Wang Na, Yang Yuanxing, Tian Xiong, Fu Hongjun, Chen Shuaishuai, Du Juping, Xu Mengyi, He Haixia, Shen Bo, Xu Jiaqin
Department of Clinical Laboratory, Taizhou Hospital of Zhejiang Province Affiliated to Wenzhou Medical University, 150 Ximen Street, Linhai, China.
Department of Endocrinology, Taizhou Hospital of Zhejiang Province Affiliated to Wenzhou Medical University, Linhai, China.
Orphanet J Rare Dis. 2025 Feb 11;20(1):66. doi: 10.1186/s13023-025-03577-8.
Gitelman syndrome (GS) is an inherited renal tubular disorder characterized by hypokalemic alkalosis and hypomagnesemia, due to biallelic pathogenic variants in the solute carrier family 12 member 3 (SLC12A3) gene encoding a sodium-chloride (Na-Cl) cotransporter (NCC). This work aimed at identifying SLC12A3 variants in the GS pedigree and reveal the effect of the mutations on protein structure and function.
Whole-exome sequencing (WES) and Sanger sequencing were performed in the pedigree. Configuration prediction of two mutant NCC proteins were achieved using SWISS-MODEL. The SLC12A3 missense mutants were generated by site-specific mutagenesis, and the protein expression, location and Na uptake activity were assessed by using the HEK293T cell line.
Genetic analysis identified novel compound heterozygous SLC12A3 variants (c.718G > A/p.E240K and c.2675T > C/p.L892P) in the patient with typical GS phenotype. Both of her parents, elder brother and her son carried the heterozygous p.L892P variant, but only the elder brother exhibited mild hypokalemia. Bioinformatics tools predicted that both mutations were highly species conserved and pathogenic. The prediction of mutant protein indicated that p.E240K and p.L892P altered protein's secondary and three-dimensional (3D) structure and stability. Functional experiments revealed decreased protein expression and Na uptake activity caused by these two variants, especially the p.L892P variant.
Our study presents the genetic and functional evidence for the novel compound heterozygous loss-of-function variants in SLC12A3 that may synergistically cuase GS, and expands the mutation spectrum of SLC12A3 variants in patients with GS.
吉特曼综合征（GS）是一种遗传性肾小管疾病，其特征为低钾性碱中毒和低镁血症，这是由于编码氯化钠（Na-Cl）共转运蛋白（NCC）的溶质载体家族12成员3（SLC12A3）基因发生双等位基因致病性变异所致。本研究旨在鉴定GS家系中的SLC12A3变异，并揭示这些突变对蛋白质结构和功能的影响。
对该家系进行全外显子测序（WES）和桑格测序。使用SWISS-MODEL对两种突变的NCC蛋白进行构象预测。通过位点特异性诱变产生SLC12A3错义突变体，并使用HEK293T细胞系评估蛋白质表达、定位和钠摄取活性。
遗传分析在具有典型GS表型的患者中鉴定出新型复合杂合SLC12A3变异（c.718G＞A/p.E240K和c.2675T＞C/p.L892P）。她的父母、哥哥和儿子均携带杂合p.L892P变异，但只有哥哥表现出轻度低钾血症。生物信息学工具预测这两种突变在物种中高度保守且具有致病性。突变蛋白预测表明，p.E240K和p.L892P改变了蛋白质的二级和三维（3D）结构及稳定性。功能实验显示，这两种变异，尤其是p.L892P变异，导致蛋白质表达和钠摄取活性降低。
我们的研究提供了SLC12A3中新型复合杂合功能丧失变异可能协同导致GS的遗传和功能证据，并扩展了GS患者中SLC12A3变异的突变谱。
