This is the first and only study in African Americans to date to show a significant association of CAD with any gene variant within the chromosome 9p21.3 locus. This SNP, rs3217989, is located within the 3′UTR of CDKN2B and is independent from the LD block previously associated with CAD in non-African ancestry populations. In our GeneSTAR cohort the protective effect of the minor allele is potent, with almost a five-fold decrease in incident CAD risk. The direction of effect was replicated and also found to be significant in a meta-analysis of two populations of Americans with similar African ancestry in North Carolina and Georgia.
We postulate that the heterogeneity in the magnitude of the odds ratio estimates in the discovery and replication samples is a function of study design. The cumulative CAD incidence in GeneSTAR was 6% and by design, GeneSTAR CAD events occurred in persons who were likey close in age-range, genetic susceptibility, and shared environment to the proband. The GeneSTAR sibling CAD events were thus likely causally more homogeneous as compared to all possible causes of CAD in the general population. In contrast, the population-based case-control studies, which are 50% cases and 50% controls by design, likely have greater variety of genetic and environmental factors among those subjects with CAD events. Given greater probable causal heterogeneity, CAD cases in these would demonstrate an odds ratio closer to null than that seen in the discovery cohort. The observation of a consistent protective effect between the discovery and replication samples supports our finding in the discovery population. Although it would have been desirable to have more replication populations of greater size, very few African American are fully phenotyped for CAD or have a GWAS. The fact that our finding remains robust in small samples supports the likelihood that it is real.
No other study has published any associations with any gene variants, identified by GWAS, and CAD in African American populations. In the only GWAS reporting CAD results in African Americans, the 9p21 polymorphisms studied were not significantly associated with CAD4
. However, in studies published to date, the statistical power has been limited4
. One study in African Americans was limited to three significant SNPs identified from a GWAS in populations of European ancestry and there was again no detectable significance15
. Our results in African American families show that rs3217989 is in a different LD block than the lead SNPs previously found in the non-protein coding region in 9p21 in other populations. However, we also acknowledge that our study had limited statistical power to detect the previously reported association of these lead SNPs, including rs10757278, with CAD. Nonetheless, we were able to show that rs3217989 was independent of rs10757278 and therefore the lack of association of rs10757278 does not diminish the significance of our primary findings.
SNP rs3217989 appears to be monomorphic in populations of European ancestry in the Human Genome Diversity Panel data (http://hgdp.uchicago.edu/cgi-bin/gbrowse/HGDP/
). Given more fragmented LD structure in African Americans, the finding of a risk allele at rs3217989 suggests a functional variant located closer to rs3217989 in persons of African ancestry than the traditional previously published 9p21 intergenic locus.
The 9p21 locus previously identified in most studies has very few gene candidates. Previous GWAS studies and subsequent replications have reported that most associations at genome-wide significance occur in a large haplotype block upstream and independent from two major genes, CDKN2A
. These genes encode protein inhibitors of cyclin-dependent kinases, p16INK4a
, respectively, expressed at high levels in endothelial and inflammatory cells, and are also thought to help regulate cell proliferation, cell aging, and apoptosis33-35
(CDKNA2BAS), which encodes a large antisense non-coding RNA, is another candidate locus in this region. ANRIL spans 126.3 kb and consists of 20 exons subjected to alternate splicing including the first two exons which appear to overlap two exons of CDKN2B36
expression has been documented in atheromatous vessels, vascular endothelial cells, monocyte-derived macrophages, and coronary smooth muscle cells11
. In a subset of individuals in the Ottawa Heart Study18
the previously identified 9p21 risk alleles were associated with ANRIL mRNA of differential lengths. Furthermore, expression of CDKN2B
was correlated with the long variant of ANRIL, suggesting that the 9p21 risk alleles may be biologically tied to atherosclerosis risk through CDKN2B
expression. Given these important findings, it is possible that the association of the CDKN2B
gene variant with CAD in GeneSTAR African Americans is related to an alteration in ANRIL. However, the GeneSTAR CDKN2B
SNP is located at the 5′ end of ANRIL
whereas the aforementioned SNPs are located more at the 3′ end of ANRIL
, from exon 13 to 20, where ANRIL
splicing variation has been shown.18
Alternatively, the variant we report in the 3′-UTR of CDKN2B
may contribute to an alteration in CDKN2B
expression and/or function independently of ANRIL
. For example, the 3′UTR could contain a regulatory binding site for transcription or stability of the coded protein.