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Lakshmi Pulagam, Nathan Morris, Penny Benchek, Peter Scacheri, Sudha Iyengar; Enhancer elements show enrichment for AMD-associated genetic variants. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6188. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Enhancers are complex yet highly coordinate cis-regulatory elements that regulate gene expression. Although age-related macular degeneration (AMD)-associated variants at >12 loci have been identified through GWAS, it is very difficult to identify mechanistic/causal variants because they co-occur with nearby markers that are in strong linkage disequilibrium (LD). The purpose of this study was to determine whether AMD-associated variants lie within enhancer elements.
We captured retina-specific enhancer profiles together with expression abundance in primary Retinal Pigment Epithelium (RPE) cells (HRPEpiC), using ChIP Seq for enhancer chromatin marks, H3K4me1 and H3K27ac and RNA Seq for expression. Paired-end sequenced data (2x100bp) was analyzed using BWA, and ChIP Seq peaks were called using MACS, after using input DNA as control. AMD-associated GWAS SNP(s) and RPE enhancer colocalization profiles were plotted. At each locus, the local LD profile surrounding the sentinel SNP was captured from HapMap2. To determine the likelihood of these results being obtained by chance alone, we randomly selected 1000 other sets of SNPs genome-wide with a similar LD profile and frequency to the GWAS hit(s), and conducted an identical test for each set.
Peak analysis of the ChIP Seq data revealed that some of the best reported AMD-association signals localize either within or in proximity of enhancer peaks. For example, variants near ARMS2/HTRA1 and CETP are either co-localized within or very close to enhancer peaks (False Discovery Rate [FDR] = 0.22%, p-value = 1.23 10-27, and FDR = 0.34%, p-value = 4.88 x 10-17 respectively). Among all loci, 35% of GWAS hits were either within an enhancer peak, or were in LD (r2 ≥ 0.8) with a variant in the enhancer. We calculated the percentage of SNP sets which were either as enriched or more enriched for enhancer profiles. Among the 1000 simulated experiments genome-wide, only 3.7% had a result as extreme or more extreme than the AMD GWAS-enhancer profiles.
Our results indicated that 35% of the AMD-associated GWAS hits localize within enhancer profiles. Comparison with 17 other cell lines from ENCODE shows that this enhancer profile is likely retina-specific. In summary, enhancer data supplemented with other epigenetic marks from ENCODE is very useful in defining mechanistic events to further understand the role of these variants in the regulation of AMD pathogenesis.
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