Patients with Fuchs' endothelial corneal dystrophy exhibit a unique signature of missplicing events in TCF4

July 15, 2017

Fuchs' endothelial corneal dystrophy (FECD) is familial, bilateral and progressive — and the most common indication for corneal transplantation in the United States. Studies have linked FECD with an expanded intronic repeat, CTG, in the transcription factor 4 (TCF4) gene in most patients in Caucasian cohorts and a smaller proportion in non-Caucasian cohorts. Up to 80 percent of patients with FECD, depending on ethnicity, exhibit trinucleotide repeat expansions.

"FECD tissue harbors focal intranuclear accumulations of the CUG repeat pre-mRNA, termed RNA foci," says Keith H. Baratz, M.D., Ophthalmology, at Mayo Clinic's campus in Rochester, Minnesota. "RNA foci colocalize with and sequester nuclear proteins, most notably splicing factors of the muscleblind (MBNL) family. A pilot study we conducted showed that trinucleotide repeat expansion in the corneal endothelium leads to sequestration of MBNL1 in RNA foci and observable changes in mRNA splicing."

Building on that study, Dr. Baratz, Michael P. Fautsch, Ph.D., with the Ophthalmology Research Unit, Eric D. Wieben, Ph.D., director of the Medical Genome Facility, and a research team at Mayo Clinic's campus in Minnesota have confirmed and validated a larger sample group to identify a core set of splicing events in human corneal endothelium that could be directly associated with FECD through CTG trinucleotide repeat expansion in the TCF4 gene. Results of their study were published in Investigative Ophthalmology & Visual Science in 2017.

RNA analysis

The research team isolated and sequenced total RNA from corneal endothelial tissue obtained during keratoplasty from 12 patients with advanced FECD (modified Krachmer grade 5 or 6) requiring corneal transplantation and four patients undergoing keratoplasty or enucleation for other indications (grade 0).

"The length of the trinucleotide repeat CTG in the TCF4 gene was determined using leukocyte-derived DNA analyzed by a combination of Southern blotting and GeneScan analysis," says Dr. Fautsch. "Commercial software — along with numerous hours of interpretation and validation by Dr. Wieben — helped to identify expression of a select set of alternatively spliced genes."

Validation of these specific alternative splicing events was performed through the use of reverse transcription polymerase chain reaction with the final gene set undergoing further analysis to identify overrepresentation in functional pathways using the web-based Panther analysis system. Outcomes include the following:

  • A unique set of alternatively spliced genes associated with patients with FECD containing a CTG trinucleotide repeat expansion sequence in the TCF4 gene was identified.
  • Differential splicing of NUMA1, PPFIBP1, MBNL1 and MBNL2 transcripts were identified in all FECD samples containing a trinucleotide repeat expansion.
  • Differentially spliced genes identified in patients with FECD were enriched for products that localize to the cell cortex, bind the cytoskeleton and have cell adhesion functions.
  • Corneal endothelial tissue from patients with FECD revealed a novel splicing event involving FGFR2.

"Corneal endothelium from patients with FECD harbors a unique signature of missplicing events due to CTG trinucleotide repeat expansion in the TCF4 gene, consistent with the hypothesis that RNA toxicity contributes to the pathogenesis of FECD," says Dr. Fautsch. "In this study, change to proteins associated with cell adhesion that may interfere with the corneal endothelial barrier function, a known event in the development of FECD, was identified as a key biological process influenced by the missplicing events."

"Divergent genetic variants lead to FECD," says Dr. Baratz. "To date, no unifying pathogenic mechanism has been identified. Nevertheless, the current mechanism involving missplicing of the candidate genes seems to hold true for patients with FECD with trinucleotide repeat expansions. Therefore, changes in gene function through alternate splicing induced by a trinucleotide repeat expansion in the TCF4 gene can be a valid pathogenic mechanism in most patients with FECD. Development of this genetic signature will be useful for identifying biochemical pathways that may contribute to the pathogenesis of the disease."

For more information

Wieben ED, et al. Trinucleotide repeat expansion in the transcription factor 4 (TCF4) gene leads to widespread mRNA splicing changes in Fuchs' endothelial corneal dystrophy. Investigative Ophthalmology & Visual Science. 2017;58:343.