Research Article

Associations of Variants within the PHACTR1, WDR12 and ANRIL Genes with Coronary Artery Disease Severity

Patrick O’Boyle1, Rohan Jayasinghe1*, Rod A Lea2,3, Heidi Sutherland2,3, Shani Stuart2,3 and Charlie1
1Department of Cardiology, Gc Hospital, Hanna Burton, Australia
2Department of Cardiology, Griffith University, Australia
3Department of Cardiology, Institute for Health and Biomedical Innovation, Australia


*Corresponding author: Rohan Jayasinghe, Department of Cardiology, Gc Hospital, Australia


Published: 16 Feb, 2017
Cite this article as: O’Boyle P, Jayasinghe R, Lea RA, Sutherland H, Stuart S, Charlie. Associations of Variants within the PHACTR1, WDR12 and ANRIL Genes with Coronary Artery Disease Severity. J Heart Stroke. 2017; 2(2): 1018.

Abstract

Objective:To conduct a stage I study to identify genetic predictors of coronary artery disease severity and develop genomic profiles for accurate classification of high- risk patients in a clinical cohort.
Methods:719 patients who underwent coronary angiography for myocardial infarction or suspected coronary artery disease were divided into cases with severe disease and controls with non-severe and no disease. Coronary artery disease severity was scored on based on; i) severity of coronary artery luminal stenosis and ii) number of diseased main coronary vessels. A case-control design was employed. We directly assessed for SNP association with coronary artery disease severity as determined by invasive coronary angiography
Results:A systematic statistical analysis strategy was used to assess the association of all genetic factors with CAD severity. Results of this analysis revealed two SNPs associated with severity of coronary artery stenosis and 3 SNPs associated with number of diseased proximal vessels. Logistic regression models were generated for each SNP factoring in related covariates – age, sex, dyslipidemia and hypertension status.
(rs12526453) in the PHACTR1 gene on chromosome 6, and (rs6725887) in the WDR12 gene on chromosome 2 were predictive of the severity of coronary artery disease luminal stenosis after adjustment for cofactors (OR=1.38 p=0.02 and OR=1.46 p= 0.048 respectively). (rs6725887) was again associated with CAD severity as determined by the number of diseased proximal vessels (OR=2.01, p=0.004). Two further SNPs were also associated with CAD severity as defined by the number of diseased proximal vessels: (rs4977574) in proximity to CDK2A & CDK2B genes on chromosome 9p21.3 (OR=1.64 p=.005); and (rs10953541) in the (BCAP29 gene on chromosome 7 (OR=1.814, p=.004).
Conclusion:We have identified four SNPs to be predictive of CAD severity as assessed by coronary angiography. Two SNPs are predictive of the severity of proximal coronary artery disease based on the extent of luminal stenosis: (rs12526453) in the PHACTR1 gene on chromosome 6 and (rs6725887) in the WDR12 gene on chromosome 2. 
(rs6725887) and a further two SNPs are predictive of triple vessel disease: (rs4977574) in the ANRIL gene in proximity to CDK2A &CCK2B on chromosome 9p21.3 and (rs10953541) in the BCAP29 gene on chromosome 7.
This discovery may pave the way for the development of a genetic diagnostic and screening tool for coronary atherosclerosis. It may also help identify targets for future gene therapeutics against atherosclerotic coronary disease.


