WO2013032883A2

WO2013032883A2 - Methods of detecting and treating cardiovascular diseases

Info

Publication number: WO2013032883A2
Application number: PCT/US2012/052199
Authority: WO
Inventors: Alexandre Francois Roy STEWART; Naif Ahmad Mahmood ALMONTASHIRI; Robert Roberts
Original assignee: Ottawa Heart Institute Research Corporation
Priority date: 2011-08-26
Filing date: 2012-08-24
Publication date: 2013-03-07
Also published as: WO2013032883A3

Abstract

The invention relates to the use of interferon a-21 for detecting the presence of or increased risk of coronary artery disease in a subject positive for a 9p21 risk allele. Diagnostic methods and kits are provided herein for detecting and reducing the risk of cardiovascular disease, including coronary artery disease, in a subject having the 9p21 risk allele by measuring interferon a-21 in the subject. The invention further relates to therapeutic methods which use interfeuron a-21 level to diagnose and treat cardiovascular diseases.

Description

METHODS OF DETECTING AND TREATING CARDIOVASCULAR DISEASES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Ser. No. 61/527,693 filed 26 August 2011, the disclosure of which is hereby incorporated by reference in its entirety as if fully set forth herein.

FIELD

[0002] The present invention is in the field of cardiovascular diseases. Particularly, the present invention relates to methods and kits for detecting, reducing the risk of, and treating cardiovascular disease, including coronary artery disease, myocardial infarction, abdominal aorta aneurysm, intracranial aneurysm restenosis and peripheral arterial disease in subjects having a 9p21 risk allele and a certain level of interferon a-21.

BACKGROUND

[0003] Coronary artery disease (CAD), also called coronary heart disease or coronary atherosclerosis, is the most common type of heart disease, affecting more than 85% of the population over the age of 50, and is a leading cause of death. CAD occurs when the arteries that supply blood to heart muscle become hardened and narrowed. This is due to the buildup of cholesterol and plaque on their inner walls. This buildup is called atherosclerosis. As plaque grows, less blood can flow through the arteries. As a result, the heart muscle cannot get the blood or oxygen it requires. This can lead to chest pain (angina) or a heart attack. Over time, CAD can also weaken the heart muscle and contribute to heart failure and arrhythmias.

[0004] Two current methods to detect CAD are coronary angiography by cardiac catherization (i.e. coronary catheterization) and 64-slice computed tomography coronary angiography (CTCA). A coronary catheterization is the conventional method to detect CAD and it is an invasive procedure to access the coronary circulation and blood filled chambers of the heart using a catheter. It is performed for both diagnostic and interventional (treatment) purposes. However, it has been increasingly recognized, since the late 1980s, that coronary catheterization does not allow the recognition of the presence or absence of coronary atherosclerosis itself, only significant luminal changes which have occurred as a result of end stage complications of the atherosclerotic process. Further, a large study has shown that cardiac catheterization has a low diagnostic yield. For example, about 39% of angiograms in high risk individuals failed to detect significant stenosis. Given that an angiogram costs about $5,000, this means there were about $500 million in unnecessary tests in the participating medical centers.

[0005] Computed tomography coronary angiography is another method to detect CAD. In this procedure an intravenous dye which contains iodine and a 64-slice CT scan is used to image the coronary arteries. While the use of catheters is not necessary (thus the term "noninvasive" test applies to this procedure), there are still some risks involved. For example, there is radiation exposure which is similar to, if not greater than, that received with a conventional coronary angiogram. A study has even suggested that 64- slice CTCA is associated with non-negligible lifetime attributable risk of cancer. Thus, new methods are required to detect and treat cardiovascular diseases, such as CAD, which are minimally invasive, effective and provide low risk to patients.

SUMMARY

[0006] Use of interferon a-21 for detecting the presence of or increased risk of cardiovascular disease in a subject positive for a 9p21 risk allele is provided herein. In a particular aspect, when the subject is homozygous for the 9p21 risk allele and the interferon a-21 level is about 30, about 40, or about 50 pg/ml or higher, than this outcome is indicative of the subject having or is at increased risk of having cardiovascular disease; or when the subject is heterozygous for the 9p21 risk allele and the interferon a-21 level is about 15, about 20 or about 25 pg/ml or higher, than this outcome is indicative of the subject having or is at increased risk of having cardiovascular disease. In a particular aspect, the cardiovascular disease is CAD.

[0007] Further provided herein is a diagnostic method for detecting the presence of or increased risk of cardiovascular disease in a subject, the method comprising performing a protein expression level test on a biological sample from the subject, wherein the subject is positive for a 9p21 risk allele; determining interferon a-21 level from the protein expression level test; wherein when the subject is homozygous for the 9p21 risk allele and the interferon a-21 level is about 30, about 40, or about 50 pg/ml or higher, than this outcome is indicative of the subject having or is at increased risk of having coronary artery disease; wherein when the subject is heterozygous for the 9p21 risk allele and the interferon a-21 level is about 15, about 20 or about 25 pg/ml or higher, than this outcome is indicative of the subject having or is at increased risk of having coronary artery disease; and communicating the interferon a-21 level to a medical practitioner.

[0008] A a method of treating or reducing the risk of cardiovascular disease in a subject in need thereof is also provided herein. The method comprises diagnosing the subject as having or at risk of having coronary artery disease when the subject is homozygous for 9p21 risk allele and the subject has an interferon a-21 level of about 30, about 40, or about 50 pg/ml or higher; or when the subject is heterozygous for 9p21 risk allele and the subject has an interferon a-21 level of about 15, about 20, or about 25 pg/ml or higher; administering treatment for cardiovascular disease to the subject.

[0009] Further a diagnostic method for detecting the presence of cardiovascular disease in a subject is provided herein. The method comprises determining interferon a- 21 level in the subject, wherein the subject is positive for a 9p21 risk allele; diagnosing the subject as having coronary artery disease when the subject is homozygous for the 9p21 risk allele and the interferon a-21 level is about 30, about 40, or about 50 pg/ml or higher, or when the subject is heterozygous for the 9p21 risk allele and the interferon a- 21 level is about 15, about 20, or about 25 pg/ml or higher.

