Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by accumulation of extracellular matrix components, especially collagen, which leads to fibrosis of the skin and internal organs. SSc shows a complex etiology and the exact cause of the disease is still unknown, however both environmental and genetic factors seem to influence the onset and outcome of the disease. Additionally, different pathophysiological processes influence the clinical heterogeneity of the disease and production of specific autoantibodies. Patients with SSc present with increased genomic instability, elevated DNA damage and impaired DNA repair mechanisms.
The aim of our study was to define whether it is possible to detect elevated endogenous double-stranded DNA damage with the Comet assay in patients with SSc. Moreover, we assessed if hydrogen peroxide induces DNA damage and evaluated the efficiency of the DNA repair mechanisms in vitro, in leukocytes of patients with SSc and healthy controls. Therefore, we tested the amount of endogenous DNA damage in blood, immediately after blood sampling, 30 minutes after the application of hydrogen peroxide and 45 minutes after the removal of hydrogen peroxide, when the DNA repair activity already began. The Comet assay is a simple and sensitive technique for the determination of DNA breaks in vitro which enables automation. We evaluated prepared blood samples of patients with SSc and healthy controls in two different ways. Semiquantitative measurement included examination of the comets under the microscope by hand and quantitative measurement consisted of taking the images of the comets and automatically analyzing them with the software tool OpenComet. The programme identifies the comets and measures their parameters, of which the most important are tail parameters (the tail length, the percentage of DNA in the tail, the tail moment and the olive moment). Finally, we determined the compliance of the semiquantitative and quantitative evaluation and possible associations of the amount of DNA breaks and efficiency of the DNA repair mechanisms with age, gender or the presence of specific autoantibodies.
We established a good correlation between semiquantitative and quantitative evaluation of the comets in the Comet assay. The amount of endogenous double-stranded DNA damage was not significantly different between patients with SSc and healthy controls. The amount of endogenous double-stranded DNA breaks and the amount of induced exogenous double-stranded DNA breaks were significantly higher in older healthy controls then in younger healthy individuals. Since age decreases the efficiency of the DNA repair mechanisms, we also demonstrated that DNA repair mechanisms were significantly less efficient in older controls, independent of gender, and the tail moment was much more profound. These changes were not observed in patients with SSc, in any of the three treatments. However, when comparing patients with different autoantibodies, we found significantly higher amount of endogenous double-stranded DNA damage in SSc patients with anticentromere antibodies than in SSc patients with anti-topoisomerase ① antibodies, indicating changes in endogenous DNA damage depend on the autoantibodies present.