Splicing Characterization of CLCNKB Variants in Four Patients With Type III Bartter Syndrome
Splicing Characterization of CLCNKB Variants in Four Patients With Type III Bartter Syndrome
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四名 III 型 Bartter 综合征患者 CLCNKB 变异体的剪接特征
DOI：
10.3389/fgene.2020.00081
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发表时间：
2020-02-21
影响因子：
3.7
通讯作者：
Zhao, Fei
中科院分区：
文献类型：
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作者：
Wang, Chunli;Han, Yuan;Zhao, Fei
目的III型巴氏综合征（Type III Bartter syndrome， BS）是由基侧氯离子通道ClC-Kb （CLCNKB）编码基因的功能缺失突变引起的，以低钾血症代谢性碱中毒和高肾素血症高醛固酮增多症为特征。在此，我们研究了4例具有Bartter综合征临床表现的中国儿童的分子缺陷。方法采用全外显子组测序（full -exome sequencing， WES）对4例患者的基因组DNA进行基因变异筛选。候选变异通过直接Sanger测序进行验证。随后进行定量PCR （qPCR）确认整个CLCNK基因缺失突变。采用基因分析和反转录PCR （RT-PCR）分析了剪接变异在体外的影响。结果本组患者发病年龄早，表现为低钠血症、低钾血症、低氯血症、反复呕吐、生长迟缓，提示Bartter综合征。遗传分析显示，所有患者都携带复合杂合或纯合截断变异的CLCNKB基因。特别是，我们发现了一个新的无义变体C . 239g > a (p.(Trp80*))，两个剪接位点变体（C .1053-1 G > a和C .1228- 2a > G），一个全基因缺失，以及一个新的同义变体C . 228a > C (p.(Arg76Arg))，位于外显子3 5 '剪接供体位点-2 bp处。此外，我们的体外迷你基因分析显示，C . 228a > C, C .1053- 1g > A和C .1228- 2a > G分别导致外显子3，外显子12和外显子13的跳跃。结论CLCNKB基因全缺失是中国III型BS患者中最常见的突变，截断和全基因缺失变异可能是导致患者更严重表型的原因。我们验证了三种剪接变异体（C . 228a > C、C .1053- 1g > A和C .1228- 2a > G）的致病作用，它们干扰了正常的mRNA剪接，表明剪接变异体在III型BS的分子基础中起着重要作用，对这些患者进行仔细的分子分析将对未来有效的个性化治疗方案至关重要。
Objective Type III Bartter syndrome (BS) is caused by loss-of-function mutations in the gene encoding basolateral chloride channel ClC-Kb (CLCNKB), and is characterized by hypokalemic metabolic alkalosis and hyperreninemic hyperaldosteronism. Here, we investigated the molecular defects in four Chinese children with clinical manifestations of Bartter syndrome. Methods The genomic DNA of the four patients was screened for gene variations using whole-exome sequencing (WES). The candidate variants were validated by direct Sanger sequencing. Quantitative PCR (qPCR) was subsequently performed to confirm the whole CLCNK gene deletion mutation. A minigene assay and reverse transcription PCR (RT-PCR) were performed to analyze the effect of splice variants in vitro. Results Our patients showed early onset age with hyponatremia, hypokalemia, hypochloremia, repeated vomiting and growth retardation, suggesting Bartter syndrome. Genetic analysis revealed that all patients carried compound heterozygous or homozygous truncating variants in the CLCNKB gene. In particular, we identified a novel nonsense variant c.239G > A (p.(Trp80*)), two splice site variants (c.1053-1 G > A and c.1228-2A > G), a whole gene deletion, and a novel synonymous variant c.228A > C (p.(Arg76Arg)) which located -2 bp from the 5′ splice donor site in exon 3. Furthermore, our in vitro minigene analysis revealed c.228A > C, c.1053-1G > A, and c.1228-2A > G cause the skipping of exon 3, exon 12, and exon 13, respectively. Conclusion Our results support that the whole CLCNKB gene deletion is the most common mutation in Chinese patients with type III BS, and truncating and whole gene deletion variants may account for a more severe phenotype of patients. We verified the pathogenic effect of three splicing variants (c.228A > C, c.1053-1G > A, and c.1228-2A > G) which disturbed the normal mRNA splicing, suggesting that splice variants play an important role in the molecular basis of type III BS, and careful molecular profiling of these patients will be essential for future effective personalized treatment options.