Introduction

Despite the broad application of proven preventive strategies, coronary artery disease (CAD) remains the leading cause of mortality worldwide.  In 2005 it killed more than 7.5 million people [1], and deaths from CAD continue to rise globally [2]. In clinical cardiology, invasive coronary angiography remains the gold standard for establishing the diagnosis and defining the severity of coronary artery disease [3]. Here we report the identification of four SNPs to be associated with the severity of CAD as assessed by coronary angiography in an Australian Caucasian population: (rs12526453) in the PHACTR1 gene on chromosome 6; (rs6725887) in the WD repeat domain 12 (WDR12) on chromosome 2; (rs4977574) in proximity to the cyclin-dependent kinase inhibitor 2A and B  (CDK2A & CDK2B) genes on chromosome 9p21.3; and (rs10953541) in the B-cell receptor-associated protein 29 (BCAP29) on chromosome 7. Our finding that these SNPS are associated with the angiographic severity of CAD but not so much with the risk of myocardial infarction (MI) indicates that their role is likely related to atherogenesis rather than plaque rupture or thrombus formation.
As well as indicating the potential mechanism of action of these genes our findings may help in developing a diagnostic screening tool for the detection of individuals at risk of severe CAD. This could potentially lead to earlier primary preventive strategies. 
CAD is characterised by lipid deposition and atherosclerotic plaque formation in the coronary arteries. Plaque formation is a result of endothelial dysfunction, oxidative stress and inflammation [4]. CAD is a multifactorial disease with well documented environmental risk factors, in particular smoking, hypertension, hypercholesterolemia, diabetes and obesity [5]. CAD is known to be heritable, with a history of premature CAD in parents conferring almost three times higher risk of CAD in their offspring [6]. The extent to which inheritance influences the risk of CAD varies greatly and so the genetic factors are thought to exert risk both directly and through gene-environment interactions [7]. Myocardial infarction results from the rupture of vulnerable plaques, thrombosis and occlusion of the coronary vessels. Plaque vulnerability is determined by the size and consistency of the atheromatous core, thickness of the fibrous cap covering the core, and ongoing inflammation within the cap [8].
Direct DNA sequencing for Mendelian forms of cardiovascular disease traits, such as familial hypercholesterolaemia, identified causative mutations in the low-density lipoprotein receptor (LDLR) gene [9]. CAD traits show complex inheritance and association study techniques such as the candidate gene approach, were initially taken to define the genetic basis of CAD.  However, candidate genes identified often failed to replicate the associations in subsequent studies [10]. The advent of high-throughput genetic testing led to the development of genome wide association study (GWAS) approach. GWAS identified the first notable genetic variant (located on chromosome 9p21.3) associated with CAD [11]. Recently, the GWAS database (www.genome.gov/26525384) has catalogued results from several large-scale GWASs of CAD patients [12] with 22 SNPs in 19 genes convincingly implicated in the risk of CAD traits.
Invasive coronary angiography is the gold standard for establishing the presence, location and severity of coronary artery disease as it provides excellent spatial and temporal resolution for the visualisation of the coronary arterial tree [3]. Most studies investigating the genetic basis of coronary artery disease were based on the clinical diagnosis of CAD traits such as myocardial infarction or angina but not coronary anatomy as assessed by angiography. It is established that myocardial infarction and acute coronary occlusion most frequently relate to plaques that cause mild to moderate coronary artery stenosis due to the propensity of a vulnerable plaque to rupture [13]. Hence studies, which identify genetic loci associated with MI, will identify genes which may influence plaque stability as well as atherogenesis and thrombosis.
Distinct morphological characteristics of CAD show different degrees of heritability. Proximal coronary artery disease has been shown to display higher degree of heritability in comparison to distal disease [14]. Hence our focus was on the degree of stenosis and the plaque distribution in the four major epicardial coronary arteries; left main, left anterior descending, left circumflex and right coronary arteries.
Our research is unique in that it goes beyond the association of SNPs with coronary artery disease traits, directly assessing for SNP association with CAD severity as assessed by invasive coronary angiography. We have identified four SNPs to be predictive of CAD severity as assessed by coronary angiography. Two SNPs are predictive of the severity of proximal coronary artery disease luminal stenosis: (rs12526453) in the PHACTR1 gene on chromosome 6 and (rs6725887) in the WDR12 gene on chromosome 2.  (rs6725887) and a further two SNPs are predictive of triple vessel disease: (rs4977574) in the ANRIL gene in proximity to CDK2A &CCK2B on chromosome 9p21.3 and (rs10953541) in the BCAP29 gene on chromosome 7.
This discovery furthers the potential of developing a genetic test for screening for the risk of severe and diffuse coronary atherosclerosis permitting early detection and effective prevention by the aggressive control of modifiable risk factors.


