Whole genome methylation profiling of retinal pigment epithelium reveals differential methylation and gene expression associated with AMD

Abstract

Purpose : Purpose: Age-related macular degeneration (AMD) is the foremost cause of blindness in the elderly. Tissue-specific epigenetic mechanisms of gene regulation are of considerable interest in AMD. We aimed to identify differentially methylated (DM) genes associated with AMD in the RPE and qualify the transcriptional activities of DM loci.

Methods : Genome-wide DNA methylation profiling (Illumina 450k BeadChip array) was performed on RPE cells from 44 human donor eyes (25 AMD and 19 normal controls). We validated the findings using bisulfite pyrosequencing in 55 RPE samples (30 AMD and 25 normal controls) including technical (n=38) and independent replicate samples (n=17). RT-qPCR and RNA sequencing was performed on independent donor RPE samples to assess gene expression changes. Transcriptional activities of loci of differential methylation were investigated using open access ENCODE data and a CpG-free luciferase reporter vector in cultured cells.

Results : Genome-wide DNA methylation profiling identified differential methylation of multiple loci including the SKI (p=1.18x10-9), GTF2H4 (p=7.03x10-7), and TNXB (p=6.30x10-6) genes in AMD. Bisulfite pyrosequencing validated the DM loci in the SKI proto-oncogene, and GTF2H4. The region surrounding the DM SKI locus displayed enrichment for histone modifications associated with gene enhancer activities in numerous cell types. We further demonstrated differential expression of SKI, GTF2H4, EIF2AK3 and TNXB in the RPE of independent AMD donors.

Conclusions : We report the largest genome-wide methylation analysis of RPE in AMD along with associated gene expression changes to date. The novel DM genes SKI and GTF2H4 regulate disease pathways implicated in AMD, including TGF beta signaling (SKI) and transcription dependent DNA repair mechanisms (GTF2H4). The novel targets identified in our study may be used for therapeutic intervention studies in AMD.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.