Differential Methylation Signatures in Severely Calcified Carotid Plaques by Genome-Wide Comprehensive Analysis
Hiroyuki Katano1, Yusuke Nishikawa1, Hiroshi Yamada1
1Department of Neurosurgery, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
Background:
The precise cellular behaviors of calcification, including its molecular and genetic activities, have not yet been fully established for carotid plaques.
Objective:
We sought specific genes with tissue-specific differential methylation associated with carotid calcification status.
Methods:
We classified eight plaques from carotid endarterectomy patients as high- or low-calcified based on their Agatston calcium scores. We analyzed differential DNA methylation and performed bioinformatics data mining.
Results:
A high correlation of average methylation levels (β-values) in promoter regions between high- and low-calcified plaque groups was observed. A principal component analysis of DNA methylation values in promoters of specimens revealed two independent clusters for high- and lowcalcified plaques. Volcano plots for methylation differences in promoter regions showed that significantly hypomethylated probes were more frequently found for high-calcified plaques than more methylated probes. Differential hypomethylation of receptor activity-modifying protein 1 (RAMP1) in high-calcified plaques was commonly extracted in both the promoter region and the cytosinephosphate- guanine (CpG) island shore region, where differential methylation had been reported to be more tissue-specific. Kyoto Encyclopedia of Genes and Genomes pathway analysis annotated a pathway associated with vascular smooth muscle contraction in the differentially methylated genes of the promoter and CpG island shore regions in high-calcified plaques.
Conclusion:
Among the extracted differentially methylated genes, hypomethylated genes were more dominant than more methylated genes. The augmentation of RAMP1 by hypomethylation may contribute to the enhancement of anti-atherosclerotic effects and hence stability in high-calcified plaques. These results contribute to our understanding of the genetic signatures associated with calcification status and cellular activity in carotid plaques.
