Reagents and methods for detection of heart failure

[0002] The present invention relates to oligonucleotides, kits, microarrays, and methods for detection of cardiovascular disease, in particular, left ventricular heart failure.

[0003] The World Health Organization has determined that cardiovascular disease (CVD) is the leading cause of death throughout the world and the leading cause of lost years of healthy life in Europe. In the U.S., CVD caused 41 per cent of all deaths in 1998 and is second only to all cancers combined in years of potential life lost. The annual number of deaths from CVD in the U.S. increased substantially between 1900 and 1970, peaking in 1963. Since 1963 the U.S. CVD death rate has changed from an increasing to a decreasing trend, and by 1995 the CVD death rate was similar to that in 1936.

[0004] The only exception to the CVD mortality decline in the U.S. is congestive heart failure (CHF). CHF is often the end stage of cardiac disease, and half of the patients diagnosed with CMF die within five years. Sudden death is common among CHF patients, occurring six to nine times more frequently than in the general population. Between 1968 and 1998, the number of deaths from CHF in the U.S. increased from approximately 10,000 to almost 50,000. Approximately five million Americans are currently living with heart failure, and 550,000 new cases are diagnosed in the U.S. each year. CHF occurs slightly more frequently among men than among women, and is twice as common among African Americans compared to Caucasian Americans, with mortality also doubled for African Americans. The number of CHF cases is expected to increase as the population ages, and as cardiac patients are able to survive and live longer with their disease. CMF has therefore become a major medical problem in the U.S.

[0005] As a symptom of underlying heart disease, heart failure is closely associated with the major risk factors of CVD: smoking, high cholesterol, hypertension, diabetes, abnormal blood sugar levels, and obesity, with hypertension and diabetes being particularly important. CMF is about twice as common for persons with hypertension as compared with normotensive individuals, and the degree of risk for heart failure appears to be directly related to the severity of high blood pressure. Persons with diabetes have a two- to eightfold greater risk for heart failure than those without diabetes, and women with diabetes have a greater risk of heart failure than men with diabetes. The presence of coronary disease is one of the greatest risks for heart failure. Muscle damage and scarring caused by heart attacks create a fivefold increase in risk of developing CHF.

[0006] Heart failure occurs when one or more of the chambers of the heart cannot keep pace with the amount of blood flowing through an individual's body. Heart failure can involve either or both sides of the heart, but in general, the left side of the heart is affected first. Each side of the heart comprises an atrium (the upper chamber) which receives blood into the heart and a ventricle (the lower chamber) which pumps blood to the body. The left ventricle supplies most of the heart's pumping power and is larger than other heart chambers, pumping oxygen-rich blood from the left atrium to the rest of the body. When the left ventricle loses its ability to contract, the heart is unable to pump with sufficient force to push enough blood into circulation. This condition is known as systolic failure. When the left ventricle loses its ability to relax, usually because of stiffness from thickening (hypertrophy), the heart cannot fill properly with blood during the resting period between each heartbeat. This condition is known as diastolic failure. In either condition, blood entering the left atrium from the lungs may back up or regurgitate, causing fluid to leak into the lungs (pulmonary edema), and blood flow throughout the body slows down, causing fluid to build up in tissues (edema). The presence of excess fluid due to heart failure is termed congestive heart failure.

[0007] The symptoms of heart failure include shortness of breath (dyspnea), fatigue, fluid accumulation, and persistent coughing that produces mucus or pink, blood-tinged sputum. Heart failure develops slowly, and the heart compensates as its pumping capacity decreases. For example, the heart may enlarge, or muscle fibers within the heart may hypertrophy to allow the heart to contract with more force and pump more blood. The heart also compensates for loss of pumping capacity by contracting more frequently to increase circulation. However, eventually the heart can no longer compensate and symptoms only appear when the condition has progressed in severity.

[0008] Physicians currently diagnose heart failure through physical examination, to detect the observable symptoms indicated above. Electrocardiography is performed to determine whether the patient's heartbeat is abnormal and to identify the cause of the heart failure. In particular, an electrocardiogram with Q-waves and poor R wave progression indicates a previous myocardial infarction, with probable left ventrical systolic dysfunction. An electrocardiogram indicating left ventricular hypertrophy may show an etiology related to hypertension, aortic stenosis, hypertrophic cardiomyopathy or dilated cardiomyopathy, and may indicate either systolic or diastolic left ventricular dysfunction. A chest x-ray may indicate pulmonary edema, pulmonary venous engorgement, or cardiac enlargement. However, heart failure from left ventricular dysfunction may be present even when a patient has a normal chest x-ray. Physicians also perform an ultrasonic examination of the heart to generate an echocardiogram, which allows measurement of systolic and diastolic ventricular contractile function, chamber size, and wall thickness. When Doppler ultrasound is used for echocardiography, valvular stenosis and regurgitation can be detected and quantified.

[0009] Lifestyle changes such as weight reduction; moderate exercise; limiting fluid intake, including alcohol intake; and cessation of smoking, are indicated for individuals with chronic CHF. Left ventricular systolic heart failure can be treated using lifestyle changes, angiotensin converting enzyme inhibitors, diuretics, and/or digoxin. The optimal treatment for left ventricular diastolic heart failure has not been determined. Since left ventricular diastolic heart failure is frequently associated with hypertension, antihypertensive therapy is frequently prescribed, and congestion is relieved with diuretics.

[0010] A need remains for detection of predisposition to heart failure in individuals who may have the condition but who do not exhibit symptoms, so that lifestyle changes can be initiated early in the progression of disease. In addition, it is important to determine predisposition to heart failure in individuals who have experienced myocardial infarction, so that appropriate treatment modalities may be initiated to limit the effects of heart failure in such individuals.

