WO2009058380A2 - Biomarkers predictive of acute coronary syndromes - Google Patents

Abstract

The present invention relates to single nucleotide polymorphisms (SNPs) useful in assessing acute coronary syndromes and identifying related cardiovascular risks in humans. In particular, the present invention relates to nucleic acid molecules containing the polymorphisms, reagents for detecting the polymorphic nucleic acid molecules and methods of using the nucleic acids as well as methods of using reagents for their detection.

Description

BIOMARKERS PREDICTIVE OF ACUTE CORONARY SYNDROMES

FIELD OF THE INVENTION

The present invention is in the field of cardiovascular disease diagnosis and therapy. In particular, the present invention relates to specific single nucleotide polymorphisms (SNPs) in the human genome, and their association with cardiovascular disease. The present invention also provides methods, assays and kits useful for detecting the presence of these SNPs.

BACKGROUND

Genetic polymorphisms can be useful in assessing Acute Coronary Syndrome (ACS) and measuring related cardiovascular risk factors in humans, including, but not limited to, angina, arterial inflammation, atherosclerosis, coronary heart disease (CHD), heart failure, hypertension, myocardial infarction, and stroke. A myocardial infarction (MI) is a type of acute coronary syndrome which is most frequently (but not always) a manifestation of CHD.

In the US, diseases of the heart are the leading cause of death, causing a higher mortality than cancer. MI is also reported to be the leading cause of death in the United States (US) as well as in most industrialized nations throughout the world. 2004 Statistics compiled by the American Heart Association show that approximately 7,200,000 men and 6,000,000 women living with some form of coronary heart disease. Each year, about

1,200,000 people suffer from a (new or recurrent) ACS-related event, and about 40 percent of them die as a result of the event.

In spite of a much better awareness of ACS presenting symptoms and an understanding of how life style choices can improve an individual's cardiovascular risk profile, many people in the US and throughout the world continue to ignore the risk factors directly and indirectly associated with ACS-related events and mechanisms to minimize such risk factors. Accordingly, there remains a need for new ways of predicting and assessing risk of ACS, as well as stratifying individuals to better treat and manage patients with a predisposition for ACS, including healthy individuals and those diagnosed with CHD, to allow for earlier treatment of individuals with genetic and/or environmental variants that predispose an individual to ACS-related cardiovascular events.

The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification. SUMMARY OF THE FNVENTION

The following disclosure and aspects thereof are provided and illustrated in conjunction with methods and compositions and are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements.

The present disclosure describes gender specific gene variations which are individually present in humans grouped by three specific cardiovascular disease states: male and female patients with early-onset MI; aged male and female patients with CHD, but no MI; and, male and female patients with no CHD and no MI. The methods and compositions disclosed herein provide for: diagnosing and evaluating the prognosis of ACS, CHD, and MI, and for the identification of patients exhibiting a predisposition to such conditions; diagnosing and monitoring patients undergoing clinical evaluation for the treatment of cardiovascular disease; monitoring the efficacy of compounds in clinical trials and the identification and therapeutic use of compounds as treatments of cardiovascular disease; utilization of gene variations to predict the efficacy of personalized medical treatment; stratifying individuals based on risk factors and ascribing differential treatment interventions based on the same.

One aspect of the methods and compositions disclosed herein is to provide genetic diagnosis of predisposition or susceptibility for cardiovascular diseases. Another related aspect is to provide treatment to reduce or prevent and/or delay the onset of disease in those predisposed or susceptible to this disease. A further aspect is to provide a means for carrying out this diagnosis.

The disclosed methods and compositions assist in the diagnosis of a disease or of certain disease states via genetic analysis which can yield useable results before onset of disease symptoms, and/or before onset of severe symptoms. Methods and compositions disclosed herein may also be of use in confirming or corroborating the results of other diagnostic methods and thus suitable for a use either as an isolated technique or in combination with other methods and apparatus for diagnosis in the form of a test on which a diagnosis may be assessed. Use of allelic association as a method for genotyping individuals allows for the investigation of a molecular genetic basis for cardiovascular diseases. Certain disease states and patient care may benefit by administration of treatment or therapy in advance of disease appearance, which is more reliably carried out if advance diagnosis of predisposition or susceptibility to disease can be identified and assessed. In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by studying the following descriptions.

DETAILED DESCRIPTION OF THE INVENTION Coronary heart disease (CHD) continues to be the largest contributor to morbidity and mortality in the developed world, with a severe manifestation of CHD being myocardial infarction (MI). MI has been shown to aggregate in studies of twins, first-degree relatives, and extended relatives, suggesting a familial and heritable component in MI risk. A few genome scans have reported genetic loci linked to MI, and a few positional candidate genes have been evaluated, but those studies have failed to replicate any of the loci that have been reported. Various functional candidate genes have been evaluated as well, but such genetic association studies have been and continue to be plagued by failures of replication.

CHD risk remains high in the developed world despite advances in the treatment of Coronary Artery Diesease and MI, including medical treatment with aspirin, cholesterol- lowering statins, hypertension-controlling ACE inhibitors and beta-blockers, and interventional therapies such as revascularization via angioplasty, antibiotic-coated stents, and coronary bypass surgery. Advances in public health education about the risks of smoking, high blood pressure, high cholesterol, physical inactivity, obesity, and other life style choices has improved, but not eliminated, the risk of CHD and ACS in developed regions of the world. Furthermore, congestive heart failure is an increasingly prevalent cardiovascular disease, largely attributable to the aforementioned treatments saving the lives of those who experience a MI, but nonetheless requiring heart function with a reduced amount of viable myocardial tissue. One of the major difficulties with MI is that among about half of patients who experience a MI, their first symptom of CHD is the MI itself, thus primary prevention remains a major potential source of benefit for those who may be at risk of MI.

ACS, CHD, and MI are caused by a combination of genetic and environmental risk factors, but known CHD risk factors (i.e., hyperlipidemia, hypertension, diabetes, smoking, inactivity, and obesity) only predict as little as 50% of individual risk and up to as much as 80% of population-level CHD risk. Each established risk factor has a strong genetic component or at least some degree of genetic regulation. However, since genetic markers that predict such intermediate phenotypes may not predict clinical endpoints, this does not necessarily indicate that MI results from these genetic factors.

The occurrence of acute MI has, however, been shown to be familial. MI clusters among first-degree relatives and is a prognostic marker for death due to CHD among first- degree relatives. It has been reported that death due to MI is familial among extended relatives and that such risk due to MI demonstrates a strong heritable component. Several studies report genomic loci for MI; however, these studies do not replicate one another and, furthermore, since the genetic polymorphisms implicated in these studies are relatively rare, the findings may not be of general importance in such a widely prevalent disease. A variety of candidate genes are also reported to be associated with MI; however, confirmatory studies often have widely variable results that fail to replicate initial reports (e.g., as in the case of the ACE gene insertion-deletion Single Nucleotide Polymorphism).

The lack of consistency of results in the linkage and association studies may be attributable to a lack of complete genetic information, low to moderately penetrant susceptibility genes, multiple genes being involved (polygenic disease where no one locus is necessary or sufficient for disease, and many combinations of loci can be sufficient), and a variety of environmental risk factors that confound some of the reported results. The studies do indicate a potential genetic component in MI and the need for further studies utilizing well-defined phenotypes in early-onset cases that provide both initial associations and confirmation of findings.

