Gitelman综合征（GS）是常见的遗传性肾小管疾病，由SLC12A3基因突变所致。虽然大量的SLC12A3基因突变被检出，但多数突变的功能及分子机制尚未得到研究。近期研究显示当点突变位于外显子剪接调控序列区时，将导致灾难性的外显子跳跃，具有重要的研究价值。我们通过分析诊治的GS患儿基因筛查结果以及已报道的98种中国人群SLC12A3基因突变，发现7种可能导致mRNA剪接异常的错义/同义突变，同时发现了3种功能尚待研究的高频突变。本研究将采用质粒构建、定点诱变、细胞转染等方法探讨SLC12A3基因3种高频突变对蛋白表达、定位及通道功能的影响。同时构建minigene剪接报告系统，验证SLC12A3外显子7种点突变对mRNA剪接的影响，通过RNA蛋白质pull-down等实验初步探讨mRNA异常剪接产生的机理，希望为GS的临床诊治和遗传咨询提供新的实验依据。

Gitelman sydrome (GS) is a common hereditary renal tubular disease which is caused by SLC12A3 gene mutation. Although a large number of SLC12A3 gene mutations have been detected, but the function and characteristic of most mutations still unknown. Recent studies have shown that when the point mutations located in the exon splicing regulation sequence following a failure of exon skipping in mature mRNA, which has important research value. By the bioinformatic analysis of our patients and 98 SLC12A3 gene point mutations which reported in Chinese GS populations, we identified 7 missense/synonymous mutations that might lead to abnormal mRNA splicing and found 3 recurrence mutations. In this study, we will investigate the function and characteristic of 3 recurrence SLC12A3 gene mutations in NCC channel by plasmid construction, site directed mutagenesis and cell transfection. Next, we will perform minigenes splicing assay in vitro to clarify the effect of 4 ESEs and 3 ESSs point mutations on the alternative mRNA transcript and use RNA pull-down assay to explore the possible mechanism leading to the abnormal splicing. Our study will provide new experimental evidence for clinical diagnosis and genetic counseling of GS.