Functionomics of novel mutations in NCC and their relevance in development of Gitelman syndrome

<p>Gitelman syndrome is a genetic disease characterized by low blood pressure and salt wasting. In most cases, Gitelman syndrome results from loss-of-function mutations in the solute carrier family 12 (SLC12A3) gene, which encodes the thiazide-sensitive sodium chloride co-transporter (NCC). At present, more than 250 distinct loss-of-function mutations have been identified in patients with Gitelman syndrome. Functional analysis has been limited by the use of only Xenopus laevis oocytes as a model system. The aim of the present study is to understand the functional consequences of these NCC mutations in mammalian cell line systems. Recently, our group published a new technique to isolate primary cells using Complex Object Parametric Analyzer and Sorter (COPAS). We have pioneered with this COPAS sorting platform to isolate and culture distal convoluted tubules (DCT) based on parvalbumin-eGFP transgenic mice model. These primary cultures exhibit several characteristics of the original epithelium including thiazide-sensitive transepithelial NaCl transport. By generating crossbred of parvalbumin-eGFP mice with NCC knockout we aim to obtain DCT primary cell line without endogenous expression of wild type NCC, which in turn will be substituted by ectopic expression of mutated NCC. Generation of this model will create an excellent platform for further functional analysis of mutant NCC proteins. Currently, out of several patients with Gitelman syndrome symptoms eight new mutations were selected. The majority of these mutations exhibited a disturbed phosphorylation and glycosylation pattern as well as diminished expression of NCC. However, functional consequences of these mutations remain to be elucidated. Overall, functional screening of many NCC mutations in DCT cultures are now possible and will also allow to unravel a complex system of NCC regulatory mechanisms.