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Gitelman’s Syndrome: characterization of a novel c.1181G>A point mutation and functional classification of the known mutations


Ravarotto, Verdiana; Loffing, Johannes; Loffing-Cueni, Dominique; Heidemeyer, Michèle; Pagnin, Elisa; Calò, Lorenzo A; Rossi, Gian Paolo (2018). Gitelman’s Syndrome: characterization of a novel c.1181G>A point mutation and functional classification of the known mutations. Hypertension Research, 41(8):578-588.

Abstract

We have investigated the mechanisms by which a novel missense point mutation (c.1181G>A) found in two sisters causes Gitelman’s syndrome by impairing the sodium chloride co-transporter (NCC, encoded by SLC12A3 gene) function. The cDNA and in vitro transcribed mRNA of either wild-type or mutated SLC12A3 were transfected into HEK293 cells and injected into Xenopus laevis oocytes, respectively. The expression, maturation, trafficking, and function of the mutated and wild-type NCC were assessed by Western blotting, immunohistochemistry and 22Na+ uptake studies. By immunoblotting of lysates from HEK293 cells and oocytes expressing wild-type NCC, two NCC-related bands of approximately 130 kDa and 115 kDa, corresponding to fully and core-glycosylated NCC, respectively, were identified. In contrast, the mutant NCC only showed a single band of approximately 115 kDa, indicating impaired maturation of the protein. Moreover, oocytes injected with wild-type NCC showed thiazide-sensitive 22Na+ uptake, which was absent in those injected with the mutant NCC. The novel mutation was discussed in the context of the functionally characterized NCC mutations causing Gitelman’s syndrome, which fit into five classes. In conclusion, the functional characterization of this novel Gly394Asp NCC and its localization on the NCC structure, alongside that of previously known mutations causing Gitelman’s syndrome, may provide novel information on the function of the different domains of the human NCC.

Abstract

We have investigated the mechanisms by which a novel missense point mutation (c.1181G>A) found in two sisters causes Gitelman’s syndrome by impairing the sodium chloride co-transporter (NCC, encoded by SLC12A3 gene) function. The cDNA and in vitro transcribed mRNA of either wild-type or mutated SLC12A3 were transfected into HEK293 cells and injected into Xenopus laevis oocytes, respectively. The expression, maturation, trafficking, and function of the mutated and wild-type NCC were assessed by Western blotting, immunohistochemistry and 22Na+ uptake studies. By immunoblotting of lysates from HEK293 cells and oocytes expressing wild-type NCC, two NCC-related bands of approximately 130 kDa and 115 kDa, corresponding to fully and core-glycosylated NCC, respectively, were identified. In contrast, the mutant NCC only showed a single band of approximately 115 kDa, indicating impaired maturation of the protein. Moreover, oocytes injected with wild-type NCC showed thiazide-sensitive 22Na+ uptake, which was absent in those injected with the mutant NCC. The novel mutation was discussed in the context of the functionally characterized NCC mutations causing Gitelman’s syndrome, which fit into five classes. In conclusion, the functional characterization of this novel Gly394Asp NCC and its localization on the NCC structure, alongside that of previously known mutations causing Gitelman’s syndrome, may provide novel information on the function of the different domains of the human NCC.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Anatomy
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Health Sciences > Internal Medicine
Life Sciences > Physiology
Health Sciences > Cardiology and Cardiovascular Medicine
Uncontrolled Keywords:Internal Medicine, Physiology, Cardiology and Cardiovascular Medicine
Language:English
Date:20 June 2018
Deposited On:12 Sep 2018 14:44
Last Modified:29 Jul 2020 07:25
Publisher:Nature Publishing Group
ISSN:0916-9636
OA Status:Closed
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1038/s41440-018-0061-1
PubMed ID:29925901

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