTGGGCCAGACGTACCTTGCTGTACTGCGTGTCCAGGC

TCCAAATGTAGGGGCAGGGRCCGAGGCARAAGTTGGCATGGTAGCCCTTG

GGCTCGTGGATCCACTTCCAGCCGAGGTCCTTGCGGAAGTCAATGTACAG

CTGCCGCACGCAGCAGTTCTTCTCCGTGGAGCTGCAGGCAGGAGAGACGC

R[A/G]

TCAGGGGCAGGGAGGGGCTACCACCATAGAAGCCACATGCCCCTCCTCCC

CAGGTGCGTGTGTCACCCTAGGTTGCCCCCRCAGCCCTATCTCCATCTGG

GTCTCCCTTGCACCCACTGTGTTAATAACAACACAAATAGTCATGATAAT

CACTAACACAGATTAAGCACTTCCTCCAGTGCTAAGAGCAATGTAATAAT

(SEQ ID NO:5)

TGFBR2 Gene:

SNP #3: RS980441

Position in Reference Sequence: 29505

Absolute Position: Chr3:30650291

Relative Position: 26950A/G

Minor Allele (G) Frequency: 49%

Assigned Name: IIPGA-TGFB2-29505

Flanking Sequence:

TTCTCAAGAGGAGCATCTCAGCTCAYAGTCAGCTGACTGTTGTCTTGGGA

TGGTGTGGGCCCACTGTTTCCAAGTCTTTTTTATTTCTTTAAGTGGAATT

AGAAAACCAGTTTTGCATGTGAAATCTCTGAATTTTAAAACTTGGTTTAT

ATTTTAAAAAGAAAAGAGTTCAGGCCAAACAGARYACATCTGTGCTCTGT

R[A/G]

TCCAGCTCTTGTACCCACTGTAGCAATTTCTGCTTTAGCTTCTCAGTCTG

TGCCCAAGTTATTTGGTTGTTAGAAACCAAGCGCCTTGGACCTCGTTATA

TTACTTTTTAAAGGGATGCCTTTCCCAAATATGGTAGTACTCTGGCCCTT

TTCCAGAGAAAATATGGAAATCATCTGTTTTGACCAATATTTTCACATGT

(SEQ ID NO: 6)

SNP #4: RS3773636 Position in Reference Sequence: 54620 Absolute Position: Chr3:30675406 Relative Position: 52065C/T Minor Allele (G) Frequency: 39% Assigned Name: IIPGA-TGFB2-54620 Flanking Sequence:

ATCTCTGGCAGGAAGACAAATTATTTTTTTACAGTATCAGACACGCGGTT TCCTGTCTATGTTTAACTTGCTAGCAGAACATCTCAGCCAGCTGGTTCCC TCTGGACCCATTGTGTCCTTAATGTCGTTGCTTTACACCATCCAGCAAAC CCACTCTGKGTACTGATCGCATGCWTTGAGCATTTTGTTGAYTTCCGTTG Y[C/T]

TTTCCGAGTAAGGGAAGTCTTAGAAATGCTGCCCTCTTTCTCCCAAATCT CATTTTRTGTTTGTTCAGCTCCAAMACCAGGGAGCTGGCTTCTTGGGGAA ATGATTAGTTCTTGCATGTAAGAAATGAAAAGAGTAAATGGTGTTTTGGT AGCTCTGGGGGATTTTAGGTAYGCTGCCTTGGAACTGTGCACTCACGCCT (SEQ ID NO: 7)

SNP #5: RS1551762 Position in Reference Sequence: 60569 Absolute Position: Chr3:30681355 Relative Position: 58014A/T Minor Allele (T) Frequency: 13% Assigned Name: IIPGA-TGFB2-60569

Flanking Sequence:

CTCTCAGCCTCTTTTATGTCCTTTGTTATGCCGACACCAGTTCCTAAAAG

GAACTTAAAACTGAACCTCCCTAATCCACTATTTTAAATAAAAGTCACTT

GGTTAAAAAAATAAAAGCATTAAATTAAGGCCAACTTGGTAAATAAAATC

CTATGTGGATCAGTTAATGTGTCTTGATAAAAAGCAAATCAACATAGCTG

W[A/T]

ATTTGGGAGATAGGCTCCTACTTTAATATTTGAAGTAGTTTGGCAATTCC

TCCACTCGGTAAAGGAGTTTAGGGTGAACATTTAGTGGGATACTAATTTA

TCCGAGCAATAGAAAGGGACGTGGATGACAGTTTTTCGCACACCTTGGTG

TATTAGTGCACTAATGAGCCTCAGATGCCAAGAGAGATTCTTGTGAAGTT

(SEQ ID NO: 8)

