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Invest Ophthalmol Vis Sci. 2017 Jan 1;58(1):343-352. doi: 10.1167/iovs.16-20900.

Trinucleotide Repeat Expansion in the Transcription Factor 4 (TCF4) Gene Leads to Widespread mRNA Splicing Changes in Fuchs' Endothelial Corneal Dystrophy.

Author information

1
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, United States.
2
Division of Biostatistics and Bioinformatics and Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota, United States.
3
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, United States.
4
Department of Experimental Pathology and Laboratory Medicine, Mayo Clinic, Rochester, Minnesota, United States.
5
Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, United States.
6
Department of Ophthalmology, Mayo Clinic, Rochester, Minnesota, United States.

Abstract

Purpose:

To identify RNA missplicing events in human corneal endothelial tissue isolated from Fuchs' endothelial corneal dystrophy (FECD).

Methods:

Total RNA was isolated and sequenced from corneal endothelial tissue obtained during keratoplasty from 12 patients with FECD and 4 patients undergoing keratoplasty or enucleation for other indications. The length of the trinucleotide repeat (TNR) CTG in the transcription factor 4 (TCF4) gene was determined using leukocyte-derived DNA analyzed by a combination of Southern blotting and Genescan analysis. Commercial statistical software was used to quantify expression of alternatively spliced genes. Validation of selected alternative splicing events was performed by using RT-PCR. Gene sets identified were analyzed for overrepresentation using Web-based analysis system.

Results:

Corneal endothelial tissue from FECD patients containing a CTG TNR expansion sequence in the TCF4 gene revealed widespread changes in mRNA splicing, including a novel splicing event involving FGFR2. Differential splicing of NUMA1, PPFIBP1, MBNL1, and MBNL2 transcripts were identified in all FECD samples containing a TNR expansion. The differentially spliced genes were enriched for products that localize to the cell cortex and bind cytoskeletal and cell adhesion proteins.

Conclusions:

Corneal endothelium from FECD patients harbors a unique signature of mis-splicing events due to CTG TNR expansion in the TCF4 gene, consistent with the hypothesis that RNA toxicity contributes to the pathogenesis of FECD. Changes to the endothelial barrier function, a known event in the development of FECD, was identified as a key biological process influenced by the missplicing events.

PMID:
28118661
PMCID:
PMC5270622
DOI:
10.1167/iovs.16-20900
[Indexed for MEDLINE]
Free PMC Article

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