One approach to solving the problem of study consistency is to enrich the genetic information available for analysis by increasing marker density. Historically, genome-wide searches for genetic loci linked to a disease phenotype have been performed using microsatellite markers that, in the past few years, have typically included about 400 markers across the genome. While such searches have provided a great deal of power from a relatively small number of microsatellite markers due to the high information content in the highly polymorphic microsatellites, biallelic (single-base variant) SNPs provide a greater deal of ease in genotyping over that of microsatellites. SNP panels have been reported to provide higher data quality, more accurate genotyping results, higher information content, and higher power to detect linkage (in cases where they are used for linkage analysis) than traditionally- used panels of microsatellite markers.

Despite their lower individual information content, SNPs are much more plentiful across the genome than microsatellite markers. Studies have shown that use of 3,000-4,000 SNPs approximates the information available from microsatellite markers. Thus, with chips 135 available now that test more than 500,000 SNP genotypes at a time, genome-wide association analyses employing such SNP chips are a means to assess risk associations for thousands of genes simultaneously. This technology utilizes very low-cost genotyping assays on high- throughput genotyping platforms using microarray chips. Due to the relatively short distances over which linkage disequilibrium (LD) exists between SNPs compared to the

140 distances (>10 centi-Morgans) across which linkage can be found for microsatellites, SNPs evaluated among unrelated individuals can potentially provide improved localization of a candidate locus to a considerably smaller region of the genome.

Identification of predisposition genes using SNP genome-wide scans is a recent occurrence, with results being reported for a variety of diseases. These studies include scans

145 for macular degeneration, esophageal cancer, bladder cancer, and prostate cancer. A genome-wide association study for MI was performed in 2002 in a small Japanese population (cases: n=94) using the investigators' own genome-wide panel of about 65,000 SNPs. While 1% of all the SNPs reached the study's threshold for attempted replication in a second population, the study ultimately identified only one validated candidate locus for MI on

150 chromosome 6p21. A subsequent report compared a Caucasian control group to the control group in that study and found a significant difference in genotype distributions for the same SNPs, suggesting that the association between 6p21 and MI may be specific to the Japanese population.

Regardless, though, it is unlikely that only one SNP is associated with MI or ACS

155 given the current understanding of ACS and genetics. It may also be that the SNP panel used in prior studies was not used or validated by other investigators and therefore may not be technically valid, or that the population of <100 cases was too small for the initial screening step of an association study. Thus there remained a need to repeat the study using a SNP chip that has been validated for accuracy of laboratory results, in different ethnic populations

160 including Caucasians, and using a larger population of cases and controls. Moreover, use of cases with early-onset MI may show a stronger genetic effect due to the disease being more etiologically homogeneous, as suggested by prior work, and this difference may be further exaggerated through the use of age-discordant controls who have grown older than the average diseased individual without developing clinical MI or any narrowing of one or more

165 coronary arteries as shown by angiography.

Gene chip technologies are available that permit genome-wide scanning for loci using an association analysis study design wherein unrelated individuals are examined for over 500,000 SNPs. While use of such chips is presently quite costly for individual genotyping, given the appropriate set of patients and clinical outcomes, an initial evaluation using a DNA

170 pooling approach (with multiple pool replicates for stability of frequency estimates) can provide a relatively inexpensive preliminary screen for the best candidate loci associated with a phenotype. A follow-up independent study can then be performed for the reduced set of SNPs that are found to be significant in the initial pooled scan.

300 patients with early-onset MI and 600 sex-matched, age-discordant controls who

175 were free from any known MI (but may or may not have had some degree of coronary artery disease) were studied. 500,568 SNPs from three DNA pools for MI patients and six pools for the matched controls (with three replicates each) were studied to determine which SNPs are associated with MI.

The study described herein applied the Affymetrix GeneChip Human Mapping 500K

180 Array Set to pooled DNA for early-onset MI cases and age-discordant sex-matched controls to discover novel Mi-associated loci and to validate previously-described candidate gene loci. One of the attractions of this approach is that the 500K SNP array allowed for the discovery of novel MI associations for SNPs across the genome, specifically for genes that have not been evaluated previously (or considered to be evaluated) for association to MI and for

185 regions devoid of protein-coding genes that may be sequences coding for regulatory RNA.

The present disclosure is based on the observations that specific alleles of a polymorphic region of certain human genes are associated with an enhanced risk for cardiovascular diseases. (See Tables 1 and 2). A listing of human gene subunits and other genes mentioned in this disclosure are summarized in Tables 1 and 2 with specific SNP ID

190 numbers as reference by the naming convention establish by the National Center for Biotechnology Information (NCBI).

As SNPs are linked to other SNPs in neighboring genes on a chromosome (Linkage Disequilibrium) those SNPs could also be used as marker SNPs. It has been reported that SNPs are linked over 100 kb, and in some cases, more than 150 kb. Reports have also

195 demonstrated that use of high-throughput SNP genotyping microarrays and pooling-based genomewide association studies are effective at identifying major genetic contributions to disease.

Definitions

200 The meaning of certain terms and phrases used in this disclosure are provided below and are intended to further inform the understanding of the subject matter disclosed herein. The term "a" or "an" entity refers to one or more of that entity; for example, "a protein" or "a nucleic acid molecule" refers to one or more of those compounds or at least one compound. As such, the terms "a" (or "an"), "one or more" and "at least one" can be used

205 interchangeably herein. As used in the specification, "a" or "an" means one or more. As used in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" mean one or more. As used herein, "another" means at least a second or more. Moreover, for the purpose of the present disclosure, it is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably.

210 "Allele" has the meaning which is commonly known in the art, that is, a genomic variant of a referent gene, including variants, which, when translated result in functional or dysfunctional (including non-existant) gene products. The term "allelic variant of a polymorphic region of a gene" refers to a region of a gene having one or several nucleotide sequence differences found in that region of the gene in certain individuals.

215 The term "biallelic" refers to a state where there are two possible bases for that SNP

(what are commonly referred to as the consensus and the polymorphic base). "Fragment" is meant to refer to any subset of the referent protein or nucleic acid molecule." "Intron," "intronic sequence" or "intronic nucleotide sequence" refers to untranslated DNA sequences between exons.

220 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. Furthermore, a compound "selected from the group consisting of refers to one or more of the compounds in the list that follows, including mixtures (i.e. combinations) of two or more of the compounds. According to the present

225 invention, an isolated, or biologically pure, protein or nucleic acid molecule is a compound that has been removed from its natural millieu. As such, "isolated" and "biologically pure" do not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from a natural source, can be produced using molecular biology techniques or can be produced by chemical synthesis.

230 "Gene" has the meaning that is commonly-known in the art, that is, a nucleic acid sequence that includes the translated sequences that code for a protein ("introns') and the untranslated intervening sequences ('exons"), and any regulatory elements ordinarily necessary to translate the protein. The term "genome" refers to the complete gene complement of an organism, contained in a set of chromosomes in eukaryotes. "Genotype"

235 has the meaning that is commonly known on the art, that is, a physical description of a nucleic acid sequence. The term linkage disequilibrium (LD) refers to a population association among alleles at two or more loci. It is a measure of co-segregation of alleles in a population. Linkage disequilibrium or allelic association is the preferential association of a particular allele or

240 genetic marker with a specific allele, or genetic marker at a nearby chromosomal location more frequently than expected by chance for any particular allele frequency in the population.

