Frequent SLC12A3 mutations in Chinese Gitelman syndrome patients: structure and function disorder

Abstract

Purposes: 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. Methods: SLC12A3 gene mutations in Chinese GS patients previously reported in the PubMed, China National Knowledge Infrastructure, and Wanfang da tabase were summarized. Predicted configurations of wild type (WT) and mutan t proteins were achieved using the I-TASSER workplace. Six missense mutations ( T60M, L215F, D486N, N534K, Q617R, and R928C) were generated by site-directed mutage nesis. 22Na+ uptake experiment was carried out in the Xenopus laevis oocyte expression system. In the study, 35 GS patients and 20 healthy volunteers underwent the thiazide test. Results: T60M, T163M, D486N, R913Q, R928C, and R959frameshift were freq uent SLC12A3 gene mutations (mutated frequency >3%) in 310 Chinese GS famili es. 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%, N534 K 41 ± 15.5%, Q617R 63 ± 22.1%, and D486N 77 ± 20.4%. In thiazide test, the net inc rease in chloride fractional excretion in 20 healthy controls was significantly higher than G S patients with or without T60M or D486N mutations. Conclusions: Frequent mutations (T60M, D486N, and R928C) and novel mutation s (L215F, N534K, and Q617R) lead to protein structure alternation and pro tein dysfunction verified by 22Na+ uptake experiment in vitro and thiazide test on the patients. Purposes: 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. Methods: SLC12A3 gene mutations in Chinese GS patients previously reported in the PubMed, China National Knowledge Infrastructure, and Wanfang da tabase were summarized. Predicted configurations of wild type (WT) and mutan t proteins were achieved using the I-TASSER workplace. Six missense mutations ( T60M, L215F, D486N, N534K, Q617R, and R928C) were generated by site-directed mutage nesis. 22Na+ uptake experiment was carried out in the Xenopus laevis oocyte expression system. In the study, 35 GS patients and 20 healthy volunteers underwent the thiazide test. Results: T60M, T163M, D486N, R913Q, R928C, and R959frameshift were freq uent SLC12A3 gene mutations (mutated frequency >3%) in 310 Chinese GS famili es. 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%, N534 K 41 ± 15.5%, Q617R 63 ± 22.1%, and D486N 77 ± 20.4%. In thiazide test, the net inc rease in chloride fractional excretion in 20 healthy controls was significantly higher than G S patients with or without T60M or D486N mutations. Conclusions: Frequent mutations (T60M, D486N, and R928C) and novel mutation s (L215F, N534K, and Q617R) lead to protein structure alternation and pro tein dysfunction verified by 22Na+ uptake experiment in vitro and thiazide test on the patients.

Keywords

• gitelman syndrome
• slc12a3 gene
• frequent mutations
• 22na+ uptake activity
• thiazide test