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Allan A. Hunter, III, Anna K. Hunter, Alyssa Cwanger, Paul A. Spechler, Zhe Zhang, Ed deZoeten, Joshua L. Dunaief; DNA Methylation Is Associated With Altered Gene Expression in AMD. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5980.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies indicate that AMD disease risk is influenced by several susceptibility loci. To assess the potential contribution of epigenetic regulation to AMD pathogenesis, we evaluated DNA methylation, a tissue specific genetic modulation that affects gene expression.
Using the Infinium HumanMethylation 27 Illumina platform, we performed DNA bisulfite sequencing to compare the methylation status between patients with AMD vs. age-matched controls. DNA was extracted from frozen retinal pigment epithelium (RPE) and neurosensory retina (NSR) from 21 post-mortem human eyes. Bisulfite conversion of genomic DNA was done using EZ DNA Methylation kit (Zymo Research). RNA was isolated with the RNeasy Mini Kit (Qiagen Inc). TaqMan gene expression assays were used for qPCR analysis.
The Illumina platform determines the methylation status of over 27,578 CpG dinucleotides spanning 14,495 genes. CpG measurements were represented as β values outputted by BeadStudio. CpG measurements of all samples were further normalized by QSPLINE method using autosomal sites. 67 sites having p<0.1 and β difference more than 0.02 were selected. We compared these results to gene expression profiles in the same tissues determined by the Affymetrix exon microarray. The data set includes 23,536 unique Entrez genes. Of the 67 genetic loci identified by the Illumina platform, 45 genes had expression changes recorded through the expression array with fold-change > 25% and p < 0.05. These exon expression results were confirmed by qPCR. Compared to controls, hypomethylation was noted in 8 genes with elevated expression levels, while hypermethylation was found in 11 genes with decreased expression. Of these 19 genes identified, some had gene function considered important in AMD pathogenesis, e.g. oxidative stress and inflammation modulation.
These data suggests that DNA methylation of cis regulatory CpG islands may play a role in regulation of genes that contribute to AMD pathogenesis.
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