The term "locus" as it is used herein refers to any specific region of DNA, a gene, a group of genes or any group of nucleotides defining a DNA region of interest.The term "marker" is intended to mean any polymorphic genomic locus. The term "mutated gene"

245 refers to an allelic form of a gene, which is capable of altering the phenotype of a subject having the mutated gene relative to a subject which does not have the mutated gene.

"Nucleic acid" and "oligonucleotide" have the meaning that is commonly-known in the art, and includes primers, probes, and oligomer fragments, and shall be generic to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), to polyribonucleotides (containing

250 D-ribose), and to any other type of polynucleotide which is an N glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base. There is no intended distinction in length between the terms "nucleic acid" and "oligonucleotide", and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double-and single-stranded RNA.

255 The term "phenotype" refers to the physical, biochemical, and physiological makeup of an organism as determined both genetically and environmentally. "Polymorphism" means a group of two or more variants of a nucleic acid or amino acid sequence. The term "polymorphic gene" refers to a gene having at least one polymorphic region. The term "polymorphic site" refers to a region in a nucleic acid at which two or more alternative

260 nucleotide sequences are observed, often in a significant number of nucleic acid samples from a population of individuals.

"Single nucleotide polymorphism" or "SNP" means a polymorphism wherein the group exists by virtue of a difference in identity of one nucleotide at a given sequence location. The location of nucleotide identity difference is usually preceded by and followed

265 by highly conserved sequences. However, more than one single nucleotide polymorphism can exist between or among the group members.

270 Description of Table 1 and Table 2

Table 1 and Table 2 disclose the SNP and associated information including the Affymetrix SNP name, silhouette score, NCBI rs number, chromosome location, gene association, SEQ ID NO, nucleotide sequence, and mutation nucleotide.

275

Acute Coronary Syndrome

Genetic polymorphisms are useful in assessing Acute Coronary Syndrome (ACS) and measuring related cardiovascular risk factors in humans, including, but not limited to, angina, arterial inflammation, atherosclerosis, coronary heart disease (CHD), chronic stress, diabetes,

280 heart failure, hypertension, ischemia/reperfusion injuries, lipid profiles, myocardial infarction, obesity, restenosis, smoking, and stroke. A myocardial infarction (MI) is a type of acute coronary syndrome which is most frequently (but not always) a manifestation of CHD. ACS includes ST segment elevation myocardial infarction (STEMI), non-ST segment elevation myocardial infarction (NSTEMI), and unstable angina (UA).

285 MI is typically defined by the death or necrosis of myocardial cells. It is a diagnosis by the existence of myocardial ischemia or ACS. MI occurs when myocardial ischemia exceeds a critical threshold and overwhelms myocardial cellular repair mechanisms that are designed to maintain normal operating function and hemostasis. Ischemia at this critical threshold level for an extended time period results in irreversible myocardial cell damage or

290 death.

Critical myocardial ischemia may occur as a result of an increased metabolic demand from the myocardium and/or decreased delivery of oxygen and nutrients to the myocardium via the coronary arteries. An interruption in the supply of oxygen and nutrients to the myocardium occurs when a thrombus or clot is superimposed on an ulcerated or unstable

295 atherosclerotic plaque and results in coronary occlusion or sub-occlusion. A high-grade (> 75 percent) fixed coronary artery stenosis due to an atherosclerotic plaque formation or a dynamic stenosis associated with coronary vasospasm can also limit the supply of oxygen and nutrients and induce MI or an ACS-related event. Conditions associated with increased myocardial metabolic demand include extremes of physical exertion, severe hypertension

300 (including forms of hypertrophic obstructive cardiomyopathy), and severe aortic valve stenosis. Other cardiac valvular pathologies and low cardiac output states associated with a decreased aortic diastolic pressure, which is the prime component of coronary perfusion pressure, can cause MI or an ACS-related event. Ml can be subcategorized on the basis of anatomic, morphologic, and diagnostic

505 clinical information. From an anatomic or morphologic standpoint, the two types of MI are transmural and nontransmural. A transmural MI is characterized by ischemic necrosis of the full thickness of the affected muscle segment(s), extending from the endocardium through the myocardium to the epicardium. A nontransmural MI is defined as an area of ischemic necrosis that does not extend through the full thickness of myocardial wall segment(s). In a

310 nontransmural MI, the area of ischemic necrosis is limited to either the endocardium or the endocardium and myocardium. It is the endocardial and subendocardial zones of the myocardial wall segment that are the least perfused regions of the heart and are most vulnerable to conditions of ischemia. An older subclassification of MI, based on clinical diagnostic criteria, is determined by the presence or absence of Q waves on an

315 electrocardiogram (ECG). However, the presence or absence of Q waves does not distinguish a transmural from a non-transmural MI as determined by pathology.

A more common clinical diagnostic classification scheme is also based on ECG findings as a means of distinguishing between two types of MI - one that is marked by ST elevation and one that is not. The distinction between an ST-elevation MI and a non-ST-

320 elevation MI also does not distinguish a transmural from a non-transmural MI. The presence of Q waves or ST segment elevation is associated with higher early mortality and morbidity; however, the absence of these two findings does not confer better long-term mortality and morbidity. The most common etiology of MI is a thrombus superimposed on a ruptured or unstable atherosclerotic plaque.

325 In general, MI can occur at any age, but its incidence rises with age. The actual incidence is dependent upon predisposing risk factors for atherosclerosis. Approximately 50 pecent of all MIs in the US occur in people younger than 65 years of age. However, it is likely that, as demographics shift and the mean age of the population increases, a larger percentage of patients presenting with MI in the future will be older than 65 years.

330 Most MIs are caused by a disruption in the vascular endothelium associated with an unstable atherosclerotic plaque that stimulates the formation of an intracoronary thrombus, which results in coronary artery blood flow occlusion. If such an occlusion persists long enough (20 to 40 minutes), irreversible myocardial cell damage and cell death will occur. The development of atherosclerotic plaque occurs over a period of years to decades

335 and has been described in five phases. In the early phase, Phase 1, lesions are small, as are commonly seen in young people, and categorized into three types as follows: type I lesions, consisting of macrophage-derived foam cells that contain lipid droplets; type II lesions, consisting of both macrophages and smooth-muscle cells and mild extracellular lipid deposits; and type III lesions, consisting of smooth-muscle cells surrounded by extracellular connective

340 tissue, fibrils, and lipid deposits.

In the advanced phase, Phase 2, lesions, although not necessarily stenotic, may be prone to rupture because of their high lipid content, increased inflammation, and thin fibrous cap. These plaques are categorized morphologically as one of two variants: type IV lesions, consisting of confluent cellular lesions with a great deal of extracellular lipid intermixed with

345 normal intima, which may predominate as an outer layer or cap; or type Va lesions, possessing an extracellular lipid core covered by an acquired fibrous cap. Phase 2 plaques can evolve into the acute phases 3 and 4. In Phase 3, the lesions are characterized by acute complicated type VI lesions, originating from ruptured (type IV or Va) or eroded lesions, and leading to mural, non-obstructive thrombosis. This process is clinically silent, but

350 occasionally may lead to the onset of angina.

