Elsevier

Atherosclerosis

Volume 220, Issue 2, February 2012, Pages 449-455
Atherosclerosis

Genetic variants at the 9p21 locus contribute to atherosclerosis through modulation of ANRIL and CDKN2A/B

https://doi.org/10.1016/j.atherosclerosis.2011.11.017Get rights and content

Abstract

Genome-wide association studies (GWAS) have identified genetic variants contributing to the risk of cardiovascular disease (CVD) at the chromosome 9p21 locus. The CVD-associated region is adjacent to the two cyclin dependent kinase inhibitors (CDKN)2A and 2B and the last exons of the non-coding RNA, ANRIL. It is still not clear which of or how these transcripts are involved in the pathogenesis of atherosclerosis.

Objective

We assessed the hypothesis that 9p21 locus polymorphisms influence the expression of the transcripts in the region (ANRIL, CDKN2A/B) and that these transcripts contribute to atherogenesis through the modulation of proliferation in VSMC.

Methods

We genotyped 18 SNPs (r2 < 0.8 and MAF > 0.05) across the region of interest: CDKN2A/B and ANRIL, encompassing the CVD-associated region. RNA and DNA were extracted from the blood of 57 volunteers (69–72 years old). Carotid ultrasound was performed in 56 subjects. CDKN2A/B and ANRIL (exons 1–2 and 17–18) expression was measured employing RT-PCR. Gene expression and cell growth were evaluated in cultured VSMC after the siRNA-mediated knock-down of ANRIL.

Results

The risk alleles for atherosclerosis-related phenotypes were consistently associated with a lower expression of ANRIL when evaluating exons 1–2. Common carotid artery stenosis was associated with a significantly lower (P < 0.01) expression of ANRIL (exons 1–2). ANRIL knock-down in VSMC caused significant variation in expression of CDKN2A/B (P < 0.05) and reduction of cell growth (P < 0.05) in vitro.

Conclusion

Disease-associated SNPs at the 9p21 locus predominantly affect the expression of ANRIL. Overall, our results suggest that several CVD-associated SNPs in the 9p21 locus affect the expression of ANRIL, which, in turn modulate cell growth, possibly via CDKN2A/B regulation.

Section snippets

Study sample

For the genetic association study between the SNPs: rs1333049, rs564398 and rs2811712 (see SNPs in green in Fig. 1), and CAD, patients with documented evidence of angiographically diagnosed coronary stenosis (including acute myocardial infarction, angina pectoris and, silent myocardial ischemia) (subjects characteristics in Supplementary Table 1 and Supplementary Methods).

For peripheral blood RNA expression analysis, we obtained whole blood samples from 57 volunteers (69–72 years old) from Asago

Confirmation of 9p21 association in the Japanese sample

We assessed the possible association of the three SNPs: rs1333049, rs564398, and rs2811712 with CAD. We confirmed the rs1333049 association with CAD in our sample (Controls CC:101; CG:224; GG:122; Cases CC:72; CG:116; GG:43; age adjusted P-value < 0.01) (Fig. 2A); however, we were not able to detect any significant association of CAD with either rs564398 or rs2811712 (see Supplementary Table 4).

Expression analysis of CDKN2A/B and ANRIL in blood

The linkage disequilibrium (LD) map for the SNPs genotyped in this study and a transcriptional

Discussion

Since the GWAs have revealed a CAD association at the 9p21 locus, researchers have hypothesized a potential regulatory role of the associated SNPs in the expression of ANRIL, CDKN2A, and CDKN2B. However, publications demonstrating a correlation between the risk genotypes and the expression of the transcripts in the locus have reported contradictory results [18], [23], [24], highlighting the need to replicate such studies to clarify true associations. Probably, this lack of consistency is due to

Acknowledgements

We would like to express our gratitude to Ms. Yuki Imaizumi and Ms. Takako Baba for their continuous support in this investigation.

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    This study was supported in part by grants-in-aid from the Program for Promotion of Fundamental Studies in National Institute of Biomedical Innovation of Japan (HR: 22-2-5), the Ministry of Education, Culture, Sports, Science, and Technology of Japan (KK: 22510211) and NOVARTIS Foundation for Gerontological Research (KK).

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