Background and objectives: The present study initially profiled and identified gene-specific promoter methylation changes in peripheral leukocytes (WBC) of patients having severe atherosclerosis. Then, the gene-specific promoter methylation changes of WBC were validated in atherosclerotic plaques by comparison with normal intima.
Methods: We first profiled and identified specific genes showing promoter methylation differences between WBC of 8 patients having severe atherosclerosis and of other 8 patients having no-atherosclerosis in extra- and intra-cranial vessels using Digital Restriction Enzyme Analysis of Methylation (DREAM) sequencing. To select candidate CpG sites from DREAM sequencing data, we used criteria; located in -1,000 bp from transcription start sites, < or >5% of methylation differences between two groups, and <0.05 false positive rate. Significance of the gene-specific promoter methylation changes identified from WBC were validated from normal intima and atherosclerotic plaques obtained from common carotid artery of 20 human cadaveric donors (male, 13, age, 62.2±14.2 years).
Results: On DREAM sequencing, CpG sites located in promoter regions of ADCK5 (no-atherosclerosis, 67.57% vs. atherosclerosis patients, 55.18%), KNDC1 (67.08% vs. 56.81%), MTNR1B (11.5% vs. 3.8%) had over 5% of the methylation differences with <0.05 false positive rate. On validation experiments using human vascular tissues, all ADCK5 (normal intima, 4.02±1.02; plaque, 4.79±1.14, p<0.05), KNDC1 (9.76±2.25 vs. 12.11±2.91, p<0.05), MTNR1B (14.08±2.93 vs. 17.53±5.07, p<0.05) showed significant higher promoter methylation in atherosclerotic plaques than normal intima.
Conclusion: The present study identified promoter methylation changes in ADCK5, KNDC1, and MTNR1B from WBC of atherosclerotic patients, and validated their significance in human atherosclerotic tissues. The three genes showed possibilities as epigenetic biomarkers for atherosclerosis, even though future validations are needed for their significance in atherosclerosis development.