Phase 4 lesions are characterized by acute complicated type VI lesions, with fixed or repetitive occlusive thrombosis. This process becomes clinically apparent in the form of an acute coronary syndrome (ACS), although not infrequently it is silent. About two-thirds of ACS are caused by occlusive thrombosis on a non-stenotic plaque, although in about one-

355 third, the thrombus occurs on the surface of a stenotic plaque. In phases 3 and 4, changes in the geometry of ruptured plaques, as well as organization of the occlusive or mural thrombus by connective tissue, can lead to the occlusive or significantly stenotic and fibrotic plaques.

Phase 5 lesions are characterized by type Vb (calcific) or Vc (fibrotic) lesions that may cause angina; however, if preceded by stenosis or occlusion with associated ischemia,

360 the myocardium may be protected by collateral circulation and such lesions may then be silent or clinically inapparent.

Methods and Compositions to Assess ACS

Preventative measures are very successful in preventing ACS, but many individuals

365 susceptible to ACS remain unidentified due to the variable phenotype and unreliable testing methods. Associations may be utilized to assess risk or susceptibility to ACS or other related conditions (diagnostics). For example, detection of the polymorphisms of the described methods in a target DNA sample may be used to determine whether an individual has an increased risk of ACS or other phenotypic trait in linkage disequilibrium (LD) with the

370 polymorphisms. In the case of an association between a set of one or more polymorphisms and an increased risk of ACS, detection of a polymorphism in an individual may justify the institution of preventative measures (e.g., modification of diet, exercise, etc.) or immediate administration of a medical treatment regimen (e.g., pharmaceutical treatment). Alternatively, a positive SNP assay as described may also be used to identify individuals who are resistant

375 to a disease or other condition.

An association may or may not be due to direct effects of the polymorphisms on the phenotypic trait of interest. For example, a polymorphism that is found to associate with a high risk of ACS-related cardiac events may affect the expression or function of a specific gene or protein directly, or may be in linkage disequilibrium with (and so predictive of)

380 another locus that affects the expression or function of a protein. As such, a polymorphism within a nucleic acid may be used for diagnosis of a disorder that is associated with a genetic locus that is linked to the polymorphism, but not necessarily within the nucleic acid. Examples of direct effects to the expression or function of a protein include, but are not limited to, a polymorphism that alters the polypeptide sequence of the protein, and a

385 polymorphism that occurs in a regulatory region (i.e., promoter, enhancer, etc.) resulting in the increased or decreased expression of the protein. However, the polymorphisms themselves need not be directly involved in the manifestation of the phenotypic trait of interest in order to serve as a means to identify genomic regions that are involved; they need only be correlated with that trait and genetically linked to the genomic region.

390 The nucleic acids as described may also be used to detect or quantify expression of an encoded gene or other genes in linkage disequilibrium with the nucleic acids in a biological specimen for use as a diagnostic marker, e.g., to predict a phenotypic characteristic such as disease susceptibility or drug responsiveness by using the described nucleic acids as probes to determine whether a particular polymorphism or a set of polymorphisms is present in the

395 genome of an organism being tested. For example, the nucleic acids may be used as oligonucleotide probes to monitor RNA or mRNA levels within the organism to be tested or a part thereof, such as a specific tissue or organ, so as to determine the expression level of the gene encoding the RNA or mRNA, where the expression level can be correlated to a particular phenotypic characteristic of the organism. Likewise, the expression of the gene

400 may be assayed at the protein level using any customary technique such as immunological methods (e.g., Western blots, radioimmune precipitation and the like) or activity based assays measuring an activity associated with the gene product. The manner in which cells are probed for the presence of particular nucleotide or polypeptide sequences is well established in the literature and does not require further elaboration here. 405 Antibodies which bind specifically to the gene products also may be used for the diagnosis of disorders characterized by their expression, or in assays to monitor patients being treated with the gene products or with agonists, antagonists or inhibitors of the gene products. Diagnostic assays for the gene products of the present invention include methods which utilize an antibody and a label to detect the gene product in human body fluids or in

410 extract of cells or tissues, such as heart muscle.

Pharmacogenomics

Associations may be used for pharmacogenomic studies and drug development. For example, since the response of individuals with ACS to different treatments varies,

415 identifying a polymorphism or sets of polymorphisms that associate with positive (or negative) response or side-effects to an administered drug or other treatment would be useful for stratifying patient populations and individualizing treatment regimens. In addition, associations may be used to develop clinical trials for new treatments for ACS and other disorders or diseases by allowing stratification of the patient population. For example, if a

420 new statin drug were to be tested for efficacy and safety, a population of individuals with ACS could be stratified based on the type of ACS that they possess. A population of individuals with ACS may be further stratified based on polymorphisms that associate with responses to different classes of drugs and thereby distinguish probable responders from nonresponders from individuals likely to have toxic side effects.

425

Alternative Methods and Kits

The present disclosure is also directed to kit or reagent systems useful for practicing the described methods. Such kits will contain a reagent combination including the particular elements required to conduct an assay according to the methods disclosed herein. The

430 reagent system is presented in a commercially packaged form, as a composition or admixture where the compatibility of the reagents will allow, in a test device configuration, or more typically as a test kit, i.e., a packaged combination of one or more containers, devices, or the like holding the necessary reagents, and in a particular embodiment may include written instructions for the performance of assays. The kit may be adapted for any configuration of

435 an assay and may include compositions for performing any of the various assay formats described herein.

Kits containing oligonucleotides and, where appropriate, reagents for detection of fluorescent, chemiluminescent, radioactive or colorimetric signals, are within the scope of the described methods. In one embodiment, a kit of is designed to allow detection of specific

440 mutations and/or polymorphisms and/or in specific sequences of target DNA, includes one or more oligonucleotide probes specific for (a) a selected mutation and/or (b) a SNP, or (c) a specific region or regions of target DNA. The probes may be labeled as described above. The kits also include a plurality of containers of appropriate buffers and reagents and instructions.

445

Alternative Methods for Identifying Polymorphic Regions

A number of methods are used for determining the presence of a predisposing mutation in an individual. Genomic DNA is isolated from the individual or individuals that are to be tested. DNA can be isolated from any nucleated cellular source such as blood, hair

450 shafts, saliva, mucous, biopsy, feces, etc. Methods for determining the molecular structure of at least one polymorphic region of a gene are known and can be used to identify specific allelic variants of the polymorphic region associated with ACS. In one embodiment, determining the molecular structure of a polymorphic region of a gene comprises determining the identity of the allelic variant. A polymorphic region of a gene, of which specific alleles

455 are associated with ACS can be located in an exon, an intron, at an intron/exon border, or in the promoter of the gene.

The described methods assist in determining whether a subject has, or is at risk, of developing ACS. Such disorders can be associated with an aberrant gene activity, e.g., abnormal binding to a form of a lipid, or an aberrant gene protein level. An aberrant gene

460 protein level can result from an aberrant transcription or post-transcriptional regulation. Thus, allelic differences in specific regions of a gene can result in differences of gene protein due to differences in regulation of expression. In particular, some of the identified polymorphisms in the human gene may be associated with differences in the level of transcription, RNA maturation, splicing, or translation of the gene or transcription product.

465 In alternate embodiments, the described methods can be characterized as comprising detecting, in a sample of cells from the subject, the presence or absence of a specific allelic variant of one or more polymorphic regions of a gene. The allelic differences can be: (i) a difference in the identity of at least one nucleotide or (ii) a difference in the number of nucleotides, which difference can be a single nucleotide or several nucleotides.

