R158Q and G212S, novel pathogenic compound heterozygous variants in SLC12A3 of Gitelman syndrome

The dysfunction of Na + -Cl − cotransporter (NCC) caused by mutations in solute carrier family12, member 3 gene ( SLC12A3 ) primarily causes Gitelman syndrome (GS). In identifying the pathogenicity of R158Q and G212S variants of SLC12A3 , we evaluated the pathogenicity by bioinformatic, expression, and localization analysis of two variants from a patient in our cohort. The prediction of mutant protein showed that p.R158Q and p.G212S could alter protein’s three-dimensional structure. Western blot showed a decrease of mutant Ncc. Immunofluorescence of the two mutations revealed a diffuse positive staining below the plasma membrane. Meanwhile, we conducted a compound heterozygous model—Ncc R156Q/G210S mice corresponding to human NCC R158Q/G212S. Ncc R156Q/G210S mice clearly exhibited typical GS features, including hypokalemia, hypomagnesemia, and increased fractional excretion of K + and Mg 2+ with a normal blood pressure level, which made Ncc R156Q/G210S mice an optimal mouse model for further study of GS. A dramatic decrease and abnormal localization of the mutant Ncc in distal convoluted tubules contributed to the phenotype. The hydrochlorothiazide test showed a loss of function of mutant Ncc in Ncc R156Q/G210S mice. These findings indicated that R158Q and G212S variants of SLC12A3 were pathogenic variants of GS.