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Thomas Rinkoski, Keith H Baratz, Uttio Roy Chowdhury, Ross A Aleff, Michael P Fautsch, Eric Wieben; Characterization of mis-splicing events in primary Fuchs corneal endothelial cell cultures harboring TCF4 repeat expansion. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5288.
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© ARVO (1962-2015); The Authors (2016-present)
The strongest genetic association with Fuchs endothelial corneal dystrophy (FECD) is with an intronic (CTG-CAG)n trinucleotide repeat (TNR) expansion in the transcription factor 4 (TCF4) gene. We have previously identified foci of TCF4 transcript accumulation in the nucleus of primary FECD tissue and corneal endothelial cell lines. These TCF4 RNA containing foci were found to sequester the splicing factor Muscleblind-like 1 (MBNL1). In this study, we profiled the transcriptome of primary normal and FECD corneal endothelial cell cultures.
cDNA was generated from poly(A) mRNA isolated from control and FECD primary cultures of corneal endothelial cells. The cDNA was blunt-ended, ligated to unique paired-end adaptors and amplified by PCR. Resulting libraries were loaded onto paired-end flow cells and sequenced with 51 x 2 bp reads on an Illumina HiSeq 2000 system using TruSeq SBS Sequencing Kit v3 and SCS v1.4.8 data collection software. The paired-end FASTQ format sequence was analyzed using Mayo Analysis Pipeline for RNA Sequencing (MAP-RSeq) to generate alignment files that were assessed using MISO software. Immunohistochemistry was performed on normal and FECD primary corneal endothelial cultures using antibodies against calnexin and CHOP.
RNASeq analysis revealed that primary cultures of FECD corneal endothelial cells harbored many of the splicing changes found in fresh corneal endothelium from FECD patients. Specifically, inclusion of alternatively spliced exons in MBNL1 and MBNL2 are among more than 40 unique splicing events that occur in primary FECD corneal endothelial cell lines when compared to primary control endothelial cell lines. Functional annotation of differential splicing events between control and FECD cells with TNR expansions reveals a statistically significant enrichment for genes involved in cytoskeletal protein binding. Additionally, primary FECD corneal endothelial cells show elevated levels of calnexin and nuclear translocation of CHOP, consistent with endoplasmic reticulum stress.
Primary FECD corneal endothelial cell cultures that harbor the TCF4 TNR expansion exhibit many of the mRNA splicing abnormalities that we have previously identified in FECD corneal endothelial tissue. These changes may result in misfolded proteins as primary FECD corneal endothelial cells show signs of endoplasmic reticulum stress.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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