Novel NCC mutants and functional analysis in a new cohort of patients with Gitelman syndrome

Abstract

Gitelman syndrome (GS) is an autosomal recessive disorder characterized by hypokalemic metabolic alkalosis in conjunction with significant hypomagnesemia and hypocalciuria. The GS phenotype is caused by mutations in the solute carrier family 12, member 3 (SLC12A3) gene that encodes the thiazide-sensitive NaCl cotransporter (NCC). We analyzed DNA samples of 163 patients with a clinical suspicion of GS by direct sequencing of all 26 exons of the SLC12A3 gene. In total, 114 different mutations were identified, 31 of which have not been reported before. These novel variants include 3 deletions, 18 missense, 6 splice site and 4 nonsense mutations. We selected seven missense mutations to investigate their effect on NCC activity and plasma membrane localization by using the Xenopus laevis oocyte expression system. The Thr392Ile mutant did not display transport activity (probably class 2 mutation), while the Asn442Ser and Gln1030Arg NCC mutants showed decreased plasma membrane localization and consequently function, likely due to impaired trafficking (class 3 mutation). Even though the NaCl uptake was hampered for NCC mutants Glu121Asp, Pro751Leu, Ser475Cys and Tyr489His, the transporters reached the plasma membrane (class 4 mutation), suggesting an effect on NCC regulation or ion affinity. The present study shows the identification of 38 novel mutations in the SLC12A3 gene and provides insight into the mechanisms that regulate NCC.

Figure 1

Multiple alignment and predicted topological localization of each NCC mutant. (a) Multiple alignment analysis shows conservation among species of the identified NCC mutant amino acids (gray bar). Gray and black letters represent conserved and non-conserved amino acids, respectively. (b) Schematic topological representation of NCC, which consists of large intracellular N- and C-terminal domains, which are located within the cell, 12 transmembrane segments (S) and a large hydrophilic extracellular loop between S7 and S8 comprising two glycosylation sites. Every dot represents one amino acid and the localization of the functionally characterized NCC mutations is denoted by a white dot (numbers 1–7).

Figure 2

Expression and functional consequence of exogenous wild-type (wt) and mutant NCC in X. laevis oocytes. (a) Western blotting analysis of the total membrane fraction of oocytes injected with H₂0, and wt or mutant NCC cRNA. (b) ²²Na⁺ uptake was measured in oocytes injected with H₂0, wt (open bars) or mutant NCC cRNA (black bars). For all groups, transport assays were also performed in the presence of NCC blocker metolazone. Uptake values were shown as thiazide-sensitive Na⁺ transport (wt was set as 100%). 121: Glu121Asp; 392: Thr392Ile; 442: Asn442Ser; 475: Ser475Cys; 489: Tyr489His; 751: Pro751Leu; 1030: Gln1030Arg; NCC glyc: glycosylated NCC; kDa: kilodalton. Data are presented as means±SEM. ^*P<0.05 indicates significant difference in relation to Wt NCC-injected oocytes.

Figure 3

Surface expression of wt or mutant NCC protein in X. laevis oocytes, assessed by measuring fluorescence using laser-scanning confocal microscopy. (a) Representative confocal images of X. laevis oocytes expressing wild-type (wt) or mutant eGFP–NCC. (b) All wt (open bar) and mutant (black bars) images were quantified by measuring pixel intensity, while wt was set to 100%. 121: Glu121Asp; 392: Thr392Ile; 442: Asn442Ser; 475: Ser475Cys; 489: Tyr489His; 751: Pro751Leu; 1030: Gln1030Arg. Data are presented as means±SEM. ^*P<0.05 indicates significant difference in relation to wt NCC-injected oocytes.