Generation and RNA Sequencing Analysis of Potential Mouse Model for Fuchs Endothelial Corneal Dystrophy Harboring CTG Trinucleotide Intronic Repeat Expansion in Tcf4

Abstract

Purpose : Intronic trinucleotide repeat expansion in the TCF4 gene is thought to be the most common causative mutation for Fuchs endothelial corneal dystrophy (FECD). However, the exact effect of CTG repeats on the pathophysiology of FECD remains unelucidated. This present study aimed to generate a CTG repeats-knock-in (KI) mouse model in Tcf4 and investigate the effect of the repeats on gene expression using RNA sequencing (RNA-Seq) analysis.

Methods : CTG (×100) repeats were knocked in the Tcf4 via the CRISPR/Cas9 system. Corneal endothelial cells were obtained from the KI mice (n = 3) and wild-type (WT) mice (n = 3), and generated mRNA-Seq libraries were sequenced using the NovaSeq 6000 Sequencing System. Differentially expressed genes (DEGs) were identified by DESeq2, and gene expression patterns were evaluated via principal component analysis (PCA) and correlation-matrix heatmap plotting. Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses were also performed.

Results : DEG analysis of transcriptomic profiles revealed that the expression of 3,707 genes (2,579 upregulated and 1,128 downregulated genes) was significantly altered in the corneal endothelium of the KI mice compared to WT mice. PCA visualized the two distinctive groups composed of KI mice and WT mice. A heatmap confirmed a hierarchical clustering of KI mice and WT mice based on gene expressions. Likewise, the correlation coefficients showed high correlations within each group of KI mice and WT mice. GO analysis demonstrated that dysregulated genes in the KI mice were associated with sensory system development, visual system development, extracellular structure organization, and extracellular matrix (ECM) organization. KEGG pathway analysis also indicated the involvement of the genes related to focal adhesion, cell adhesion molecules, and ECM-receptor interaction.

Conclusions : Our in vivo mouse model added evidence that intronic trinucleotide expansion in TCF4 might be involved in the pathophysiology of FECD. We succeeded in generating a mouse model harboring repeat expansion which is the potential research tool for FECD.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.