Identification of novel hyper- or hypomethylated CpG sites and genes associated with atherosclerotic plaque using an epigenome-wide association study

DNA methylation is an important epigenetic modification that has been implicated in the pathogenesis of atherosclerosis. Although previous studies have identified various CpG sites and genes whose methylation is associated with atherosclerosis in populations with European or Mexican ancestry, the genome-wide pattern of DNA methylation in the atherosclerotic human aorta is yet to be elucidated in Japanese individuals. In the present study, a genome-wide analysis of DNA methylation at ~853,000 CpG sites was performed using 128 postmortem aortic intima specimens obtained from 64 Japanese patients. To avoid the effects of interindividual variation, intraindividual paired comparisons were performed between atheromatous plaque lesions and corresponding plaque-free tissue for each patient. Bisulfite-modified genomic DNA was analyzed using a specific microarray for DNA methylation. DNA methylation at each CpG site was calculated as the β value, where β = (intensity of the methylated allele)/(intensity of the methylated allele + intensity of the unmethylated allele + 100). Bonferroni's correction for statistical significance of association was applied to compensate for multiple comparisons. The methylation of 2,679 CpG sites differed significantly (P<5.86×10(−8)) between atheromatous plaque lesions and the corresponding plaque-free intima, with 2,272 and 407 CpG sites in atheromatous plaques being hyper- or hypomethylated, respectively. A total of 5 hypermethylated CpG sites in atheromatous plaques were demonstrated to have a difference in β value of >0.15 (plaque lesion-plaque-free intima) and 11 had a β ratio of >1.50 (plaque/plaque-free intima). A further 15 and 17 hypomethylated CpG sites in atheromatous plaques were observed to have a difference in β value of <−0.15 or a β ratio of <0.67, respectively. According to these limits, a total of 16 novel genes that were significantly hyper- or hypomethylated in atheromatous plaque lesions compared with the plaque-free intima were identified in the present study. The results of the present study suggest that the methylation of these genes may contribute to the pathogenesis of atherosclerosis in the Japanese population.