Functional analysis of XRCC4 mutations in reported microcephaly and growth defect patients in terms of radiosensitivity.
Functional analysis of XRCC4 mutations in reported microcephaly and growth defect patients in terms of radiosensitivity.
复制标题
XRCC4突变的功能分析在放射敏感性方面报告的小头畸形和生长缺陷患者。
DOI：
10.1093/jrr/rrab016
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发表时间：
2021-05-12
影响因子：
2
通讯作者：
Matsumoto Y
中科院分区：
文献类型：
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作者：
Asa ADDC;Wanotayan R;Sharma MK;Tsukada K;Shimada M;Matsumoto Y
非同源末端连接是DNA双链断裂（DSB）修复的主要途径之一，也参与免疫系统中的V（D）J重组。因此，非同源末端连接（NHEJ）蛋白的突变被发现与人类以及模型动物的免疫缺陷相关。据报道，几名患有XRCC4突变的人类患者表现出小头畸形和生长缺陷，但出乎意料地表现出正常的免疫功能。在这里，为了评估这些疾病相关的XRCC4突变在放射敏感性方面的功能，我们在XRCC4缺陷的小鼠M10细胞中产生了表达这些突变体的稳定转染子，并通过集落形成测定法测量了它们的放射敏感性。V83_S105del、R225X和D254Mfs*68的表达水平与野生型XRCC4相似，而W43R、R161Q和R275X的表达水平甚至高于野生型XRCC4。DNA连接酶IV在具有这些突变体的转染子中的表达水平与野生型XRCC4转染子中的表达水平相当。与野生型XRCC4转染子相比，V83S_S105del转染子和D254Mfs*68转染子在较小程度上显示出显著增加的放射敏感性。与野生型XRCC4转染子相比，W43 R、R161 Q、R225 X和R275 X转染子显示出轻微但统计学显著的放射敏感性增加。当与绿色荧光蛋白（GFP）融合表达时，R225 X、R275 X和D254 Mfs *68定位于细胞质，而其它突变体定位于细胞核。这些结果共同表明，患者中XRCC4的缺陷可能主要是由于蛋白质数量不足和功能受损，强调了XRCC4的DSB修复功能在正常发育中的重要性。
Non-homologous end joining is one of the main pathways for DNA double-strand break (DSB) repair and is also implicated in V(D)J recombination in immune system. Therefore, mutations in non-homologous end-joining (NHEJ) proteins were found to be associated with immunodeficiency in human as well as in model animals. Several human patients with mutations in XRCC4 were reported to exhibit microcephaly and growth defects, but unexpectedly showed normal immune function. Here, to evaluate the functionality of these disease-associated mutations of XRCC4 in terms of radiosensitivity, we generated stable transfectants expressing these mutants in XRCC4-deficient murine M10 cells and measured their radiosensitivity by colony formation assay. V83_S105del, R225X and D254Mfs*68 were expressed at a similar level to wild-type XRCC4, while W43R, R161Q and R275X were expressed at even higher level than wild-type XRCC4. The expression levels of DNA ligase IV in the transfectants with these mutants were comparable to that in the wild-type XRCC4 transfectant. The V83S_S105del transfectant and, to a lesser extent, D254Mfs*68 transfectant, showed substantially increased radiosensitivity compared to the wild-type XRCC4 transfectant. The W43R, R161Q, R225X and R275X transfectants showed a slight but statistically significant increase in radiosensitivity compared to the wild-type XRCC4 transfectant. When expressed as fusion proteins with Green fluorescent protein (GFP), R225X, R275X and D254Mfs*68 localized to the cytoplasm, whereas other mutants localized to the nucleus. These results collectively indicated that the defects of XRCC4 in patients might be mainly due to insufficiency in protein quantity and impaired functionality, underscoring the importance of XRCC4’s DSB repair function in normal development.