SNP #6: Reference Sequence (RS): Not available in dbSNP

Position in Reference Sequence: 67305

Absolute Position: Chr3:30688091

Relative Position: 64750A/T

Minor Allele (A) Frequency: 27%

Assigned Name: IIPGA-TGFBR2-67305 Flanking Sequence:

GCATCCCTGAAATAAAAATTAACAATATCGTATCTACAAAAACTATGCAG

ATGCTAAAATCTATAGATGCTCAGGCATGAACCCACTTCCTGACAGTACTTACCT

ACCACATCCAACTCCTTCTCTCCTTGTTTTGTTTCCCCA

W[T/A]

CAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCATATTTCAAGTGACAGG

CATCAGCCTCCTGCCACCACTGGGAGTTGCCATATCTGTCATCATCATCTTCTAC

TGCTACCGCGTTAACCGGCAGCAGAAGCTGAGTTC (SEQ ID NO: 9)

SNP #7: RS2276767 Position in Reference Sequence: 87000 Absolute Position: Chr3:30707786 Relative Position: 84445 A/C Minor Allele (A) Frequency: 20% Assigned Name: IIPGA-TGFB2-87000

Flanking Sequence:

AAGCCCAGCAGAGGATCATTGTGCTGTCAGGACCTATTGTGATCAGATAG

CACCAGAGCCAAAAAGTATGGTAGGTTTTSAGCACAGCTGCCACCAGCAC

AAACCCCCCACCACCCTTTCCACATGGAACTGGCTGGCCTGCAGCAGCAG

GCACTCAGTCAGCACATGTTAAATGCACAGGCACTTTTGGACCCTGCTTG

M[A/C]

ACTCACTATAGCAACAAGGTCAGCAGGCCACCTTGCCTTCCGCGGAGCCC

ACCAACTCATGGTGCCCTTTGGATCTCTTTCCYGCTACAGGGCATCCAGA

TGGTGTGTGAGACGTTGACTGAGTGCTGGGACCACGACCCAGAGGCCCGT

CTCACAGCCCAGTGTGTGGCAGAACGCTTCAGTGAGCTGGAGCATCTGGA

(SEQ ID NO: 10)

[0088] The assay is based on genetic codes of the patient and assesses the inherited generic risk for MI. It determines lifelong risk, in contrast to serum blood (protein) marker, which assesses the risk only at the time of measurement. Based on the design of the study, the assay represents genetic markers that have been validated in both initial (discovery) and second (validation) cohort of patients with atherosclerosis at risk for MI.

[0089] Theoretically, a patient with established CAD, especially one with moderate lesion, would be a candidate for further risk stratification using assay. At the present, that may constitute up to 20%-30% of patients undergoing a coronary angiogram, representing 300,000 patients in the U.S. annually. Example 2

[0090] We determined that the Toll-like receptor 2 gene located on chromosome 4 q32, the Transforming Growth Factor Beta 1 (TGFBl) gene located on chromosome 19 ql3 1-2 and Transforming Growth Factor Beta Receptor 2 (TGFBR21) gene located on chromosome 3p22 are partially responsible for the pathogenesis of coronary artery disease lesion progression in reversed saphenous vein grafts placed as conduits during coronary bypass graft surgery. Using a candidate gene approach, we identified highly statistically significant relationships after adjustment for other factors known to affect bypass graft patency of the T16934A mutation (p=0.01), the SNPs C20743T in TGFBl gene and T64750A in TGFB R2 gene (p<.001 and p= .02 respectively) with bypass graft patency at 5 and 10 years after surgery.

SNP Info: RS4696480 Position in Reference Sequence: 540 Absolute Position: Chr4:155184753 Relative Position: 16934A/T Minor Allele (A) Frequency: 39% Assigned Name: IIPGA-TLR2-540

Flanking Sequence

AGTTGACAGCATGTTTTGGGGGTTTCTAAGCTATCAGTTAATTTTTTGGGGTACA

GTTTTATTGATAAACTAACAGAGTAGAAAAAATCTAATGGAATACCCCCAAATTT

AAAAGAGGGCAAGAAAAGAGAGACAATAGAACATAAAACAAATGGGACAAGAATA

AAGTACATAGTTGTCACAGTCCCTTGGGTGCTGCTGTAACAAAATACCTGAGACT

GGGTAATTTACAAAGAACAGAAATTTATCCATTCATGGTTCTGGAGTCTGGGAAG

TCCAAGATTGAAGGGCTGCATCTGG

W[A/T]

