Repeat-associated non-ATG (RAN) translation in fuchs’ endothelial corneal dystrophy

Elisabetta Soragni, Lina Petrosyan, Tommy A. Rinkoski, Eric D. Wieben, Keith H. Baratz, Michael P. Fautsch, Joel M. Gottesfeld

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

PURPOSE. The strongest genetic association with Fuchs’ endothelial corneal dystrophy (FECD) is the presence of an intronic (CTG·CAG)n trinucleotide repeat (TNR) expansion in the transcription factor 4 (TCF4) gene. Repeat-associated non-ATG (RAN) translation, an unconventional protein translation mechanism that does not require an initiating ATG, has been described in many TNR expansion diseases, including myotonic dystrophy type 1 (DM1). Given the similarities between DM1 and FECD, we wished to determine whether RAN translation occurs in FECD. METHODS. Antibodies against peptides in the C-terminus of putative RAN translation products from TCF4 were raised and validated by Western blotting and immunofluorescence (IF). CTG·CAG repeats of various lengths in the context of the TCF4 gene were cloned in frame with a 3× FLAG tag and transfected in human cells. IF with antipeptide and anti-FLAG antibodies, as well as cytotoxicity and cell proliferation assays, were performed in these transfected cells. Corneal endothelium derived from patients with FECD was probed with validated antibodies by IF. RESULTS. CTG·CAG repeats in the context of the TCF4 gene are transcribed and translated via non-ATG initiation in transfected cells and confer toxicity to an immortalized corneal endothelial cell line. An antipeptide antibody raised against the C-terminus of the TCF4 poly-cysteine frame recognized RAN translation products by IF in cells transfected with CTG·CAG repeats and in FECD corneal endothelium. CONCLUSIONS. Expanded CTG·CAG repeats in the context of the third intron of TCF4 are transcribed and translated via non-ATG initiation, providing evidence for RAN translation in corneal endothelium of patients with FECD.

Original languageEnglish (US)
Pages (from-to)1888-1896
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume59
Issue number5
DOIs
StatePublished - Apr 2018

Keywords

  • Corneal endothelial cells
  • Fuchs’ dystrophy
  • Microsatellite repeats
  • RAN translation
  • Trinucleotide repeat expansion

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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