RNA Toxicity and Missplicing in the Common Eye Disease Fuchs Endothelial Corneal Dystrophy

Jintang Du,Ross A Aleff,Elisabetta Soragni,Krishna Kalari,Jinfu Nie,Xiaojia Tang,Jaime Davila,Jean-Pierre Kocher,Sanjay V Patel,Joel M Gottesfeld,Keith H Baratz,Eric D Wieben

doi:10.1074/jbc.m114.621607

Abstract

Fuchs endothelial corneal dystrophy (FECD) is an inherited degenerative disease that affects the internal endothelial cell monolayer of the cornea and can result in corneal edema and vision loss in severe cases. FECD affects ∼5% of middle-aged Caucasians in the United States and accounts for >14,000 corneal transplantations annually. Among the several genes and loci associated with FECD, the strongest association is with an intronic (CTG·CAG)n trinucleotide repeat expansion in the TCF4 gene, which is found in the majority of affected patients. Corneal endothelial cells from FECD patients harbor a poly(CUG)n RNA that can be visualized as RNA foci containing this condensed RNA and associated proteins. Similar to myotonic dystrophy type 1, the poly(CUG)n RNA co-localizes with and sequesters the mRNA-splicing factor MBNL1, leading to missplicing of essential MBNL1-regulated mRNAs. Such foci and missplicing are not observed in similar cells from FECD patients who lack the repeat expansion. RNA-Seq splicing data from the corneal endothelia of FECD patients and controls reveal hundreds of differential alternative splicing events. These include events previously characterized in the context of myotonic dystrophy type 1 and epithelial-to-mesenchymal transition, as well as splicing changes in genes related to proposed mechanisms of FECD pathogenesis. We report the first instance of RNA toxicity and missplicing in a common non-neurological/neuromuscular disease associated with a repeat expansion. The FECD patient population with this (CTG·CAG)n trinucleotide repeat expansion exceeds that of the combined number of patients in all other microsatellite expansion disorders.

Highlights

Expansion of intronic (CTG1⁄7CAG)n repeats in TCF4 is found in most Fuchs endothelial corneal dystrophy (FECD) patients
Among the several genes and loci associated with FECD, the strongest association is with an intronic (CTG1⁄7CAG)n trinucleotide repeat expansion in the TCF4 gene, which is found in the majority of affected patients
The CTG1⁄7CAG trinucleotide repeat (TNR) are located in an intron just downstream of a major upstream promoter used in the corneal endothelium, and the repeats are actively transcribed in both patients and controls

Summary

Background

Expansion of intronic (CTG1⁄7CAG)n repeats in TCF4 is found in most Fuchs endothelial corneal dystrophy (FECD) patients. RNA Toxicity in Fuchs Endothelial Corneal Dystrophy the possibility that RNA toxicity might play a role in the pathogenesis of this common autosomal dominant disorder, as it does in several of the relatively rare neurodegenerative and neuromuscular repeat expansion diseases, such as myotonic dystrophy types 1 and 2 (DM1 and DM2) [8], fragile X-associated tremor/ataxia syndrome (FXTAS) [9], and C9ORF72-associated amyotrophic lateral sclerosis and frontotemporal dementia (C9ORF72 ALS/FTD) [10] In each of these diseases, expanded microsatellite DNA sequences are found in noncoding regions of various genes, including 5Ј-UTRs (FXTAS), introns (DM2 and C9ORF72 ALS/FTD), and 3Ј-UTRs (DM1) (reviewed in Ref. 11), and these repetitive elements are transcribed into toxic gain-of-function RNAs. In the case of DM1, the (CTG1⁄7CAG)n repeats in the 3Ј-UTR of the serine/threonine protein kinase gene DMPK (dystrophia myotonica protein kinase) are transcribed into poly(CUG) mRNA, which disrupts normal cellular processes at the level of mRNA processing by sequestering the splicing regulator MBNL1 (muscleblind-like 1Ϫ) [8, 12]. Our results suggest potential novel therapeutic approaches to the treatment of FECD

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION