Transcriptomic Profiling of Posterior Polymorphous Corneal Dystrophy

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. Overall design: Cornea endothelial tissue from two patients with PPCD, ages 15 (P5) and 5 (P6), were obtained when the patients underwent a Descemet membrane endothelial keratoplasty (P5) or a penetrating keratoplasty (PK) (P6) . The Descemet membrane and endothelium were placed onto a piece of filter paper and submerged in TRI Reagent (Thermo Fisher, Waltham, MA) for RNA isolation. Human cornea tissue from two age-matched donors, ages 6 and 17, were obtained from commercial eye banks. Descemet's membrane and corneal endothelium were stripped from the donor corneas using a published technique and placed into TRI Reagent for RNA isolation. To assess the effect of decreased ZEB1 protein on gene expression in primary human corneal endothelial cells (pHCEnC), confluent pHCEnC cultures were transfected with 10nM anti-ZEB1 siRNA (rGrGrCrCrUrArCrArArUrArArCrUrArGrCrArUrUrUrGrUTG) or scrambled siRNA (OriGene Technologies, Rockville, MD) using Lipofectamine LTX (Life Technologies) per the manufacturer's instructions. Performed in triplicates at 24,48, 72 and 96 hours post-transfection, the transfected cells were lysed in either Tri Reagent (Sigma-Aldrich, St. Louis, MO, USA), or radioimmunoprecipitation assay (RIPA) buffer for RNA and protein isolation, respectively. RNA from 24 hr and 72 hrs post-transfection with ZEB1 siRNA (n=3) was sequenced by RNAseq. RNA from pHCEnC transfected with CNTL (scramble) siRNA at 24 hours post-tranfection (n=3) was also collected and processed for RNAseq.