470 In other detection methods, it is necessary to first amplify at least a portion of a gene prior to identifying the allelic variant. Amplification can be performed according to methods known in the art. In one embodiment, genomic DNA of a cell is exposed to two PCR primers and amplification for a number of cycles sufficient to produce the required amount of amplified DNA.

475 Methods using PCR amplification can be performed on the DNA from a single cell, although it is convenient to use at least about 10⁵ cells. Where large amounts of DNA are available, the genomic DNA is used directly. Alternatively, the region of interest is cloned into a suitable vector and grown in sufficient quantity for analysis, or amplified by conventional techniques. Of particular interest is the use of the polymerase chain reaction

480 (PCR) to amplify the DNA that lies between two specific primers. The use of the polymerase chain reaction is well known in the art. A detectable label may be included in the amplification reaction. Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6- FAM), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxy-X -rhodamine

485 (ROX), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), radioactive labels (e.g. ³²P, ³⁵S, ³H; etc.). The label may be a two stage system, where the amplified DNA is conjugated to biotin, haptens, etc., having a high affinity binding partner, e.g. avidin, specific antibodies, etc., where the binding partner is conjugated to a detectable label. The label may be conjugated to

490 one or both of the primers. Alternatively, the pool of nucleotides used in the amplification is labeled, so as to incorporate the label into the amplification product.

Primer pairs are selected from the genomic sequence using conventional criteria for selection. The primers in a pair will hybridize to opposite strands, and will collectively flank the region of interest. The primers will hybridize to the complementary sequence under

495 stringent conditions, and will generally be at least about 16 nucleotides in length, and may be 20, 25 or 30 nucleotides in length. The primers will be selected to amplify the specific region of the gene suspected of containing the predisposing mutation. Typically the length of the amplified fragment will be selected so as to allow discrimination between repeats of 3 to 7 units. Multiplex amplification may be performed in which several sets of primers are

500 combined in the same reaction tube, in order to analyze multiple exons simultaneously. Each primer may be conjugated to a different label.

Alternative amplification methods include: self sustained sequence replication, transcriptional amplification system, Q-Beta Replicase, or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques

505 well known to those of skill in the art. In general, detection of a SNP may be by any method known in the art. In a particular embodiment, detection is conducted by allele-specific probe hybridization, allele-specific primer extension, allele-specilic amplification, DNA sequencing, 5' nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, single-stranded conformation polymorphism and the like. These detection schemes may be 510 useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

Statistical Analysis

General characteristics of the study cases and controls were described using means

515 and standard deviations for continuous variables, and proportions for discrete and categorical variables. Variables described include age, sex, hypertension, hyperlipidemia, diabetes, family history of CHD, renal failure, and congestive heart failure. Since case and control patients were chosen as age-discordant and differed based on MI event status, it was expected that many, if not all, of these variables differed significantly between cases and controls. As

520 a consequence, it is not clear whether any differences in these variables have meaning with regard to the genetic purposes of the study, but statistical testing of the differences between cases and controls were performed for informative purposes using the Chi-square test for discrete variables and Student's T-test for continuous variables.

While genetic association analysis is a powerful method for detecting genetic

525 contributors to disease risk, the traditional Chi-square test is not the most powerful test for genome-wide SNP data that compared allele frequencies to phenotypic disease states. This is especially the case for pooled DNA since the standard Chi-square test assumes that the variance of the allele frequency difference results only from sampling variation. In studies of SNP data, and in pooled DNA specifically, allele frequencies may differ not only due to

530 sampling error but also due to population stratification (population history), evolutionary influences, errors introduced by the DNA pooling process, and errors in genotype allele- calling. Because of these additional sources of variation, the Chi-square test were not used in this study and instead the SNP associations to the MI endpoint were tested using a silhouette scoring method.

535 In the described SNP genotyping assay, the genotype assignment was based on scatter plots of signals corresponding to two SNP alleles. The three clusters that define the genotypes were well separated and the distances between the data points within a cluster were short. A "Silhouette score" is a graphical aid for interpretation and validation of data clusters that provides a measure of how well a data point was classified when it was assigned to a

540 cluster. Thus a Silhouette score was used as a qualitative measure for SNP genotyping results and for objective comparison of the performance of SNP assays under varying conditions, phenotypes, and circumstances. A Silhouette score condenses the quality of the genotype assignment for each SNP assay into a single numeric value, which ranges from 1.0, when the genotype assignment is unequivocal, down to -1.0, when the genotype assignment is arbitrary.

545 A Silhouette score close to 1.0 is obtained when the average distance from a data point to the other data points within its own cluster is smaller than the average distances to all data points in the closest cluster. A Silhouette score close to zero indicates that the data-point could equally well have been assigned to the neighboring cluster. A negative Silhouette score is obtained when the cluster assignment has been arbitrary and the data point is actually closer

550 to the neighboring cluster than to the other data points within its own cluster. Thus the

Silhouette score condenses the cluster quality for each SNP assay into a single measure that ranges from 1.0 to -1.0. When calculating the Silhouette score, the distance between data points can be measured either in one dimension, for example on the x-axis, or in two dimension using vectors. According to the approach described herein, a Silhouette score for

555 a SNP assay > 0.50 was considered significant.

The primary analysis compared the female case pool to the female control pool and each of the male case pools separately to their corresponding control pools. This allowed for the determination of gender-specific SNP associations to MI and provided the potential to replicate the associations between the groups. Evaluation of associations was performed for

560 additive, recessive, and dominant models.

Although the silhouette scoring method provided a powerful test of association, many of the sources of variation were reduced, thus preventing loss of information. First, sampling error was reduced by enrolling a large number of patients in the study population and by ensuring that it was a representative random sample of the overall population being studied.

565 In this study, a relatively large number of early-onset acute MI patients were included (n=300) from a population representative of those who experience such cardiovascular events. Population stratification can be dealt with through several methods, including the use of genomic control SNPs, use of pedigree-based analysis, or examination of racially homogeneous groups in the analysis. Because this study examined all SNPs for potential

570 association, none of them are definitively not associated with MI and thus would not be candidates for use as genomic controls. Pedigree-based analysis was not possible in this study population of unrelated individuals. Further subdivision of the population into racially homogeneous groups was also not possible as the study population is about 95% Caucasian. Since pooling of DNA samples can introduce error due to mixing unequal DNA volumes 575 from the patients who compose the pool, or due to inconsistencies in the PCR amplification reactions, multiple replicates of each DNA pool were tested. The results for each SNP's allele frequencies from each pool replicate were averaged to decrease the error variance potentially introduced by the pooling process. Genotyping error is increasingly a small issue in genetic studies since improvements in technology make allele call accuracy very high. For 580 example, the Affymetrix IOOK GeneChip has been shown to have 99.73% accuracy for genotyping; although this suggests that each chip will misclassify about 314, this error should be random across the 116,204 SNPs and the use of multiple replicates of each DNA pool would effectively eliminate this source of error. Finally, for DNA pooling projects, evolutionary forces exhibited in LD between SNPs cannot be addressed since the resulting 585 genotyping data are allele frequencies for the pools, and are not individual genotypes for each patient. However, the use of DNA pooling as an initial screen of potentially associated SNPs is an efficient study design for genetic association analysis.

TABLE 1 MALE NON SMOKERS

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UJ U)

Table 2 FEMALE NON SMOKERS

*0 to

K)

O

O

U)

590 Examples

The following examples are included to demonstrate particular embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute particular modes for

595 its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still a like or similar result will be obtained without departing from the spirit and scope of the invention.