[0011] The present inventors have found that predisposition to left ventricular heart failure may be detected by analysis of certain polymorphisms in the nucleic acid set forth in SEQ ID NO:1. Specifically, the presence of an A nucleotide at position 24801 of SEQ ID NO:1 (the sense strand), or a T nucleotide at the corresponding position of the complement of SEQ ID NO:1, is indicative of a predisposition to left ventricular diastolic heart failure. Furthermore, the presence of a T nucleotide at position 24941 of SEQ ID NO:1, or an A at the corresponding position in the complement of SEQ ID NO:1, is also indicative of predisposition to left ventricular diastolic heart failure. In addition, the presence of an A nucleotide at position 32614 of SEQ ID NO:1 (the sense strand), or a T nucleotide at the corresponding position of the complement of SEQ ID NO:1, is indicative of a predisposition to left ventricular systolic heart failure in individuals who have experienced a myocardial infarction.

[0012] Moreover, individuals who possess the SEQ ID NO:1 haplotype characterized by a G nucleotide at position 24801 of SEQ ID NO:1; a C nucleotide at position 24941 of SEQ ID NO:1; a C nucleotide at position 27645 of SEQ ID NO:1; a C nucleotide at position 32163 of SEQ ID NO:1; and a G nucleotide at position 32614 of SEQ ID NO:1 are at lower risk of developing left ventricular diastolic heart failure.

[0013] The nucleic acid of SEQ ID NO:1 (nucleotides 1-38653 of GenBank Accession No. AC004923) is the genomic sequence of a gene of unknown function currently described as hUNC93B1 (Kashuba, et al., submitted for publication). The hUNC93B1 gene is located on chromosome 11q13, and the mRNA (GenBank Accession No. AJ271326, SEQ ID NO:50) is expressed at high levels in cardiac tissue (Id.). Without wishing to be bound by any theory, it is believed that the hUNC93B1 gene may encode a twelve transmembrane transporter protein.

[0014] In one embodiment, the invention provides a sequence determination oligonucleotide complementary to a polymorphic region within a nucleic acid having a sequence as set forth in SEQ ID NO:1, wherein the region corresponds to a polymorphic site selected from the group consisting of position 24801 of SEQ ID NO:1, position 24941 of SEQ ID NO:1, position 27645 of SEQ ID NO:1, position 32163 of SEQ ID NO:1, and position 32614 of SEQ ID NO:1.

[0015] In yet another embodiment, the invention provides a microarray comprising at least one oligonucleotide complementary to a polymorphic region in the nucleic acid set forth in SEQ ID NO:1, wherein the region corresponds to a polymorphic site selected from the group consisting of position 24801 of SEQ ID NO:1, position 24941 of SEQ ID NO:1, position 27645 of SEQ ID NO:1, position 32163 of SEQ ID NO:1, and position 32614 of SEQ ID NO:1.

[0016] In another embodiment, the invention provides the oligonucleotide primer pairs useful for amplification of a polymorphic region in the nucleic acid of SEQ ID NO:1 from a biological sample, wherein the region corresponds to a polymorphic site selected 10 from the group consisting of position 24801 of SEQ ID NO:1, position 24941 of SEQ ID NO:1, position 27645 of SEQ ID NO:1, position 32163 of SEQ ID NO:1, and position 32614 of SEQ ID NO:1.

[0017] In another embodiment, the invention provides a kit comprising at least one oligonucleotide primer pair complementary to a polymorphic region of the nucleic acid of SEQ ID NO:1, wherein the region corresponds to a polymorphic site selected from the group consisting of position 24801 of SEQ ID NO:1, position 24941 of SEQ ID NO:1, position 27645 of SEQ ID NO:1, position 32163 of SEQ ID NO:1, and position 32614 of SEQ ID NO:1.

[0018] The invention is also embodied in a method of diagnosing predisposition to left ventricular diastolic heart failure in a human, said method comprising the steps of obtaining a nucleic acid sample from the human; detecting the presence or absence of at least one allelic variant of a polymorphic region in a nucleic acid having a sequence as set forth in SEQ ID NO:1 in the sample, wherein the polymorphic region corresponds to the polymorphic site at position 24801 of SEQ ID NO:1.

[0019] The invention is also embodied in a method of diagnosing predisposition to left ventricular diastolic heart failure in a human, said method comprising the steps of obtaining a nucleic acid sample from the human; and detecting the presence or absence of at least one allelic variant of a polymorphic region in a nucleic acid having a sequence as set forth in SEQ ID NO:1 in the sample, wherein the polymorphic region corresponds to the polymorphic site at position 24941 of SEQ ID NO:1.

[0020] In another embodiment, the invention provides a method of diagnosing predisposition to left ventricular systolic heart failure in a human who has experienced a myocardial infarction, said method comprising the steps of obtaining a nucleic acid sample from the human; and detecting the presence or absence of at least one allelic variant of a polymorphic region in a nucleic acid having a sequence as set forth in SEQ ID NO:1 in the sample, wherein the polymorphic region corresponds to the polymorphic site at position 32614 of SEQ ID NO:1.

[0021] In a further embodiment, the invention provides a method of diagnosing predisposition to left ventricular diastolic heart failure in a human comprising the steps of obtaining a nucleic acid sample from the human; and detecting the presence or absence of a haplotype of the nucleic acid having a sequence as set forth in SEQ ID NO:1, said haplotype being characterized by: a G nucleotide at position 24801 of SEQ ID NO:1; a C nucleotide at position 24941 of SEQ ID NO:1; a C nucleotide at position 27645 of SEQ ID NO:1; a C nucleotide at position 32163 of SEQ ID NO:1; and a G nucleotide at position 32614 of SEQ ID NO:1.

[0022] FIG. 1 sets forth the sequence of the nucleic acid of SEQ ID NO:1, with the polymorphic sites at positions 24801, 24941, 27645, 32163, and 32614 in bold capital type. The transcription start site of the nucleic acid of SEQ ID NO:1 is indicated in italics, introns are depicted in lower case type, and exons are depicted in bold lower case type.

[0023] FIG. 2 sets forth the sequences of certain oligonucleotides of the invention, which are correlated with the polymorphic site the oligonucleotides are designed to detect. Polymorphic sites in these oligonucleotides are indicated in bold capital type.