[0010] A kit for detecting the presence of or increased risk of cardiovascular disease is also provided herein. The kit comprises a reagents for a protein expression level assay and instructions for detecting and determining the level of interferon a-21 in a biological sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0012] Figure 1 displays interferon a-21 and CDKN2A/2B protein expression levels in primary cultures of human aortic smooth muscle cells are associated with 9p21 CAD risk locus. A: Positive controls for immunoblot analysis. Human aortic smooth muscle cells (SMCs) were homozygous non-risk (GG). B: interferon a-21 was positively correlated and CDKN2A, CDKN2B and IFNW1 were negatively correlated with 9p21 risk allele (CC). An antibody that recognizes all interferon-α isoforms showed no overall difference of interferon-α expression with 9p21 risk allele. C: Quantification of the protein levels by densitometry, normalized to GAPDH. N= 6 independent primary cultures (2 homozygous non-risk, 2 heterozygous, 2 homozygous risk). See Example 1.

[0013] Figure 2 displays interferon a-21 and CDKN2A protein expression levels in peripheral blood lymphocytes of patients with CAD according to carrier status for 9p21 risk alleles. A:, Protein extracts from 6 individuals homozygous for the non-risk (GG) genotype, 6 heterozygous (GC) and 6 homozygous for the risk genotype (CC) were analyzed by immunoblot. B: Quantification revealed interferon a-21 was positively correlated and CDKN2A negatively correlated with the 9p21 risk allele. See Example 1.

[0014] Figure 3 is a graphical representation demonstrating that serum interferon a- 21 levels (pg/ml) are influenced by the 9p21 risk allele in angiographically-defined cases and controls for CAD. Of 331 genotyped CAD cases, 124 were homozygous for the risk allele (CC), 109 were heterozygous and 98 were homozygous for the non-risk allele (GG). Of the 231 genotyped controls, 76 were homozygous risk, 78 heterozygous and 77 homozygous non-risk. P-values obtained by linear regression using log₁₀-transformed values without adjustment for covariates. See Example 2.

[0015] Figure 4 is a graphical representation demonstrating that 9p21 risk allele and CAD interact to elevate plasma interferon a-21 levels. Plasma interferon a-21 is expressed as a box plot of mean and median + 25th and 75th percentile. See Example 3.

[0016] Figure 5 is a graphical representation demonstrating that the 9p21 risk genotype specifically elevates interferon a-21 expression. See Example 3.

[0017] Figure 6 is a graphical representation demonstrating plasma interferon a-21 (pg/ml) versus time. This demonstrates that plasma interferon a-21 levels do not change significantly overtime and thus are most likely not elevated by acute infection. See Example 3. DETAILED DESCRIPTION

[0018] The 9p21 risk allele is a genetic variant on chromosome 9 at 9p21.3 and is associated with the risk of CAD. A number of studies have identified SNPs at this locus which are associated with the risk of CAD. For example, see McPherson R., et al. Science 316: 1488-1491 (2007) which is hereby incorporated by reference in its entirety.

[0019] In fact, the 9p21 locus is tagged by several linked (co-inherited) SNPs and covers a 53,000 bp region that overlaps the 3' region of a long non-coding RNA (LncRNA) of unknown function called CDKN2BAS or ANRIL. The 9p21 risk allele is carried by 75% of the European population and confers risk for CAD independently of known risk factors. The 9p21 locus also contributes to the risk of intracranial and abdominal aortic aneurysms, ischemic stroke, peripheral arterial disease and late onset vascular dementia.

[0020] Interferon a-21 is a well characterized cytokine belonging to the type 1 interferon family, which binds to the interferon-a receptor (IFNAR) that consists of IFNAR 1 and IFNAR2 chains. The present inventors have found that an increased level of interferon a-21 reflects expression of the 9p21 risk allele and development of CAD. Thus, use of interferon a-21 for detecting cardiovascular diseases or the increased risk of cardiovascular diseases in individuals with a 9p21 risk genotype is provided herein. For example, use of interferon a-21 for detecting the presence of or increased risk of cardiovascular diseases such as coronary atherosclerosis, CAD, myocardial infarction, abdominal aortic aneurysm, intracranial aneurysm, and peripheral arterial disease are provided herein.

[0021] Additionally or alternatively, new methods are provided herein for detecting and reducing the risk of cardiovascular diseases, such as CAD, in patients having the 9p21 risk allele. Further, treatments are provided herein which take advantage of measuring interferon a-21 levels in patients having the 9p21 risk allele in order to diagnose and treat patients. The present methods have many advantages over the current options for detecting, diagnosing and treating cardiovascular diseases, such as CAD. For example, the methods disclosed herein are minimally invasive methods as only a blood sample needs to be drawn from the subject. Additionally, the methods disclosed herein do not present risks associated with radiation and catheterization.

[0022] Furthermore, a diagnostic method is provided herein for detecting the presence or increased risk of cardiovascular disease in a subject. The method comprises performing a protein expression level test on a biological sample from the subject, wherein the subject is positive for a 9p21 risk allele; and determining interferon a-21 level from the protein expression level test.

[0023] The term "cardiovascular disease" as used herein, refers to the class of diseases that involve the heart or blood vessels (arteries and veins). Non-limiting examples of cardiovascular diseases include, for example, coronary atherosclerosis, CAD, myocardial infarction, abdominal aortic aneurysm, intracranial aneurysm, and peripheral arterial disease.

[0024] In a particular aspect, the methods are used to detect CAD or the increased risk of CAD. As used herein, the term CAD is intended to embrace coronary atherosclerosis which causes CAD. It is not intended to embrace cardiac events, such as blood clots.

[0025] As noted above, a protein expression level test is performed on a biological sample from a subject having a 9p21 risk allele to determine an amount of interferon a-21 in the biological sample. A variety of known methods/tests can be used for detecting (i.e. measuring or determining) protein expression levels in a biological sample. One example is an immunoassay. Non-limiting examples of immunoassays include an immunosorbant assay, a Western blot, an immunoprecipitation, and an immunofluorescent assay. In a particular embodiment, an immunosorbant assay is used, such as an ELISA, to measure interferon a-21 level. Such analysis methods can be obtained as commercially available kits. Additionally or alternatively, a DNA aptamer assay or protein-mass spectrometry may be used to measure interferon a-21 level.

