Splicing Characterization of CLCNKB Variants in Four Patients With Type III Bartter Syndrome

Abstract

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.

Keywords: CLCNKB; abnormal RNA splicing; classical Bartter syndrome; hypokalemia; synonymous variant.

Figure 1

CLCNKB gene variants identified in type III Bartter syndrome patients. (A) Direct sequencing showing four point variants of the CLCNKB gene (arrows), the c.228G > A show only C because another allele is deleted; the wild-type sequence is also shown. (B) CLCNKB gene qPCR analysis of patient 3 showed a heterozygous loss of exons 2, 10 and 20 in CLCNKB. (C) CLCNKB qPCR analysis of patient 4 showed a homozygous loss of exons 2, 10 and 20 in CLCNKB, while both parents appeared heterozygous.

Figure 2

Effect of CLCNKB gene c.228A > C variant by Minigene assays. (A) RT-PCR amplified products of hybrid minigene transcripts in HEK293 cells. The transcripts produced by the hybrid minigene are schematically shown, and the arrows show the primers used to amplify (inset) (Wang et al., 2018b). (B) Exon 3 and adjacent structures of the CLCNKB gene. The arrow shows the location of the splice site variant c.228A > C in exon 3. (C) Gel electrophoresis of the RT-PCR product of minigene transcripts in HEK293 cell. Lane 1: marker; Lane 2: pSPL3 (263 bp); Lane 3: E3-WT (392 bp and 263 bp); Lane 4: c.228A > C (392 bp and 263 bp). The two fragments were directly sequenced (right panel). (D) Gel electrophoresis of the RT-PCR product of minigene transcripts in Hela cell. Lane 1: marker; Lane 2: pSPL3 (263 bp); Lane 3: E3-WT (392 bp and 263 bp); Lane 4: c.228A > C (392 bp and 263 bp). (E) Quantification of the splicing percentage in HEK293 and Hela cells was densitometrically calculated on a molar basis as the percentage of exclusion (%) = (lower band/[lower band + upper band]) x 100. Error bars represent SEM (n=3). **P < 0.01, unpaired Student's t-test.

Figure 3

Effect of CLCNKB gene c.1053-1G > A and c.1228-2A > G variants by Minigene assays. (A) Gel electrophoresis of the RT-PCR product of c.1053-1G > A transcripts in HEK293 cell. Lane 1: marker; Lane 2: pSPL3 (263 bp); Lane 3: E12-WT (437bp); Lane 4: c.1053-1 G > A (263 bp). The two fragments were directly sequenced (right panel). (B) Gel electrophoresis of the RT-PCR product of c.1228-2A > G transcripts in HEK293 cell. Lane 1: marker; Lane 2: pSPL3 (263 bp); Lane 3: E13-WT (333 bp and 263 bp); Lane 4: c.1228-2 A > G (263 bp). The two fragments were directly sequenced (right panel). (C) Gel electrophoresis of the RT-PCR product of minigene transcripts in Hela cell. (D) Quantification of the splicing percentage in HEK293 and Hela cells was densitometrically calculated on a molar basis as the percentage of exclusion (%) = (lower band/[lower band + upper band]) x 100. Error bars represent SEM (n=3). **P < 0.01, unpaired Student's t-test.