[0024] FIG. 3 sets forth the sequences of certain oligonucleotide primer pairs designed to amplify polymorphic regions of the nucleic acid of SEQ ID NO:1.

[0025] The U.S. patents and publications referenced herein are hereby incorporated by reference.

[0026] Examples 1 through 3 below demonstrate associations between certain polymorphic regions in SEQ ID NO:1 and measurements of physical parameters of left ventricular heart failure. One such parameter, the echocardiographic ratio of peak velocities of the early (E) and a trial (A) contributions to left ventricular filling (the “E/A ratio”) is a measurement of left ventricular diastolic function.

[0027] The present inventors have observed a correlation between E/A ratio and polymorphic variants at position 24801 of SEQ ID NO:1. Specifically, an A nucleotide at position 24801 of SEQ ID NO:1 is associated with lower E/A ratio (p=0.04836). Thus individuals who exhibit a homozygous A/A genotype at position 24801 of SEQ ID NO:1 have a higher risk of developing left ventricular diastolic heart failure, as compared to individuals who are heterozygous A/G or homozygous G/G at position 24801.

[0028] In addition, the present inventors have discovered a correlation between E/A ratio and polymorphic variants at position 24941 of SEQ ID NO:1, wherein the presence of a T nucleotide at that position is associated with lower E/A ratio (p=0.00170). Individuals who exhibit a homozygous T/T genotype at position 24941 of SEQ ID NO:1 have a higher risk of developing left ventricular diastolic heart failure, as compared to individuals who are heterozygous T/C or homozygous C/C at position 24941.

[0029] Moreover, the haplotype characterized by a G nucleotide at position 24801 of SEQ ID NO:1; a C nucleotide at position 24941 of SEQ ID NO:1; a C nucleotide at position 27645 of SEQ ID NO:1; a C nucleotide at position 32163 of SEQ ID NO:1; and a G nucleotide at position 32614 of SEQ ID NO:1 is associated with increased E/A ratio (p=0.00020). Individuals who are homozygous for this haplotype have significantly lower risk of developing left ventricular diastolic heart failure.

[0030] Another echocardiographic test for heart failure is the ejection fraction, which measures the amount of blood pumped out when the heart contracts, thus indicating left ventricular performance. Example 3 indicates that for those individuals who have suffered a myocardial infarction, an association exists between the polymorphic variant at position 32614 of SEQ ID NO:1 and ejection fraction (p=0.0365). Specifically, the presence of an A nucleotide at position 32614 of SEQ ID NO:1 is correlated with a low ejection fraction or risk of left ventricular heart failure.

[0031] The various embodiments of the present invention for the first time allow physicians to determine predisposition to left ventricular heart failure in a human using a simple diagnostic test performed on a biological sample from the individual. In accordance with the present invention, the sequence of one or more polymorphic regions of SEQ ID NO:1 is determined and risk of left ventricular heart failure is assessed. Using this information, physicians can intervene in the disease process of heart failure at a much earlier time than was previously possible, advising lifestyle changes or prescribing drug modalities which will reduce or delay progression of the disease. This early intervention will result in decreased morbidity and mortality from heart failure, ultimately impacting on economic sequence of the disease such as hospitalization expenses and lost years of healthy life.

[0032] For the purposes of the invention, certain terms are defined as follows. “Oligonucleotide” means a nucleic acid molecule preferably comprising from about 8 to about 50 covalently linked nucleotides. More preferably, an oligonucleotide of the invention comprises from about 8 to about 35 nucleotides. Most preferably, an oligonucleotide of the invention comprises from about 10 to about 25 nucleotides. In accordance with the invention, the nucleotides within an oligonucleotide may be analogs or derivatives of naturally occurring nucleotides, so long as oligonucleotides containing such analogs or derivatives retain the ability to hybridize specifically within the polymorphic region containing the targeted polymorphism. Analogs and derivatives of naturally occurring oligonucleotides within the scope of the present invention are exemplified in U.S. Pat. Nos. 4,469,863; 5,536,821; 5,541,306; 5,637,683; 5,637,684; 5,700,922; 5,717,083; 5,719,262; 5,739,308; 5,773,601; 5,886,165; 5,929,226; 5,977,296; 6,140,482; WO 00/56746; WO 01/14398, and the like. Methods for synthesizing oligonucleotides comprising such analogs or derivatives are disclosed, for example, in the patent publications cited above and in U.S. Pat. Nos. 5,614,622; 5,739,314; 5,955,599; 5,962,674; 6,117,992; in WO 00/75372, and the like. The term “oligonucleotides” as defined herein includes compounds which comprise the specific oligonucleotides disclosed herein, covalently linked to a second moiety. The second moiety may be an additional nucleotide sequence, for example, a tail sequence such as a polyadenosine tail or an adaptor sequence, for example, the phage M13 universal tail sequence, and the like. Alternatively, the second moiety may be a non-nucleotidic moiety, for example, a moiety which facilitates linkage to a solid support or a label to facilitate detection of the oligonucleotide. Such labels include, without limitation, a radioactive label, a fluorescent label, a chemiluminescent label, a paramagnetic label, and the like. The second moiety may be attached to any position of the specific oligonucleotide, so long as the oligonucleotide retains its ability to hybridize to the polymorphic regions described herein.

[0033] A polymorphic region as defined herein is a portion of a genetic locus that is characterized by at least one polymorphic site. A genetic locus is a location on a chromosome which is associated with a gene, a physical feature, or a phenotypic trait. A polymorphic site is a position within a genetic locus at which at least two alternative sequences have been observed in a population. A polymorphic region as defined herein is said to “correspond to” a polymorphic site, that is, the region may be adjacent to the polymorphic site on the 5′ side of the site or on the 3′ side of the site, or alternatively may contain the polymorphic site. A polymorphic region includes both the sense and antisense strands of the nucleic acid comprising the polymorphic site, and may have a length of from about 100 to about 5000 base pairs. For example, a polymorphic region may be all or a portion of a regulatory region such as a promoter, 5′ UTR, 3′ UTR, an intron, an exon, or the like. A polymorphic or allelic variant is a genomic DNA, cDNA, mRNA or polypeptide having a nucleotide or amino acid sequence that comprises a polymorphism. A polymorphism is a sequence variation observed at a polymorphic site, including nucleotide substitutions (single nucleotide polymorphisms or SNPs), insertions, deletions, and microsatellites. Polymorphisms may or may not result in detectable differences in gene expression, protein structure, or protein function. Preferably, a polymorphic region of the present invention has a length of about 1000 base pairs. More preferably, a polymorphic region of the invention has a length of about 500 base pairs. Most preferably, a polymorphic region of the invention has a length of about 200 base pairs.

