Genome-wide analysis of DNA methylation in human atherosclerosis

Abstract

Purpose: Epigenetic modifications, especially changes in DNA methylation at gene promoters, have been implicated in the pathogenesis of various complex diseases. Given that cellular patterns of DNA methylation are affected by environmental and dietary factors as well as genetic variants, knowledge of the DNA methylome of atherosclerotic lesions may provide insight into the molecular mechanisms and interindividual outcome variability of atherosclerotic diseases. To date, however, analysis of DNA methylation in atherosclerosis has been limited to a relatively small number of selected candidate genes. We therefore examined the pattern of DNA methylation in the atherosclerotic human aorta at the genome-wide level. Methods: A total of 48 human postmortem aortic intima specimens was studied. To avoid the effects of interindividual variation, we performed intraindividual paired comparisons between atheromatous plaque lesions and corresponding plaque-free tissue in 24 subjects. The tissue samples were frozen at -80°C immediately after dissection. DNA methylation was analyzed in bisulfite-modified genomic DNA with a DNA methylation-specific microarray that includes 485,553 CpG sites distributed throughout the genome (HumanMethylation450 BeadChip, Illumina). To compensate for multiple comparisons, we applied Bonferroni's correction and adopted a criterion of P < 1.0 × 10-7 for statistical significance of association between changes in DNA methylation and atherosclerosis. Results: DNA methylation was significantly increased at 16 CpG sites in 11 genes and significantly decreased at 29 CpG sites in 20 genes in atheromatous plaque. Three of the hypermethylated genes (MAP4K4, ZEB1, FYN) and three of the hypomethylated genes (HECA, EBF1, NOD2) have been implicated in atherosclerosis or cardiovascular disease. Hyper- or hypomethylation of DNA is associated with down- or up-regulation of gene transcription, respectively. We therefore examined the effects of suppression of MAP4K4, ZEB1, or FYN expression with short hairpin RNAs or of overexpression of HECA, EBF1, or NOD2 on gene expression in cultured HEK293 cells with the use of DNA microarrays (HumanHT-12 v4 Expression BeadChip, Illumina). The expression of several atherosclerosis-related genes was significantly up- or down-regulated by these manipulations. Conclusions: MAP4K4, ZEB1, and FYN were significantly hypermethylated, whereas HECA, EBF1, and NOD2 were hypomethylated, in atheromatous plaque lesions compared with plaque-free intima. Our findings thus suggest that epigenetic mechanisms may contribute to the pathogenesis of atherosclerosis.