[0026] The biological sample from the subject may be whole blood, serum, or plasma. [0027] Additionally, the diagnostic methods can further comprise communicating the interferon a-21 level determined from the protein expression level test on a biological sample to a medical practitioner. This communication can be done to facilitate diagnosis of cardiovascular disease, treatment and/or whether further diagnostic procedures should be undertaken. Thus, the diagnostic method may further comprise facilitating treatment options for the subject and/or a further diagnostic procedure for the subject, such as, but not limited to, coronary angiography by cardiac catherization (i.e. coronary catheterization) and/or computed tomography coronary angiography (CTCA), such as 64- slice or 128-slice CTCA.

[0028] Additionally, any of the methods described herein can further comprise determining if the subject is positive for a 9p21 risk allele. As noted above, a 9p21 risk allele is a genetic marker that has been associated with the development of CAD. This marker allele can be detected in a test sample, such as genomic DNA, from a subject by known methods such as hybridization methods, including, but not limited to, Southern analysis, Northern analysis, and/or in situ hybridizations. The presence of a specific 9p21 allele can be indicated by sequence-specific hybridization of a nucleic acid probe specific for the 9p21 allele. A sequence- specific probe can be directed to hybridize to genomic DNA, RNA or cDNA. One of skill in the art would know how to design such a probe so that sequence specific hybridization will occur only if a particular allele is present in a genomic sequence from a test sample. Other representative methods of nucleic acid analysis include, for example, direct manual sequencing, automated fluorescent sequencing, single-stranded conformation polymorphism assays, claimed denaturing gel electrophoresis, denaturing gradient gel electrophoresis, mobility shift analysis, restriction enzyme analysis, and allele- specific PCR.

[0029] In the methods of the present invention, the subject is positive for a 9p21 risk allele. The subject may be determined to be homozygous or heterozygous for the 9p21 risk allele.

[0030] The interferon a-21 level or amount in a subject having a 9p21 risk allele can be determined to be above a certain threshold compared to a control, such as a subject homozygous for non-risk allele. For example, if the interferon a-21 level is elevated in a subject having a 9p21 risk allele compared to a control sample, than this is indicative of cardiovascular disease, such as CAD, or to the subject being at a greater risk of developing cardiovascular disease. This information can then be used to diagnose and/or treat the subject.

[0031] If the subject is homozygous for the 9p21 risk allele and the interferon a-21 level is about 30, or about 40, or about 50 pg/ml or higher, than this outcome is indicative of the subject having or is at increased risk of developing cardiovascular disease, such as CAD. Alternatively, if the subject is heterozygous for the 9p21 risk allele and the interferon a-21 level is about 15, about 20 or about 25 pg/ml or higher, than this outcome is indicative of the subject having or is at increased risk of developing cardiovascular disease, such as CAD.

[0032] In a particular aspect, if the subject is homozygous for the 9p21 risk allele and the interferon a-21 level is about 30 pg/ml or higher, than this outcome is indicative of the subject having or is at increased risk of developing cardiovascular disease, such as CAD. Alternatively, if the subject is heterozygous for the 9p21 risk allele and the interferon a-21 level is about 15 pg/ml or higher, than this outcome is indicative of the subject having or is at increased risk of developing cardiovascular disease, such as CAD.

[0033] In another particular aspect, if the subject is homozygous for the 9p21 risk allele and the interferon a-21 level is about 40 pg/ml or higher, than this outcome is indicative of the subject having or is at increased risk of developing cardiovascular disease, such as CAD. Alternatively, if the subject is heterozygous for the 9p21 risk allele and the interferon a-21 level is about 20 pg/ml or higher, than this outcome is indicative of the subject having or is at increased risk of developing cardiovascular disease, such as CAD.

[0034] In a further particular aspect, if the subject is homozygous for the 9p21 risk allele and the interferon a-21 level is about 50 pg/ml or higher, than this outcome is indicative of the subject having or is at increased risk of developing cardiovascular disease, such as CAD. Alternatively, if the subject is heterozygous for the 9p21 risk allele and the interferon a-21 level is about 25 pg/ml or higher, than this outcome is indicative of the subject having or is at increased risk of developing cardiovascular disease, such as CAD.

[0035] Additionally, a method of treating or reducing the risk of cardiovascular disease, such as CAD, in a subject is provided. The method comprises diagnosing the subject as having or at risk of having cardiovascular disease, such as CAD, if the subject is homozygous for 9p21 risk allele and the subject has an interferon a-21 level of about 30, about 40 or about 50 pg/ml or higher; or if the subject is heterozygous for 9p21 risk allele and the subject has an interferon a-21 level of about 15, about 20, or about 25 pg/ml or higher; and administering treatment for cardiovascular disease, such as CAD, to the subject.

[0036] Non-limiting examples of agents for treating cardiovascular disease, such as CAD, include cholesterol-modifying drugs, anti-coagulants, vasodilators, anti- hypertension drugs and anti-diabetic drugs.

[0037] Non-limiting examples of cholesterol-modifying drugs (hypolipidemic agents or anti-hyperlipidemic agents) include for example, statins (such as HMG-CoA reductase inhibitors), fibrates (amphipathic carboxylic acids that lower cholesterol), niacin, bile acid sequestrants, and anti-diabetics.

[0038] Non-limiting examples of anti-coagulants (blood thinners) include aspirin, heparin and coumarins (e.g. warfarin).

[0039] Non-limiting examples of anti-hypertension drugs include alpha-adrenoceptor antagonists (alpha-blockers), angiotensin converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), beta₂-adrenoceptor agonists (p₂-agonists), calcium-channel blockers (CCBs), centrally acting sympatholytics, vasodilators (such as nitroglycerin), endothelin receptor antagonists, ganglionic blockers, phosphodiesterase inhibitors, Potassium-channel openers, and renin inhibitors.

[0040] Non-limiting examples of anti-diabetic drugs include biguanides, such as metformin, thiazolidinediones and insulin.