600 Materials and Clinical Sampling Methods

Patients undergoing coronary arteriography who have given written informed consent were enrolled in a cardiovascular registry, designated the database registry of the Intermountain Heart Collaborative Study (IHCS), and have had a blood sample drawn. Blood samples were separated into plasma and DNA fractions and frozen at -80⁰C for future

605 testing. For each consenting patient, clinical and angiographic variables were collected and entered into the IHCS database. Patients also provided various epidemiologic information in a comprehensive cardiovascular survey.

Patient inclusion in the registry of the IHCS was based primarily on the willingness of a patient to consent to participation. Patients were included regardless of age, sex, race, or

610 disease status. The process for obtaining consent and collection of the data involves individually approaching patients as they prepare for procedures in the cardiac catheterization laboratory. While the willingness of a patient to participate in the registry may introduce some self-selection bias, any such bias should be very minimal — if it exists — since the only risk to the patient is a blood draw that is performed as an adjunct to other more invasive

615 procedures.

Patients included in the registry ranged in age from 18 to 95 years of age (mean: 63 years), 66% are male, and cardiac risk factors include an average BMI of 29 kg/m², 59% have hypertension, 55% have hyperlipidemia, 20% are diabetic, 20% are smokers, and 40% have a family history of early CHD. The initial clinical presentation at the time of hospitalization

620 included 16% acute MI, 30% unstable angina including angina at rest, and 55% with stable exertional angina. Coronary stenoses and degree of stenosis were reported by the patient's cardiologist from angiographic evidence collected from cardiac catheterization. About 65% of patients were found to have angiographically significant coronary artery disease (defined as >70% stenosis in at least one coronary artery), 10% had mild/moderate CHD (10-69% 625 stenoses), and 25% had a normal coronary angiogram. This study utilized DNA samples and clinical data from 900 patients, including 300 patients who presented with an acute MI and were confirmed to have significant CAD, and 300 age-discordant, sex-matched patients who were found to have angiographically-normal coronaries.

630 Clinical Data

Baseline clinical and angiographic variables were obtained at baseline and are included in the IHCS cardiovascular research database. They included age, sex, diabetes mellitus, hypertension, dyslipidemia, smoking, family history of early coronary heart disease, renal insufficiency, heart failure, and other clinically relevant information. Diabetes was

635 physician-diagnosed based on a history of fasting blood sugar >126 mg/dL or presence of antidiabetic therapy. Hypertension was physician-diagnosed based on a history of a systolic blood pressure > 140 or a diastolic blood pressure >90 mmHg or the presence of antihypertensive therapy. Dyslipidemia was physician-diagnosed based on a fasting cholesterol >200, LDL >130 (>100 if CHD or DM present), triglycerides >100, and/or HDL

640 <40 mg/dL. Family history was considered positive if a first-order relative suffered cardiovascular death, MI, or coronary revascularization before age 65. Renal insufficiency was defined as serum creatinine >2.0 mg/dL. Heart failure was physician-defined as noted on an angiographic data form or a hospital discharge summary. Tobacco use was considered present in active smokers or those with a smoking history of >10 pack-years.

645

Study Population and Clinical Endpoint

Case patients were those with at least one angiographically-confirmed coronary stenosis of 10-100% and a clinically-confirmed MI. The study MI endpoint was defined based on CK-MB values and electrocardiogram, with either the combination of CK-MB >6

650 mg/dl and CK-MB index >3% or the development of new Q-waves on electrocardiogram. Adjudication of the MI events were performed by one of two cardiologists. All acute MI patients were selected to include only those younger than the age of 65 at the time of their MI. An MI was accepted as an endpoint if it occurred at the time of the patient's index hospitalization in which they were entered into the cardiac catheterization research database

655 or if it occurred during longitudinal follow-up after successful discharge from that first hospitalization. An MI was also considered an event if it occurred prior to the time of enrollment into the database registry and was documented as per study criteria.

Patients free from any prior MI, acute MI at database enrollment, or follow-up MI were considered as potential controls. Controls were free of MI at baseline presentation and

660 as far into the future as follow-up was available (up to 14 years). Control patients were matched 2:1 to MI case patients by sex, but were also qualified based on age criteria. Age- based selection of controls were based on the control patient's age being discordant by being 20-30 years older than the case, with enrollment of the control patient into the cardiac catheterization research database at or after the age of 65 years. Controls included patients

665 regardless of CHD status since genetic markers among controls may differ from MI cases both due to the CHD status of MI patients and the actual occurrence of the MI.

All patients with a smoking history and those who were current smokers were excluded from consideration as potential MI cases or as matched control patients to reduce non-genetic confounding. This exclusion criterion allowed for the elimination of one of the

670 strongest environmental risk factors for CAD and MI.

Genetic Factors

At the time of angiography, 20 mL blood samples were collected in EDTA tubes and refrigerated at 4⁰C. Within 24 hours, samples were centrifuged and DNA extracted from the 675 white cell buffy coat using an automated DNA processing platform (AutoPure LS, Qiagen,

Valenica, CA). The DNA and the plasma from the samples were frozen and stored at -8O⁰C until testing.

For study testing, genetic samples were tested using the Affymetrix GeneChip

Mapping 500K Set (Affymetrix, Santa Clara, CA). This SNP array tested the genotypes of 680 500,568 SNPs spread across all 23 chromosomes in the genome and comprehensively covering almost all regions of each chromosome. To decrease the cost, time, and labor involved in genotyping multiple individual samples, the well-established technique of creating pools of DNA from many individuals was utilized. DNA pools were established with approximately 50 patients per group. Separate pools were created for males and females, and 685 for cases and controls. Thus, a total of six pools were studied. Furthermore, each pool was run in triplicate on separate arrays for validation purposes. Thus, the total number of DNA pools tested in the study is 18. 690 Affymetrics GENECHIP Array Assays

In one embodiment, allele specific hybridization is employed using probes overlapping the polymorphic site and having about 5, 10, 20, 25, or 30 nucleotides around the polymorphic region. In one embodiment, several probes capable of hybridizing specifically to allelic variants are attached to a solid phase support, e.g., a "chip." Oligonucleotides can

695 be bound to a solid support by a variety of processes, including lithography. For example a chip can hold up to 250,000 oligonucleotides (GeneChip, Affymetrix, Santa Clara, CA). Mutation detection analysis using these chips including oligonucleotides, also termed "DNA probe arrays" is well known in the art. 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

700 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.

GeneChip microarrays disclosed herein consisted of small DNA fragments (referred to as probes), chemically synthesized at specific locations on a coated quartz surface. The

705 precise location where each probe is synthesized is called a feature, and millions of features were contained on one array. By extracting and labeling nucleic acids from experimental samples, and then hybridizing those prepared samples to the array, the amount of label can be monitored at each feature, enabling a wide range of applications on a whole-genome scale — including genotyping.

710 The GeneChip Human Mapping 500K Array Set enabled wholegenome association studies across different populations. The Mapping 500K Array Set is comprised of two arrays, each capable of genotyping on average 250,000 SNPs. One array uses the Nsp I restriction enzyme (-262,000 SNPs), while the second uses Sty I (-238,000 SNPs).

