The chromosome 9p21.3 locus has recently attracted much attention because of the consistent associations observed between SNPs at this locus and CVD-related traits. As in the FHS17
and other3, 7
studies, we found common SNPs at this locus to be associated with CAC, a well-validated marker of atherosclerosis and predictor of MI. The novel contribution of the present study is our demonstration that some of these MI/CAD-associated alleles are also associated with increased platelet aggregation, an important risk factor for both MI and stroke. We found the chromosome 9p SNPs to be associated across three different populations to two different platelet aggregation phenotypes, two reflecting whole blood platelet aggregation in response to collagen and one reflecting threshold aggregation concentration in response to epinephrine agonist in platelet rich plasma. On the one hand, the differences among the platelet aggregation phenotypes among the three studies argue for the robustness of the genotype association with platelet aggregation, although on the other hand, one could also say that no single phenotype association has been replicated. Although differences in the platelet aggregation measures and their scales preclude direct comparison of effect sizes among the three studies, there is a suggestion that the platelet aggregation association may be stronger in the Amish (with collagen dose of 0.5 μg/ml) than in GeneStar (with collagen dose of 1.0 μg/ml). A stronger association in Amish may reflect increased sensitivity to differences in aggregation at a lower dose of collagen agonist. This speculation is supported by the fact that higher doses of collagen cause a more robust platelet activation response than lower doses, including secretion of platelet granules and thromboxane release, which are absent or incomplete at lower doses. It is thus possible that higher doses of collagen may overwhelm gene-association signals of subtle difference in platelet function that are pathophysiologically relevant and are discoverable using lower doses.
The association with platelet aggregation is intriguing, suggesting increased platelet reactivity leading to thrombosis may be a mechanism whereby this locus contributes to MI/CAD. It seems unlikely that the SNP-platelet reactivity association can be explained by associations of these SNPs with CAC since there was virtually no correlation between CAC and platelet aggregation in the Amish. Association with platelet reactivity adds to the growing number of traits that SNP variation at this locus has been shown to influence – a list that now includes in addition to MI/CAD and stroke, abdominal aortic and intracranial aneurysms,9
and familial melanoma.33
Moreover, a SNP in a different LD block approximately 10 kb centromeric to this MI/CAD-associated region has been consistently associated with type 2 diabetes mellitus.9, 34–36
The diabetes-associated locus appears to be entirely distinct from the MI/CAD-associated locus.
Because SNPs in the MI/CAD associated region are in relatively high LD, it has been difficult to pinpoint the specific causal SNPs. In our Amish population, associations with CAC quantity extended over an 86 kb region that includes two large LD blocks, similar to the pattern seen in other Caucasian populations.19
However, the most highly associated SNPs in each block were not independently associated with CAC, suggesting that the association could be driven by a single (yet to be identified) variant marked by both SNPs somewhere in this region.
Our data suggest that variant(s) affecting CAC are associated with SNPs in LD with both Blocks 1 and 2, while variants associated with platelet reactivity are associated most strongly to SNPs in Block 1. There are no annotated protein-coding genes that map to the CAC and platelet aggregation-associated SNPs in Block 1, although this region does map to a recently identified noncoding RNA, called ANRIL
Noncoding RNAs can alter expression of associated protein-coding genes through a number of mechanisms.38, 39
Such mechanisms include the knockdown of messenger RNAs and the alteration of gene transcription via the recruitment of chromatin-modifying enzymes or epigenetic silencing. ANRIL
has been shown to be expressed in vascular endothelial cells, monocyte-derived macrophages, coronary smooth muscle cells and other cell types known to be affected by atherosclerosis, making it a strong candidate gene for the chromosome 9p disease associations reported.40
Recently, Jarinova and colleagues have reported that a conserved sequence within this locus has enhancer activity and that the associated haplotype alters the regulatory sequence of ANRIL
and changes expression levels of this gene. Through additional experiments, these authors then showed that ANRIL
expression changes correlated with changes in expression of other genes, particularly those in pathways associated with cell proliferation.41
Genotype associations with differential ANRIL
gene expression have also been independently reported other groups.42, 43
Thus, risk alleles in the 9p21.3 region may act by altering ANRIL
expression levels and in turn potentially influence a wide variety of vascular responses. Ultimately, a molecular profiling of the appropriate locus sequences is mandatory to identify the causal variant, which reliably associates with CAD-related phenotypes. Understanding more fully the molecular basis underlying the 9p21.3 association may have important implications for understanding, preventing, and treating heart disease.
In summary, we confirm that common SNPs on chromosome 9p21.3 are associated with CAC, a well-recognized subclinical marker of CAD and a predictor of MI. We find that one mechanism underlying the well-replicated association of this locus with MI/CAC is likely to involve increased platelet reactivity, also a risk factor for CVD events. This observation is consistent with the observation that this locus is a risk factor for thromboembolic disease more generally, including stroke. Additional studies, including in subjects from other ethnic groups and with varying degrees of CVD severity, to further define the importance of platelet reactivity in those who carry the at-risk genotype may provide mechanistic insights towards personalized medicine through genotype-specific interventions that target platelet reactivity.