[0041] Additionally, the methods can further comprise diagnosing the subject as having or at risk of having cardiovascular disease, such as CAD, with additional diagnostic procedures such as coronary angiography or computed tomography coronary angiography, in addition to interferon a-21 level in the subject.

[0042] Additionally or alternatively, a diagnostic method for detecting the presence of cardiovascular disease, such as CAD, in a subject is provided comprising determining interferon a-21 level in the subject, wherein the subject is positive for a 9p21 risk allele; and diagnosing the subject as having cardiovascular disease, such as CAD, when the subject is homozygous for the 9p21 risk allele and the interferon a-21 level is about 30, about 40, or about 50 pg/ml or higher, or when the subject is heterozygous for the 9p21 risk allele and the interferon a-21 level is about 15, about 20, or about 25 pg/ml or higher.

[0043] Additionally, the method can further comprise administering treatment for cardiovascular disease, such as CAD, to the subject with, for example, any of the agents mentioned herein.

[0044] Additionally, the method can further comprise diagnosing the subject as having CAD by coronary angiography or computed tomography coronary angiography.

[0045] Use of interferon a-21 for prophylactic treatment of cardiovascular diseases in individuals with a 9p21 risk genotype is also provided herein.

[0046] In certain aspects, prophylactic treatment can be performed using the methods of the invention. For example, interferon a-21 level in the subject can be used to determine the risk of the subject to develop cardiovascular disease, such as CAD. Prophylactic treatment includes reducing the risk or likelihood that a subject will develop cardiovascular disease or reducing the susceptibility of a subject from developing cardiovascular disease.

[0047] In a particular aspect, if the subject is homozygous for 9p21 risk allele and the subject has an interferon a-21 level of about 30, about 40, or about 50 pg/ml or higher; or if the subject is heterozygous for 9p21 risk allele and the subject has an interferon a-21 level of about 15, about 20, or about 25 pg/ml or higher, then prophylactic treatment is determined and given or administered to the subject. Cardiovascular disease-treating agents, such as those examples provided herein, can be administered to a subject for such prophylactic treatment. Additionally or alternatively, life style changes can be implemented, such as diet and exercise, as prophylactic treatment to reduce the risk of developing cardiovascular disease, such as CAD.

[0048] Kits useful in the methods of the invention are provided herein which comprise components useful in any of the methods described herein. For example, the kits can provide reagents for assays to be used in combination with the methods of the present invention. In one aspect, the invention pertains to a kit for assaying a biological sample from a subject to detect cardiovascular disease or an increased risk of cardiovascular disease, wherein the kit comprises reagents for a protein expression level assay, such as an immunoassay, to detect the level of interferon a-21 in a biological sample; and instructions for detecting and determining the level of interferon a-21 in the biological sample. The immunoassay can be any immunoassay as mentioned herein, for example an immunosorbant assay. Further, the cardiovascular disease can be coronary atherosclerosis, CAD, myocardial infarction, abdominal aortic aneurysm, intracranial aneurysm, and peripheral arterial disease. In a particular aspect, the cardiovascular disease is CAD.

[0049] Additionally, the kit may comprise reagents, including probes as described herein, for nucleic acid analysis on a biological sample to detect a 9p21 risk allele. For example, a kit is contemplated for use in detecting or reducing the risk of cardiovascular diseases, such as CAD, which contain reagents for both a protein expression level assay, such as an immunosorbant assay, to detect the level of interferon a-21 in a biological sample, and reagents for a nucleic acid assay to detect the presence of a 9p21 risk allele in a biological sample.

Further Embodiments

[0050] Embodiment 1. Use of interferon a-21 for detecting the presence of or increase risk of cardiovascular disease in a subject positive for a 9p21 risk allele.

[0051] Embodiment 2. The use according to embodiment 1 wherein the cardiovascular disease is selected from the groups consisting of coronary atherosclerosis, CAD, myocardial infarction, abdominal aortic aneurysm, intracranial aneurysm, and peripheral arterial disease. [0052] Embodiment 3. A diagnostic method for detecting the presence of or increased risk of cardiovascular disease in a subject, the method comprising performing a protein expression level test on a biological sample from the subject, wherein the subject is positive for a 9p21 risk allele; determining interferon a-21 level from the protein expression level test; wherein if the subject is homozygous for the 9p21 risk allele and the interferon a-21 level is about 30, about 40, or about 50 pg/ml or higher, than this outcome is indicative of the subject having or is at increased risk of having cardiovascular disease; wherein if the subject is heterozygous for the 9p21 risk allele and the interferon a-21 level is about 15, about 20, or about 25 pg/ml or higher, than this outcome is indicative of the subject having or is at increased risk of having cardiovascular disease.

[0053] Embodiment 4. The method according to embodiment 3, further comprising communicating the interferon a-21 level to a medical practitioner.

[0054] Embodiment 5. The method according to embodiment 3 or 4, further comprising determining if the subject is positive for the 9p21 risk allele.

[0055] Embodiment 6. The method according to embodiment 3, 4 or 5, wherein the protein expression level assay test is an immunoassay or a DNA aptamer assay or protein-mass spectrometry.

[0056] Embodiment 7. The method according to embodiment 6, wherein the immunoassay is selected from the group consisting of an immunosorbant assay, a Western blot, an immunoprecipitation, and an immunofluorescent assay.

[0057] Embodiment 8. The method according to embodiment 3, 4, 5, 6 or 7 wherein the biological sample is whole blood, serum or plasma.

[0058] Embodiment 9. The method according to embodiment 3, 4, 5, 6, 7 or 8 wherein the cardiovascular disease is selected from the group consisting of coronary atherosclerosis, CAD, myocardial infarction, abdominal aortic aneurysm, intracranial aneurysm, and peripheral arterial disease.

[0059] Embodiment 10. A method of treating or reducing the risk of cardiovascular disease in a subject in need thereof, the method comprising diagnosing the subject as having or at risk of having cardiovascular disease when the subject is homozygous for 9p21 risk allele and the subject has an interferon a-21 level of about 30, about 40 or about 50 pg/ml or higher; or when the subject is heterozygous for 9p21 risk allele and the subject has an interferon a-21 level of about 15, about 20, or about 25 pg/ml or higher; administering treatment for cardiovascular disease to the subject.

