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Margaret M DeAngelis, Margaux A Morrison, Jeeyun Ahn, Evangeline E Tsironi, Silvestri Giuliana, M Elizabeth Hartnett, Joan W Miller, Kyu Hyung Park, Ivana K Kim, Lindsay Farrer; Genetic and epigenetic evidence for a pathway involving lipid metabolism and HTRA1 in AMD pathophysiology.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2185.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the previously reported hypothetical network of age related macular degeneration (AMD)-associated genes based on pathway analysis of Affy 6.0 microarray gene expression data from lymphoblastoid cell lines (LCLs) of those with and without AMD.
We genotyped TagSNPs from genes within the pathway (RGS13, CXCL13, RPS6KA2, ABCA1, and NLRP2), and performed gene-gene interaction analysis of the TagSNPs and previously reported AMD-associated SNPs from genes within the pathway (ARMS2, HTRA1, ROBO1, and RORA) in 4 cohorts (n = 2,224) representing all subtypes of AMD of varying ethnicities. We performed ChIP-Seq on DNA from LCLs of patients with and without AMD. To interrogate methylation status, bisulfite sequencing was performed on DNA and tissue from the retina and RPE/choroid of fresh donor eyes with and without AMD. This fresh eye tissue was also used to perform differential gene expression analysis using RT-PCR.
Employing a cases-only approach and meta-analysis, we found significant gene-gene interaction between the previously reported AMD genes ARMS2, HTRA1, ROBO1, and RORA and TagSNPs within ABCA1, RGS13, and RPS6KA2 (p<.01). ChIP-Seq analysis did not reveal any significant binding results. Three methylated CpG sites within ABCA1 were significantly associated with decreased risk of all AMD subtypes (p<.05). Gene expression analysis using fresh donor eye tissue revealed significantly decreased RGS13 and RORA expression within the macular retina tissue of AMD eyes compared to normal (p=.009 and .001, respectively). In the macula RPE of AMD eyes, significantly decreased expression was observed for both ABCA1 and ROBO1 compared to those without AMD (p=.016 and .039, respectively). All other genes examined, while expressed in both the macula and extra macula retina and RPE/choroid, did not show differential expression between AMD and normal eyes (n = 18). Further IPA pathway analysis of the significant genes showed this pathway to be involved in lipid metabolism.
We describe several lines of evidence, including gene-gene interaction, gene expression data, and tissue specific methylation that support the role of this previously reported hypothetical network in AMD pathogenesis. Further pathway analysis with these genes, including HTRA1, showed they may be involved in lipid metabolism and underlie AMD pathophysiology.
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