目的 回顾性分析2015 年8 月至2022 年11 月南京医科大学附属儿童医院收治的20 例Gitelman综合征患儿临床症状及基因突变情况,初步探讨中国人群高频突变D486N致病分子机制.方法 收集患儿的临床资料及SLC12A3基因变异情况等,在人胚胎肾293T细胞(HEK293T)中分别过表达野生型和变异型SLC12A3 基因,使用蛋白免疫印迹法和免疫荧光技术分别检测肾噻嗪敏感性钠-氯协同转运体(NCC)的表达水平和亚细胞定位,探讨SLC12A3 基因高频突变D486N对NCC蛋白表达和定位的影响.结果 本研究期间共收集到20 例Gitelman综合征患者,患儿均表现为低血钾症,共筛查到26 种SLC12A3 基因突变,错义变异13 种、同义变异1 种、无义变异 1 种、移码变异 4 种、剪接位点变异7 种.其中4 种突变p.T235K、c.1096-1G>A、p.A464A、c.2660+1_2660+2insT为新发突变.结论 本研究初步发现中国人群高频突变 D486N影响 NCC 总蛋白和膜蛋白的表达,并影响 NCC 蛋白的膜表达.本研究的实验结果可为Gitelman综合征遗传咨询及诊治提供实验依据.
Objective This study aimed at conducting retrospective analysis of the clinical symptoms and genetic mutations in 20 children with Gitelman syndrome treated at the Affiliated Children's Hospital of Nanjing Medical University from August 2015 to November 2022 and also explored the molecular mechanism of the pathogenic high-frequency mutation D486N in the Chinese population.Methods We collected the clinical manifestations,growth and development status,laboratory examination results,and SLC12A3 gene variations of the patients.We distinguished the wild-type and mutant SLC12A3 genes overexpressed in human embryonic kid-ney 293T cells(HEK293T).We used protein immunoblotting to detect the expression level of NCC,and used immunofluorescence techniques to examine the subcellular localization of NCC.In addition,we investigated the impact of the high-frequency SLC12A3 gene mutation D486N on NCC protein expression and localization.Results In the 20 patients with Gitelman syndrome,all of them had hypokalemia.We indemnified twenty-six SLC12A3 gene mutations,13 of which are missense mutation,1 of which synonymous mutation,1 nonsense muta-tion,4 frameshift mutation,and 7 splicing site mutation.Among them,four mutations(p.T235K,c.1096-1G>A,p.A464A,and c.2660+1_2660+2insT)were novel mutations.Conclusions We found the preliminary evidence that the high-frequency mutation D486N in the Chinese population affected the expression of total and membrane-bound NCC protein and influenced the membrane localization of NCC protein.The findings of this study provides experimental evidence for genetic counseling,diagnosis,and treatment of Gitelman syndrome.