[0060] Embodiment 11. The method according to embodiment 10 wherein the treatment is one or more drugs selected from the group consisting of a cholesterol- modifying drug, an anti-coagulant drug, an anti-hypertensive drug, a vasodilator and an anti-diabetic drug.

[0061] Embodiment 12. The method according to embodiment 10 or 11 further comprising diagnosing the subject as having or at risk of having coronary artery disease by coronary angiography or computed tomography coronary angiography.

[0062] Embodiment 13. The method according to embodiment 10, 11 or 12 wherein the cardiovascular disease is selected from the group consisting of coronary atherosclerosis, CAD, myocardial infarction, abdominal aortic aneurysm, intracranial aneurysm, and peripheral arterial disease.

[0063] Embodiment 14. A diagnostic method for detecting the presence of cardiovascular disease in a subject, the method comprising determining interferon a-21 level in the subject, wherein the subject is positive for a 9p21 risk allele; diagnosing the subject as having cardiovascular disease when the subject is homozygous for the 9p21 risk allele and the interferon a-21 level is about 30, about 40, or 50 pg/ml or higher, or when the subject is heterozygous for the 9p21 risk allele and the interferon a-21 level is about 15, about 20, or about 25 pg/ml or higher.

[0064] Embodiment 15. The method according to embodiment 14 further comprising administering treatment for coronary artery disease to the subject.

[0065] Embodiment 16. The method according to embodiment 15, wherein the treatment is one or more drugs selected from the group consisting of a cholesterol- modifying drug, an anti-coagulant drug, an anti-hypertensive drug, a vasodilator and an anti-diabetic drug. [0066] Embodiment 17. The method according to embodiment 14, 15 or 16 further comprising diagnosing the subject as having coronary artery disease by coronary angiography or computed tomography coronary angiography.

[0067] Embodiment 18. The method according to embodiment 14, 15, 16 or 17 wherein the cardiovascular disease is selected from the group consisting of coronary atherosclerosis, CAD, myocardial infarction, abdominal aortic aneurysm, intracranial aneurysm, and peripheral arterial disease.

[0068] Embodiment 19. A kit for detecting the presence of or increased risk of cardiovascular disease, the kit comprising a protein expression level assay and instructions for detecting and determining the level of interferon a-21 in a biological sample.

[0069] Embodiment 20. The kit according to embodiment 19 wherein the protein expression level assay is an immunoassay or a DNA aptamer assay.

[0070] Embodiment 21. The kit according to embodiment 20 wherein the immunoassay is immunoassay is an immunosorbant assay, a Western blot, an immunoprecipitation, and an immunofluorescent assay.

[0071] Embodiment 22. The kit according to embodiment 19, 20 or 21 wherein the cardiovascular disease is selected from the group consisting of coronary atherosclerosis, CAD, myocardial infarction, abdominal aortic aneurysm, intracranial aneurysm, and peripheral arterial disease.

EXAMPLES

[0072] The following examples are merely illustrative, and do not limit this disclosure in any way.

[0073] The present inventors have discovered that serum and plasma interferon a-21 levels were highest in individuals homozygous for the 9p21 risk allele and were three times higher in subjects with documented coronary artery disease than in controls. These results suggest that interferon a-21 contributes to the risk of coronary artery disease associated with the 9p21 risk allele, and can be used to detect cardiovascular diseases such as CAD. [0074] Example 1 - Peripheral Blood Lymphocytes & Primary Human Aortic Smooth Muscle Cells

[0075] Materials and Methods

[0076] Study Participants. The Research Ethics Board of the University of Ottawa Heart Institute approved this study and written informed consent was obtained from all study participants. Cases consisted of males with disease onset below the age of 55 years or females below the age of 65 years with at least one epicardial stenosis >50 . Controls were patients who had undergone coronary angiography and were found to have minimal coronary stenosis (<30%). Controls were matched for the age of consent of cases and for gender. Individuals with diabetes mellitus were excluded from the study. Serum and dual aliquots of buffy coat were obtained from all participants. In all cases, one aliquot was processed for DNA extraction and genotyping. Buffy coat samples from the other aliquot were processed for immunoblot analysis, 6 from each genotype, were selected at random from 5,211 subjects of the Ottawa Heart Genomics Study.

[0077] Genotyping. DNA samples were genotyped for the 9p21 risk locus (rsl333049) on Affymetrix 6.0 SNP arrays, as described or on Affymetrix Axiom arrays.

[0078] Processing of Patien s Peripheral Blood Lymphocytes for Immunoblotting. Blood was collected in EDTA-tubes and then centrifuged for 10 minutes at 3000 rpm, the buffy coat containing peripheral blood lymphocytes was isolated and processed for protein extraction. Lymphocytes were lysed in RIPA buffer (4.25 mM Tris, pH8.0, 135 mM NaCl, 1% IGEPAL CA-630, 1% SDS 0.5% deoxycholate) in the presence of protease and phosphatase inhibitors (Roche Molecular Systems, Branchburg, NJ). Lysates were washed with the Ni-NTA agarose beads (Invitrogen, R901-01) to remove residual hemoglobin contamination. Clear supernatants were collected and assayed for protein concentration by Bradford assay before SDS- PAGE and immunoblot analysis.

[0079] Primary Human Aortic Smooth Muscle Cells (SMCs). Human aortic smooth muscle cell (SMC) primary cultures were obtained and cultured in smooth muscle growth medium (cat.311-500) from Cell Applications, Inc. (San Diego, CA). Protein lysates were isolated from human aortic SMCs from six Caucasian donors, two homozygous for the 9p21 non-risk allele of rsl333049 (#1841, #1576), two heterozygotes (#1596, #2531), and two homozygous (#8222, #1426) for the risk allele.

