Minigene splicing assays reveal new insights into exonic variants of the SLC12A3 gene in Gitelman Syndrome.

Gitelman syndrome (GS) is a kind of salt-losing tubular disease, most of which is caused by SLC12A3 gene variants, and missense variants account for the majority. Recently, the phenomenon of exon skipping, in which exonic variants disrupt normal pre-mRNA splicing, has been related to a variety of diseases. The purpose of this study was to identify the effect of previously presumed missense SLC12A3 variants on pre-mRNA splicing using bioinformatics tools and minigenes. The results revealed that, among ten candidate variants, six variants (c.602G>A, c.602G>T, c.1667C>T, c.1925G>A, c.2548G>C and c.2549G>C) led to complete or incomplete exon skipping by affecting exonic splicing regulatory elements and/or disturbing canonical splice sites. It is worth mentioning that this is the largest study on pre-mRNA splicing of SLC12A3 exonic variants. In addition, our study emphasizes the importance of detecting splicing function at the mRNA level in GS and indicates that minigene analysis is a valuable tool for splicing functional assays of variants in vitro.

Frequent SLC12A3 mutations in Chinese Gitelman syndrome patients: structure and function disorder

Purposes: This study was conducted to identify the frequent mutations from reported Chinese Gitelman syndrome (GS) patients, to predict three-dimensional structure change of human Na-Cl co-transporter (hNCC), and to test the activity of these mutations and some novel mutations in vitro and in vivo. Methods: SLC12A3 gene mutations in Chinese GS patients previously reported in the PubMed, CNKI and Wanfang database were summarized. Predicted configurations of wild type (WT) and mutant proteins were achieved using the I-TASSER workplace. Six missense mutations (T60M, L215F, D486N, N534K, Q617R and R928C) were generated by site-directed mutagenesis. 22Na+ uptake experiment was carried out in the Xenopus laevis oocyte expression system. 35 GS patients and 20 healthy volunteers underwent the thiazide test. Results: T60M, T163M，D486N, R913Q, R928C and R959 frameshift were frequent SLC12A3 gene mutations (mutated frequency >3%) in 310 Chinese GS families. The protein’s three-dimensional structure was predicted to be altered in all mutations. Compared with WT hNCC, the thiazide-sensitive 22Na+ uptake was significantly diminished for all 6 mutations: T60M 22±9.2%, R928C 29±12%, L215F 38±14%, N534K 41±15.5%, Q617R 63±22.1% and D486N 77±20.4%. In thiazide test, the net increase in chloride fractional excretion in 20 healthy controls was significantly higher than GS patients with or without T60M or D486N mutations. Conclusions: Frequent mutations (T60M, D486N, R928C) and novel mutations (L215F, N534K and Q617R) lead to protein structure alternation and protein dysfunction verified by 22Na+ uptake experiment in vitro and thiazide test on patients.

A novel compound heterozygous variant of the SLC12A3 gene in Gitelman syndrome with diabetes and the choices of the appropriate hypoglycemic drugs: a case report

Background Gitelman syndrome (GS) is an autosomal recessive tubulopathy caused by mutations of the SLC12A3 gene. It is characterized by hypokalemic metabolic alkalosis, hypomagnesemia and hypocalciuria. It is universally known that both hypokalemia and hypomagnesemia can influence insulin secretion and insulin resistance, but the exact mechanisms require further study. We identified a novel deletion variant of the SLC12A3 gene and discussed the appropriate hypoglycemic drugs in Gitelman syndrome (GS) patients with type 2 diabetes. Case presentation A 55-year-old diabetic female patient was hospitalized for evaluation because of paroxysmal general weakness and numbness of extremities for one year. We suspected that she was suffering from GS by initial estimation. Direct Sanger sequencing was used to analyze the causative gene SLC12A3 of GS. Oral glucose tolerance test (OGTT) was carried out to assess the glucose metabolism and insulin resistance status. Genetic analysis revealed that she was a compound heterozygote for a recurrent missense mutation c.179C > T and a novel deletion c.1740delC in SLC12A3, thus her diagnosis of GS was confirmed. The patient was treated with potassium chloride (3.0 g/d) and magnesium chloride (element magnesium 350 mg/d) on the basis of initial treatment of diabetes with hypoglycemic drug (Repaglinide, 3.0 mg/day). However, she developed frequent hypoglycemia after one week. OGTT showed that her glucose metabolism and insulin resistance much improved after potassium and magnesium supplemental therapy. Then we changed the hypoglycemic agent to a dipeptidyl peptidase-4 (DPP-4) inhibitor (Trajenta 5 mg/d), since then her blood glucose level remained normal during two-year of follow-up. Conclusion We have identified a novel deletion of the SLC12A3 gene and discussed the appropriate hypoglycemic drugs in Gitelman syndrome (GS) patients with type 2 diabetes. We suggested that attention need to be paid to blood glucose monitoring in GS patients, especially when hypokalemia and hypomagnesemia are corrected. Besides, the insufficient blood volume and serum electrolyte disturbance should also be taken into consideration in the selecting hypoglycemic drugs for GS patients.