[0034] A haplotype as defined herein is a representation of the combination of polymorphic variants in a defined region within a genetic locus on one of the chromosomes in a chromosome pair. A genotype as used herein is a representation of the polymorphic variants present at a polymorphic site.

[0035] A polymorphic region of the present invention comprises a portion of SEQ ID NO:1 corresponding to at least one of the polymorphic sites identified above. That is, a polymorphic region of the invention may include a nucleotide sequence surrounding and/or including any of the polymorphic sites at positions 24801, 24941, 27645, 32163, and 32614 of SEQ ID NO:1. Polymorphic regions in the antisense nucleic acid complementary to SEQ ID NO:1 are also encompassed in the present invention, wherein the region includes a nucleotide sequence surrounding and/or including any of the antisense positions corresponding to positions 24801, 24941, 27645, 32163, and 32614 in the complement of SEQ ID NO:1. For example, a polymorphic region corresponding to the polymorphic site at position 24801 of SEQ ID NO:1 may comprise a sequence as set forth in any of SEQ ID NO:2; SEQ ID NO:7; SEQ ID NO:12; SEQ ID NO:13; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:32; or SEQ ID NO:37. A polymorphic region corresponding to the polymorphic site at position 24941 of SEQ ID NO:1 may comprise a sequence as set forth in any of SEQ ID NO:3; SEQ ID NO:8; SEQ ID NO:14; SEQ ID NO:15; SEQ ID NO:24; SEQ ID NO:25; SEQ ID NO:33; or SEQ ID NO:38. A polymorphic region corresponding to the polymorphic site at position 27645 of SEQ ID NO:1 may comprise a sequence as set forth in any of SEQ ID NO:4; SEQ ID NO:9; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:34; or SEQ ID NO:39. A polymorphic region corresponding to the polymorphic site at position 32163 of SEQ ID NO:1 may comprise a sequence as set forth in any of SEQ ID NO:5; SEQ ID NO:10; SEQ ID NO:18; SEQ ID NO:19; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:35; or SEQ ID NO:40. A polymorphic region corresponding to the polymorphic site at position 32614 of SEQ ID NO:1 may comprise a sequence as set forth in any of SEQ ID NO:6; SEQ ID NO:11; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:30; SEQ ID NO:31; SEQ ID NO:36; or SEQ ID NO:41.

[0036] In certain embodiments of the invention, oligonucleotides are used as probes for the polymorphic regions in the nucleic acid having the sequence set forth in SEQ ID NO:1. Such regions include the regions believed to be the putative promoter, that is, the region 5′ to the transcription start site (nucleotide 26116 of SEQ ID NO:1), intron 3 (nucleotide 27179 to 30519 of SEQ ID NO:1), intron 6 (nucleotide 31842 to 32400 of SEQ ID NO:1), and intron 7 (nucleotide 32529 to 33414 of SEQ ID NO:1), as depicted in FIG. 1. These oligonucleotides may also be termed “sequence determination oligonucleotides” within the scope of the invention, and may be used to determine the presence or absence of a particular nucleotide at a particular polymorphic site within the nucleic acid of SEQ ID NO:1. Specific oligonucleotides of the invention include any oligonucleotide complementary to any of the polymorphic regions described above.

[0037] Those of ordinary skill will recognize that oligonucleotides complementary to the polymorphic regions described herein must be capable of hybridizing to the polymorphic regions under conditions of stringency such as those employed in primer extension-based sequence determination methods, restriction site analysis, nucleic acid amplification methods, ligase-based sequencing methods, methods based on enzymatic detection of mismatches, microarray-based sequence determination methods, and the like. The oligonucleotides of the invention may be synthesized using known methods and machines, such as the ABI™3900 High Throughput DNA Synthesizer and the Expedite™ 8909 Nucleic Acid Synthesizer, both of which are available from Applied Biosystems (Foster City, Calif.).

[0038] The oligonucleotides of the invention may be used, without limitation, as in situ hybridization probes or as components of diagnostic assays. Numerous oligonucleotide-based diagnostic assays are known. For example, primer extension-based nucleic acid sequence detection methods are disclosed in U.S. Pat. Nos. 4,656,127; 4,851,331; 5,679,524; 5,834,189; 5,876,934; 5,908,755; 5,912,118; 5,976,802; 5,981,186; 6,004,744; 6,013,431; 6,017,702; 6,046,005; 6,087,095; 6,210,891; WO 01/20039; and the like. Primer extension-based nucleic acid sequence detection methods using mass spectrometry are described in U.S. Pat. Nos. 5,547,835; 5,605,798; 5,691,141; 5,849,542; 5,869,242; 5,928,906; 6,043,031; 6,194,144, and the like. The oligonucleotides of the invention are also suitable for use in ligase-based sequence determination methods such as those disclosed in U.S. Pat. Nos. 5,679,524 and 5,952,174, WO 01/27326, and the like. The oligonucleotides of the invention may be used as probes in sequence determination methods based on mismatches, such as the methods described in U.S. Pat. Nos. 5,851,770; 5,958,692; 6,110,684; 6,183,958; and the like. In addition, the oligonucleotides of the invention may be used in hybridization-based diagnostic assays such as those described in U.S. Pat. Nos. 5,891,625; 6,013,499; and the like.

