Functional severity of CLCNKB mutations correlates with phenotypes in patients with classic Bartter's syndrome

Abstract

Mutations in the CLCNKB gene encoding the human voltage-gated chloride ClC-Kb (hClC-Kb) channel cause classic Bartter's syndrome (BS). In contrast to antenatal BS, classic BS manifests with highly variable phenotypes. The functional severity of the mutant channel has been proposed to explain this phenomenon. Due to difficulties in the expression of hClC-Kb in heterologous expression systems, the functional consequences of mutant channels have not been thoroughly examined, and the genotype-phenotype association has not been established. In this study, we found that hClC-Kb, when expressed in human embryonic kidney (HEK) cells, was unstable due to degradation by proteasome. In-frame fusion of green fluorescent protein (GFP) to the C-terminus of the channel may ameliorate proteasome degradation. Co-expression of barttin increased protein abundance and membrane trafficking of hClC-Kb and markedly increased functional chloride current. We then functionally characterized 18 missense mutations identified in our classic BS cohort and others using HEK cells expressing hClC-Kb-GFP. Most CLCNKB mutations resulted in marked reduction in protein abundance and chloride current, especially those residing at barttin binding sites, dimer interface and selectivity filter. We enrolled classic BS patients carrying homozygous missense mutations with well-described functional consequences and clinical presentations for genotype-phenotype analysis. We found significant correlations of mutant chloride current with the age at diagnosis, plasma chloride concentration and urine calcium excretion rate. In conclusion, hClC-Kb expression in HEK cells is susceptible to proteasome degradation, and fusion of GFP to the C-terminus of hClC-Kb improves protein expression. The functional severity of the CLCNKB mutation is an important determinant of the phenotype in classic BS.

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

“…The functional severity of the mutant channel has been proposed to explain this phenomenon. Cheng et al established a genotypephenotype association and revealed that the functional severity of CLCNKB genotypes correlated with age at onset, plasma chloride concentration, and urine calcium excretion rate (Cheng et al, 2017). Keck et al also reported that CLCNKB mutations with milder functional outcomes were linked to older age at diagnosis of classic BS (Keck et al, 2013).…”

Paving the way for Bartter syndrome type 3 drug discovery: a hope from basic research

“…In this issue of The Journal of Physiology , Cheng et al . () significantly contribute toward advancing our understanding of the phenotype/genotype correlation and of the precise pathogenic mechanisms associated with BS type 3. As a first step, the authors collected classic BS patients carrying homozygous missense mutations with previously described functional consequences and clinical features.…”

Salt-Losing Tubulopathies in Children: What’s New, What’s Controversial?

“…[39][40][41][42][43] There are some data for CLCNKB suggesting that mutation type may influence the phenotype, with mutations affecting the Barttin-binding site, the dimerization interface, or the selectivity filter causing more severe dysfunction. 44 Yet, the most common mutation in CLCNKB is a whole-gene deletion, which can be associated with the whole phenotypic spectrum. One recent review of 30 patients with BS type 3 found no evidence for a genotypephenotype association, 45 whereas in a larger series of 115 patients an association of complete loss-of-function mutations with age at onset was seen.…”