Analyzing Presymptomatic Tissue to Gain Insights into the Molecular and Mechanistic Origins of Late-Onset Degenerative Trinucleotide Repeat Disease

Abstract

How genetic defects trigger the molecular changes that cause late-onset disease is important for understanding disease progression and therapeutic development. Fuchs' endothelial corneal dystrophy (FECD) is an RNA-mediated disease caused by a trinucleotide CUG expansion in an intron within the TCF4 gene. The mutant intronic CUG RNA is present at 1-2 copies per cell, posing a challenge to understand how a rare RNA can cause disease. Late-onset FECD is a uniquely advantageous model for studying how RNA triggers disease because; 1) Affected tissue is routinely removed during surgery; 2) The expanded CUG mutation is one of the most prevalent disease-causing mutations, making it possible to obtain pre-symptomatic tissue from eye bank donors to probe how gene expression changes precede disease; and 3) The affected tissue is a homogeneous single cell monolayer, facilitating accurate transcriptome analysis. Here we use RNA sequencing (RNAseq) to compare tissue from individuals who are pre-symptomatic (Pre_S) to tissue from patients with late stage FECD (FECD_REP). The abundance of mutant repeat intronic RNA in Pre_S and FECD_REP tissue is elevated due to increased half-life in a corneal cell-specific manner. In Pre_S tissue, changes in splicing and extracellular matrix gene expression foreshadow the changes observed in advanced disease and predict the activation of the fibrosis pathway and immune system seen in late-stage patients. The absolute magnitude of splicing changes is similar in presymptomatic and late stage tissue. Our data identify gene candidates for early drivers of disease and biomarkers that may represent diagnostic and therapeutic targets for FECD. We conclude that changes in alternative splicing and gene expression are observable decades prior to the diagnosis of late-onset trinucleotide repeat disease. 3 3 6 6

TCF4trinucleotide repeat expansions and UV irradiation increase susceptibility to ferroptosis in Fuchs endothelial corneal dystrophy

“…RNA-Seq datasets of corneal endothelial samples from 47 patients with FECD and 21 donor controls were obtained from publicly available datasets (SRA Accession Numbers: PRJNA445238, PRJNA524323, PRJNA597343). Pre-symptomatic controls with TCF4 trinucleotide repeats were excluded from the analysis (28,29,51). Reads were aligned and gene counts were made using STAR (52), data quality was assessed using FastQC, gene expression was normalized, batch corrected, and determined using EdgeR (53).…”

Trinucleotide Repeat-Targeting dCas9 as a Therapeutic Strategy for Fuchs’ Endothelial Corneal Dystrophy

“…By the examination of endothelial tissue of presymptomatic individuals harboring the TCF4 triplet repeat expansion mutation, we revealed that the presence of expanded CUG repeat RNA triggers mis-splicing of MBNL-sensitive exons and upregulation of fibrosis genes early in the disease course. 24 Therefore, we feel that the mutant CUG repeat RNA species has advantages as a target for drug development to halt FECD disease pathogenesis at its molecular origins.…”