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Keith H Baratz, Santanu Bhattacharya, Cindy Bahler, Ross A Aleff, Elisabetta Soragni, Jintang Du, Sanjay V Patel, Eric Wieben, Joel M Gottesfeld, Michael P Fautsch; Identification of CUG-containing RNA foci in cultured Fuchs endothelial corneal dystrophy cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2576. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Approximately 75% of Fuchs endothelial corneal dystrophy (FECD) cases in the United States are associated with a CTG trinucleotide repeat expansion in the transcription factor 4 (TCF4) gene. We have recently identified the accumulation of poly-CUG RNA transcripts and splicing cofactors (RNA foci) in corneal endothelium from FECD patients, indicating RNA toxicity due to transcribed CTG repeats as a possible cause of the disease. In this study, we evaluated human primary corneal endothelial cell (HCEC) cultures from FECD and normal patients for RNA foci in order to validate HCEC culture as a model to study RNA toxicity in FECD.
HCECs were obtained from excised Descemet membrane/endothelium during endothelial keratoplasty or from eye bank eyes and incubated overnight in 8% FBS in Opti-MEM. Cells were released from Descemet membrane by incubating in 0.02% EDTA for 1 hour with agitation. The cells were pelleted by centrifugation, washed in culture medium, pelleted again, and re-suspended in culture medium. Cells were plated into collagen type IV coated 6-well plates and incubated at 37°C in a humidified atmosphere with 5% CO2. Cell lines (n=4 normal, n=6 FECD) were probed with a CTG complementary repeat sequence (Cy3-(CAG)10) containing a fluorescent tag and visualized using a laser scanning confocal microscope. Normal and FECD RNA was isolated, made into cDNA and amplified by PCR to examine expression patterns for RNA toxicity candidate markers MBNL1, INF2, SORBS1 and ADD3. RALGAPA1 was used as a control splicing event that is independent of RNA toxicity.
Nuclear RNA foci consisting of poly-CUG transcripts were present in cultured HCECs from FECD but not normal controls. Splicing patterns of MBNL1, INF2, SORBS1 and ADD3 differed between FECD and normal cells, but RALGAPA1 splicing did not differ.
Primary human FECD endothelial cells but not normal endothelial cells harbor RNA foci. The splicing pattern of INF2, SORBS1, ADD3 and RALGAPA1 was consistent with that seen in other repeat expansion diseases. These results validate FECD cell culture as a model to examine the role of RNA toxicity due to RNA foci development as a possible cause of FECD.
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