Novel compound heterozygous CLCNKB gene mutations (c.1755A>G/c.848_850delTCT) cause classic Bartter syndrome
Novel compound heterozygous CLCNKB gene mutations (c.1755A>G/c.848_850delTCT) cause classic Bartter syndrome
复制标题
新型复合杂合 CLCNKB 基因突变 (c.1755A > G/c.848_850delTCT) 导致经典 Bartter 综合征
DOI：
10.1152/ajprenal.00077.2017
复制
发表时间：
2018-10-01
影响因子：
4.2
通讯作者：
Zhang, Aihua
中科院分区：
文献类型：
--
作者：
Wang, Chunli;Chen, Ying;Zhang, Aihua
编码基底外侧氯离子通道ClC-Kb的CLCNKB基因的失活变体引起典型的Bartter综合征，其特征在于低钾代谢性贫血和高肾素性高醛固酮血症。在此，我们鉴定了一个中国家庭中的两个cBS兄弟姐妹，由于新的复合杂合CLCNKB突变（c.848_850delTCT/c.1755A>G）而表现出低钾血症。通过扩增和测序患者的基因组DNA证实复合杂合性。同义突变c.1755A>G（Thr 585 Thr）位于外显子15中距5'剪接供体位点+2bp处。进一步的转录本分析表明，这种单核苷酸突变导致从先证者和他的母亲的cDNA中排除外显子15。此外，我们还研究了c.848_850delTCT（Delta TCT）和外显子15缺失（Delta E15）突变在体外的表达和蛋白运输变化。Delta E15突变显著降低了ClC-Kb的表达，并导致一个低分子量条带（类似于55 kDa）捕获在内质网中，而Delta TCT突变体仅降低了总ClC-Kb蛋白和质膜ClC-Kb蛋白的表达，但不影响亚细胞定位。最后，我们用全细胞膜片钳技术研究了突变的生理功能，发现Δ E15或Δ TCT突变降低了ClC-Kb/barttin通道的电流。这些结果表明，CLCNKB基因的复合缺陷突变是两个cBS同胞的分子机制。
In-activated variants in CLCNKB gene encoding the basolateral chloride channel ClC-Kb cause classic Bartter syndrome characterized by hypokalemic metabolic alkalosis and hyperreninemic hyperaldosteronism. Here, we identified two cBS siblings presenting hypokalemia in a Chinese family due to novel compound heterozygous CLCNKB mutations (c.848_850delTCT/c.1755A>G). Compound heterozygosity was confirmed by amplifying and sequencing the patient's genomic DNA. The synonymous mutation c.1755A>G (Thr585Thr) was located at +2 bp from the 5' splice donor site in exon 15. Further transcript analysis demonstrated that this single nucleotide mutation causes exclusion of exon 15 in the cDNA from the proband and his mother. Furthermore, we investigated the expression and protein trafficking change of c.848_850delTCT (Delta TCT) and exon 15 deletion (Delta E15) mutation in vitro. The Delta E15 mutation markedly decreased the expression of ClC-Kb and resulted in a low-molecular-weight band (similar to 55 kDa) trapping in the endoplasmic reticulum, while the Delta TCT mutant only decreased the total and plasma membrane ClC-Kb protein expression but did not affect the subcellular localization. Finally, we studied the physiological functions of mutations by using whole cell patch-clamp and found that the Delta E15 or Delta TCT mutation decreased the current of the ClC-Kb/barttin channel. These results suggested that the compound defective mutations of the CLCNKB gene are the molecular mechanism of the two cBS siblings.