Frequent SLC12A3 mutations in Chinese Gitelman syndrome patients: structure and function disorder.
Frequent SLC12A3 mutations in Chinese Gitelman syndrome patients: structure and function disorder.
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DOI：
10.1530/ec-21-0262
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发表时间：
2022-01-27
影响因子：
2.9
通讯作者：
Chen L
中科院分区：
文献类型：
--
作者：
Jiang L;Peng X;Zhao B;Zhang L;Xu L;Li X;Nie M;Chen L
本研究旨在鉴定中国Gitelman综合征（GS）患者中常见的突变，预测人钠-氯共转运蛋白（hNCC）的三维结构变化，并在体外和体内检测这些突变和一些新突变的活性。总结了PubMed、中国国家知识基础设施和万方数据库中既往报告的中国GS患者的SLC 12 A3基因突变。使用I-TASSER workplace实现野生型（WT）和突变蛋白的预测构型。通过定点诱变产生六个错义突变（T60 M、L215 F、D486 N、N534 K、Q617 R和R928 C）。在非洲爪蟾卵母细胞表达系统中进行22 Na+摄取实验。在这项研究中，35例GS患者和20名健康志愿者进行了噻嗪试验。在310个GS家系中，SLC 12 A3基因的突变频率>3%，其中T60 M、T163 M、D486 N、R913 Q、R928 C和R959为常见的移码突变。预测蛋白质的三维结构在所有突变中都会改变。与WT hNCC相比，所有六种突变的噻嗪敏感性22 Na+摄取均显著降低：T60 M 22 ± 9.2%、R928 C 29 ± 12%、L215 F 38 ± 14%、N534 K 41 ± 15.5%、Q617 R 63 ± 22.1%和D486 N 77 ± 20.4%。在噻嗪试验中，20名健康对照者的氯排泄分数的净增加显著高于有或无T60 M或D486 N突变的GS患者。通过体外22 Na+摄取实验和患者噻嗪试验证实了常见突变（T60 M、D486 N和R928 C）和新突变（L215 F、N534 K和Q617 R）导致蛋白质结构改变和蛋白质功能障碍。
This study was conducted to identify the frequent mutations from reported Chinese Gitelman syndrome (GS) patients, to predict the three-dimensional structure change of human Na–Cl co-transporter (hNCC), and to test the activity of these mutations and some novel mutations in vitro and in vivo. SLC12A3 gene mutations in Chinese GS patients previously reported in the PubMed, China National Knowledge Infrastructure, and Wanfang database were summarized. Predicted configurations of wild type (WT) and mutant proteins were achieved using the I-TASSER workplace. Six missense mutations (T60M, L215F, D486N, N534K, Q617R, and R928C) were generated by site-directed mutagenesis. 22Na+ uptake experiment was carried out in the Xenopus laevisoocyte expression system. In the study, 35 GS patients and 20 healthy volunteers underwent the thiazide test. T60M, T163M, D486N, R913Q, R928C, and R959frameshift were frequent SLC12A3 gene mutations (mutated frequency >3%) in 310 Chinese GS families. The protein’s three-dimensional structure was predicted to be altered in all mutations. Compared with WT hNCC, the thiazide-sensitive 22Na+ uptake was significantly diminished for all six mutations: T60M 22 ± 9.2%, R928C 29 ± 12%, L215F 38 ± 14%, N534K 41 ± 15.5%, Q617R 63 ± 22.1%, and D486N 77 ± 20.4%. In thiazide test, the net increase in chloride fractional excretion in 20 healthy controls was significantly higher than GS patients with or without T60M or D486N mutations. Frequent mutations (T60M, D486N, and R928C) and novel mutations (L215F, N534K, and Q617R) lead to protein structure alternation and protein dysfunction verified by 22Na+ uptake experiment in vitro and thiazide test on the patients.