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.