This analysis of several distinct case-control studies provides evidence for consistent associations between a locus on chromosome 9p21.3 and CAD and MI. The magnitude and direction of the effect in each of the present study samples are consistent with the 4 previous genome-wide analyses that uniformly identified this locus as the strongest genetic signal for CAD.3-6
Together, the comprehensive replication across multiple samples evaluated in the present studies provides unequivocal evidence that variants at this locus increase the risk of CAD and MI in individuals of European ancestry.
We observed associations of the chromosome 9p21.2 locus with CAD in both cross-sectional case-control studies and subjects who developed the disease in the prospective PRIME study. Likewise, we found similar associations in men and women and in various subgroups defined by age or other cardiovascular risk factors. We also confirmed that the associations are independent of these risk factors.
The striking consistency of the association across this broad range of subjects, coupled with the high frequency of the risk allele and the substantial increase in the probability to develop the disease by each allele, suggests that genotyping for this locus could have important clinical utility in risk prediction in the future if the findings are supported in further large population-based studies. However, it also is evident from the large number of SNPs in this region with highly significant association with CAD that the causally responsible variant and the related mechanism remain to be identified. The high LD within the region is the most likely explanation of this finding. The region on chromosome 9p21.3 associated with CAD is located in 2 blocks of strong intrablock and interblock LD.3,4
We have previously shown that the association signal was stronger for a haplotype (ACAC) derived from 4 SNPs (the last one being the rs1333049 SNP) across the 2 blocks. Here, we confirm this finding by showing that the effect of the lead SNP, rs1333049, was restricted mainly to the ACA- haplotypic background. This result suggests that the lead SNP, rs1333049, alone does not fully explain the association with CAD. Deep resequencing of cases carrying this haplotype is required to obtain the full spectrum of variation in this region and to identify the causal variant(s) that affect risk.
If the effect of the locus is due largely to a single variant, we also show evidence suggesting that the most likely mode of inheritance is additive. This information may help to define the role of chromosome 9p21.3 variants in algorithms for cardiovascular risk prediction.
Beyond the 7 case-control samples, we undertook a meta-analysis of all previously published replication data. The findings from this analyses based on 12 004 cases and 28 949 controls confirm the strength of the association of the locus with the risk of CAD and provide reliable and robust estimates of the per-allele risk associated with the locus. These findings provide a firm rationale for further interrogation of the locus.
The validity of our findings is further strengthened by virtually identical estimates of the ORs and the 95% CIs from the FE and RE models (). The FE model provides more impressive P
values because of the crucial assumption that the ORs of all the different studies are identical. In contrast to FE models, RE models allow for between-study variabilities resulting, for example, from differences in ascertainment schemes or inclusion and exclusion criteria. This statistic is more conservative, and P
values from RE models are therefore substantially larger than P
values from FE models () or the originally combined P
values reported by Samani et al.3
Although this broad replication offers unprecedented information for a better understanding of the molecular genetic architecture of CAD, the underlying mechanism is as yet elusive. The region is defined by 2 flanking recombination hotspots and contains the coding sequences of genes for 2 cyclin-dependent kinase inhibitors, CDKN2A (encoding p16INK4a) and CDKN2B (encoding p15INK4b). These genes are known to play an important role in the regulation of the cell cycle and belong to a family of genes that have been implicated in the pathogenesis of atherosclerosis through their role in transforming growth factor-β–induced growth inhibition. However, the most strongly associated SNPs lie considerably upstream of these genes, and the nearest signal is 10 kb upstream of CDKN2B. Although an effect through regulation of one or both of the cyclin genes is possible, other explanations need to be considered, including the involvement of the MTAP gene or expression sequence tags located in the region.
Our studies, like the previous studies, have analyzed primarily subjects of north European origin.23
In fact, data from the HapMap consortium suggest that the frequency of the rs1333049-C allele differs from 0.17 in Yoruba to 0.48 in Han Chinese to 0.51 in Japanese to 0.49 in Europeans.25
Thus, the risk of CAD and MI related to the chromosome 9p21.3 locus may vary among different ethnic groups. Indeed, the role of this locus in other ethnic groups remains to be investigated.
The present study is focused on large case-control samples with CAD and MI. Although these related pheno-types display similar patterns of association, further studies are needed in a wider scope of atherosclerotic complications to fully understand the pathophysiological role of the chromosome 9p21.3 locus. Moreover, larger prospective studies are needed to precisely define the incremental information obtained when risk variants at chromosome 9p21.3 are included in algorithms predicting CAD events such as the Framingham or the Prospective Cardiovascular Münster (PROCAM) study risk scores.