Changes In Promoter Methylation In Aire1 And Alox12: Novel Epigenetic Markers In Atherosclerotic Tissues

Objective: The present study aimed to identify and validate specific genes developing changes in promoter methylation in atherosclerotic plaques. Approach and Results: We initially comprehensively profiled DNA methylation signatures via Solexa sequencing, using amplicons prepared via methylated-CpG island amplification (MCA) in genomic DNA obtained from 5 plaques acquired through carotid endarterectomy (CEA) to identify genes with altered promoter methylation. The changes in promoter methylation of these genes were validated via bisulfite pyrosequencing from plaques and non-plaque intima of the common carotid artery (CCA) harvested from 20 cadavers as well as from 27 CEA plaques and 5 human umbilical vein endothelial cell (HUVEC) lines. Expression changes based on changes in promoter methylation in these genes were evaluated in 20 CEA plaques and in 2 HUVEC cell lines after demethylation with 5-aza-2'-deoxycytidine. Upon MCA-Solexa sequencing, five genes ( AIRE1 , ALOX12 , FANK1 , NETO1 , and SERHL2 ) displayed changes in promoter methylation. Of these, AIRE1 and ALOX12 displayed significantly greater promoter methylation in plaques rather than in the non-plaque intima of the CCA. Furthermore, promoter methylation was greater in AIRE1 in CEA plaques than in HUVECs, while that in ALOX12 was greater in HUVEC cells than in CEA plaques. Concurrently, gene expression levels decreased with an increase in promoter methylation in these genes in CEA plaques. After DNA demethylation in HUVEC cells, AIRE1 and ALOX12 expression levels increased by 23 and 52 folds. Conclusions: Changes in promoter methylation in AIRE1 and ALOX12 were identified and validated in atherosclerotic plaques, thereby establishing novel epigenetic markers for atherosclerosis.