Methods

Study population
This study was conducted collaboratively between the cardiology department of the Gold Coast Hospital, Queensland and The Genomics Research Centre (GRC) at Griffith University, Queensland Australia. The study was approved by the institutional ethics committee and all subjects gave informed written consent. 719 Caucasian subjects who underwent coronary angiography between 2006 and 2012 were selected. The study population was homogeneous consisting only white Caucasians.
Coronary angiography
The indications for coronary angiography included acute coronary syndrome (diagnosed by clinical history, electrocardiographic findings and elevated cardiac troponin [15]), pre-surgical cardiac assessment, work-up for valve surgery and stable angina. The full spectrum of clinically manifested coronary artery disease was represented in this population as follows: ST elevation myocardial infarction (STEMI) (5.7%) and Non-ST elevation myocardial infarction (NSTEMI) (26.0%); Unstable Angina (5.0%); Stable Angina (21.1%); and other reasons (42.1%) including positive cardiac stress testing, positive functional imaging and pre-operative assessment. Coronary angiography was carried out using a Siemans Axiom Artis image intensifier (Siemens inc. Sweden) according to the Judkins’ technique using standard 6 French gauge right and left coronary catheters with the images acquired at a frame count of 15 per second and the orthogonal projections were made according to a standard institutional protocol and images of the four main coronary arteries: the left main coronary artery (LMCA), the left anterior descending artery (LAD), the left circumflex artery (LCx) and the right coronary artery (RCA) were acquired. Two independent workers analysed and reported the findings of angiography on two separate occasions for cross-validation. The lesions in the main coronary arteries were recorded as a percentage of the stenosis of the vessel (determined by visual estimation of the diameter of the narrowing compared to the proximal normal vessel). The number of diseased main vessels and the number of lesions in each main vessel were recorded. The reader reporters of the angiograms were blinded to the genotyping results of the patients.
In our study the coronary artery disease severity was measured utilising two steps: In the first step, the vessel score, was defined as the number of diseased main vessels with >50% luminal diameter stenosis.   A point was allocated to each diseased vessel: single vessel disease (1 point), two vessel disease (2 points) or triple vessel disease (3 points).  Stenosis of the left main coronary artery (LMCA) was considered more severe and was attributed a score of 2 points (LMCA divides into two main branches the left anterior descending and the left circumflex). In the second step the scoring was based on the number of lesions in each vessel. Patient’s were dichotomised into those with no or mild disease (those with less than 30% diameter stenosis were defined as non-severe disease) and severe disease (one or more lesion of 50% or more diameter luminal stenosis in a main coronary artery was defined as severe disease). This approach aimed at identifying genetic markers associated with the morphological characteristics of CAD which have clinical significance and which demonstrate the highest heritability. Therefore by focusing our scoring on proximal disease we intend to enhance our prospect of identifying genetic predictors of heritability and severity.
Risk factors
