R158Q and G212S, novel pathogenic compound heterozygous variants in <i>SLC12A3</i> of Gitelman syndrome

doi:10.1007/s11684-022-0963-9

Front. Med.

2022, Vol. 16

Issue (6) : 932-945 https://doi.org/10.1007/s11684-022-0963-9

RESEARCH ARTICLE

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

Zongyue Li^1,^2,³, Huixiao Wu^1,^2,³, Shuoshuo Wei^1,^2,³, Moke Liu^1,^2,³, Yingzhou Shi^1,^2,³, Mengzhu Li^1,^2,³, Ning Wang^2,^3,⁴, Li Fang^2,^3,⁴, Bo Xiang^2,^3,⁴, Ling Gao^1,^2,^3,⁴, Chao Xu^1,^2,^3,⁴(

), Jiajun Zhao^1,^2,^3,⁴(

)

¹. Department of Endocrinology, Shandong Provincial Hospital, Shandong University, Jinan 250021, China
². Shandong Provincial Key Laboratory of Endocrinology and Lipid Metabolism, Jinan 250021, China
³. Shandong Institute of Endocrine and Metabolic Disease, Jinan 250021, China
⁴. Department of Endocrinology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan 250021, China

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Abstract

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 Mg²⁺ 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.

Keywords Gitelman syndrome mouse model compound heterozygous hypokalemia Slc12a3

Corresponding Author(s): Chao Xu,Jiajun Zhao

Just Accepted Date: 23 September 2022 Online First Date: 15 November 2022 Issue Date: 16 January 2023

Cite this article:

Zongyue Li,Huixiao Wu,Shuoshuo Wei, et al. R158Q and G212S, novel pathogenic compound heterozygous variants in SLC12A3 of Gitelman syndrome[J]. Front. Med., 2022, 16(6): 932-945.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-022-0963-9
https://academic.hep.com.cn/fmd/EN/Y2022/V16/I6/932

Tab.1 Clinical characterization of the patient

Fig.1 Effects of SLC12A3 variants on NCC expression and localization in vitro. (A) Schematic diagram of NCC and variants identified in our study. NCC is represented as a 12-transmembrane protein with intracytoplasmic amino and carboxyl termini. The sites of variants are denoted by arrows and solid dot. (B) Cross-species conservation of SLC12A3 around R158 and G212. (C) Protein structure prediction of WT and mutant SLC12A3. (D) Upper, immunoblot of NCC using cell surface fraction and cytoplasmic fraction from MDCT transfected with WT-NCC, R158Q-NCC, and G212S-NCC cDNA. Na⁺/K⁺ ATPase was used as a loading control for the cell surface fraction. For cytoplasmic NCC, GAPDH was used as a loading control. Lower, bar chart shows the relative cell surface and cytoplasmic NCC protein levels in MDCT cells after being transfected with R158Q-NCC and G212S-NCC cDNA compared with WT-NCC. Data represent mean ± SEM of three independent experiments. *P < 0.05, ***P < 0.001, vs. WT. (E) Immunofluorescence analyses of MDCT cells transfected with WT-NCC, R158Q-NCC, and G212S-NCC cDNA. WT-NCC immunostained at the membrane and cytoplasm, whereas R158-NCC and G212S-NCC showed cytoplasmic immunostaining under the membrane.

Fig.2 Body weight and blood and urine biochemistries of Ncc R156Q and G210S knock-in mice. (A–F) Body weight (g), urine (μL/day), serum K⁺ (mmol/L), serum Mg²⁺ (mmol/L), urine Ca²⁺/Cr (mmol/mmol), and fractional excretion of K⁺(%) in WT (n = 12), Ncc^R158Q/+(n = 9), Ncc^G210S/+(n = 12), Ncc^R158Q/R158Q(n = 7), Ncc^G210S/G210S(n = 12), and Ncc^R156Q/G210S(n = 12) mice.

Tab.2 Blood pressure level and blood and urine biochemistries of Ncc R156Q and G210S knock-in mice

Fig.3 Renal Ncc expression and localization in Ncc R156Q and G210S knock-in mice. (A) RT-PCR analysis of Ncc mRNA. Quantitative gene expression of Ncc in WT, heterozygotes (Ncc^R158Q/+, Ncc^G210S/+), homozygotes (Ncc^R158Q/R158Q, Ncc^G210S/G210S), and compound heterozygotes (Ncc^R156Q/G210S, n = 3) was relative to control (control = 100%, **P < 0.01 versus WT). (B) An antibody recognizing the N-terminal of Ncc is applied to detect Ncc expression. Analysis on Ncc expression by Western blotting in WT, heterozygote (Ncc^R158Q/+, Ncc^G210S/+), homozygote (Ncc^R158Q/R158Q, Ncc^G210S/G210S), and compound heterozygote (Ncc^R156Q/G210S) mice. (C) Immunofluorescence for Ncc in WT, heterozygote (Ncc^G210S/+), homozygote (Ncc^R158Q/R158Q), and compound heterozygote (Ncc^R156Q/G210S) mice. (D) All images were quantified by measuring fluorescence intensity. The ROI of cell membrane fluorescence intensity of wild-type mice accounted for the total fluorescence intensity, which was set to 100%. Data are presented as mean ± SEM. **P < 0.01 and ***P < 0.01 indicate significant difference in relation to wild-type mice. (E) Immunohistochemical staining for Ncc in WT, heterozygote (Ncc^R158Q/+, Ncc^G210S/+), homozygote (Ncc^R158Q/R158Q, Ncc^G210S/G210S), and compound heterozygote (Ncc^R156Q/G210S) mice.

Fig.4 Gender effect on the phenotype of NCC^R156QG210S mice. (A) serum K⁺, serum Mg²⁺, fractional excretion of K⁺, fractional excretion of Mg²⁺, and 24 h excretion of Ca^{2 +} in urine of male and female WT and CH mice. *P < 0.05, **P < 0.01 versus WT. (B) Immunohistochemical staining of Ncc in male (♂) and female (♀) compound heterozygous (NccR156Q/G210S) mice.

Fig.5 Expression of the magnesium channel and epithelial calcium channels in male and female NCC^R156Q/G210S mice. (A) Gene expression analysis of Slc12a3, Trpm6, Trpv5, and Trpv6 in male and female WT and CH mice by qPCR. *P < 0.05, **P < 0.01, ***P < 0.001 versus WT. (B) Analysis on the magnesium channel and epithelial calcium channels by Western blotting in male and female NCC^R156Q/G210S mice. (C) Quantitative analysis of the magnesium channel and epithelial calcium channel levels was presented as the percentage of control (control = 100%). *P < 0.05, **P < 0.01, ***P < 0.001 versus WT.

Fig.6 Effect of hydrochlorothiazide (HCTZ) administration on urine amount and Na⁺, K⁺, and Ca^{2 +} excretion in Ncc^R156Q/G210S mice. After a single dose of HCTZ (12.5 mg/kg) was intraperitoneally administered to WT and CH (compound heterozygotes, Ncc^R156Q/G210S) mice, urine was collected for 4 h. FENa, FEK, and FECa represented the fractional excretion of Na⁺, K⁺, and Ca²⁺, respectively. In response to HCTZ, significantly increased urine amount (A), FEK (B), and FENa (C) were observed in WT but blunted in CH mice. No significant difference in Ca²⁺/Cr was observed (D). *P < 0.05, **P < 0.01, ****P < 0.0001.

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