Abstract
Purpose :
Fuchs Endothelial Corneal Dystrophy (FECD), a late-onset oxidative stress disorder, is the most common cause of corneal endothelial (CE) degeneration and is genetically associated with CTG repeat expansion in TCF4. We previously reported accumulation of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) damage causing CE cell death in FECD. Specifically, mtDNA damage was a prominent finding in development of FECD phenotype in the ultraviolet-A (UVA) light-induced FECD mouse model. In this study, we investigated whether deficiency in DNA repair pathways leads to accumulation of toxic DNA lesions in FECD.
Methods :
Differential expression profiles of DNA repair genes were analyzed by real-time PCR arrays in which 84 genes and five housekeeping genes were tested. Total RNA was extracted from Descemet’s membrane-CE stripped from age-matched normal donors (n=4) and FECD specimens (n=8) stratified as either with TCF4 repeat expansion (> 50 CTG repeats; TCF4+) or without expansion (< 50 CTG repeats; TCF4-). cDNA was subjected to real-time PCR analysis on Human DNA Repair RT2 Profiler plates. Change in mRNA expression of <0.5 or >2.0-fold in FECD relative to normal was set as the cutoff value for down- and upregulation. Total protein was extracted from dissected CE of 7-9 week-old mice irradiated with 1000 J/cm2 UVA at 1 day and western blotting was carried out to determine Lig3 and XPC levels.
Results :
FECD specimens showed significant downregulation (<0.5-fold; p< 0.05) of 8 genes and upregulation (> 2-fold, p <0.05) of 7 genes belonging to base excision repair (BER), nucleotide excision repair (NER) and mismatch repair pathways, compared to normal donors. Interestingly, BER genes Neil1, Smug1 and PNKP were preferentially downregulated in TCF4+ FECD. Key mtDNA repair genes Lig3 (BER) and XPC (NER) were downregulated in both TCF4+ and TCF4- specimens. In FECD model, UVA irradiation reduced Lig3 (2.5-fold, p<0.05) and XPC (3-fold, p<0.05) levels in mouse CE, correlating with the increased mtDNA and not nDNA damage at that time-point.
Conclusions :
Our findings indicate impaired BER and NER DNA repair pathways that are important for the repair of ROS-induced mtDNA damage noted in FECD. Downregulation of mtDNA repair genes Lig3 and XPC in FECD specimens (with and without repeats) and UVA-based in vivo model suggest DNA repair deficiency is an important contributor of the final common pathway of FECD pathogenesis.
This is a 2020 ARVO Annual Meeting abstract.