All clinical data was obtained during the index admission. Each patients had the standard coronary risk factors recorded. 62.7% of the study population were male, 50.5% were hypertensive, 57.8% had hypercholesterolaemia, 21.3% had diabetes, 5.3% were current smokers and 20.6% of patients had a positive family history (CAD in male first-degree relative, or father less than 55, or female first-degree relative or mother less than 65).
Genotyping
5ml EDTA blood samples were collected from femoral artery or the radial artery at the time of arterial puncture for vascular access.  Genomic DNA was extracted and purified from peripheral blood lymphocytes using astandard salting-out procedure [16]. 22 previously identified SNPs [17] were genotyped in the 719 patients. SNPs were selected from a search of the GWAS database for SNPs associated with CAD in 2011. Genotypes for each SNP was determined by restriction-fragment 140 length polymorphism (RFLP) analysis of restriction enzyme-digested PCR products on agarose gels. A 20-"l PCR reaction mix contained 1× PCR buffer, 1.75 mM MgCl2, 0.2 mM dNTPs, 0.15 "M of each primer, 20–40 ng of genomic DNA and 1.5 U of GoTaq® (Promega). Thermocycler conditions were an initial denaturation at 95°C for 10 min, followed by 35 cycles of 95°C for 45 s, 60°C for 45 s, 72°C for 45 s and a final extension step of 72°C for 7 min. Specific primers were used to amplify each region containing the SNPs and restriction enzymes (NEB) chosen that would differentiate the two alleles after separation on 3% agarose gels.
Statistical analysis For the primary exploratory analysis, a bivariate correlation analysis of all SNPs was performed against severity (as determined by the number of vessels involved and the severity of stenosis) using Spearman's statistic [18]. 2 SNPS were shown to be associated with severity of stenosis: (rs12526453, OR=1.42 p = 0.01) and (rs6725887, OR=1.51 p = 0.031). Four SNPs were shown to be associated with triple vessel disease in the primary exploratory analysis (rs10953541, OR=1.77 p=0.005), (rs12526453 OR=1.52, p=0.014), (rs4977574, OR=1.66 p=0.003) and (rs6725887 OR=2.05 P=0.003).
For the analysis of the severity of luminal stenosis, the severity was dichotomised into 0 (Normal, which is <30% stenosis) and 1 (significant disease, >50% stenosis in 1 or more proximal vessel). A Logistic regression model was generated for the two SNPs identified in the severity score primary analysis.  Age, sex, family history, dylipidaemia, hypertension, smoking and diabetes were factored in as co-variates.
Results
Results of this analysis showed (rs12526453) in the PHACTR1 gene on chromosome 6, and (rs6725887) in the WD repeat domain 12 (WDR12) on chromosome 2 to be predictive for the severity of coronary artery disease (as determined by the severity of luminal stenosis) after adjustment for cofactors (OR=1.38 p=0.02 and OR=1.46 p= 0.048 respectively). 
For the analysis of the number of vessels involved, the number of vessels was dichotomised into 0 (no vessels, n=226) and 1 (1,2 or 3 vessels, n=493). A Logistic regression model was generated for the three SNPs identified in the vessel score primary analysis. Again, age, sex, family history, dylipidaemia, hypertension, smoking and diabetes were factored in as co-variates. 
Results of this analysis again showed (rs6725887) in the WDR12 gene on chromosome 2 to be associated with CAD severity as defined by the number of vessels involved (OR=2.01, p=0.004). Two further SNPs were also associated with CAD severity as defined by the vessel score: (rs4977574) in proximity to CDK2A & CDK2B genes on chromosome 9p21.3 (OR=1.64 p=.005); and (rs10953541) in the B-cell receptor-associated protein 29 (BCAP29) on chromosome 7 (OR=1.814, p=.004).


