1例Ⅲ型Bartter综合征患儿CLCNKB基因复合杂合变异功能分析

Functional analysis of compound heterozygous variations in the CLCNKB gene in a patient with Bartter syndrome typeⅢ

目的分析1例Ⅲ型Bartter综合征患儿CLCNKB基因复合杂合变异的功能特性,探索囊性纤维化跨膜传导调节剂VX-809化合物对CLCNKB基因错义突变的挽救作用。方法回顾性分析2019年9月2日至南京医科大学附属儿童医院肾脏内科就诊的1例Ⅲ型Bartter综合征患儿的临床表现、生长发育情况、实验室检查结果及CLCNKB基因变异情况等。在人胚胎肾293细胞(HEK293)中分别转染野生型和变异型CLCNKB基因,并运用蛋白免疫印迹法检测各组ClC-Kb蛋白的表达水平,运用非配对t检验比较野生型与突变型之间蛋白表达的差异。同时,利用免疫荧光技术检测ClC-Kb的亚细胞定位,并使用囊性纤维化跨膜传导调节剂VX-809化合物处理过表达突变体的细胞。结果患儿为2岁个8个月的女性,表现为低钾血症、低氯血症、代谢性碱中毒、肾性盐消耗,高血压四项及血压均正常。基因检测结果显示患儿为CLCNKB基因复合杂合型变异。在HEK293细胞中转染2种突变(p.F213C和p.Y466Mfs*13)质粒,发现突变型ClC-Kb蛋白表达均低于野生型(P均<0.01),其中移码突变导致了一个低分子量带(~5.5×10^(4))。免疫荧光定位实验示p.F213C和p.Y466Mfs*13变异体滞留于细胞质内。VX-809处理p.F213C突变转染后的HEK293细胞后,可呈剂量依赖性地显著增加ClC-Kb蛋白的膜表达。结论CLCNKB基因2种变异体导致ClC-Kb蛋白表达降低和定位出现异常,VX-809化合物可在一定程度上纠正Ⅲ型Bartter综合征患儿CLCNKB基因变异体的蛋白表达缺陷,可能对于治疗Ⅲ型Bartter综合征有新的应用前景。

Objective To explore the functional characteristics of a patient of Bartter syndrome type III and compound heterozygous mutations in CLCNKB gene and explore the rescue effect of cystic fi-brosis transmembrane conductance regulator modulator compound VX-809 on CLCNKB gene missense variant.Methods A retrospective analysis was conducted for a hospitalized patient of Bartter syndrome type III on September 2,2019.Clinical characteristics,growth and development status,laboratory findings and genetic data were reviewed.Wild-type and variant CLCNKB genes were separately transfected into hu-man embryonic kidney 293 cells(HEK293)and the expression levels of ClC-Kb protein in each group de-tected by Western blot.The differences in protein expression between wild-type and variant type were compared by unpaired t-test.Additionally,immunofluorescent stain was utilized for examining the subcel-lular localization of ClC-Kb protein.And cystic fibrosis transmembrane conductance regulator modulator compound VX-809 was employed for treating cells after transfecting with a over-expressing variant in CLCNKB gene.Results This 32-month-old girl presented with hypokalemia,hypochloremia,metabolic alkalosis,renal salt wasting and normal blood pressure in all four extremities.Genetic testing results re-vealed compound heterozygous variations in CLCNKB gene.Transfection of two variant plasmids(p.F213C&p.Y466Mfs13)into HEK293 cells indicated that the expression of variant ClC-Kb protein was significantly lower than that of wild-type(P<0.01 for both).Frameshift variant resulted in a lower molecu-lar weight band(~5.5×10^(4)).Immunofluorescent localization assays revealed that both variants were re-tained in cytoplasm.Treatment of HEK293 cells transfected with p.F213C variant with VX-809 resulted in a dose-dependent significant spike in membrane expression of ClC-Kb protein.Conclusion Two vari-ants of CLCNKB gene result in lower expression and abnormal localization of ClC-Kb protein.Compound VX-809 may partially correct the protein expression defects caused by CLCNKB gene variants in patients of Bartter syndrome type III.It hints at new therapeutic potentials for Bartter syndrome type III.