Transcriptomic Profiling of Posterior Polymorphous Corneal Dystrophy

Abstract

PurposeTo investigate the molecular basis of posterior polymorphous corneal dystrophy (PPCD) by examining the PPCD transcriptome and the effect of decreased ZEB1 expression on corneal endothelial cell (CEnC) gene expression.MethodsNext-generation RNA sequencing (RNA-seq) analyses of corneal endothelium from two PPCD-affected individuals (one with PPCD3 and one of unknown genetic cause) compared with two age-matched controls, and primary human CEnC (pHCEnC) transfected with siRNA-mediated ZEB1 knockdown. The expression of selected differentially expressed genes was validated by quantitative polymerase chain reaction (qPCR) and/or assessed by in situ hybridization in the corneal endothelium of four independent cases of PPCD (one with PPCD3 and three of unknown genetic cause).ResultsExpression of 16% and 46% of the 104 protein-coding genes specific to ex vivo corneal endothelium was lost in the endothelium of two individuals with PPCD. Thirty-two genes associated with ZEB1 and 3 genes (BMP4, CCND1, ZEB1) associated with OVOL2 were differentially expressed in the same direction in both individuals with PPCD. Immunohistochemistry staining and RNA-seq analyses demonstrated variable expression of type IV collagens in PPCD corneas. Decreasing ZEB1 expression in pHCEnC altered expression of 711 protein-coding genes, many of which are associated with canonical pathways regulating various cellular processes.ConclusionsIdentification of the altered transcriptome in PPCD and in a cell-based model of PPCD provided insight into the molecular alterations characterizing PPCD. Further study of the differentially expressed genes associated with ZEB1 and OVOL2 is expected to identify candidate genes for individuals with PPCD and without a ZEB1 or OVOL2 mutation.

ZEB1 insufficiency causes corneal endothelial cell state transition and altered cellular processing

“…Nevertheless, as ZEB1 is robust at mediating EMT in disparate cell types, we concluded that the “background” gene expression was not likely to play a significant role in our study, although this remains a limitation of our model. We demonstrated that the ZEB1 +/- CEnC possessed a gene expression profile similar to that observed in PPCD, with many epithelial-associated genes demonstrating either increased or ectopic expression in both [17]. Concurrently, we showed that some corneal endothelial associated genes were downregulated in ZEB1 +/- CEnC, similar to that observed in PPCD.…”

“…Herein, we validated the ZEB1 monoallelic knockout cell line as a cell-based model of PPCD using a transcriptomics approach, and provide evidence (transcriptomic and cell function) to support our hypothesis that a novel MET-like process, termed endothelial to epithelial transition (EnET), best explains the PPCD phenotype. Importantly, key findings from the transcriptomic profiling of human PPCD endothelium [17] were recapitulated in the ZEB1 knockout cell line, further supporting the utility of the CRISPR-Cas9-mediated knockout of ZEB1 in CEnC to gain a better understanding of the molecular factors central to the pathogenesis of PPCD. In addition, based on the evidence provided here for EnET, we propose a corollary to the EMT/MET paradigm, in which EnET is classified as a MET subtype that is characteristic of PPCD.…”

“…Prior to utilizing the CEnC line to study the effects of ZEB1 knockout on cellular processes, we determined the extent to which the line recapitulated one of the primary molecular hallmarks of PPCD endothelium: an ectopic/increased expression of epithelial-specific (and/or associated) genes (Fig 1A). We identified 1715 differentially expressed genes in PPCD endothelium compared to age-matched controls, of which 920 were upregulated and 795 were downregulated [17]. Comparison of the differentially expressed genes in PPCD with genes highly-associated with ex vivo corneal epithelial cells (evCEpC) or with ex vivo corneal endothelial cells (evCEnC) demonstrated that 26% (65/249) of evCEpC genes were upregulated and 37% (40/108) of evCEnC genes were downregulated in PPCD endothelium, significantly different from the expected percentages due to chance alone (p<0.01) (Fig 1A and S1 Fig).…”

“…Together, these reports provided the first indication that the corneal endothelial cells had gained an epithelial-like phenotype given that the observed morphologic/ultrastructural features and gene expression changes were consistent with such a phenotype. The first transcriptomic study characterizing the gene expression changes in PPCD was reported in 2017, and provided evidence for a widespread increase or ectopic expression of epithelial-associated genes [17]. The genotypes that have been associated with PPCD have also proved to be strong evidence for an MET-like transition of an endothelial to an epithelial phenotype [16].…”

“…A smaller percentage of affected individuals demonstrate non-coding mutations in OVOL2 and GRHL2 , presumably as a result of ectopic expression of either gene in the corneal endothelium, with subsequent repression of ZEB1 transcription [13–16]. As a consequence of ZEB1 insufficiency, various epithelial-like features are observed in PPCD corneal endothelium, including a stratified organization, desmosomal intracellular junctions, and expression of an epithelial-like transcriptomic profile, including increased/ectopic expression of epithelial-associated keratins and cadherins (e.g., CDH1 ), and decreased expression of CDH2 [12, 17, 18]. Recently we reported that reduced ZEB 1 expression in a cell-based model of PPCD using short-interfering RNA (siRNA) targeting ZEB1 resulted in significantly increased CEnC apoptosis and barrier function [18], consistent with prior reports of ZEB1 reduction leading to increased cell death [19, 20] and increased cell barrier function [21–23].…”