Results

Results of logistic regression analysis for the severity of coronary artery luminal stenosis showed (rs12526453) in the PHACTR1 gene on chromosome 6, and (rs6725887) in the WDR12 gene on chromosome 2 to be predictive for the severity of coronary artery disease after adjustment for cofactors (OR=1.38 p=0.02 and OR=1.46 p= 0.048 respectively). 
Results of logistic regression analysis for the number of proximal coronary artery vessels involved again showed (rs6725887) in the WDR12 gene to be associated with CAD severity (OR=2.01, p=0.004). Two further SNPs were also associated with CAD severity as defined by the vessel score: (rs4977574) in proximity to CDK2A & CDK2B genes on chromosome 9p21.3 (OR=1.64 p=.005); and (rs10953541) in the B-cell receptor-associated protein 29 (BCAP29) on chromosome 7 (OR=1.814, p=.004).

Discussion

Our study evaluated the association between 22 SNPs and CAD severity as assessed by the gold standard investigation, invasive coronary.  Results of our analysis have shown four SNPs in total to be associated with disease severity.
One of the strengths of our study design is that we identify cases based on angiographic evidence of both severity and the most heritable morphological characteristics of CAD. Other case-control comparison studies often have variability in study enrolment, in particular controls are chosen from healthy individuals in the general population whose coronary anatomy is not known (and therefore may well be harbouring sub-clinical atherosclerosis). Each of our controls has angiographic evidence of the percentage of luminal stenosis in their coronary arteries and our study design excludes individuals from the general population who may have CAD but are asymptomatic.
Variants of phosphatase and actin regulator 1 gene (PHACTR1) demonstrated association with early onset MI in a study published by the MI genetics consortium [19]. PHACTR1 has also been associated with severity of coronary stenosis in a Lebanese cohort (OR = 1.37). SNP (rs9349379) in their study was associated with the severity of atherosclerosis in the Lebanese study but not replicated in our study. Although rs12526453 did not show association in the Lebanese exploratory set it did show association in their replication set [20]. Multiple GWASs in the Chinese Han population have not shown rs12526453 and PHACTR1 to be associated with CAD or MI. In the Chinese studies the minor allele frequency (based on HapMap data) of rs12526453 was 0% and none of the 113 SNPs in or near PHACTR1 showed significant association (p <0.01) for coronary artery disease [21,22]. Although their study sizes were small in comparison to the European studies it suggests that PHACTR1 may not play a role in the Chinese population.
The pathophysiology of the PHACTR1 in coronary artery disease remains to be elucidated. PHACTR1 has been proposed to be a key regulator of endothelial cell function [23] and is a regulator protein of protein phosphatise 1 (PP1), an enzyme that regulates endothelial nitric oxide (eNO) [24]. eNO is an important modulator of coronary artery disease and has been shown to influence thrombogenicity and inflammation [25]. Mutations in the PHACTR1 may contribute to atherosclerotic plaque formation by leading to endothelial dysfunction. Our finding that rs12526453 is associated with coronary artery disease severity but not associated with MI is suggestive that the role of PHACTR1 is in atherogenesis (plaque formation) and not plaque rupture or thrombosis.
Rs 4977574 has previously been shown to be associated with coronary artery disease severity in an Italian study [26] and also with MI [19,27]. Rs4977574 is located within the ANRIL gene that encodes a large non-coding RNA, its pathophysiology is unknown but its role in CAD has been implicated in several expression studies. The ANRIL promoter region contains binding sites for zinc-finger proteins that are critical for the transcription of CDKN2Aand CDKN2B and in the mouse, deletion of the 9p21 orthologous non-coding region including the 3′ region of ANRIL affects proliferation of vascular cells with severely reduced expression of CDKN2A and CDKN2B [27].
At least 11 chromosomal regions have been identified as associated with the risk of MI beyond that of traditional risk factors, with each risk allele increasing the risk of MI by a relatively small margin (10-30%) [28]. Myocardial infarction and coronary occlusion most frequently evolve from mild to moderate stenosis whilst severe atherosclerotic coronary artery disease is less likely to be the cause of MI_ENREF_8 [8]. Severe disease is clinically significant due to the associated symptoms, morbidity, mortality and therapeutic options such as stent insertion or coronary artery bypass surgery.
Conventional risk factors do not explain the full extent of coronary artery disease pathophysiology. Our work shows the importance of using the phenotype information gained from coronary angiography to begin to identify the mechanism of action of the risk genes identified in large scale GWASs. Data from coronary angiography will also improve the development and accuracy of genetic tools for identifying individuals at risk of severe coronary artery disease. This could eventually enable earlier prevention and would be a leap forward in the personalisation of medicine based on one’s genetic risk. The identification of novel genetic links and elucidation of their pathophysiology will be essential for the development for new therapies for the prevention and treatment of coronary artery disease.


Conclusion

In conclusion, we have identified of four SNPs (rs12526453) in the PHACTR1 gene on chromosome 6, (rs6725887) in the WDR12 gene on chromosome 2, (rs4977574) in the ANRIL gene on chromosome 9p21.3 and (rs10953541) in the BCAP29 gene on chromosome 7 to be predictive of CAD severity as assessed by coronary angiography. This discovery may pave the way for the development of a genetic diagnostic and screening tool for coronary atherosclerosis. It may also help identify targets for future gene therapeutics against atherosclerotic coronary disease.