[0039] The oligonucleotides of the invention may also be used as components of a diagnostic microarray. Methods of making and using oligonucleotide microarrays suitable for diagnostic use are disclosed in U.S. Pat. Nos. 5,492,806; 5,525,464; 5,589,330; 5,695,940; 5,849,483; 6,018,041; 6,045,996; 6,136,541; 6,142,681; 6,156,501; 6,197,506; 6,223,127; 6,225,625; 6,229,911; 6,239,273; WO 00/52625; WO 01/25485; WO 01/29259; and the like. Preferably, the microarray of the invention comprises at least one oligonucleotide complementary to a polymorphic region of SEQ ID NO:1, wherein the region corresponds to a polymorphic site selected from the group consisting of position 24801 of SEQ ID NO:1, position 24941 of SEQ ID NO:1, and position 32614 of SEQ ID NO:1. More preferably, the microarray of the invention comprises an oligonucleotide complementary to a polymorphic region corresponding to position 24801 of SEQ ID NO:1, an oligonucleotide complementary to a polymorphic region corresponding to position 24941 of SEQ ID NO:1, and an oligonucleotide complementary to a polymorphic region corresponding to position 32614 of SEQ ID NO:1. Most preferably, the microarray of the invention comprises an oligonucleotide complementary to a polymorphic region corresponding to position 24801 of SEQ ID NO:1, an oligonucleotide complementary to a polymorphic region corresponding to position 24941 of SEQ ID NO:1, an oligonucleotide complementary to a polymorphic region corresponding to position 27645 of SEQ ID NO:1, an oligonucleotide complementary to a polymorphic region corresponding to position 32163 of SEQ ID NO:1, and an oligonucleotide complementary to a polymorphic region corresponding to position 32614 of SEQ ID NO:1. In a specific embodiment, the oligonucleotides of the microarray of the invention are complementary to any or all of the polymorphic regions selected from the group consisting of SEQ ID NO:2; SEQ ID NO:7; SEQ ID NO:12; SEQ ID NO:13; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:32; and SEQ ID NO:37 (corresponding to the polymorphic site at position 24801 of SEQ ID NO:1); the polymorphic regions selected from the group consisting of SEQ ID NO:3; SEQ ID NO:8; SEQ ID NO:14; SEQ ID NO:15; SEQ ID NO:24; SEQ ID NO:25; SEQ ID NO:33; and SEQ ID NO:38 (corresponding to the polymorphic site at position 24941 of SEQ ID NO:1); the polymorphic regions selected from the group consisting of SEQ ID NO:4; SEQ ID NO:9; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:34; and SEQ ID NO:39 (corresponding to the polymorphic site at position 27645 of SEQ ID NO:1); the polymorphic regions selected from the group consisting of SEQ ID NO:5; SEQ ID NO:10; SEQ ID NO:18; SEQ ID NO:19; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:35; and SEQ ID NO:40 (corresponding to the polymorphic site at position 32163 of SEQ ID NO:1); and the polymorphic regions selected from the group consisting of SEQ ID NO:6; SEQ ID NO:11; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:30, SEQ ID NO:31; SEQ ID NO:36; and SEQ ID NO:41 (corresponding to the polymorphic site at position 32614 of SEQ ID NO:1).

[0040] The invention is also embodied in oligonucleotide primer pairs suitable for use in the polymerase chain reaction (PCR) or in other nucleic acid amplification methods. Each oligonucleotide primer pair of the invention is complementary to a polymorphic region of the nucleic acid of SEQ ID NO:1. Thus an oligonucleotide primer pair of the invention is complementary to a polymorphic region characteristic of at least one of the polymorphic sites at positions 24801, 24941, 27645, 32163, and 32614 of SEQ ID NO:1. Those of ordinary skill will be able to design suitable oligonucleotide primer pairs using knowledge readily available in the art, in combination with the teachings herein. Specific oligonucleotide primer pairs of this embodiment include the oligonucleotide primer pairs set forth in SEQ ID NO:42 and SEQ ID NO:43, which are suitable for amplifying the polymorphic region corresponding to the polymorphic sites at positions 24801 and 24941 of SEQ ID NO:1; the oligonucleotide primer pairs set forth in SEQ ID NO:44 and SEQ ID NO:45, which are suitable for amplifying the polymorphic region corresponding to the polymorphic site at position 27645 of SEQ ID NO:1; the oligonucleotide primer pairs set forth in SEQ ID NO:46 and SEQ ID NO:47, which are suitable for amplifying the polymorphic region corresponding to the polymorphic site at position 32163 of SEQ ID NO:1; the oligonucleotide primer pairs set forth in SEQ ID NO:48 and SEQ ID NO:49, which are suitable for amplifying the polymorphic region corresponding to the polymorphic site at position 32614 of SEQ ID NO:1. Those of skill will recognize that other oligonucleotide primer pairs suitable for amplifying the polymorphic regions of the nucleic acid of SEQ ID NO:1 can be designed without undue experimentation. In particular, oligonucleotide primer pairs suitable for amplification of larger portions of SEQ ID NO:1 would be preferred for haplotype analysis.

[0041] Each of the PCR primer pairs of the invention may be used in any PCR method. For example, a PCR primer pair of the invention may be used in the methods disclosed in U.S. Pat. Nos. 4,683,195; 4,683,202, 4,965,188; 5,656,493; 5,998,143; 6,140,054; WO 01/27327; WO 01/27329; and the like. The PCR pairs of the invention may also be used in any of the commercially available machines that perform PCR, such as any of the GeneAmpo Systems available from Applied Biosystems.

