Higher incidence of death in multi-vessel coronary artery disease patients associated with polymorphisms in chromosome 9p21

Coronary heart disease (CHD), including acute myocardial infarction, are leading causes of morbidity and mortality worldwide [1]. Lifestyle and environmental factors play an important role in their development, but genetic inheritance appears to be strongly involved [2]. Recently, genome-wide association studies have revealed single nucleotide polymorphisms (SNP) on chromosome 9p21 that confer susceptibility to coronary artery disease (CAD) and myocardial infarction (MI) in some populations such as Caucasians from Northern Europe [3], North American [4], Italian [5] and Belgian [6]. Among the Han Chinese population, Lin et al. observed that the chromosome 9p21 had a significant association with carotid atherosclerosis in a gender-specific manner [7]. Moreover, meta-analysis of the relationship between chromosome 9p21.3 polymorphisms and CAD has provided stronger evidences for the association [8, 9].

9p21.3 is a chromosomal region relatively replete of open reading frames (ORF) and the closest genes are a cluster consisting of CDKN2A-ARF-CDKN2B (cyclin-dependent kinase N2A and N2B), MTAP, and ANRIL. The linkage disequilibrium (LD) structure of this genomic locus varies depending on the studied population, and different LD blocks are described between associated SNP and ORF in the region. This locus has been associated with tumor suppression, cell proliferation, senescence, apoptosis [10, 11], all features implicated in atherogenesis and now with CAD [12]. The mechanisms involved remain unclear but studies in mice have provide direct evidence that the CAD risk interval has a pivotal role in regulation of cardiac Cdkn2a/b expression, and suggest that this region affects CAD progression by altering the dynamics of vascular cell proliferation [13].

At the same time, genetic variants around the same genes were associated with an increased risk of type 2 diabetes mellitus [14–16] atherothrombosis [17] and ischemic stroke [18]. All these data suggest that there may be common pathogenic mechanisms involved in these apparently disparate diseases. On the other hand, Plant et al. showed that there was no association between rs10757278 and stroke status based on the presence or absence of angiographically demonstrated CAD in non-stroke controls. They supposed an association of this genomic region with ischemic stroke independent of its effect on CAD, suggesting an additional stroke-specific pathophysiological relationship [19].

In this report, we analyzed whether the rs10757274, rs2383206, rs10757278 and rs1333049 polymorphisms are associated with cardiovascular events in a group of 611 patients enrolled in the MASS II [20, 21], a study that compared therapeutic strategies for individuals with multi-vessel CAD established. This will explore the association of 9p21 genetic variants in individuals with already established and severe CAD.

Significant associations between rs10757274, rs2383206, rs10757278 and rs1333049 polymorphisms and diabetes and coronary artery disease were observed in patients with well-known multi-vessel CAD. Furthermore, our results suggested a significant difference in overall mortality and death from cardiac causes according to the rs2383206 in this patient sample that already presents well established risk factors and multivessel coronary artery disease.

Currently, genome-wide association identified a single region on chromosome 9p21.3 associated with CHD and MI risks [3, 4] independently of traditional risk factors including age, gender, obesity, smoking, hypertension and hyperlipidemia [8, 9]. Besides, Musunuru et al. have proposed that risk alleles at 9p21.3 locus may have pleiotropic effects on MI, CAD and stroke risks, possibly through their influence on platelet reactivity [23]. Helgadottir et al. [4] identified the rs10757278 that showed significant association with MI risk and that approximately 21% of individuals in the population are homozygous for this variant with estimated risk of suffering MI equal 1.64 times as great as that of non-carriers. McPherson et al. [3] identified 2 SNPs in the same genomic region, rs10757274 and rs2383206, both associated with risk, in 6 independent studies comprising more than 23,000 individuals and with a population-attributable fraction of 10% to 15%. Moreover, the Wellcome Trust Case Control Consortium [24] identified rs1333049 in this region, also showing strong association with CHD risk and a study that combined the Wellcome Trust Case Control Consortium data with data from Germany also confirmed the chromosome 9 region and identified 6 additional novel candidate genes for CHD [12].

In our study, we observed the association of the SNP rs2383206 to CAD within a group of patients with multi-vessel coronary disease that can reveal higher incidence of death depending on its genotype. The reports cited here have provided evidences of increased risk or susceptibility to develop CAD in case–control studies and our findings have showed significant differences of cardiovascular events within a sick population.

Regarding baseline clinical characteristics, our results showed association of all studied SNPs with prevalence of type 2 diabetes mellitus in these patients. In fact, the GG genotypes for the rs10757274, rs2383206 and rs10757278 suggested protection while the GG genotype for the rs1333049 indicated risk. Recent reports have discussed the association of these loci on chr9p21 with type 2 diabetes mellitus suggesting the sharing of pathogenic mechanisms with heart disease [14–16]. Studying the association between the SNP rs2383206 and CAD, Doria et al. observed increased CAD risk in the presence of poor glycemic control in type 2 diabetes [25]. Gori et al. [26] have confirmed that rs2891168 and rs10811661 are independently associated with MI and type 2 diabetes, respectively in an Italian population as reported by the PROCARDIS group [27].

In a recent study, Wang et al. observed a significant association of homozygous CC genotype for the rs1333049 with CAD in non-diabetic Chinese Han population but not in type 2 diabetic patients. Besides, they have demonstrated that CC or GC genotype carriers had a more severe plaque progression than GG genotype carriers [28]. The GRACE genetics study has provided evidence that the association of rs1333049 with recurrent MI or cardiac death was independent of all other risk factors [29]. Another recent report showed that the 9p21 variant rs10116277 is independently associated with all-cause mortality after primary CABG surgery in Whites and significantly improves the predictive value of the logistic EuroSCORE [30]. Here, we find that not only the rs1333049 CC genotype is more associated with serious CAD but also rs10757278 GG genotype; both are related to triple-vessel disease.

Finally, with respect to the end points, there was higher incidence of overall mortality in patients with GG genotype for rs2383206, which remained significantly associated with a 1.75-fold increased risk of overall mortality (CI 95% = 1.08 – 2.82) even after adjustment of a Cox multivariate model. The association of this SNP with death can be observed in the survival curves where not only overall mortality was affected but cardiac death too.

There are some limitations in our study. First, we were not able to perform replication studies in a similar sample because there are very few studies similar to MASSII in the literature. However, our main findings are in accordance to previous studies including a current study published in this journal which confirmed a strong association of the 9p21.3 locus with MI particularly in patients with a positive family history [30]. Second, our sample size only provide 80% power to detect an association with overall mortality with an effect size of 3.4-3.9 for the polymorphisms and correction for multiple testing was not performed. We recognize that such effects are highly unlikely for common genetic variants and reaffirm the need for the development of increased samples of multivessel CAD patients with long-time follow-up period for the identification of more modest effects. In addition, we observed that diabetes and smoking did not have significant effects in the multivariate analysis for overall mortality, as well as gender and hypertension, probably because our sample is composed of more severe patients. Third, it is not possible to completely exclude the interaction between the use of concomitant drugs, other genetic markers, ethnicity and other covariates on our findings [31–34].

Our data are in accordance to previous evidence that chromosome 9p21 genetic variation may constitute a genetic modulator in the cardiovascular system in different scenarios, including in patients with established CAD, by revealing an association between the rs2383206 and higher incidence of overall mortality and death from cardiac causes in patients with multi-vessel CAD.