Background and objective

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.

Materials and methods

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.

Results

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.

Conclusion

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.

中文翻译：

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通过彗星试验诊断放射敏感性严重联合免疫缺陷病 (RS-SCID)，骨髓移植管理。

背景与目的

严重联合免疫缺陷病（SCID）是一种罕见的遗传性严重免疫缺陷，其中 T 细胞和 B 细胞功能受损。SCID 以 X 连锁隐性或常染色体隐性形式遗传，并且对放射敏感或放射抗性。Artemis（DCLRE1C基因）、DNA 连接酶 IV、DNA-PKC 和 Cernunnos/XLF 蛋白被认为是 NHEJ（非同源末端连接）蛋白，参与双链 DNA 断裂的修复过程，它们的突变会导致对细胞放射敏感性。诊断放射敏感性测定对于临床 BMT（骨髓移植）病症的管理很重要，例如应使用何种调理剂和剂量。

材料和方法

在这项研究中，检查了五名 SCID 患者和健康对照。培养皮肤成纤维细胞。X 射线照射后，细胞要么进行克隆形成试验，要么孵育以允许 DNA 修复并通过碱性彗星试验进行检查。最后，对DCLRE1C、RAG-1和RAG-2基因进行测序。

结果

通过克隆测定，三名患者被检测为对放射敏感且 NHEJ 基因（如DCLRE1C基因）可能发生突变。在所有三名患者中，通过彗星试验测量的尾部 DNA 百分比与其他两名患者和对照组显着不同（p 值 < 0.05）。通过Sanger测序，在其中一名放射敏感患者中检测到DCLRE1C基因突变，在两名放射抗性患者中检测到RAG-1和RAG-2基因突变。

结论

我们的研究结果表明，彗星试验是一种诊断放射敏感型 SCID 的快速技术，非常适合在 BMT 之前及时诊断 RS-SCID。