Jiang Lanping, Peng Xiaoyan, Zhao Bingbin, Zhang Lei, Xu Lubin, Li Xuemei, Nie Min, Chen Limeng
Department of Nephrology, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Department of Nephrology & Key Laboratory of Nephrology, National Health Commission and Guangdong Province, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
Endocr Connect. 2022 Jan 27;11(1):e210262. doi: 10.1530/EC-21-0262.
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.
本研究旨在确定已报道的中国吉特曼综合征（GS）患者中的常见突变，预测人钠氯共转运体（hNCC）的三维结构变化，并在体外和体内测试这些突变以及一些新突变的活性。
总结了先前在PubMed、中国知网和万方数据库中报道的中国GS患者的SLC12A3基因突变情况。使用I-TASSER工作平台获得野生型（WT）和突变蛋白的预测构象。通过定点诱变产生六个错义突变（T60M、L215F、D486N、N534K、Q617R和R928C）。在非洲爪蟾卵母细胞表达系统中进行22Na+摄取实验。在本研究中，35例GS患者和20名健康志愿者接受了噻嗪类药物试验。
T60M、T163M、D486N、R913Q、R928C和R959移码是310个中国GS家系中常见的SLC12A3基因突变（突变频率>3%）。预测所有突变都会改变蛋白质的三维结构。与WT hNCC相比，所有六个突变的噻嗪类敏感22Na+摄取均显著降低：T60M为22±9.2%，R928C为29±12%，L215F为38±14%，N534K为41±15.5%，Q617R为63±22.1%，D486N为77±20.4%。在噻嗪类药物试验中，20名健康对照者的氯排泄分数净增加显著高于有或无T60M或D486N突变的GS患者。
常见突变（T60M、D486N和R928C）和新突变（L215F、N534K和Q617R）导致蛋白质结构改变和蛋白质功能障碍，这在体外22Na+摄取实验和对患者的噻嗪类药物试验中得到了验证。