[0042] The invention is also embodied in a kit comprising at least one oligonucleotide primer pair of the invention. Preferably, the kit of the invention comprises at least three oligonucleotide primer pairs, wherein each primer pair is complementary to a different polymorphic region of the nucleic acid of SEQ ID NO:1. More preferably, the kit of the invention comprises at least four oligonucleotide primer pairs suitable for amplification of polymorphic regions corresponding to positions 24801, 24941, 27645, 32163, and 32614 of SEQ ID NO:1. Most preferably, the kit of the invention comprises at least five oligonucleotide primer pairs suitable for amplification of polymorphic regions corresponding to positions 24801, 24941, 27645, 32163, and 32614 of SEQ ID NO:1. This embodiment may optionally further comprise a sequence determination oligonucleotide for detecting a polymorphic variant at any or all of the polymorphic sites corresponding to positions 24801, 24941, 27645, 32163, and 32614 in SEQ ID NO:1. The kit of the invention may also comprise a polymerizing agent, for example, a thermostable nucleic acid polymerase such as those disclosed in U.S. Pat. Nos. 4,889,818; 6,077,664, and the like. The kit of the invention may also comprise chain elongating nucleotides, such as dATP, dTTP, dGTP, dCTP, and dITP, including analogs of dATP, dTTP, dGTP, dCTP and dfIP, so long as such analogs are substrates for a thermostable nucleic acid polymerase and can be incorporated into a growing nucleic acid chain. The kit of the invention may also include chain terminating nucleotides such as ddATP, ddTTP, ddGTP, ddCTP, and the like. In a preferred embodiment, the kit of the invention comprises at least one oligonucleotide primer pair, a polymerizing agent, chain elongating nucleotides, at least one sequence determination oligonucleotide and at least one chain terminating nucleotide. The kit of the invention may optionally include buffers, vials, microtiter plates, and instructions for use.

[0043] Methods of diagnosing predisposition to left ventricular heart failure in a human are also encompassed by the present invention. In the methods of the invention, the presence or absence of at least one polymorphic variant of the nucleic acid of SEQ ID NO:1 is detected to determine or diagnose such a predisposition. Specifically, in a first step, a nucleic acid is isolated from biological sample obtained from the human. Any nucleic-acid containing biological sample from the human is an appropriate source of nucleic acid for use in the methods of the invention. For example, nucleic acid can be isolated from blood, saliva, sputum, urine, cell scrapings, biopsy tissue, and the like. In a second step, the nucleic acid is assayed for the presence or absence of at least one allelic variant of any or all of the polymorphic regions of the nucleic acid of SEQ ID NO:1 described above. Preferably, the polymorphic regions on both chromosomes in the chromosome pair of the human are assayed in the method of the invention, so that the zygosity of the individual for the particular polymorphic variant may be determined.

[0044] Any method may be used to assay the nucleic acid, that is, to determine the sequence of the polymorphic region, in this step of the invention. For example, any of the primer extension-based methods, ligase-based sequence determination methods, mismatch-based sequence determination methods, or microarray-based sequence determination methods described above may be used, in accordance with the present invention. Alternatively, such methods as restriction fragment length polymorphism (RFLP) detection, single strand conformation polymorphism detection (SSCP), PCR-based assays such as the Taqman® PCR System (Applied Biosystems) may be used.

[0045] In accordance with one method of the invention, predisposition to left ventricular diastolic heart failure is diagnosed by determining the identity of the nucleotide at position 24801 of SEQ ID NO:1. In this method, an A nucleotide at position 24801 of SEQ ID NO:1, or a T nucleotide at the corresponding position of the complement of SEQ ID NO:1, is indicative of greater risk of developing left ventricular diastolic heart failure. Conversely, the presence of a G nucleotide at position 24801 of SEQ ID NO:1, or of a C nucleotide at the corresponding position of the complement of SEQ ID NO:1, is indicative of a lower risk of developing left ventricular diastolic heart failure. In a further step, the zygosity of the individual may be determined, wherein a homozygous A/A genotype at position 24801 of SEQ ID NO:1 or T/T at the corresponding position of the complement of SEQ ID NO:1, indicates greatest risk for developing left ventricular diastolic heart failure. A person whose genotype is homozygous G/G at position 24801 of SEQ ID NO:1 or C/C at the corresponding position of the complement of SEQ ID NO:1 is at least risk for developing left ventricular diastolic heart failure. An individual whose genotype is heterozygous A/G at position 24801 of SEQ ID NO:1 or T/C at the corresponding position of the complement of SEQ ID NO:1 is at intermediate risk for developing left ventricular diastolic heart failure.

[0046] Alternatively, predisposition to left ventricular diastolic heart failure is diagnosed by determining the identity of the nucleotide at position 24941 of SEQ ID NO:1. In this method, a T nucleotide at position 24941, of SEQ ID NO:1, or an A nucleotide at the corresponding position of the complement of SEQ ID NO:1, is indicative of greater risk of developing left ventricular diastolic heart failure. Conversely, the presence of a C nucleotide at position 24941 of SEQ ID NO:1, or of a G nucleotide at the corresponding position of the complement of SEQ ID NO:1, is indicative of a lower risk of developing left ventricular diastolic heart failure. In a further step, the zygosity of the individual may be determined, wherein a homozygous T/T genotype at position 24941 of SEQ ID NO:1 or A/A at the corresponding position of the complement of SEQ ID NO:1, indicates greatest risk for developing left ventricular diastolic heart failure. A person whose genotype is homozygous C/C at position 24941 of SEQ ID NO:1 or GIG at the corresponding position of the complement of SEQ ID NO:1 is at least risk for developing left ventricular diastolic heart failure. An individual whose genotype is heterozygous T/C at position 24941 of SEQ ID NO:1 or A/G at the corresponding position of the complement of SEQ ID NO:1 is at intermediate risk for developing left ventricular diastolic heart failure.

[0047] In another method of the invention, risk of left ventricular heart failure is assessed by determining the haplotype of the individual for various polymorphic positions within SEQ ID NO:1. For example, individuals who possess the SEQ ID NO:1 haplotype characterized by a G nucleotide at position 24801 of SEQ ID NO:1; a C nucleotide at position 24941 of SEQ ID NO:1; a C nucleotide at position 27645 of SEQ ID NO:1; a C nucleotide at position 32163 of SEQ ID NO:1; and a G nucleotide at position 32614 of SEQ ID NO:1 are at lower risk of development of left ventricular diastolic heart failure. This haplotype may alternatively be detected on the antisense strand, or complement of SEQ ID NO:1 as a C nucleotide at the position corresponding to position 24801 of the complement of SEQ ID NO:1; a G nucleotide at the position corresponding to position 24941 of the complement of SEQ ID NO:1; a G nucleotide at the position corresponding to position 27645 of the complement of SEQ ID NO:1; a G nucleotide at the position corresponding to position 32163 of the complement of SEQ ID NO:1; and a C nucleotide at the position corresponding to position 32614 of the complement of SEQ ID NO:1. Individuals who are homozygous for allelic variants comprising this haplotype are at particularly low risk of developing left ventricular diastolic heart failure.

