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

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

Zongyue Li et al.

References

[1] Melander O, Orho-Melander M, Bengtsson K, Lindblad U, Râstam L, Groop L, Hulthén UL. Genetic variants of thiazide-sensitive NaCl-cotransporter in Gitelman’s syndrome and primary hypertension. Hypertension 2000; 36(3): 389–394

[2] Gitelman HJ, Graham JB, Welt LG. A new familial disorder characterized by hypokalemia and hypomagnesemia. Trans Assoc Am Physicians 1966; 79: 221–235

[3] Tago N, Kokubo Y, Inamoto N, Naraba H, Tomoike H, Iwai N. A high prevalence of Gitelman’s syndrome mutations in Japanese. Hypertens Res 2004; 27(5): 327–331

[4] Ravarotto V, Loffing J, Loffing-Cueni D, Heidemeyer M, Pagnin E, Calò LA, Rossi GP. Gitelman’s syndrome: characterization of a novel c.1181G>A point mutation and functional classification of the known mutations. Hypertens Res 2018; 41(8): 578–588

[5] Riveira-Munoz E, Chang Q, Godefroid N, Hoenderop JG, Bindels RJ, Dahan K, Devuyst O; Belgian Network for Study of Gitelman Syndrome. Transcriptional and functional analyses of SLC12A3 mutations: new clues for the pathogenesis of Gitelman syndrome. J Am Soc Nephrol 2007; 18(4): 1271–1283

[6] Sabath E, Meade P, Berkman J, de los Heros P, Moreno E, Bobadilla NA, Vázquez N, Ellison DH, Gamba G. Pathophysiology of functional mutations of the thiazide-sensitive Na-Cl cotransporter in Gitelman disease. Am J Physiol Renal Physiol 2004; 287(2): F195–F203

[7] Miao Z, Gao Y, Bindels RJ, Yu W, Lang Y, Chen N, Ren H, Sun F, Li Y, Wang X, Shao L. Coexistence of normotensive primary aldosteronism in two patients with Gitelman’s syndrome and novel thiazide-sensitive Na-Cl cotransporter mutations. Eur J Endocrinol 2009; 161(2): 275–283

[8] Glaudemans B, Yntema HG, San-Cristobal P, Schoots J, Pfundt R, Kamsteeg EJ, Bindels RJ, Knoers NV, Hoenderop JG, Hoefsloot LH. Novel NCC mutants and functional analysis in a new cohort of patients with Gitelman syndrome. Eur J Hum Genet 2012; 20(3): 263–270

[9] Valdez-Flores MA, Vargas-Poussou R, Verkaart S, Tutakhel OA, Valdez-Ortiz A, Blanchard A, Treard C, Hoenderop JG, Bindels RJ, Jeleń S. Functionomics of NCC mutations in Gitelman syndrome using a novel mammalian cell-based activity assay. Am J Physiol Renal Physiol 2016; 311(6): F1159–F1167

[10] Blanchard A, Bockenhauer D, Bolignano D, Calò LA, Cosyns E, Devuyst O, Ellison DH, Karet Frankl FE, Knoers NV, Konrad M, Lin SH, Vargas-Poussou R. Gitelman syndrome: consensus and guidance from a Kidney Disease: Improving Global Outcomes (KDIGO) Controversies Conference. Kidney Int 2017; 91(1): 24–33

[11] Schultheis PJ, Lorenz JN, Meneton P, Nieman ML, Riddle TM, Flagella M, Duffy JJ, Doetschman T, Miller ML, Shull GE. Phenotype resembling Gitelman’s syndrome in mice lacking the apical Na⁺-Cl^– cotransporter of the distal convoluted tubule. J Biol Chem 1998; 273(44): 29150–29155

[12] Loffing J, Vallon V, Loffing-Cueni D, Aregger F, Richter K, Pietri L, Bloch-Faure M, Hoenderop JG, Shull GE, Meneton P, Kaissling B. Altered renal distal tubule structure and renal Na(+) and Ca(2+) handling in a mouse model for Gitelman’s syndrome. J Am Soc Nephrol 2004; 15(9): 2276–2288

[13] Yang SS, Lo YF, Yu IS, Lin SW, Chang TH, Hsu YJ, Chao TK, Sytwu HK, Uchida S, Sasaki S, Lin SH. Generation and analysis of the thiazide-sensitive Na⁺-Cl^– cotransporter (Ncc/Slc12a3) Ser707X knockin mouse as a model of Gitelman syndrome. Hum Mutat 2010; 31(12): 1304–1315