GAGGGTCATCTGGCTACATTATAACATGATGGAAAGCATCACATGGTGAGAGAGA

GCAAGAGAGGACAGAACTTACTTTTATAACAAACTCATTCTCACAATAAACTGCT

CCCTTGGTGATAACATTAATCCATTTATGAGGGTACAGCCTTCATAACCTAATCA

CCTCTTAAAAGGTTCCACCTCTCAGCTCGCAGTGAGCCGAGATCATGCCACTGCA

CTCCAGCCTGGGTGACAGAGCGAGACTCTGTCTCAAAACAAACAAACAAACAAAA

ATTCCACCTCTCAACACTGTTGCAT (SEQ ID NO: 11) TGFBl Gene

SNP Info: RS8179181

Position in Reference Sequence: 24086

Absolute Position: Chrl9:46530046

Relative Position: 20743C/T

Minor Allele (T) Frequency: 16%

Assigned Name: IIPGA-TGFB 1-24086

Flanking Sequence

TCTCGTAGCCCGGTGGGCCAGACGTACCTTGCTGTACTGCGTGTCCAGGC

TCCAAATGTAGGGGCAGGGRCCGAGGCARAAGTTGGCATGGTAGCCCTTG

GGCTCGTGGATCCACTTCCAGCCGAGGTCCTTGCGGAAGTCAATGTACAG

CTGCCGCACGCAGCAGTTCTTCTCCGTGGAGCTGCAGGCAGGAGAGACGC

R[A/G]

TCAGGGGCAGGGAGGGGCTACCACCATAGAAGCCACATGCCCCTCCTCCC

CAGGTGCGTGTGTCACCCTAGGTTGCCCCCRCAGCCCTATCTCCATCTGG

GTCTCCCTTGCACCCACTGTGTTAATAACAACACAAATAGTCATGATAAT

CACTAACACAGATTAAGCACTTCCTCCAGTGCTAAGAGCAATGTAATAAT

(SEQ ID NO: 5)

Reference Sequence (RS): Not available in dbSNP Position in Reference Sequence: 67305 Absolute Position: Chr3:30688091 Relative Position: 64750A/T Minor Allele (A) Frequency: 27% Assigned Name: IIPGA-TGFBR2-67305

Flanking Sequence:

GCATCCCTGAAATAAAAATTAACAATATCGTATCTACAAAAACTATGCAG

ATGCTAAAATCTATAGATGCTCAGGCATGAACCCACTTCCTGACAGTACTTACCT

ACCACATCCAACTCCTTCTCTCCTTGTTTTGTTTCCCCA

W[T/A]

CAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCATATTTCAAGTGACAGG

CATCAGCCTCCTGCCACCACTGGGAGTTGCCATATCTGTCATCATCATCTTCTAC

TGCTACCGCGTTAACCGGCAGCAGAAGCTGAGTTC (SEQ ID NO: 9)

[0091] We discovered a novel disease-causing gene for coronary artery disease in saphenous vein grafts placed for bypass surgery. Over 300,000 patients in the United States annually receive coronary bypass graft surgery using reverse saphenous veins. Within 10 years, about 60% develop angina, 15% heart attacks and 10-15% expire, with each of these events often related to development of obstructive disease in the saphenous vein grafts (Campeau L, Lesperance J, Hermann J, Corbara F, Grondin CM, Bourassa MG. Loss of the improvement of angina between 1 and 7 years after aorta-coronary bypass surgery. Circulation 1979;60(suppl I):I-1). (CASS Principal Investigators and Their Associates: Myocardial infarction and mortality in the coronary artery surgery (CASS) randomized trial. N Engl J Med 1984;310:750.)

Claims

Having described the invention the following is claimed:

1. A method of identifying a subject at increased risk of developing cardiovascular disorder comprising: obtaining a biological sample containing a nucleic acid from the subject; determining the presence of at least one polymorphism in at least one of a TGFBl gene, TGFB R2 gene, or TLR2 gene, the polymorphism including at least one nucleotide at one or more nucleotide positions selected from the group consisting of nucleotide position 18831 of SEQ ID NO: 1, nucleotide position 24086 of SEQ ID NO: 1, nucleotide position 29505 of SEQ ID NO: 2, nucleotide position 54620 of SEQ ID NO: 2, nucleotide position 60569 of SEQ ID NO: 2, nucleotide position 67305 of SEQ ID NO: 2, nucleotide position 87000 of nucleotide NO: 2, and nucleotide position 540 of SEQ ID NO: 3, wherein the presence of the at least one polymorphism is indicative of an increased risk of a cardiovascular disorder.

