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Jingwen Cai, Michelle Dianne Drewry, Kristin Perkumas, Michael Dismuke, Iris Navarro, Michael A Hauser, W Daniel Stamer, Yutao Liu; DNA methylation in Human SC Cells with Glaucoma. Invest. Ophthalmol. Vis. Sci. 2016;57(12):804.
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© ARVO (1962-2015); The Authors (2016-present)
Schlemm’s canal (SC) endothelial cells contribute to the generation of conventional outflow resistance in normal and ocular hypertensive eyes, a major risk factor for developing glaucoma. This study aims to identify differentially methylated regions (DMRs) in primary cultures of human SC cells.
We examined the methylation pattern of 11 primary cultures of human cells (2 glaucoma / 5 normal SC cells, and 4 normal trabecular meshwork (TM) cells); which were isolated and characterized using established protocols. DNA methylation was profiled using Illumina Human Methylation 450 BeadChip. Raw data were extracted and exported using Illumina GenomeStudio software. After quantile normalization, DNA methylation data was analyzed using R package RnBeads in Bioconductor. DMRs were filtered with p<1E-5, methylation change at least 0.1, and false discovery rate <0.05. Nearest genes within each CpG site were annotated using R package FDb.InfiniumMethylation.hg19. Gene ontology and pathway analysis were performed using WebGestalt.
Cluster analysis of DMRs indicated a significant segregation between non-glaucoma SC cells and TM cells, and also between SC cases and controls. After quality control, we identified 298 highly significant DMRs. Among them, 71 DMRs were within 1kb of a nearby gene and 129 DMRs were within 5kb of a nearby gene. The ontology analysis demonstrated significant enrichment in positive regulation of cell adhesion, negative regulation of endothelial cell proliferation, stress fiber and actin filament bundle. KEGG pathway analysis showed enrichment in cell adhesion and gap junctions. Several glaucoma-related genes were identified in the list, including THBS1, PLEKHA7, COL24A1, and LAMA3. Integrated with our previous expression study of human SC cells with glaucoma, 4 genes showed similar results in methylation and expression, including hypermethylated TGFBR3 and RHOU, plus hypomethylated NET1 and ANKLE2.
This study represents the first genome-wide DNA methylation profiling in human primary SC cells. We have identified many DMRs in glaucoma SC cells that may be associated with SC cell adhesion, extracellular matrix turnover and cell proliferation, which may contribute to the increased outflow resistance in glaucoma patients.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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