[Functional characterization of SLC12A1 gene variants in 3 patients with Bartter syndrome type Ⅰ].

Objective: To clarify the molecular basis of patients with Bartter syndrome type I and explore the therapeutic effect of trafficking-defective variations by chemical chaperone 4-Phenylbutyric acid(4-PBA). Methods: The clinical characteristics, laboratory findings and genetic data of 3 patients diagnosed with Bartter syndrome type I who were admitted to Department of Nephrology, Children's Hospital of Nanjing Medical University from 2017 to 2018 were retrospectively analyzed. Wild type and variant SLC12A1 gene constructs were transiently overexpressed in HEK293 cells. Western blotting was used to detect the expression levels of Na+-K+-2Cl-cotransporter(NKCC2) protein. Immunofluorescent staining was applied to investigate the subcellular localization of NKCC2 protein. In addition, the effect of the chemical chaperone 4-PBA on the expression and localization of the SLC12A1 gene variants was investigated. Unpaired t test was used for statistical analysis of 4-PBA treatment. Results: All the 3 patients (2 males and 1 female), aged 3.0, 4.0 and 1.2 years, respectively. All patients had antenatal onset with polyhydramnios and were born prematurely. After birth, all patients presented with hypochlorine alkalosis accompanied by hypokalemia and hyponatremia. Sequencing analysis revealed that the 3 patients were homozygotes or compound heterozygotes for variants in the SLC12A1 gene. In HEK293 cells, the surface expression of NKCC2 in 3 variants (p.L463S, p.L479V, p.507-510del) are all lower than in wild type (0.718±0.039, 0.287±0.081, 0.025±0.156 vs. 1.001±0.028, t=5.92, 8.35, 30.49, all P<0.01). Moreover, the total protein expression of p.L479V and p.507-510del group were all lower than that in wild type group (0.630±0.032, 0.043±0.003 vs. 1.000±0.111, t=3.21, 8.65, all P<0.05). 4-PBA treatment increased the mature protein expression level of the p.L463S and p. L479V group in 4-PBA treatment group are all higher than the untreated group (0.459±0.018 vs. 1.123±0.024, 0.053±0.012 vs. 1.256±0.037, t=2.75, 18.35, all P<0.05). Cytoplasmic retention of the L479V and 507-510del variants were observed by immunofluorescent staining. 4-PBA treatment could rescue a number of NKCC2 L479V variants to the membrane. Conclusions: The 3 SLC12A1 variants cause expression or subcellular localization defects of the protein. The findings that plasma membrane expression and activity can be rescued by 4PBA might help to develop novel therapeutic strategy for Bartter syndrome type Ⅰ.目的： 分析Ⅰ型Bartter综合征患儿SLC12A1基因变异的功能特性，探索分子伴侣类药物4-苯丁酸钠对SLC12A1基因变异体的纠正作用。 方法： 回顾性分析2017至2018年南京医科大学附属儿童医院收治的3例Ⅰ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SLC12A1基因变异情况等，在人胚胎肾293细胞（HEK293）中分别过表达野生型和变异型SLC12A1基因，运用蛋白免疫印迹法检测各组Na+-K+-2Cl-共同转运体（NKCC2）的表达水平，免疫荧光技术检测NKCC2的亚细胞定位，同时用分子伴侣类药物4-苯丁酸钠处理转染SLC12A1基因变异体的细胞，运用非配对t检验比较处理前后药物对变异体的纠正作用。 结果： 3例患儿中男2例、女1例，分别为3.0、4.0、1.2岁。均表现为早产，母亲孕期羊水多，生后出现低氯性碱中毒，伴低钾、低钠血症等。一代测序结果显示3例患儿为SLC12A1基因纯合或复合杂合型变异。在HEK293细胞中发现3种变异体NKCC2（p.L463S、p.L479V和p.507-510del）膜蛋白表达均低于野生型（0.718±0.039、0.287±0.081、0.025±0.156比1.001±0.028，t=5.92、8.35、30.49，均P<0.01），p.L479V和p.507-510del总蛋白表达均低于野生型（0.630±0.032、0.043±0.003比1.000±0.111，t=3.21、8.65，均P<0.05）。4-苯丁酸钠处理组p.L463S、p.L479V成熟型蛋白表达水平均高于未处理组（0.459±0.018比1.123±0.024、0.053±0.012比1.256±0.037，t=2.75、18.35，均P<0.05）。免疫荧光实验示p.L479V和p.507-510del 2种变异体滞留于细胞质内，且4-PBA可以促进p.L479V变异体转运到细胞膜上。 结论： SLC12A1基因3种变异体导致蛋白表达或定位异常，4-苯丁酸钠可在一定程度上纠正Ⅰ型Bartter综合征患儿SLC12A1基因变异体的表达和定位缺陷，可能为临床治疗Ⅰ型Bartter综合征提供新的治疗靶点。.