Investigation of transcriptional dysregulation events driving Posterior Polymorphous Corneal Dystrophy type 1

Abstract

Purpose : Posterior Polymorphous Corneal Dystrophy (PPCD) is associated with a dysregulated cell state, induced by mutations that alter the levels of epithelial mesenchymal transition (EMT)-regulating transcription factors ZEB1, GRHL2 or OVOL2. Here we investigate the transcriptomic signature of dysregulation in PPCD type 1 (PPCD1) corneal endothelial cells (CECs) to identify biomarkers of the disease mechanism as potential therapeutic targets.

Methods : We cultured primary CECs from five individuals affected with PPCD1 and seven controls. All PPCD1 individuals were confirmed to harbour the same regulatory mutation in the OVOL2 promoter (NM_021220:c.−370T>C), previously hypothesised to lead to ectopic expression of OVOL2 within the corneal endothelium. Total RNA was extracted from each primary culture using NucleoSpin RNA XS isolation kit (Macherey Nagel). We enriched mRNA using oligo(dT) beads and paired-end Illumina RNA sequencing was performed using a stranded library preparation. Transcriptomic analysis was performed using DESeq2, rMATS and IsoformSwitchAnalyzeR programs to investigate differential gene expression and splicing events in PPCD1 CECs compared to control CECs.

Results : Utilizing the EMT Gene Database, 279 EMT-associated genes were found to be highly dysregulated (false discovery rate (FDR) corrected p-value <.05; Log-2 fold change > 1) in PPCD1 including CDH1, OVOL2, GRHL2, GATA6, members of the mir200 family, numerous keratins, and the epithelial splice regulator ESRP1. Alternative splicing was further identified for ESRP1 targets, CD44 and FGFR2.

Conclusions : Our study confirms the aberrant upregulation of OVOL2 in PPCD1 and suggests that EMT-associated genes and pathways are significantly disrupted in PPCD1 CECs. Our data suggests that overexpression of the epithelial cell type-specific splicing regulator, ESRP1, induces aberrant splicing of CD44 and FGFR2. We hypothesise that this mechanism contributes to the ‘epithelialisation’ of the corneal endothelium observed in PPCD1 and may act as a target for future therapeutic interventions.

This is a 2021 ARVO Annual Meeting abstract.