[0080] Antibodies and Immunoblot Analyses. Mouse anti-CDKN2A (sc-9968), anti-CDKN2B (sc-171798), anti-MTAP (sc-100782), anti-IFN-ω (sc-51978) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA) and diluted 1: 1,000 in blocking solution. Rabbit IFNa21 (SAB1401202, Sigma) was diluted 1: 10,000 in blocking solution. Goat anti-mouse IgG (HAF007, R&D Systems, Minneapolis, MN) and goat anti-rabbit IgG (#31460, Piercenet, Rockford, IL) were diluted in 1: 15,000 in blocking solution before application. Where available, positive control cell lysates for IFNa21 were purchased from Novus Biologicals (Littleton, CO) and for MTAP, CDKN2A and CDKN2B from Santa Cruz Biotechnology. Quantification of protein levels, normalized to GAPDH, was performed using ImageQuant TL V2005 software. Per genotype, N=6 for the PBL and two for human aortic SMCs. Relative protein levels were expressed as means + SEM. The same samples were tested for the expression of five proteins relative to 9p21 allele dosage and considered significant with a Bonferroni corrected p<0.01.

[0081] Results. To test whether the levels of proteins encoded by genes in the vicinity of long-range enhancers is influenced by sequences at the 9p21 locus, immunoblot analysis was carried out using genotyped human primary aortic smooth muscle cells (SMCs) (Figure 1, n=6). These cells were cultured under identical conditions to the same confluence. Homozygosity for the 9p21 risk allele was associated with reduced expression of CDKN2A. CDKN2B showed a dose-dependent decrease in expression associated with each risk allele of the 9p21 locus. MTAP expression was not affected. An antibody that recognizes all interferon-a isoforms indicated no difference in expression related to the 9p21 risk allele. However, interferon a-21 levels were positively correlated with the number of 9p21 risk alleles in an additive fashion, whereas interferon a-1 showed a dose-dependent decrease in expression in human aortic SMCs with each copy of the 9p21 risk allele. The absence of an overall increase in interferon a indicates that the elevated expression of interferon a-21 is likely specific to this isoform of interferon a. [0082] The association between the 9p21 risk locus and these gene products in protein extracts from peripheral blood lymphocytes (PBL) of patients with coronary artery disease (see Table 1 for clinical characteristics) was examined. The results of immunoblot analyses are presented in Figure 2 (n=18). CDKN2A protein levels were markedly reduced in patients homozygous for 9p21 risk allele, as observed for SMCs. CDKN2B protein expression was below the threshold of detection in PBL. As in SMCs, the levels of MTAP did not associate with 9p21 risk genotype (n=3 per genotype). Unlike for SMCs, interferon a-1 levels were not different in PBLs of different genotypes suggesting that the expression of interferon a-1 may be cell type specific. In contrast, similar to the observed association in SMCs, interferon a-21 protein levels increased proportional to the number of 9p21 risk alleles, indicating an additive effect.

Table 1. Characteristics of coronary artery disease patients stratified by 9p21.3 rs 1333049 genotype for lymphocyte protein expression analysis by immunoblot

MI (%) 4 (66.7) 2 (33.3) 3 (50.0) 0.513

PCI (%) 2 (33.3) 3 (50.0) 1 (20.0) 0.472

CABG (%) 1 (16.7) 1 (16.7) 4 (66.7) 0.105

The continuous variables were presented as mean + SD. P values were obtained by χ² test for categorical variables or Student t test for continuous variables, (b-) prefix indicates baseline values, (r-) prefix indicates recent values. MI, myocardial infarction; PCI, percutaneous intervention; CABG, coronary artery bypass graft. All patients were on statin therapy.

[0083] Example 2 - Serum

[0084] Interferon a-21 Enzyme-linked immunosorbent assay (ELISA). Serum concentrations of human interferon a-21 were measured by ELISA using a commercial kit (cat. #E96966Hu, USCN Life Science Inc, Wuhan, P.R. China) according to the manufacturer's instructions. The kit employs a mouse monoclonal antibody specific for human interferon a-21 adsorbed to 96 well plates. Details of the sensitivity and inter- assay variability are available in the online supplement.

[0085] Statistical Analysis. Continuous variables are presented as mean + standard deviation (SD). Differences across genotype groups were examined using the χ test for categorical variables, Student's t-test, and one-way analysis of variance (ANOVA) for continuous variables. A linear regression model was used to test the relation between the 9p21.3 risk locus and serum interferon a-21 levels. The odds ratio (OR) of CAD and its 95% CI for individuals homozygous for the 9p21.3 risk allele exposed to higher serum interferon a-21 were calculated with logistic regression analysis. Because of skewed distributions, the values for serum interferon a-21 were log-transformed before the analyses. All statistical analyses were 2-sided and performed with the statistical software SPSS (version 17.0, SPSS Inc, Chicago, IL). A P value <0.05 was considered statistically significant. Power calculations were made using the freeware software Power*G3.

[0086] Given that interferon a-21 is a secreted cytokine, serum levels of interferon a- 21 where investigated to see if they would reflect the cellular levels detected by immunoblot analysis. Using serum samples from 331 genotyped coronary artery disease cases (124 homozygous risk, 109 heterozygotes and 98 homozygous for the non-risk genotype of rs 1333049), interferon a-21 levels were measured by ELISA. See Table 1 for clinical characteristics of coronary artery disease cases and controls. Consistent with immunoblot analysis of PBLs, serum from patients homozygous for the 9p21 risk allele exhibited the highest levels of interferon a-21 (345+179.7 pg/ml), heterozygotes had intermediate levels (35.8+30.3), whereas those homozygous for the non-risk allele had even lower concentrations of serum interferon a-21 (12.6+11.0) (Table 1, Figure 3, p=4.2 x 10^"93).

[0087] Assuming that the mean serum interferon a-21 levels for CC risk allele carriers would be 4 times lower but with the same standard deviation as in CAD cases, it was estimated that a total sample size of 165 (55 per genotype) had 80% power to replicate a linear trend association using an F test with a p<0.05. Interferon a-21 in serum samples from 231 controls (76 CC, 78 GC, and 77 GG) with minimal angiographically-defined coronary disease (<30% stenosis in any vessel) was measured. Again, a very robust association between serum interferon a-21 levels and 9p21 risk on

genotype was observed (p=2.7 x 10^" ). Interestingly, control subjects who were homozygous carriers of the risk genotype (CC) exhibited levels of interferon a-21 (118.9+48.9 pg/ml) that were one third those of cases with coronary artery disease of the same genotype (345.0+179.7 pg/ml). However, serum interferon a-21 levels in controls homozygous for the non-risk allele were similar (9.0+4.2 pg/ml) to those of cases of the same genotype (12.6+11.0 pg/ml).