Total genomic DNA (250 ng) was digested with a restriction enzyme (Nsp I or Sty I)

715 and ligated to adaptors that recognize the cohesive four base-pair (bp) overhangs. All fragments resulting from restriction enzyme digestion, regardless of size, were substrates for adaptor ligation. A generic primer that recognizes the adaptor sequence was used to amplify adaptor-ligated DNA fragments. PCR conditions were optimized to preferentially amplify fragments in the 200 to 1,100 bp size range. The amplified DNA was then fragmented,

720 labeled, and hybridized to a GeneChip.

All SNPs on the GeneChip Human Mapping 500K Array Set went through a rigorous screening and validation process. Optimal SNPs were selected and tiled on arrays based on accuracy, call rate, and linkage disequilibrium analysis in three populations across the genome. The median physical distance between SNPs was 2.5 kb and the average distance

725 between SNPs was 5.8 kb. The average heterozygosity of these SNPs is 0.30. Eighty-five percent of the human genome was within 10 kb of an SNP. Extensive annotation for each SNP was provided in both GeneChip Genotyping Analysis Software (GTYPE) and the NetAffx Analysis Center. This annotation combines data from multiple sources within the public domain and consolidates it into a single database. SNP annotation included dbSNP ID,

730 nearest gene, physical map location, cytoband, and allele frequencies in multiple populations.

Each array in the Mapping 500K Array Set required only 250 ng genomic DNA as starting material. Whole-genome amplified material prepared by the Qiagen REPLI-g kits (Qiagen, Valenica, CA) were used with the Mapping 500K Array Set. The GeneChip Human Mapping 500K Array Set was used in conjunction with GeneChip Genotyping Analysis

735 Software (GTYPE), which uses an automated, model-based genotype-calling algorithm that provides a confidence score for each individual genotype. The Dynamic Model algorithm (DM), generates a QC call rate for each array. A call rate of greater than or equal to 93 percent (at a DM confidence threshold of 0.33), when using good quality DNA, was used to determine whether a sample should be repeated or used for downstream analysis. The

740 Bayesian Robust Linear Model with Mahalanobis distance classifier (BRLMM) algorithm is an extension of the Robust Linear Model with Mahalanobis distance classifier (RLMM) algorithm developed for Mapping IOOK Set arrays, and uses data from multiple arrays to make genotype calls. It provides a significant improvement over DM in two important areas: it improves overall performance (call rates and accuracy), and it equalizes the performance of

745 homozygous and heterozygous genotypes. The BRLMM Analysis Tool (BAT) enabled analysis using the BRLMM algorithm. BAT is available as a standalone software tool from the Affymetrix web site (Affymetrix Inc., Santa Clara, CA).

GTYPE incorporates advanced functionality that supports multiple options for data export, including export by chromosome, filtering by allele frequency, Hardy- Weinberg

750 Equilibrium, and Mendelian-error. Each array in the Mapping 500K Array Set included more than 6.5 million features, each consisting of more than one million copies of a 25-bp oligonucleotide probe of a defined sequence, synthesized in parallel by proven photolithographic manufacturing. Each SNP was interrogated by 6- or 10- probe quartets where each probe quartet is comprised of a perfect match and a mismatch probe for each

755 allele. In total, there were 24 or 40 different 25-bp oligonucleotides per SNP. Two reagent kits were developed and validated for use in conjunction with the Mapping 500K Array Set. One kit is specific to the Nsp I restriction enzyme while the other is designed for the Sty I restriction enzyme. Both kits contained validated and qualified reagents for the most critical steps in the GeneChip Mapping Assay. This included the PCR primer and adaptor necessary

760 to selectively amplify a portion of the human genome, reagents to fragment and label the PCR products, and several control reagents. Fifty SNPs on both the Nsp I and Sty I arrays served as built-in controls for the Mapping 500K Array Set. GTYPE leverages these controls in the Sample Mismatch Report to cross-check genotypes from the same sample on each array to verify that both arrays remain together from DNA preparation to data analysis. In

765 addition, GTYPE uses additional controls to cross-check Mapping 500K arrays with previous generation arrays to allow customers who are using the Mapping 500K Array Set to increase power in ongoing studies.

In one aspect of the present disclosure, methods are provided for determining a predisposition to ACS in an individual. The methods comprise an analysis of genomic DNA

770 in an individual for one or more SNPs which confer an increased susceptibility to ACS.

Individuals are screened by analyzing their genomic DNA for the presence of one or more SNPs.

Example 1

775 In this example, 300 patients suffering from early onset MI as examined by a cardiologist matched in age and sex and without any signs of cardiovascular risk were analyzed per the methods of the present invention. Reference Chart 1 and 2 for results of this analysis.

780 Example 2

In this example, 300 aged-matched patients without MI as examined by a cardiologist matched in age and sex and with CHD were analyzed per the methods of the present invention. Reference Chart 1 and 2 for results of this analyis.

785 Example 3

In this example, 300 patients with and without MI and without CHD as examined by a cardiologist matched in age and sex and without any signs of cardiovascular risk were analyzed per the methods of the present invention. Reference Chart 1 and 2 for results of this analysis.

790 Example 4

In this study, nine male-specific DNA pools were analyzed per the methods described above. Silhouette scores for MI compared with all controls ranged from -0.04 to 0.58, except

795 for one SNP with score=0.67 (rs3120784 (SEQ ID NO: 1) located on chromosome Iq32.2).

Evaluation of a second set of non-smokers sampled from the sample population tested the association of rs3120784 (with genotypes GG, GA, AA) in individually genotyped samples for early-onset MI cases compared with older controls.

Among males, genotype frequencies appeared to be GG: 89.6% (n= 1381), GA: 9.5%

800 (n=147), AA: 0.9% (n=14) and MI was present in 33.2% (458 of n=1381), 29.3% (43 of n=147), and 0.0% (0 of n=14) of GG, GA, and AA genotypes, respectively (Armitage test of trend: p-trend=0.029, univariate odds ratio=0.69 per A allele, 95% confidence interval=0.49, 0.97).

Multivariate analysis adjusting for hypertension, hyperlipidemia, diabetes, family

805 history of early coronary heart disease, and body mass index showed that significance appeared to be retained (odds ratio=0.67 per A allele, 95% confidence interval=0.48, 0.95; p- trend=0.025). For individual comparisons, GA compared with GG appeared to have an odds ratio=0.83, 95% confidence interval=0.57, 1.21 (p=0.34) and AA compared with GG appeared to have an odds ratio=0.00 (confidence interval and p-value not calculated).

810 Among females, rs3120784 genotypes GG, GA, and AA had frequencies of 87.6%

(n=514), 11.2% (n=66), and 1.2% (n=7), respectively, with MI in 24.7% (127 of n=514), 21.2% (14 of n=66), and 28.6% (2 of n=7) for GG, GA, and AA, respectively (p-trend=0.70).

As the heterozygote among females had a similar down-ward trend as in males compared with GG and the odds ratio was similar (0.82 in females for GA vs. GG), and

815 because the AA genotype among females had such a small sample size, the data from males and females were combined for an overall analysis.

Genotype frequencies overall appeared to be GG: 89.0% (n=1895), GA: 10.0% (n=213), and 1.0% (n=21). Percentage in each genotype with MI was, for GG: 30.9% (585 of n=1895), GA: 26.8% (57 of n=213), and 9.5% (2 of n=21), with odds ratio=0.74 (95%

820 confidence interval=0.55, 0.98; p-trend=O.O33). Adjustment for sex, hypertension, hyperlipidemia, diabetes, family history of early coronary heart disease, and body mass index resulted in an odds ratio for the SNP of 0.74 per A allele (95% confidence interval: 0.55, 0.99; p-trend=0.039). For individual genotype comparisons with multivariate adjustment, GA vs. GG had an adjusted odds ratio=0.84 (95% confidence interval=0.61, 1.17; p=0.30) and AA vs.

