Abstract
Purpose :
Fuchs endothelial corneal dystrophy (FECD) is a genetic, age-related corneal endothelial (CE) degeneration from lifelong exposure to oxidative stress. We previously reported heightened nuclear (nDNA) and mitochondrial (mtDNA) damage to be a hallmark of FECD. This was recapitulated in the ultraviolet-A (UVA) light-induced FECD mouse model which showed early stage mtDNA and late stage nDNA damage to contribute to degenerative CE phenotype. In this study, we investigated whether impaired DNA repair pathways can cause the build-up of deleterious DNA lesions in FECD
Methods :
Differential expression profiles of 84 DNA repair and 5 housekeeping genes were analyzed by real-time (RT)PCR arrays. Total RNA was extracted from Descemet’s membrane-CE stripped from age-matched normal donors (n=4) and FECD specimens (n=8). cDNA was subjected to RTPCR on Human DNA Repair RT Profiler plates. A change of <0.5 or >2.0-fold mRNA expression in FECD relative to normal was set as the cutoff for down- and upregulation. Total protein was extracted from CE of 7-9 weeks C57BL/6 mice irradiated with 1000 J/cm2 UVA at 1 day and western blotting was carried out to assess LIG3 and XPC levels
Results :
FECD specimens differentially expressed (p<0.05) 8 nucleotide excision repair (NER), 5 base excision repair (BER), 4 double strand break (DSB) repair and 4 mismatch repair (MMR) pathway genes compared to normal donors. While the total 11 upregulated (>2-fold; p<0.05) genes were equally distributed between the 4 pathways, the set of 10 downregulated (<0.5-fold; p<0.05) genes predominantly (8 out of 10 genes) belonged to NER or BER pathways. NER gene XPC and BER gene LIG3 were downregulated (0.2-fold; p<0.005 and 0.36-fold; p<0.005) in FECD specimens and were also reduced (0.3-fold; p<0.05 and 0.4-fold; p<0.05) in mouse CE cells after UVA irradiation; correlating to the early time-point of mtDNA damage seen in UVA mouse model of FECD. Differentially expressed DSB and MMR pathways were primarily upregulated (3 out of 4 genes each) in FECD compared to normal donors
Conclusions :
Our findings indicate that deficient NER and BER pathways may trigger the accumulation of unrepaired DNA lesions in FECD. Downregulation of XPC and lig3 may contribute to the mtDNA damage and mitochondrial dysfunction seen in FECD. This is the first study to provide evidence that DNA repair pathways are deficient in FECD and may be important when designing therapeutics for degenerative cell loss
This is a 2021 ARVO Annual Meeting abstract.