Identification of fifteen novel mutations in the SLC12A3 gene encoding the Na-Cl Co-transporter in Italian patients with Gitelman syndrome

Abstract

The SLC12A3 gene encodes the thiazide-sensitive Na-Cl co-transporter (NCCT) expressed in the apical membrane of the distal convoluted tubule of the kidney. Inactivating mutations of this gene are responsible for Gitelman syndrome (GS), a disorder inherited as an autosomal recessive trait. We searched for SLC12A3 gene mutations in 21 Italian patients with the clinical and biochemical features of GS (hypokalemia, hypomagnesemia, metabolic alkalosis, hypocalciuria, and the absence of nephrocalcinosis). All coding regions with their intron-exon boundaries were analyzed using PCR and SSCP techniques followed by sequencing analysis. We identified 21 different mutations evenly distributed throughout the gene without any mutation hot-spot. Fifteen are novel variants, including 12 missense mutations, one deletion, one deletion-insertion and one splice site mutation: R158Q, T163M, W172R, G316V, G374V, G463E, A464T, S615W, V677M, R852S, R958G, C985Y, 2114-2120delACCAAGT, 2144-2158delGCCTTCTACTCGGATinsTG, and 531-2A>G.

Gitelman’s Syndrome Associated with Chondrocalcinosis: A Case Study from the Azores Islands (Portugal)

“…In the present study the specific involvement of this cotransporter in the etiology of this disorder is further substantiated by the finding that the proband is homozygous for the S615L variation. The S615L identified in this study was previously described by Cruz and co-workers [18] in a study involved 36 kindreds from the United States, Canada and England and later reported in a study by Syrén et al [22]. Although the SLC12A3 variations reported in previous studies are distributed throughout the whole protein [4,23], the study of Lemmink (1998) indicates that the carboxyterminal end represents a hot spot for variations [4].…”

Rare independent mutations in renal salt handling genes contribute to blood pressure variation

“…In addition, we included one further variant in NCCT found once in FHS, R861C, which has previously been reported as disease-causing 23,24 and was found in 4 of our European Caucasian GS subjects (homozygous in 1). This non-conservative substitution is at a position conserved among all vertebrates.…”

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

“…Human eGFP-NCC cotransporter function was determined by assessing tracer 22 Na + uptake (PerkinElmer Life Sciences, Groningen, The Netherlands) in groups of 25 oocytes following a standard protocol of 30 min of incubation in a Cl À -free ND96 medium containing 1 mM ouabain, 0.1 mM amiloride and 0.1 mM bumetanide, following 1 h of uptake in K + -free, NaCl medium (40 mM NaCl, 56 mM sodium-gluconate, 4 mM CaCl 2 , 1 mM MgCl 2 and 5 mM Hepes/Tris, pH 7.4) containing 1 mM ouabain, 0.1 mM amiloride, 0.1 mM bumetanide and 2 m Ci of 22 Na + /ml. 20 As a control, one group of waterinjected oocytes has been included to determine the basal unspecific tracer 22 Na + uptake in all experiments.…”

“…20 As a control, one group of waterinjected oocytes has been included to determine the basal unspecific tracer 22 Na + uptake in all experiments.…”

“…The functionality of each mutant NCC transporter was investigated using the X. laevis oocyte heterologous expression system. To compare 22 Na + uptake between wild-type and each mutant NCC, all groups of oocytes were injected with 10 ng cRNA/oocyte. At the same day of the NCC-mediated 22 Na + uptake experiments, in parallel, microinjected X. laevis oocytes were selected to determine total membrane NCC protein expression using western blotting analysis.…”