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Doug D Chung, Ricardo F Frausto, Benjamin Lin, E. Maryam Maryam Hanser, Anthony J Aldave; Transcriptomic Profiling of Posterior Polymorphous Corneal Dystrophy. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5648.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the molecular basis of posterior polymorphous corneal dystrophy (PPCD) by examining the transcriptome in affected individuals and the effect of decreased ZEB1 expression on corneal endothelial gene expression.
RNA-seq analysis was performed on corneal endothelium from individuals with PPCD, age-matched controls, and primary corneal endothelial cell cultures (pHCEnC) transfected with siRNA targeting ZEB1. Transcriptomic analyses were performed, including differential gene expression, gene ontology and pathway. The expression of selected differentially expressed genes was validated by qPCR and/or assessed by in situ hybridization in the corneal endothelium of independent cases of PPCD.
Corneal endothelium from two individuals with PPCD expressed (RPKM > 0.1) 83.7% (87/104) and 53.8% (56/104) of the 104 protein coding genes specific to ex vivo corneal endothelial cells. Comparing gene expression in the corneal endothelium of these affected individuals, one with and one without a truncating ZEB1 mutation, to controls revealed 5049 and 5952 differentially expressed (fold-change ≥ 2 and RPKM values ≥ 0.1) protein coding genes. Thirty-two genes associated with ZEB1 and three genes (BMP4, CCND1, ZEB1) associated with OVOL2 were differentially expressed in the same direction in both individuals with PPCD. Corroborating our RNAseq results, in situ hybridization in corneas from affected individuals demonstrated increased transcript levels of BMP4 and CCND1 and lower ZEB1 levels compared to a control cornea. RNA-seq analysis and immunohistochemical staining demonstrated variable expression of each type IV collagen in corneas from affected individuals. Decreasing ZEB1 expression in pHCEnC resulted in altered expression (fold-changes ≥ 1.25, p-values <0.05, and RPKM values ≥ 0.1) of 711 protein coding genes that are associated with canonical pathways involved in cell proliferation, migration, adhesion, and morphology.
Identification of the altered transcriptome in PPCD and in a cell-based ZEB1 knockdown model of PPCD provides insights into the molecular alterations that characterize PPCD. Further study of the differentially expressed protein coding genes that are known to interact with ZEB1 and OVOL2 is expected to identify other candidate genes for PPCD that may harbor mutations in affected individuals without a ZEB1 or OVOL2 mutation.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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