2. The method of claim 1 , the cardiovascular disorder comprising myocardial infarction in subjects with established atherosclerosis.

3. The method of claim 2, comprising determining the presence of one or more of the following nucleotides selected from the group consisting of: a G at a position corresponding to nucleotide position 18831 of SEQ ID NO: 1, or a C at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 24086 of SEQ ID NO: 1, or an A at a position corresponding to a complementary position thereof; a G at a position corresponding to nucleotide position 29505 of SEQ ID NO: 2, or a C at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 54620 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 60569 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof an A at a position corresponding to nucleotide position 67305 of SEQ ID NO: 2, or a T at a position corresponding to a complementary position thereof; and an A at a position corresponding to nucleotide position 87000 of nucleotide NO: 2 or a T at a position corresponding to a complementary position thereof.

4. The method of claim 2, wherein one or more of the single nucleotide polymorphisms define a haplotype indicative of the cardiovascular disorder, the haplotype comprising: an A at a position corresponding to nucleotide position 18831 of SEQ ID NO: 1, or a T at a position corresponding to a complementary position thereof; and a T at a position corresponding to nucleotide position 24086 of SEQ ID NO: 1, or an A at a position corresponding to a complementary position thereof.

5. The method of claim 2, wherein one or more of the single nucleotide polymorphisms define a haplotype indicative of the cardiovascular disorder, the haplotype comprising: an A at a position corresponding to nucleotide position 29505 of SEQ ID NO: 2, or a T at a position corresponding to a complementary position thereof; a C at a position corresponding to nucleotide position 54620 of SEQ ID NO: 2, or a G at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 60569 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof; an T at a position corresponding to nucleotide position 67305 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof; and an A at a position corresponding to nucleotide position 87000 of nucleotide or a T at a position corresponding to a complementary position thereof.

6. The method of claim 2, wherein one or more of the single nucleotide polymorphisms define a haplotype indicative of the cardiovascular disorder, the haplotype comprising: an A at a position corresponding to nucleotide position 18831 of SEQ ID NO: 1, or a T at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 24086 of SEQ ID NO: 1, or an A at a position corresponding to a complementary position thereof; an A at a position corresponding to nucleotide position 29505 of SEQ ID NO: 2, or a T at a position corresponding to a complementary position thereof; a C at a position corresponding to nucleotide position 54620 of SEQ ID NO: 2, or a G at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 60569 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof; an T at a position corresponding to nucleotide position 67305 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof; and an A at a position corresponding to nucleotide position 87000 of nucleotide or a T at a position corresponding to a complementary position thereof.

7. The method of claim 1, the cardiovascular disorder comprising pathogenesis of coronary artery disease lesion progression in saphenous vein grafts placed in the subject for bypass graft surgery.

8. The method of claim 7, comprising determining the presence of one or more of the following nucleotides selected from the group consisting of: a T at a position corresponding to nucleotide position 24086 of SEQ ID NO: 1, or an A at a position corresponding to a complementary position thereof; an A at a position corresponding to nucleotide position 67305 of SEQ ID NO: 2, or a T at a position corresponding to a complementary position thereof; and a T at a position corresponding to nucleotide position 540 of SEQ ID NO: 3 or an A at a position corresponding to a complementary position thereof.

9. A method of assessing the risk of a myocardial infarction in a subject with established atherosclerosis comprising: obtaining a biological sample containing a nucleic acid from the subject; determining the presence of at least one polymorphism in at least one of a TGFBl gene or TGFB R2 gene, the polymorphism including at least one nucleotide at one or more nucleotide positions selected from the group consisting of nucleotide position 18831 of SEQ ID NO: 1, nucleotide position 24086 of SEQ ID NO: 1, nucleotide position 29505 of SEQ ID NO: 2, nucleotide position 54620 of SEQ ID NO: 2, nucleotide position 60569 of SEQ ID NO: 2, nucleotide position 67305 of SEQ ID NO: 2, and nucleotide position 87000 of nucleotide NO: 2. wherein the presence of the at least one polymorphism is indicative of an increased risk of a myocardial.

