Quantification of Double Stranded DNA Breaks and Telomere Length as Proxies for Corneal Damage and Replicative Stress in Human Keratoconus Corneas

Abstract

Purpose: The pathogenesis of keratoconus (KC) is multifactorial, and associated with oxidative stress and subsequent DNA damage. We investigate differences in DNA damage and replicative stress in patients with KC, and in healthy and diseased controls. Methods: We obtained 64 corneal buttons from 27 patients with KC after corneal transplant surgery, 21 with a decompensated graft (DG), and 16 healthy controls (HC). The amount of intact Alu elements per genome copy as measured by quantitative polymerase chain reaction (qPCR) was used to quantify intact DNA. Telomere length was measured as a proxy for replicative stress. In addition, telomerase reverse transcriptase (hTERT) gene expression level was assessed. Results: Mean (6 standard deviation [SD]) DNA damage was similar between the KC (5.56 6 14.08), DG (3.16 6 8.22), and HC (3.51 6 6.66) groups (P = 0.807). No associations were found between DNA damage and patient age (P = 0.523), atopic constitution (P = 0.240), or contact lens wear (P = 0.393). Telomere length differed (P = 0.034), most notably in the KC group, and hTERT was not detected in any corneal sample. Three cross-linked (CXL) KC corneas did not contain significantly more DNA damage (☓2.6, P = 0.750). Conclusions: Based on these findings, differences in actual corneal DNA damage in KC could not be identified, and the longer telomere length in KC did not support replicative stress as a major etiologic factor in the pathogenesis of KC. Future longitudinal investigations on KC etiology should assess progressively early cases to better comprehend the cellular and molecular processes preceding the archetypical morphologic changes. Translational Relevance: The standard treatment for progressive keratoconus promotes the crosslinking of collagen fibers through ultraviolet radiation and the subsequent formation of reactive oxygen species. Our study helps to underline the safety of this treatment approach.