Methylation of p15INK4b and Expression of ANRIL on Chromosome 9p21 Are Associated with Coronary Artery Disease

BACKGROUND Genome-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 FINDINGS The methylation status of six candidate genes (BAX, BCL-2, TIMP3, p14(ARF), p15(INK4b) and p16(INK4a)) 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 p15(INK4b) significantly increased in CAD patients (p = 0.006). To validate and dissect the methylation percentage of each target CpG site at p15(INK4b), pyrosequencing was performed, finding CpG +314 and +332 remarkably hypermethylated in CAD patients. Further investigation determined that p15(INK4b) 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 p15(INK4b) and p16(INK4a) 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 p15(INK4b) methylation is associated with CAD and ANRIL expression. The epigenetic changes in p15(INK4b) methylation and ANRIL expression may involve in the mechanisms of chromosome 9p21 on CAD development.