Abstract
DNA damage is a constant event in every cell caused by exogenous factors such as ultraviolet and ionizing radiation (UVR/IR) and intercalating drugs, or endogenous metabolic and replicative stress. Proteins of the DNA damage response (DDR) network sense DNA lesions and induce cell cycle arrest, DNA repair, and apoptosis. Genetic defects of DDR or DNA repair proteins can be associated with immunodeficiency, bone marrow failure syndromes, and cancer susceptibility. Although various diagnostic tools are available to evaluate DNA damage, their quality to identify DNA repair deficiencies differs enormously and depends on affected pathways. In this study, we investigated the DDR biomarkers γH2AX (Ser139), p-ATM (Ser1981), and p-CHK2 (Thr68) using flow cytometry on peripheral blood cells obtained from patients with combined immunodeficiencies due to non-homologous end-joining (NHEJ) defects and ataxia telangiectasia (AT) in response to low-dose IR. Significantly reduced induction of all three markers was observed in AT patients compared to controls. However, delayed downregulation of γH2AX was found in patients with NHEJ defects. In contrast to previous reports of DDR in cellular models, these biomarkers were not sensitive enough to identify ARTEMIS deficiency with sufficient reliability. In summary, DDR biomarkers are suitable for diagnosing NHEJ defects and AT, which can be useful in neonates with abnormal TREC levels (T cell receptor excision circles) identified by newborn screening. We conclude that DDR biomarkers have benefits and some limitations depending on the underlying DNA repair deficiency.
Highlights
Every cell is constantly exposed to DNA damage caused by external factors such as ionizing (IR) and ultraviolet radiation (UVR), chemicals — including alkylating drugs, or by endogenous factors such as replicative and metabolic stress
After culture for 4 days to recover from metabolic stress, Peripheral blood mononuclear cells (PBMCs) were treated with 2 Gy of IR
The γH2AX response was investigated by flow cytometry in CD45+CD3+ T and CD45+CD3−CD56dimCD16+ NK lymphocytes before IR, and 1 h, 4 h, 8 h, and 24 h after DNA damage was induced (Figure S1)
Summary
Every cell is constantly exposed to DNA damage caused by external factors such as ionizing (IR) and ultraviolet radiation (UVR), chemicals — including alkylating drugs, or by endogenous factors such as replicative and metabolic stress. While these insults may result in both DNA single-strand breaks (SSBs) and double-strand breaks (DSBs), the latter are more critical in terms of cell survival and mutation probability. Journal of Clinical Immunology to protect the DNA from any persisting damage, known as DNA damage response (DDR) If this system fails, apoptosis, senescence, or introduction of chromosomal breaks and mutations potentially leading to neoplastic transformation are the consequences [3]
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