[0048] In yet another method of the invention, risk of left ventricular systolic heart failure in individuals having suffered a myocardial infarction is assessed by determining the identity of the nucleotide at position 32614 of SEQ ID NO:1. An A nucleotide at position 32614 of SEQ ID NO:1 or a T nucleotide at the corresponding position of the complement of SEQ ID NO:1, is indicative of greater risk of developing left ventricular systolic heart failure. Conversely, the presence of a G at position 32614 of SEQ ID NO:1, or a C at the corresponding position of the complement of SEQ ID NO:1, is indicative of a lower risk of developing left ventricular systolic heart failure. In a further step, the zygosity of the individual may be determined, wherein a homozygous A/A genotype at position 32614 of SEQ ID NO:1 or T/T at the corresponding position of the complement of SEQ ID NO:1, indicates greatest risk for developing left ventricular systolic heart failure. A person whose genotype is homozygous G/G at position 32614 of SEQ ID NO:1 or C/C at the corresponding position of the complement of SEQ ID NO:1 is at least risk for developing left ventricular systolic heart failure. An individual whose genotype is heterozygous A/G at position 32614 of SEQ ID NO:1 or T/C at the corresponding position of the complement of SEQ ID NO:1 is at intermediate risk for developing left ventricular systolic heart failure.

[0049] The examples set forth below are provided as illustration and are not intended to limit the scope and spirit of the invention as specifically embodied therein.

[0050] During the Uppsala Longitudinal Study of Adult Men (ULSAM) re-examination 1990-94 (n=1221) (www.pubcare.uu.se/ULSAM), a sub-study including a comprehensive echocardiographic assessment of cardiac morphology, systolic and diastolic function in 583 individuals was performed (Andren B, Lind L, Hedenstiema G, Lithell H. (1998) Echocardiography 5:433-450, “Left ventricular diastolic function in a population sample of elderly men”, hereinafter Andren et al.). In 475 subjects the technical quality of the assessment was sufficient to calculate all indicators of cardiac function and morphology. The ECGs of this subset were analysed thoroughly and systematically, and biochemical markers of subclinical heart failure were also analyzed. Of these 475, DNA samples were available from 395 subjects. The results set forth in the Examples are based on 330 of these individual, from whom informed consent for genetic analysis was obtained.

[0051] Andren et al. performed a comprehensive echocardiographic examination using a Hewlett-Packard Sonos 1500 cardiac ultrasound unit with a 2.5 MHz transducer. Left ventricular dimensions (interventricular septum thickness, posterior wall thickness, end diastolic diameter) were measured in 2D-guided M-mode using a leading edge to leading edge convention. Left ventricular volumes including ejection fraction (EF) were calculated with the Teichholz formula. Left ventricular mass was determined from M-mode measurements according to the American Society of Echocardiography. Measurements of left ventricular filling were used to estimate diastolic function. Peak velocities of the early (E) and a trial (A) contributions to LV filling were recorded, and the E/A ratio was calculated.

[0052] Office blood pressure was measured in the right arm with the subject in the supine position after resting for 10 minutes, and then after the subject had been standing for 2 minutes. The values were recorded twice and to the nearest even figure. The means of the two values are given for each blood pressure. The cuff size was 12.35 cm or 15.45 cm depending on the arm circumference. Systolic (SBP) and diastolic (DBP) blood pressures were defined as Korotkoff phases I and V, respectively.

[0053] The ambulatory blood pressure measuring device Accutracker II (Suntech Medical Instruments, Raleigh, N.C.) was attached to the subjects' non-dominant arm by a skilled laboratory technician. Systolic and diastolic blood pressures were measured every 30 minutes during daytime (0600-2300) and every hour during the night over a 24 hour period. After November 1993, the blood pressure was measured every 20 minutes during the whole 24-hour period.

[0054] Data were edited to a limited extent. All readings of zero, all heart rate readings <30, diastolic blood pressure readings >170 mm Hg, systolic blood pressure readings >270 and <80 mmHg, and all readings where the difference between systolic and diastolic blood pressure was less than 10 mm Hg were omitted. Mean arterial blood pressure (MAP) was calculated over the whole measurement period according to the formula MAP=2/3*DBP+1/3*SBP.

[0055] A standard 12-lead ECG was recorded at 50 mm/s and 10 mm/mV and evaluated according to the Minnesota code by one experienced physician who was unaware of the other data collected from the subjects.

[0056] A power calculation was performed in which the objective was to estimate the power to detect a SNP/endpoint association using DNA samples and clinical information from the study population of Example 1. The power was estimated as a function of realistic combinations of percent phenotype variation, explained by a quantitative trait locus (QTL), that is, a hidden causative SNP, and the degree of linkage disequilibrium (LD) between an assumed number of five SNPs and the QTL. Assumed genetic effects of 10-25% were considered, and a range of LD's between the selected genetic positions was used in the power calculation. The result indicated that, in the case of high LD, an effect of 13% phenotypic variation could be detected with 80% power. In the case of lower LD, the effect would have to be 25% or higher for the study to achieve 80% power. The use of haplotype information was not considered in the power calculation. Primary and secondary end point variables (phenotypes) were selected based on clinical relevance, coverage, and inter-correlations. The ejection fraction (representing systolic function), E/A ratio (diastolic function), left ventricular mass index and relative wall thickness (left ventricular geometry) and Qt-max (arrhythmia) where chosen as primary endpoints. Table 1 shows that these variables have high coverage in the study population, and Table 2, depicting the Pearson correlation coefficients between primary endpoints, shows that their inter-correlations are low, even if the within-subject variability is taken into account. The E/A ratio was adjusted for heart rate at the time of measurement in all analyses. Also, the assumed effects of a QTL for the assumptions of 10 and 25 percent explained variance are shown, converted to a mean difference between two genotypes for each endpoint.