[0088] Multiple linear regression analysis showed that the rsl33049 genotype of the 9p21 coronary artery disease risk locus was the strongest predictor of serum interferon a- 21 level. Adjusting for sex, smoking history, BMI, and age at consent, the likelihood of coronary artery disease associated with a 100 pg/ml serum increase in interferon a-21 levels among individuals homozygous for the risk allele of 9p21 was significant, p=8.8 x 10^"11, odds ratio and 95% confidence interval: 3.27 (2.29-4.68). In other words, for every 100 pg/ml increase in serum interferon a-21 levels, the likelihood of significant coronary artery disease as defined by coronary angiography is increased more than 3 fold. The coronary artery disease association with elevated interferon a-21 levels was similar for male and female patients (see Table 2).

Table 2. Multiple linear regression analysis for predicting the level of interferon a-

21 (logio-transformed before analyses)

*Statistical model: rsl333049, Age, gender, BMI, smoking, Hypertension were entered as predictors into the multiple linear regression model.

[0089] To test whether the levels of interferon a-21 were associated with the severity of coronary artery disease, serum interferon a-21 levels in individuals homozygous for the 9p21 risk allele were correlated with the number of affected coronary arteries with >50 stenosis. Although a clear association with the presence or absence of coronary artery disease was noted, we did not observe a correlation with disease severity beyond 1 vessel disease. In other words, in the sample studied, among carriers of the 9p21 risk allele, the elevated serum levels of interferon a-21 were indicative of the presence but not the severity of clinically significant coronary artery disease, as measured by the number of affected coronary arteries. [0090] A strong correlation between the 9p21 coronary artery disease risk locus and elevated serum levels of interferon a-21 exists.

[0091] The long-range enhancer region between IFNW1 and IFNA21 that is influenced by the 9p21 risk locus appears to be associated with increased levels of interferon a-21. In accord with previous data, the opposite effect was seen for CDKN2A, with homozygosity for 9p21 risk locus being associated with markedly lower expression in both PBLs and human aortic SMCs. Recent studies have provided functional confirmation that CDKN2BAS directly imposes transcriptional repression on the CDKN2A/CDKN2B locus by forming a stable complex with the Polycomb Repressive Complex (PRC1 and PRC2). That the 9p21 risk allele reduces CDKN2A and CDKN2B protein expression while increasing interferon a-21 levels would have a net effect to promote vascular smooth muscle cell proliferation and increase coronary artery disease. Indeed, the human aortic smooth muscle cells exhibited markedly different proliferation rates, with the cells bearing the 9p21 non-risk allele having the slowest rate of proliferation under the same culture conditions (data not shown).

[0092] The 9p21 locus-regulated enhancer downstream of interferon a-21 (IFNA21) was associated with increased interferon a-21 protein expression in PBLs, and also in human aortic SMCs, an effect that would be expected to augment a pro-inflammatory response relevant to atherosclerosis.

[0093] The observation that elevated serum interferon a-21 levels are associated with clinically important coronary artery disease among patients homozygous for the 9p21 risk allele, but not with the severity of coronary artery disease, as measured by the number of affected coronary arteries, is consistent with the idea that interferon a-21 is associated with disease initiation, but not disease progression. The finding that elevated serum levels of interferon a-21 are specific for the 9p21 risk genotype provides novel insight into the pathogenesis of atherosclerosis. Importantly, clinically significant coronary atherosclerosis was shown to be associated with markedly elevated serum interferon a-21 levels in individuals homozygous for the risk allele. Therefore, interferon a-21 levels can be utilized to detect coronary artery disease in individuals homozygous for the 9p21 risk allele, who make up 25% of the population, and those heterozygous. [0094] Example 3 - combined serum/plasma

[0095] In addition to the serum studied in Example 2, studies were also performed on blood plasma. The results were the same whether the biological sample was serum or plasma, therefore the data from serum Example 2 was combined with plasma data.

[0096] Plasma Interferon alpha-21 is increased in patients with the 9p21 risk variant and CAD

[0097] Plasma levels of interferon omega- 1, alpha-21, and alpha-4 were measured with ELISA assays based on monoclonal antibodies to the various interferons. These proteins are encoded by genes on chromosome 9 in the vicinity of the sequence regulated by the 9p21 enhancer.

[0098] The controls (n=231) were individuals for which coronary angiography showed minimal coronary artery obstruction (<30%) and the CAD cases (n=595) had clinically significant CAD>50 obstruction. Individuals with the 9p21 risk genotype exhibited markedly increased plasma levels of IFNA21 (Fig.4). In individuals with minimal disease (clinical controls), plasma levels of IFNA21 are elevated in heterozygotes (HET) with one risk allele compared to those homozygote for the non-risk allele (NR) and even more so in homozygotes with two copies of the risk allele (R). For cases of clinically significant CAD, the plasma INFA21 levels were also elevated for each copy of the 9p21 risk allele. Importantly, the effect of the 9p21 risk allele to elevate IFNA21 is amplified by coronary artery disease, being nearly three times higher in CAD cases than in controls who are homozygous for the risk allele, but without significant CAD as determined by coronary angiography.

[0099] The 9p21 risk genotype specifically elevates interferon alpha-21 (IFNA21) expression:

[00100] In contrast to the robust association of interferon alpha-21, interferon omega- 1 (IFNW1) was not associated with 9p21 risk genotype in 230 CAD cases and interferon alpha-4 (IFNA4) was only modestly associated with 9p21 risk genotype in 176 CAD cases (Fig. 5A, 5B). We observed no cross-reactivity of the assays for IFNA21, IFNW1 (Fig. 5A) and IFNA4 (Fig.5B). Thus, the 9p21 risk locus selectively activates IFNA21 expression, and this effect is amplified in cases with clinically significant CAD. The p- values were obtained by linear regression using logio-transformed values without adjustment for covariates. We confirmed the 9p21 risk genotype and CAD interaction was not due to a non-specific inflammatory mechanism by showing C-reactive protein was not associated with 9p21 risk genotype in either cases or controls (Fig.5C, mean + standard deviation).

