Diagnosis of radiosensitive severe combined immunodeficiency disease (RS-SCID) by Comet Assay, management of bone marrow transplantation.

Abstract

Severe combined immunodeficiency disease (SCID) is a rare inherited severe immunodeficiency, in which functions of T cells and B cells are impaired. SCID is inherited either in X-linked recessive, or autosomal recessive forms, and is either radiosensitive or radioresistant. Artemis (DCLRE1C gene), DNA ligase IV, DNA-PKC, and Cernunnos/XLF proteins are regarded as NHEJ (Non-Homologous End-Joining) proteins that are involved in the repair process of double-strand DNA breaks and their mutations would lead to cellular radiosensitivity. Diagnostic radiosensitivity assays are important for the management of clinical BMT (Bone Marrow Transplantation) conditions, such as what conditioning agents and doses should be used. In this study, five SCID patients and healthy controls were examined. Skin fibroblasts were cultured. After X-irradiation, cells either underwent clonogenic assay or incubated to allow DNA repair and examined by the alkaline comet assay. Finally, DCLRE1C, RAG-1, and RAG-2 genes sequenced. By clonogenic assay, three patients were detected as radiosensitive with possible mutations in NHEJ genes such as DCLRE1C gene. The percentage of DNA in the tail measured by comet assay, in all three patients, was significantly different from the two other patients and the control group (p-value < 0.05). By using Sanger sequencing, a mutation in DCLRE1C gene was detected in one of the radiosensitive patients and two mutations in RAG-1, and RAG-2 genes were detected in the two radioresistant patients. Our findings suggest that comet assay is a fast technique for the diagnosis of the radiosensitive form of SCID and is very suitable for the timely diagnosis of RS-SCID before BMT.