Cardiovascular disease including stroke is the most frequent cause of death and a major cause of disability worldwide 1
. This study demonstrates that the recently identified locus for CAD on chromosome 9p21.3 is also implicated in risk of atherosclerotic stroke. This relationship between the locus and risk of stroke appears to be mediated through mechanisms that are not dependent on the presence of MI, CAD, or other vascular risk factors. The observed ORs are substantial and the population attributable risk (PAR) is high, suggesting that the 9p21.3 region is a major locus for atherosclerotic stroke. Our findings add to recent observations in patients with abdominal aortic aneurysms (AAA) and intracranial aneurysms (IA) 13
. Together these findings reveal a key role of the chromosome 9p21.3 region in arterial disease.
The validity of our findings is supported by several observations. First, associations between SNPs on 9p21 and atherosclerotic stroke were detected in both screening samples and were subsequently confirmed in the meta-analysis of the overall sample totalling 4376 IS cases and 4305 controls. Second, the ORs for the lead SNP (rs1537378) were remarkably consistent across subgroups including various populations from different geographical regions and ethnic backgrounds ( and Supplementary Figure 2
). Third, associations in the overall sample were robust when we controlled for potential confounders as well as intermediate variables. Fourth, the signal was strictly confined to a single stroke subtype both in the screening samples and in the overall population suggesting that the signal is clearly delimited and not related to stroke in general.
Significant associations were found with several SNPs covering a genomic interval of more than 100kb. It is evident from the distribution of associated SNPs in this region that the causally responsible variant remains to be identified. The genetic interval overlaps with exons 18-24 of ANRIL
, a newly annotated gene encoding a large antisense non-coding RNA 25
has recently been shown to be expressed in human atheromatous vessels including both AAA and carotid endarteriectomy (CEA) samples 12
. It was further found to be expressed in isolated vascular endothelial cells, monocyte-derived macrophages and coronary smooth muscle cells all of which have a role in atherosclerosis. As for most non-coding RNAs the cellular function of ANRIL
is still unknown. However, it appears from the above findings that ANRIL
represents a good candidate for atherosclerosis risk. The genetic interval on 9p21 further overlaps with exon 5 of a splicing variant of the methylthioadenosine phosphorylase (MTAP)
gene and is relatively close to the coding sequences of genes for two cyclin-dependent kinase inhibitors CDKN2a
(encoding p16INK4a) and CDKN2B
(encoding p15INK4b). These genes play a key role in regulating cell proliferation, cell senescence, and apoptosis 26
, and may be implicated in atherosclerosis through their role in transforming growth factor (TGF)-β-induced growth inhibition 27,28
. Of note, there is evidence for a coordinated transcriptional regulation of ANRIL
as well as other genes in the 9p21.3 region 25
. Further work is needed to determine, whether the association between atherosclerotic stroke and the 9p21 region is mediated through these genes or other pathways possibly through long-range regulatory effects.
Apart from demonstrating an association with atherosclerotic stroke we also replicated the reported association between 9p21.3 and both MI and CAD. The associated alleles and the mode of inheritance (recessive model, data not shown) match well with those previously reported for this phenotype and the OR are close to those found in recent studies 6,13
Stroke, most notably atherosclerotic stroke, shares common risk factors and mechanisms with MI and CAD 15
. Further, there is substantial comorbidity between stroke, CAD, and MI. Between 20 and 40% of stroke patients have an abnormal cardiac stress response with the rate being highest in patients with atherosclerotic stroke 15,29
. Carotid intima-media thickness (IMT), a quantitative marker, and intermediate phenotype for early atherosclerosis strongly correlates with both CAD and risk of MI 30,31
. Thus, one might speculate, that the 9p21.3 region contributes to vascular disease by promoting atherosclerosis.