References

  1. Fuster V, Kelly BB. Promoting Cardiovascular Health in the Developing World. A Critical Challenge to Achieve Global Health. 2010.
  2. Mathers CD, Loncar D. Projections of global mortality and burden of disease from 2002 to 2030. PLoS med. 2006; 3: e442.
  3. Kohsaka S, Makaryus AN. Coronary Angiography Using Noninvasive Imaging Techniques of Cardiac CT and MRI. Curr cardiol Rev. 2008; 4: 323-330.
  4. Libby P, Theroux P. Pathophysiology of coronary artery disease. Circulation. 2005; 111: 3481-3488.
  5. Torpy JM, Burke AE, Glass RM. JAMA patient page. Coronary heart disease risk factors. JAMA. 2009; 302: 2388.
  6. Lloyd-Jones DM, Nam BH, D'Agostino RB, Levy D, Murabito JM, Wang TJ, et al. Parental cardiovascular disease as a risk factor for cardiovascular disease in middle-aged adults: a prospective study of parents and offspring. JAMA. 2004; 291: 2204-2211.
  7. Cambien F, Tiret L. Genetics of cardiovascular diseases: from single mutations to the whole genome. Circulation. 2007; 116: 1714-1724.
  8. Falk E, Shah PK, Fuster V. Coronary plaque disruption. Circulation. 1995; 92: 657-671.
  9. Lehrman MA, Schneider WJ, Südhof TC, Brown MS, Goldstein JL, Russell DW. Mutation in LDL receptor: Alu-Alu recombination deletes exons encoding transmembrane and cytoplasmic domains. Science. 1985; 227: 140-146.
  10. McPherson R. A gene-centric approach to elucidating cardiovascular risk. Circ Cardiovasc Genet. 2009; 2: 3-6.
  11. Welcome Trust Case Control Consortium. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature. 2007; 447: 661-678.
  12. Hindorff LA, Sethupathy P, Junkins HA, Ramos EM, Mehta JP, Collins FS, et al. Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proc Natl Acad Sci U S A. 2009; 106: 9362-9367.
  13. Finn AV, Nakano M, Narula J, Kolodgie FD, Virmani R. Concept of vulnerable/unstable plaque. Arterioscler Thromb Vasc Biol. 2010; 30: 1282-1292.
  14. Fischer M, Broeckel U, Holmer S, Baessler A, Hengstenberg C, Mayer B, et al. Distinct heritable patterns of angiographic coronary artery disease in families with myocardial infarction. Circulation. 2005; 111: 855-862.
  15. Thygesen K, Alpert JS, White HD. Universal definition of myocardial infarction. Eur Heart J. 2007; 28: 2525-2538.
  16. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988; 16: 1215.
  17. Schunkert H, Schunkert H, König IR, Kathiresan S, Reilly MP, Assimes TL, et al. Large-scale association analysis identifies 13 new susceptibility loci for coronary artery disease. Nat Genet. 2011; 43: 333-338.
  18. Wauters E, Carruthers KF, Buysschaert I, Dunbar DR, Peuteman G, Belmans A, et al. Influence of 23 coronary artery disease variants on recurrent myocardial infarction or cardiac death: the GRACE Genetics Study. Eur Heart J. 2012; 34: 993-1001.
  19. Kathiresan S, Voight BF, Purcell S, Musunuru K, Ardissino D, Mannucci PM, et al. Genome-wide association of early-onset myocardial infarction with single nucleotide polymorphisms and copy number variants. Nat Genet. 2009; 41: 334-341.
  20. Hager J, Kamatani Y, Cazier JB, Youhanna S, Ghassibe-Sabbagh M, Platt DE, et al. Genome-wide association study in a Lebanese cohort confirms PHACTR1 as a major determinant of coronary artery stenosis. PloS One. 2012; 7: e38663.
  21. Wang F, Xu CQ, He Q, Cai JP, Li XC, Wang D, et al. Genome-wide association identifies a susceptibility locus for coronary artery disease in the Chinese Han population. Nat Genet. 2011; 43: 345-349.
  22. Chen X, Li S, Yang Y, Yang X, Liu Y, Liu Y, et al. Genome-wide association study validation identifies novel loci for atherosclerotic cardiovascular disease. J Thromb Haemost. 2012; 10: 1508-1514.
  23. Jarray R, Allain B, Borriello L, Biard D, Loukaci A, Larghero J, et al. Depletion of the novel protein PHACTR-1 from human endothelial cells abolishes tube formation and induces cell death receptor apoptosis. Biochimie. 2011; 93: 1668-1675.
  24. Mount PF, Kemp BE, Power DA. Regulation of endothelial and myocardial NO synthesis by multi-site eNOS phosphorylation. J Mol Cell Cardiol. 2007; 42: 271-279.
  25. Massion PB, Feron O, Dessy C, Balligand JL. Nitric oxide and cardiac function: ten years after, and continuing. Circ Res. 2003; 93: 388-398.
  26. Rivera NV, Carreras-Torres R, Roncarati R, Viviani-Anselmi C, De Micco F, Mezzelani A, et al. Assessment of the 9p21.3 locus in severity of coronary artery disease in the presence and absence of type 2 diabetes. BMC Med Genet. 2013; 14: 11.
  27. Saade S, Baptiste Cazier J, Ghassibe-Sabbagh M, Youhanna S, Badro DA, Kamatani Y, et al. Large scale association analysis identifies three susceptibility loci for coronary artery disease. PloS One. 2011; 6: e29427.
  28. Schunkert H, Erdmann J, Samani NJ. Genetics of myocardial infarction: a progress report. Eur Heart J. 2010; 31: 918-925.