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Mutations affecting the sodium-chloride cotransporter (NCC) in the distal convoluted tubule of the nephron are causative of Gitelman's syndrome (GS), a rare autosomal recessive disease characterized by electrolytic alterations similar to those induced by high dose thiazide treatment. Notably, the co-existence of hypomagnesemia and hypocalciuria is a feature of GS which is a distinguish hallmark from another hypokalemic renal tubulopathy, the Bartter's syndrome (BS). Commonly GS is heterozygous compound with an estimated prevalence of 1:40000 and can be silent for years before the revealing in the early adulthood. Recognizing the genetic background is fundamental for the screening and the diagnosis of the disease, as recently studies showed that mutation affecting regulators of renal salt handling are underestimated in the general population. In a registry of BS/GS based at our University we discovered a novel point mutation (c.1204G>A which codify for an amino acid exchange G394D) in the sodium-chloride cotransporter NCC (SLC12A3) in a young woman with hypokalemia, hypomagnesemia and hypocalciuria associated to muscle pain and cramps. The present study aimed to investigate how this mutation affects NCC function by using a molecular biology approach and providing functional evidences. After a prior screening with bioinformatics tools predicting the possible pathogenicity of the mutation, were created different expression vectors with either the wild-type (wt-NCC) or the mutated G394D-NCC sequences. DNA and in-vitro transcribed RNA were afterwards transfected in a human embryonic kidney cells line (HEK293) and injected into oocytes deriving from Xenopus Laevis frog respectively. In transfected HEK 293 cells, wildtype NCC was detected by immunoblotting as two bands at approximately 130 kDa and 115 kDa corresponding to fully and core-glycosylated NCC, respectively. In contrast, G394D-NCC was seen as a single band at about 115 kDa only, suggesting an impaired maturation of the mutated protein. Similar findings were made in the oocyte expression system. Confocal microscopy on the oocytes, did also show a strong cell surface localization of wildtype NCC while mutated NCC was retained at intracellular compartments. Consistently, a decent thiazide-sensitive 22Na+ uptake into injected oocytes was only found for wildtype but not mutated NCC. Taken together all the findings in this study, a novel GS point mutation has been characterized to diminish NCC function by impairing trafficking of the protein to the cell surface. The absence of any mature glycosylation form of G394D-NCC suggests that the mutation impairs protein folding leading to a retention of NCC in the endoplasmic reticulum.

SP022MOLECULAR CHARACTERIZATION OF A NOVEL MUTATION IN THE RENAL NACL COTRANSPORTER CAUSING GITELMAN'S SYNDROME BY IMPAIRING TRANSPORTER TRAFFICKING