[14] Yang SS, Fang YW, Tseng MH, Chu PY, Yu IS, Wu HC, Lin SW, Chau T, Uchida S, Sasaki S, Lin YF, Sytwu HK, Lin SH. Phosphorylation regulates NCC stability and transporter activity in vivo. J Am Soc Nephrol 2013; 24(10): 1587–1597

[15] Shao L, Lang Y, Wang Y, Gao Y, Zhang W, Niu H, Liu S, Chen N. High-frequency variant p.T60M in NaCl cotransporter and blood pressure variability in Han Chinese. Am J Nephrol 2012; 35(6): 515–519

[16] Shen Q, Chen J, Yu M, Lin Z, Nan X, Dong B, Fang X, Chen J, Ding G, Zhang A, Gao C, Miao L, Xu Y, Jiang X, Bai H, Zhuang J, Gao X, Xu H; for Chinese Children Genetic Kidney Disease Database (CCGKDD). Multi-centre study of the clinical features and gene variant spectrum of Gitelman syndrome in Chinese children. Clin Genet 2021; 99(4): 558–564

[17] Huang CL, Yang SS, Lin SH. Mechanism of regulation of renal ion transport by WNK kinases. Curr Opin Nephrol Hypertens 2008; 17(5): 519–525

[18] Liu T, Wang C, Lu J, Zhao X, Lang Y, Shao L. Genotype/phenotype analysis in 67 Chinese patients with Gitelman’s syndrome. Am J Nephrol 2016; 44(2): 159–168

[19] Vargas-Poussou R, Dahan K, Kahila D, Venisse A, Riveira-Munoz E, Debaix H, Grisart B, Bridoux F, Unwin R, Moulin B, Haymann JP, Vantyghem MC, Rigothier C, Dussol B, Godin M, Nivet H, Dubourg L, Tack I, Gimenez-Roqueplo AP, Houillier P, Blanchard A, Devuyst O, Jeunemaitre X. Spectrum of mutations in Gitelman syndrome. J Am Soc Nephrol 2011; 22(4): 693–703

[20] Zhong F, Ying H, Jia W, Zhou X, Zhang H, Guan Q, Xu J, Fang L, Zhao J, Xu C. Characteristics and follow-up of 13 pedigrees with Gitelman syndrome. J Endocrinol Invest 2019; 42(6): 653–665

[21] Li C, Zhou X, Han W, Jiang X, Liu J, Fang L, Wang H, Guan Q, Gao L, Zhao J, Xu J, Xu C. Identification of two novel mutations in SLC12A3 gene in two Chinese pedigrees with Gitelman syndrome and review of literature. Clin Endocrinol (Oxf) 2015; 83(6): 985–993

[22] Jayasinghe K, Stark Z, Kerr PG, Gaff C, Martyn M, Whitlam J, Creighton B, Donaldson E, Hunter M, Jarmolowicz A, Johnstone L, Krzesinski E, Lunke S, Lynch E, Nicholls K, Patel C, Prawer Y, Ryan J, See EJ, Talbot A, Trainer A, Tytherleigh R, Valente G, Wallis M, Wardrop L, West KH, White SM, Wilkins E, Mallett AJ, Quinlan C. Clinical impact of genomic testing in patients with suspected monogenic kidney disease. Genet Med 2021; 23(1): 183–191

[23] Syrén ML, Tedeschi S, Cesareo L, Bellantuono R, Colussi G, Procaccio M, Alì A, Domenici R, Malberti F, Sprocati M, Sacco M, Miglietti N, Edefonti A, Sereni F, Casari G, Coviello DA, Bettinelli A. Identification of fifteen novel mutations in the SLC12A3 gene encoding the Na-Cl Co-transporter in Italian patients with Gitelman syndrome. Hum Mutat 2002; 20(1): 78

[24] Wang F, Shi C, Cui Y, Li C, Tong A. Mutation profile and treatment of Gitelman syndrome in Chinese patients. Clin Exp Nephrol 2017; 21(2): 293–299

[25] Budakoti M, Panwar AS, Molpa D, Singh RK, Büsselberg D, Mishra AP, Coutinho HDM, Nigam M. Micro-RNA: the darkhorse of cancer. Cell Signal 2021; 83: 109995

[26] Bicknell AA, Ricci EP. When mRNA translation meets decay. Biochem Soc Trans 2017; 45(2): 339–351