[00101] INFA21 levels are not elevated by an acute infection:

[00102] Eight (8) CAD cases, whose plasma was drawn at different times, ranging from 1 to nearly 5 years apart, were examined. It was found that IFNA21 levels do not change significantly when sampled at different times, as shown in Fig.6, providing further evidence that INFA21 levels are not elevated by an acute infection.

[00103] Preliminary sensitivity of interferon alpha-21 (IFNA21) to detect CAD:

[00104] Using a cut-off of 50 pg/ml, the diagnostic sensitivity of the IFNA21 ELISA to detect CAD among homozygous carriers of the 9p21 risk genotype was 95%. Assuming that individuals homozygous for the 9p21 risk allele with angiographically proven CAD and plasma IFNA21 level below 50 pg/ml are false negatives. At this cutoff for heterozygotes, the IFNA21 ELISA had a sensitivity of 52% to detect CAD. However, using a cut-off of 25 pg/ml, sensitivity of the assay was 98.5% for homozygous risk allele carriers and 79% for heterozygotes. Our preliminary results identify the IFNA21 ELISA as a highly sensitive assay at predicting clinically significant CAD at this cut-off. Thus, in 75% of the population with European ancestry that carries the 9p21 risk allele, our preliminary data suggests we can detect CAD with a sensitivity of greater than 85%.

[00105] The foregoing description of the embodiments has been provided for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure. Individual elements or features of a particular embodiment are generally not limited to that particular embodiment, but, where applicable, are interchangeable and can be used in a selected embodiment, even if not specifically shown or described. The same may also be varied in many ways. Such variations are not to be regarded as a departure from the disclosure, and all such modifications are intended to be included within the scope of the disclosure.

[00106] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

WHAT IS CLAIMED IS:

1. Use of interferon a-21 for detecting the presence of or increased risk of coronary artery disease in a subject positive for a 9p21 risk allele.

2. The use according to Claim 1, wherein when the subject is homozygous for the 9p21 risk allele and the interferon a-21 level is about 30, about 40, or about 50 pg/ml or higher, than this outcome is indicative of the subject having or is at increased risk of having coronary artery disease; or when the subject is heterozygous for the 9p21 risk allele and the interferon a-21 level is about 15, about 20 or about 25 pg/ml or higher, than this outcome is indicative of the subject having or is at increased risk of having coronary artery disease.

3. A diagnostic method for detecting the presence of or increased risk of coronary artery disease in a subject, the method comprising performing a protein expression level test on a biological sample from the subject, wherein the subject is positive for a 9p21 risk allele; determining interferon a-21 level from the protein expression level test; wherein when the subject is homozygous for the 9p21 risk allele and the interferon a-21 level is about 30, about 40, or about 50 pg/ml or higher, than this outcome is indicative of the subject having or is at increased risk of having coronary artery disease; wherein when the subject is heterozygous for the 9p21 risk allele and the interferon a-21 level is about 15, about 20 or about 25 pg/ml or higher, than this outcome is indicative of the subject having or is at increased risk of having coronary artery disease; communicating the interferon a-21 level to a medical practitioner.

4. The method of Claim 3, further comprising determining if the subject is positive for the 9p21 risk allele.

5. The method of Claim 3, wherein the protein expression level test is an immunoassay, a DNA aptamer assay or protein-mass spectrometry.

6. The method of Claim 5, wherein the immunoassay is selected from the group consisting of an immunosorbant assay, a Western blot, an immunoprecipitation, and an immunofluorescent assay.

7. The method of Claim 3, wherein the biological sample is whole blood, serum or plasma.

8. A method of treating or reducing the risk of coronary artery disease in a subject in need thereof, the method comprising diagnosing the subject as having or at risk of having coronary artery disease when the subject is homozygous for 9p21 risk allele and the subject has an interferon a-21 level of about 30, about 40, or about 50 pg/ml or higher; or when the subject is heterozygous for 9p21 risk allele and the subject has an interferon a-21 level of about 15, about 20, or about 25 pg/ml or higher; administering treatment for coronary artery disease to the subject.

9. The method of Claim 8, wherein the treatment is one or more drugs selected from the group consisting of a cholesterol-modifying drug, an anti-coagulant drug, an antihypertensive drug, a vasodilator and an anti-diabetic drug.

10. The method of Claim 8, further comprising diagnosing the subject as having or at risk of having coronary artery disease by coronary angiography or computed tomography coronary angiography.

11. A diagnostic method for detecting the presence of coronary artery disease in a subject, the method comprising determining interferon a-21 level in the subject, wherein the subject is positive for a 9p21 risk allele; diagnosing the subject as having coronary artery disease when the subject is homozygous for the 9p21 risk allele and the interferon a-21 level is about 30, about 40, or about 50 pg/ml or higher, or when the subject is heterozygous for the 9p21 risk allele and the interferon a-21 level is about 15, about 20, or about 25 pg/ml or higher.

12. The method of Claim 11, further comprising administering treatment for coronary artery disease to the subject.

13. The method of Claim 12, wherein the treatment is one or more drugs selected from the group consisting of a cholesterol-modifying drug, an anti-coagulant drug, an antihypertensive drug, a vasodilator and an anti-diabetic drug.

14. The method of Claim 11, further comprising diagnosing the subject as having coronary artery disease by coronary angiography or computed tomography coronary angiography.

15. A kit for detecting the presence of or increased risk of coronary artery disease, the kit comprising a reagents for a protein expression level assay and instructions for detecting and determining the level of interferon a-21 in a biological sample.

16. The kit of Claim 15, wherein the protein expression level assay is an immunoassay or a DNA aptamer assay.

17. The kit of Claim 16, wherein the immunoassay is an immunosorbant assay, a Western blot, an immunoprecipitation, and an immunofluorescent assay.

18. The kit of Claim 15, further comprising reagents for a nucleic acid assay to detect the presence of a 9p21 risk allele and instructions for detecting the presence of a 9p21 risk allele in a biological sample.