However, several observations suggest the mechanisms are more complex. First, in this study associations with atherosclerotic stroke and CAD/MI were found to be independent from each other ( and Supplementary Table 4
) suggesting that the underlying processes do not run strictly in parallel. Second, the two lead SNPs for CAD (rs10757278-G) and atherosclerotic stroke (rs1537378) identified here are more than 70kb apart. Accumulating data suggests that these two lead SNPs are likely to represent the same signal, each serving as a marker for the same causal variant 11
. Thus, the difference in associated SNPs may just be coincidental. Alternatively however, this observation might reflect differences in the genetic architecture of atherosclerotic stroke and CAD/MI with regard to the 9p21.3 region as previously documented for CAD and type 2 diabetes 13
. Third, it was recently shown that the 9p21.3 region is also implicated in risk of AAA and IA which are pathologically distinct from atherosclerosis 13
. In fact, atherosclerosis is not considered a risk factor for IA 32,33
. Together, these findings raise the possibility that the chromosome 9p21 region has a broader role in large artery disease possibly by impacting on vascular remodelling or repair rather than atherosclerosis per se 34-36
. This concept would agree with results from a recent study that found no association of the rs1333049 genotype with carotid IMT in 3572 population-based subjects from Finland 37
. Further studies are needed to determine the mechanisms by which the chromosome 9p21.3 region affects vascular risk. Importantly, the observed effects of 9p21 on atherosclerotic stroke were found to be independent from established risk factors for cardiovascular disease. This finding agrees with similar data for CAD and MI showing that the effects of 9p21 on MI and CAD are not mediated by known vascular risk factors 7,9
The strongest associations were seen with rs1537378. This SNP was also significant in the overall group of IS patients and in patients with stroke of “undetermined” aetiology. Most likely these associations likewise reflect an association with atherosclerotic stroke as both categories include patients with atherosclerotic stroke and there was no significant signal for any of the other stroke subtypes including cardioembolic stroke and small vessel stroke. In accord with this, OR were in the same direction but lower than for atherosclerotic stroke, which would be expected if the results were driven by atherosclerotic stroke.
Our findings might resolve some of the inconsistencies among recent studies that have looked for an association between sequence variants on 9p21 and IS risk 13,16,17
. These studies provided no 13,16
or marginally 17
significant evidence for an independent association between SNPs on 9p21.3 and IS as a whole. However, sample sizes were limited (between 249 and 705 IS cases) and stroke subtypes such as atherosclerotic stroke were not considered separately. The current study documents the need for large sample sizes particularly when the ORs are moderate and associations are limited to stroke subtypes.
This study also has potential limitations. First, genotyping involved different methods and platforms which in theory might have impacted on our findings. However, we consider this possibility very unlikely for the following reasons: i), cases and controls were numerically well balanced on all three platforms; ii), ORs were similar across all sites and platforms (); iii), ORs were similar for six neighbouring SNPs, which can thus be considered technical replicates. Second, samples were ascertained through different protocols without central phenotyping. Conceivably this might have affected the results. Yet again, we found no evidence for a center effect suggesting that differences in case ascertainment and phenotyping are less relevant. Finally, we cannot exclude that CAD and MI remained underdiagnosed in our stroke patients which in turn could have contributed to the association with stroke. However, a major bias seems unlikely. For one, this study replicated the known association with CAD and MI, which suggests that the level of phenotyping was good. Second, there was no evidence for an interaction between any of the SNPs and CAD or MI in multivariate analyses when considering stroke as the dependent variable. Regardless of this relationship the association between SNPs on 9p21 region and atherosclerotic stroke remains considerable and important.
The consistency of associations across a broad range of subjects and vascular conditions in conjunction with the substantial increase in IS risk particularly in homozygous individuals suggests that genotyping of this locus might have considerable clinical utility in risk prediction provided that the current findings are supported in further large population-based studies. Future studies will determine whether genotyping of the 9p21 region adds to already established scores for cardiovascular risk such as the Framingham risk score 38
. Identifying individuals at risk for cardiovascular events may impact on preventive strategies. In this regard, the current findings might have therapeutic implications.
In conclusion, this study reveals an unexpectedly broad role of the chromosome 9p21 region in arterial disease. Identification of the molecular pathways and biological mechanisms might offer new perspectives for therapeutic interventions.