Journal Article

Functional evaluation of novel compound heterozygous variants in SLC12A3 of Gitelman syndrome

Tian, Xiong, Chen, Shuaishuai, He, Haixia, Xu, Jiaqin, Xu, Mengyi, Shen, Bo, Fu, Hongjun, Yang, Yuanxing, Wang, Na, Du, Juping

2025

Overview

Background 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. Methods 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. Results 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. Conclusion 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.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Alkalosis / Bioinformatics / Chloride / DNA sequencing / Exome Sequencing / Female / Gene mutation / Gene mutations / Genetic analysis / Genetic aspects / Gitelman syndrome / Gitelman Syndrome - genetics / Gitelman Syndrome - metabolism / HEK293 Cells / Heterozygote / Hormones / Hospitals / Human Genetics / Humans / Hypokalemia / Hypomagnesemia / Kidney diseases / Loss-of-function / Male / Medical research / Medicine / Medicine & Public Health / Medicine, Experimental / Metabolism / Missense mutant / Mutagenesis / Mutation / Mutation, Missense / Nucleotide sequencing / Patients / Pedigree / Pharmacology/Toxicology / Phenotypes / Physiological aspects / Potassium / Protein expression / Protein structure / Proteins / Site-directed mutagenesis / SLC12A3 / Sodium / Solute Carrier Family 12, Member 3 - genetics / Solute Carrier Family 12, Member 3 - metabolism / Structure-function relationships / Type 2 diabetes / Whole genome sequencing / Wildlife conservation