Abstract
BackgroundGenome-wide association studies have identified that multiple single nucleiotide polymorphisms on chromosome 9p21 are tightly associated with coronary artery disease (CAD). However, the mechanism linking this risk locus to CAD remains unclear.Methodology/Principal FindingsThe methylation status of six candidate genes (BAX, BCL-2, TIMP3, p14ARF, p15INK4b and p16INK4a) in 205 patients and controls who underwent coronary angiography were analyzed by quantitative MethyLight assay. Rs10757274 was genotyped and expression of INK4/ARF and antisense non-coding RNA in the INK4 locus (ANRIL) was determined by real-time RT-PCR. Compared with controls, DNA methylation levels at p15INK4b significantly increased in CAD patients (p = 0.006). To validate and dissect the methylation percentage of each target CpG site at p15INK4b, pyrosequencing was performed, finding CpG +314 and +332 remarkably hypermethylated in CAD patients. Further investigation determined that p15INK4b hypermethylation prevalently emerged in lymphocytes of CAD patients (p = 0.013). The rs10757274 genotype was significantly associated with CAD (p = 0.003) and GG genotype carriers had a higher level of ANRIL exon 1–5 expression compared among three genotypes (p = 0.009). There was a stepwise increase in p15INK4b and p16INK4a methylation as ANRIL exon 1–5 expression elevated (r = 0.23, p = 0.001 and r = 0.24, p = 0.001, respectively), although neither of two loci methylation was directly linked to rs10757274 genotype.Conclusions/Significance p15INK4b methylation is associated with CAD and ANRIL expression. The epigenetic changes in p15INK4b methylation and ANRIL expression may involve in the mechanisms of chromosome 9p21 on CAD development.
Highlights
Genome-wide association studies (GWAS) have found that single nucleotide polymorphisms (SNPs) on chromosome 9p21 (Chr9p21) affect susceptibility to coronary artery disease (CAD) in Caucasian population [1,2,3,4], and these associations have been reproduced in other populations [5,6,7,8]
Previous studies show that both deletion of Chr9p21 locus and repression of INK4/ARF or antisense non-coding RNA in the INK4 locus (ANRIL) expression have their impacts on atherosclerosis [10,11]
Our data indicated that p15INK4b methylation was an important event in atherosclerosis, and such potential bridge between genotype and p15INK4b methylation might be mediated by altered expression of ANRIL
Summary
Genome-wide association studies (GWAS) have found that single nucleotide polymorphisms (SNPs) on chromosome 9p21 (Chr9p21) affect susceptibility to coronary artery disease (CAD) in Caucasian population [1,2,3,4], and these associations have been reproduced in other populations [5,6,7,8]. Most of SNPs are highly correlated and located within a roughly 53-kb linkage disequilibrium (LD) region in which a long non-coding RNA, known as antisense non-coding RNA in the INK4 locus (ANRIL), is transcribed. Previous studies show that both deletion of Chr9p21 locus and repression of INK4/ARF or ANRIL expression have their impacts on atherosclerosis [10,11]. Genome-wide association studies have identified that multiple single nucleiotide polymorphisms on chromosome 9p21 are tightly associated with coronary artery disease (CAD). The mechanism linking this risk locus to CAD remains unclear
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