June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Characterization of gene mis-splicing events in Fuchs endothelial corneal dystrophy primary cell cultures
Author Affiliations & Notes
  • Tommy A. Rinkoski
    Ophthalmology, Mayo Clinic, Rochester, Minnesota, United States
  • Cindy K. Bahler
    Ophthalmology, Mayo Clinic, Rochester, Minnesota, United States
  • Xiaojia Tang
    Division of Biostatistics and Bioinformatics and Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota, United States
  • Krishna R Kalari
    Division of Biostatistics and Bioinformatics and Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota, United States
  • Keith Baratz
    Ophthalmology, Mayo Clinic, Rochester, Minnesota, United States
  • Sanjay V Patel
    Ophthalmology, Mayo Clinic, Rochester, Minnesota, United States
  • Michael P Fautsch
    Ophthalmology, Mayo Clinic, Rochester, Minnesota, United States
  • Eric Wieben
    Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, United States
  • Footnotes
    Commercial Relationships   Tommy Rinkoski, None; Cindy K. Bahler, None; Xiaojia Tang, None; Krishna Kalari, None; Keith Baratz, None; Sanjay Patel, None; Michael Fautsch, None; Eric Wieben, None
  • Footnotes
    Support  NIH Grants EY26490 and EY25071, MN Partnership for Biotechnology and Medical Genomics, Research to Prevent Blindness, and Mayo Foundation
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1433. doi:
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      Tommy A. Rinkoski, Cindy K. Bahler, Xiaojia Tang, Krishna R Kalari, Keith Baratz, Sanjay V Patel, Michael P Fautsch, Eric Wieben; Characterization of gene mis-splicing events in Fuchs endothelial corneal dystrophy primary cell cultures. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1433.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : We have previously identified a mis-splicing gene signature in Fuchs endothelial corneal dystrophy (FECD) tissue that associates with the CTG trinucleotide repeat (TNR) expansion in the transcription factor 4 (TCF4) gene. In this study, we characterized RNA splicing events in primary cultures of FECD cells to determine if this model retains the characteristic mis-splicing gene signature of FECD.

Methods : Human Fuchs corneal endothelial cells were obtained from tissue removed at endothelial keratoplasty. Control corneal endothelial cells were obtained from normal donor eyes. Cells were dissociated from Descemet membrane in 0.02% EDTA and placed in 6-well collagen IV coated plates with Joyce’s media. Upon confluence, Joyce’s media was replaced with maturation media (50:50 F12 and M199 with 5% fetal bovine serum, 20ug/ml ascorbic acid, 1X ITS and 1X Pen/Strep) for a minimum of 5 days. Total RNA isolated from confluent control (n=2) and FECD cell cultures (n=4) were processed into cDNA libraries using the TruSeq Stranded Total RNA Library kit. The resulting libraries were minimally amplified and quantified for sequencing at 3 samples/lane using a HiSeq4000 (Illumina) sequencer and paired end 101 bp reads. The paired-end FASTQ format sequence was analyzed using Mayo Analysis Pipeline for RNA Sequencing (MAP-RSeq) to generate alignment files. Pairwise comparisons were performed with MISO software.

Results : RNASeq analysis of FECD cultured cells revealed mis-splicing of 15 genes that were previously identified in FECD corneal endothelial tissue containing a TCF4 TNR expansion sequence. These genes included MBNL1, MBNL2, NUMA1, PPFIBP1, VEGFA, FGFR1, CD46, BZRAP1 and NHSL1. Pathway analysis of the mis-spliced gene set showed statistically significant enrichment of genes associated with cytoskeletal binding proteins SYNE1, ABI1, CLASP1, INF2, KIF13A and PLEKHM2. Cell lines from FECD patients without TCF4 TNR expansion did not share these mis-splicing events.

Conclusions : Primary Fuchs corneal endothelial cell lines containing a TCF4 TNR expansion sequence exhibit an RNA mis-splicing profile similar to Fuchs corneal endothelial tissue. These results lend validity to using FECD primary cell culture models to study molecular aspects of the disease.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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