10. The method of claim 9, comprising determining the presence of one or more of the following nucleotides selected from the group consisting of: a G at a position corresponding to nucleotide position 18831 of SEQ ID NO: 1, or a C at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 24086 of SEQ ID NO: 1, or an A at a position corresponding to a complementary position thereof; a G at a position corresponding to nucleotide position 29505 of SEQ ID NO: 2, or a C at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 54620 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 60569 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof an A at a position corresponding to nucleotide position 67305 of SEQ ID NO: 2, or a T at a position corresponding to a complementary position thereof; and an A at a position corresponding to nucleotide position 87000 of nucleotide NO: 2 or a T at a position corresponding to a complementary position thereof.

11. The method of claim 9, wherein one or more of the single nucleotide polymorphisms define a haplotype indicative of the cardiovascular disorder, the haplotype comprising: an A at a position corresponding to nucleotide position 18831 of SEQ ID NO: 1, or a T at a position corresponding to a complementary position thereof; and a T at a position corresponding to nucleotide position 24086 of SEQ ID NO: 1, or an A at a position corresponding to a complementary position thereof.

12. The method of claim 9, wherein one or more of the single nucleotide polymorphisms define a haplotype indicative of the cardiovascular disorder, the haplotype comprising: an A at a position corresponding to nucleotide position 29505 of SEQ ID NO: 2, or a T at a position corresponding to a complementary position thereof; a C at a position corresponding to nucleotide position 54620 of SEQ ID NO: 2, or a G at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 60569 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof; an T at a position corresponding to nucleotide position 67305 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof; and an A at a position corresponding to nucleotide position 87000 of nucleotide or a T at a position corresponding to a complementary position thereof.

13. The method of claim 9, wherein one or more of the single nucleotide polymorphisms define a haplotype indicative of the cardiovascular disorder, the haplotype comprising: an A at a position corresponding to nucleotide position 18831 of SEQ ID NO: 1, or a T at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 24086 of SEQ ID NO: 1, or an A at a position corresponding to a complementary position thereof; an A at a position corresponding to nucleotide position 29505 of SEQ ID NO: 2, or a T at a position corresponding to a complementary position thereof; a C at a position corresponding to nucleotide position 54620 of SEQ ID NO: 2, or a G at a position corresponding to a complementary position thereof; a T at a position corresponding to nucleotide position 60569 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof; an T at a position corresponding to nucleotide position 67305 of SEQ ID NO: 2, or an A at a position corresponding to a complementary position thereof; and an A at a position corresponding to nucleotide position 87000 of nucleotide or a T at a position corresponding to a complementary position thereof.

14. A method of identifying a subject at increased risk of coronary artery disease lesion progression following bypass surgery using saphenous vein grafts comprising: obtaining a biological sample containing a nucleic acid from the subject; determining the presence of at least one polymorphism in at least one of a TGFB 1 gene, a TGFB R2 gene or TLR2 gene, the polymorphism including at least one nucleotide at one or more nucleotide positions selected from the group consisting of, nucleotide position 24086 of SEQ ID NO: 1, nucleotide position 67305 of SEQ ID NO: 2, and nucleotide position 540 of SEQ ID NO: 3, wherein the presence of the at least one polymorphism is indicative of an increased risk of coronary artery disease lesion progression in the subject following bypass surgery using saphenous vein grafts.

15. The method of claim 14, comprising determining the presence of one or more of the following nucleotides selected from the group consisting of: a T at a position corresponding to nucleotide position 24086 of SEQ ID NO: 1, or an A at a position corresponding to a complementary position thereof; an A at a position corresponding to nucleotide position 67305 of SEQ ID NO: 2, or a T at a position corresponding to a complementary position thereof; and a T at a position corresponding to nucleotide position 540 of SEQ ID NO: 3 or an A at a position corresponding to a complementary position thereof.

16. A kit for identifying a subject at increased risk of developing cardiovascular disorder: at least one hybridization probe that is capable of identifying a polymorphism of at least one of a TGFBl gene, TGFB R2 gene, or TLR2 gene, the polymorphism including at least one nucleotide at one or more nucleotide positions selected from the group consisting of nucleotide position 18831 of SEQ ID NO: 1, nucleotide position 24086 of SEQ ID NO: 1, nucleotide position 29505 of SEQ ID NO: 2, nucleotide position 54620 of SEQ ID NO: 2, nucleotide position 60569 of SEQ ID NO: 2, nucleotide position 67305 of SEQ ID NO: 2, nucleotide position 87000 of nucleotide NO: 2, and nucleotide position 540 of SEQ ID NO: 3.