[0057] The following variables were selected as secondary end-points: N-atrial natriuretic peptide (ANP) and AV-plane displacement (representing systolic function), isovolumetric relaxation time (IVRT) (diastolic function), Cornell and Sokolow-Lyon ECG criteria for left ventricular hypertrophy (left ventricular geometry) and QT-dispersion (arrhythmia). IVRT values were adjusted for heart rate as with E/A ratio.

[0058] To identify polymorphic positions in the nucleic acid characterized by SEQ ID NO:1, twenty-two (22) fragments covering selected regions of the gene were amplified (GeneAmp™ PCR Systems 9700, Applied Biosystems) using genomic DNA from 15-30 unrelated anonymous individuals. The 3′-end of the gene was avoided, since that region is extremely homologous to other sequences in the human genome and there is risk of unspecific amplification. After amplification, detection of genetic variation in the nucleic acid of SEQ ID NO:1 was performed using solid phase sequencing (AutoLoad™ Solid-Phase Sequencing kit, Amersham Pharmacia Biotech) and gel electrophoresis on ALFexpress™ sequencers (Amersham Pharmacia Biotech).

[0059] Six SNP's were detected with a minor allele frequency of ≧10% in the investigated individuals and five of these were included in the main study, see Table 3. Fragments covering each of the five SNP's were amplified from genomic DNA from the 330 individuals. SNP detection was subsequently performed using the PSQ 96 platform (Pyrosequencing AB, Uppsala, Sweden).

[0060] Genotyping was successful and only a few samples for each SNP failed, as shown in Table 4. Three samples failed to give result in all five polymorphic positions. In order to detect any genotyping anomalies, Hardy-Weinberg, haplotype analysis and duplicate control genotyping were performed. No deviation in genotyping result could be seen in the 100 duplicates checked. Hardy-Weinberg calculations were performed and the frequency of the three different genotypes for each SNP did not differ significantly from expected values.

[0061] Haplotype analysis could be performed on a total of 310 individuals. This analysis was performed using software based on maximum likelihood methodology and using the EM algorithm of Excoffier et al. (1995), Mol Biol Evol. 12:921-927. In total 8 likely haplotype were identified by the program. Two of these occurred only once in the study population and may not be true haplotypes but the result of incorrect genotyping in one of the polymorphic positions in the two individuals. The characterization and frequency of each haplotype is presented in Table 5. From the haplotype information two different kinds of variables were created: one variable was formed as a haplotype combination variable (HTYPE). This variable has the value H1/H2 when the subject has haplotypes 1 ans 2, etc. Variables H1, H2, H3 and H4 are haplotype annotations that denote the number of copies of that particular haplotype for the subject, e.g., for a subject with haplotype H1/H2 the variables H1, H2, H3 and H4 will be 1, 1, 0 and 0, respectively. Each of these variables can thus take on the values 0, 1 or 2. Only the four most frequent haplotypes were considered when those variables were formed. In all, ten genotype variables were considered (five SNP variables and five haplotype variables).

[0062] A discrepancy was noted between the protein encoded by SEQ ID NO:1, which was derived from GenBank Accession No. AC004923, and the protein encoded by the mRNA of Kashuba et al. (GenBank Accession No. AJ271326, SEQ ID NO:50). Specifically, in exon 7 SEQ ID NO:50 contains an insertion of a cytosine nucleotide at position 928, which is not present in the corresponding position in exon 7 of SEQ ID NO:1 (between positions 32505 and 32506). The mRNA of SEQ ID NO:50 is believed to be the correct sequence.

[0063] Table 6 sets forth the results of the genetic analysis of the five SNPs and the five haplotype variables for the primary endpoints selected as phenotypes. It can readily be seen that the E/A ratio indicator of left ventricular diastolic function is strongly associated with the polymorphic variant at position 24801 of SEQ ID NO:1; the polymorphic variant at position 24941 of SEQ ID NO:1, and haplotype H3.

[0064] Table 7 sets forth the results of the genetic analysis of the five SNPs and the five haplotype variables for the secondary endpoint phenotypes. It can readily be seen that Cornell ECG indicator of left ventricular hypertrophy is strongly associated with the polymorphic variant at position 24801 of SEQ ID NO:1 and with haplotype H3. Table 7 also indicates an apparent association between the QT dispersion indicator of cardiac arrhythmia and haplotype H4.

[0065] Table 8 indicates that for the E/A ratio a significant result (using the permutation test disclosed in Churchill, G. A. and Doerge, R. W. (1994) Genetics138, 963-971) was achieved for the H3 haplotype variable (p=0.0002):

[0066] A group of subjects homozygous for H3 has thus approximately 34% increased level of E/A ratio compared to a group of non-carriers for this haplotype.

[0067] Table 9 indicates that a significant association was observed between ECG criteria according to Cornell and the polymorphic variant present at position 24801 of SEQ ID NO:1 (p=0.001):

[0068] Systolic function was also evaluated among different SEQ ID NO:1 genotypes in the 53 subjects who had suffered a myocardial infarction prior to the echocardiographic examination. The mean values of ejection fraction were significantly different for polymorphic variants at position 32614 of SEQ ID NO:1: A/A 0.56, A/G 0.62, and G/G 0.68 (p=0.0365). When “low ejection fraction” was defined as an EF value below 50%, the prevalence was 37% for individuals with the A/A genotype compared with 20% for A/G and 11% for GIG individuals (p=ns). These data therefore are consistent with a protective effect on post-myocardial infarction left ventricular performance by the G allele at position 32614 of SEQ ID NO:1.

[0069] While the invention has been described in terms of the specific embodiments set forth above, those of skill will recognize that the essential features of the invention may be varied without undue experimentation and that such variations are within the scope of the appended claims.