825 GG resulted in an adjusted odds ratio=0.21 (95% confidence interval=0.05, 0.92; p=0.038). In the 9 female-specific DNA pools, silhouette scores appeared to range from -0.04 to 0.54, with the best SNP being rs2173369 on chromosome 10q21.3. In the second population, replication was not achieved. Genotypes for rs2173369 were AA, AG, and GG and genotyping was performed among N=862 individuals. Among females in the replication

830 population, genotype frequencies were AA: 91.6% (n=240), AG: 8.0% (n=21), and GG: 0.4% (n=l), and MI was found in 22.1% (53 of 240), 28.6% (6 of 21), and 0% (0 of 1) of AA, AG, and GG, respectively (p-trend=0.68). Among males, frequencies appeared to be 88.5%, 11.2%, and 0.3% for AA, AG, and GG, respectively, and MI was found in 39.0% (207 of n=531), 38.8% (26 of n=67), and 50.0% (1 of n=2) for AA, AG, and GG, respectively (p-

835 trend=0.94). Overall, results were similar (MI=33.7%, 36.4%, and 33.3% for AA, AG, and GG, respectively; p-trend=0.65).

While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions, and sub- 840 combinations thereof. It is therefore intended that the following claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions, and sub- combinations as are within their true spirit and scope.

845

850

855

Claims

860 CLAIMS

1. A method of assessing cardiovascular risk in a male patient, the method comprising:

a. testing a DNA sample from a patient for the presence of a single

865 nucleotide polymorphism (SNP) in any one of the nucleotide sequences of

SEQ ID NOS: 1-17

b. providing a plurality of reference SNP profiles, each associated with a cardiovascular risk, wherein the SNPs from the DNA sample and each

870 reference profile has a pluarilty of values, each value representing a quantifiable risk of acute coronary syndrome (ACS);

c. and selecting the reference profile most similar to the sample SNP to thereby assess the cardiovascular risk for the patient.

875

2. The method as claimed in claim 1 wherein said testing uses genomic DNA for the DNA sample.

3. The method as claimed in claim 2, further comprising isolating genomic DNA from the 880 white cell buffy coat of a blood sample using an automated DNA processing platform.

4. The method as claimed in claim 1, wherein said testing comprises carrying out polymerase chain reaction amplification of the genomic DNA with subsequent sequence analysis.

885 5. The method as claimed in claim 1, wherein said cardiovascular risk is increased.

6. The method as claimed in claim 5, wherein a SNP is detected in any one of the nucleotide sequences of SEQ ID NOS: 2, 5, 8-9 or 16.

890 7. The method as claimed in claim 1, wherein said cardiovascular risk is decreased.

8. The method as claimed in claim 7, wherein a SNP is detected in any one of the nucleotide sequences of SEQ ID NOS: 1, 3, 4, 6-7, 10- 15 or 17.

895 9. A method for identifying a male patient at risk of developing acute coronary syndrome (ACS) on the basis of genetic predisposition, comprising;

testing a DNA sample from the patient for the presence of a single nucleotide polymorphism (SNP) in any one of the nucleotide sequences of SEQ ID NOS: 2, 5, 8, 9 or 900 16; wherein the presence in said DNA sample of a tested SNP indicates that the person is at risk of developing ACS.

10. The method as claimed in claim 9, wherein said testing uses genomic DNA for the DNA sample.

905

11. The method as claimed in claim 10, further comprising isolating genomic DNA from the white cell buffy coat of a blood sample using an automated DNA processing platform.

12. The method as claimed in claim 9, wherein said testing comprises carrying out

910 polymerase chain reaction amplification of the genomic DNA with subsequent sequence analysis.

13. The method as claimed in claim 9, wherein a SNP is detected in any one of the nucleotide sequences of SEQ ID No: 2, SEQ ID No: 5, SEQ ID No: 8, SEQ ID NO: 9 and

915 SEQ ID NO: 16 is detected.

14. A computer readable medium comprising a plurality of digitally-encoded correlations selected from the genotype correlations identified in TABLE 1 and TABLE 2, wherein each correlation of the plurality has a value representing a risk for an acute coronary syndrome

920 (ACS).

15. A method of assessing cardiovascular risk in a female patient, the method comprising:

a. testing a DNA sample from a patient for the presence of a single

925 nucleotide polymorphism (SNP) in any one of the nucleotide sequences of

SEQ ID NOS: 18-25; b. providing a plurality of reference SNP profiles, each associated with a cardiovascular risk, wherein the SNPs from the DNA sample and each

930 reference profile has a pluarilty of values, each value representing a quantifiable risk of acute coronary syndrome (ACS);

c. and selecting the reference profile most similar to the sample SNP to thereby assess the cardiovascular risk for the patient.

935

16. The method as claimed in claim 15, wherein said testing uses genomic DNA for the DNA sample.

17. The method as claimed in claim 16, further comprising isolating genomic DNA from the 940 white cell buffy coat of a blood sample using an automated DNA processing platform.

18. The method as claimed in claim 15, wherein said testing comprises carrying out polymerase chain reaction amplification of the genomic DNA with subsequent sequence analysis.

945

19. The method as claimed in claim 15, wherein said cardiovascular risk is increased.

20. The method as claimed in claim 19, wherein a SNP is detected in any one of the nucleotide sequences of SEQ ID NOS: 20 or 23-25.

950

21. A method for identifying a female patient at risk of developing acute coronary syndrome (ACS) on the basis of genetic predisposition, comprising;

testing a DNA sample from the patient for the presence of a single nucleotide 955 polymorphism (SNP) in any one of the nucleotide sequences of SEQ ID NOS: 20, 23, 24-25; wherein the presence in said DNA sample of a tested SNP indicates that the person is at risk of developing ACS.

22. The method as claimed in claim 21 wherein said testing uses genomic DNA for the DNA 960 sample.

23. The method as claimed in claim 22, farther comprising isolating genomic DNA from the white cell buffy coat of a blood sample using an automated DNA processing platform.

965 24. The method as claimed in claim 23, wherein said testing comprises carrying out polymerase chain reaction amplification of the genomic DNA with subsequent sequence analysis.

25. A method for identifying a decreased cardiovascular risk in a male patient, comprising; 970 detecting a single nucleotide polymorphism (SNP) in any one of nucleotide sequences of SEQ ID NOS: 1, 3-4, 6-7, 10-15 or 17 in said individual's nucleic acids, wherein the presence of said SNP is correlated with a decreased risk of cardiovascular disease in said individual. 975

26. The method of claim 25, wherein said detection is performed by a process selected from the group consisting of allele-specific probe hybridization, allele-specific primer extension, allele-specific amplification, DNA sequencing, 5 '-nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis and single-stranded conformation

980 polymorphism.

27. A method for identifying a decreased cardiovascular risk in a female patient, comprising;

detecting a single nucleotide polymorphism (SNP) in any one of the nucleotide 985 sequences of SEQ ID NOS: 18-19, or 21-22 in said patient's nucleic acids, wherein the presence of said SNP is correlated with a decreased cardiovascular risk in said patient.

990

995