[27] Fujimura J, Nozu K, Yamamura T, Minamikawa S, Nakanishi K, Horinouchi T, Nagano C, Sakakibara N, Nakanishi K, Shima Y, Miyako K, Nozu Y, Morisada N, Nagase H, Ninchoji T, Kaito H, Iijima K. Clinical and genetic characteristics in patients with Gitelman syndrome. Kidney Int Rep 2019; 4(1): 119–125

[28] Godefroid N, Riveira-Munoz E, Saint-Martin C, Nassogne MC, Dahan K, Devuyst O. A novel splicing mutation in SLC12A3 associated with Gitelman syndrome and idiopathic intracranial hypertension. Am J Kidney Dis 2006; 48(5): e73–e79

[29] Riveira-Munoz E, Chang Q, Bindels RJ, Devuyst O. Gitelman’s syndrome: towards genotype-phenotype correlations? Pediatr Nephrol 2007; 22(3): 326–332 doi:10.1007/s00467-006-0321-1

[30] Tseng MH, Yang SS, Hsu YJ, Fang YW, Wu CJ, Tsai JD, Hwang DY, Lin SH. Genotype, phenotype, and follow-up in Taiwanese patients with salt-losing tubulopathy associated with SLC12A3 mutation. J Clin Endocrinol Metab 2012; 97(8): E1478–E1482

[31] Verlander JW, Tran TM, Zhang L, Kaplan MR, Hebert SC. Estradiol enhances thiazide-sensitive NaCl cotransporter density in the apical plasma membrane of the distal convoluted tubule in ovariectomized rats. J Clin Invest 1998; 101(8): 1661–1669

[32] Nieves JW, Komar L, Cosman F, Lindsay R. Calcium potentiates the effect of estrogen and calcitonin on bone mass: review and analysis. Am J Clin Nutr 1998; 67(1): 18–24

[33] Park S, Kang S, Kim DS. Severe calcium deficiency increased visceral fat accumulation, down-regulating genes associated with fat oxidation, and increased insulin resistance while elevating serum parathyroid hormone in estrogen-deficient rats. Nutr Res 2020; 73: 48–57

[34] McKane WR, Khosla S, Burritt MF, Kao PC, Wilson DM, Ory SJ, Riggs BL. Mechanism of renal calcium conservation with estrogen replacement therapy in women in early postmenopause—a clinical research center study. J Clin Endocrinol Metab 1995; 80(12): 3458–3464

[35] Dick IM, Devine A, Beilby J, Prince RL. Effects of endogenous estrogen on renal calcium and phosphate handling in elderly women. Am J Physiol Endocrinol Metab 2005; 288(2): E430–E435

[36] Nijenhuis T, Vallon V, van der Kemp AW, Loffing J, Hoenderop JG, Bindels RJ. Enhanced passive Ca²⁺ reabsorption and reduced Mg²⁺ channel abundance explains thiazide-induced hypocalciuria and hypomagnesemia. J Clin Invest 2005; 115(6): 1651–1658

[37] Chubanov V, Gudermann T. Trpm6. Handb Exp Pharmacol 2014; 222: 503–520

[38] Flores-Aldama L, Vandewege MW, Zavala K, Colenso CK, Gonzalez W, Brauchi SE, Opazo JC. Evolutionary analyses reveal independent origins of gene repertoires and structural motifs associated to fast inactivation in calcium-selective TRPV channels. Sci Rep 2020; 10(1): 8684

[39] Pumroy RA, Fluck EC 3rd, Ahmed T, Moiseenkova-Bell VY. Structural insights into the gating mechanisms of TRPV channels. Cell Calcium 2020; 87: 102168

[40] Yang SS, Morimoto T, Rai T, Chiga M, Sohara E, Ohno M, Uchida K, Lin SH, Moriguchi T, Shibuya H, Kondo Y, Sasaki S, Uchida S. Molecular pathogenesis of pseudohypoaldosteronism type II: generation and analysis of a Wnk4(D561A/+) knockin mouse model. Cell Metab 2007; 5(5): 331–344

Acknowledgements

The authors thank all researchers and subjects who participated in this study. This work was supported by grants from the National Natural Science Foundation of China (No. 81974124), Special Funds for Taishan Scholar Project (No. tsqn20161071) and Academic Promotion Program of Shandong First Medical University (No. 2019RC015).

Compliance with ethics guidelines

Zongyue Li, Huixiao Wu, Shuoshuo Wei, Moke Liu, Yingzhou Shi, Mengzhu Li, Ning Wang, Li Fang, Bo Xiang, Ling Gao, Chao Xu, and Jiajun Zhao declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all patients for being included in the study. All institutional and national guidelines for the care and use of laboratory animals were followed.

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