HARVARD DEPARTMENT OF OPHTHALMOLOGY
Category : Basic and Translational Research Candidate : Neha Deshpande Poster #: B5
Deregulated DNA Repair in Fuchs Endothelial Corneal Dystrophy
Neha Deshpande , Shazia Ashraf , Geetha Melangath , Shivakumar Vasanth , Marianne Price , Francis Price Jr ., Ula V . Jurkunas
Purpose : Fuchs endothelial corneal dystrophy ( FECD ) is an age-related disorder involving corneal endothelial ( CE ) degeneration resulting from lifelong exposure to oxidative stress . We previously reported heightened accumulation of nuclear ( nDNA ) and mitochondrial ( mtDNA ) damage to be a hallmark of FECD . This finding 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 explore impaired DNA repair pathways that may drive the build-up of deleterious 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 ). cDNA was subjected to real-time PCR analysis on Human DNA Repair RT 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- or upregulation . Total protein was extracted from CE stripped from 7-9 weeks old C57BL / 6 mice irradiated with 1000 J / cm2 UVA at 1 day and western blotting was carried out to assess DNA repair protein levels .
Results : FECD specimens differentially expressed ( p < 0.05 ) 6 nucleotide excision repair ( NER ), 7 base excision repair ( BER ), 3 double strand break ( DSB ) repair , 2 mismatch repair ( MMR ) and 1 unclassified pathway gene compared to normal donors . Both MMR genes belonged to the upregulated group . A majority of the deregulated genes predominantly belonged to NER and BER pathways ( 8 out of 11 downregulated , and 5 out of 8 upregulated genes ). NER gene XPC and BER gene lig3 were both downregulated ( 0.20-fold ; p < 0.005 and 0.32-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 the FECD mouse CE model 1 day post UVA irradiation , correlating to the early time-point mtDNA damage shown earlier .
Conclusions : Our findings indicate that of the 4 major pathways involved in DNA repair ; NER , BER and DSB pathways feature in the sets of both up and down regulated genes , while MMR pathway features only in the set of upregulated genes in FECD . Deficient NER and BER pathways may trigger the accumulation of unresolved DNA damage in FECD . Downregulation of XPC and lig3 may contribute to the early mtDNA damage observed in the UVA-mouse model of FECD .