J Korean Med Sci.  2013 Jun;28(6):821-826. 10.3346/jkms.2013.28.6.821.

Translational Read-Through of a Nonsense Mutation Causing Bartter Syndrome

• Cho HY
• Lee BH
• Cheong HI

• KMID: 2158027
• DOI: http://doi.org/10.3346/jkms.2013.28.6.821

Abstract

Bartter syndrome (BS) is classified into 5 genotypes according to underlying mutant genes and BS III is caused by loss-of-function mutations in the CLCNKB gene encoding for basolateral ClC-Kb. BS III is the most common genotype in Korean patients with BS and W610X is the most common CLCNKB mutation in Korean BS III. In this study, we tested the hypothesis that the CLCNKB W610X mutation can be rescued in vitro using aminoglycoside antibiotics, which are known to induce translational read-through of a nonsense mutation. The CLCNKB cDNA was cloned into a eukaryotic expression vector and the W610X nonsense mutation was generated by site-directed mutagenesis. Cultured polarized MDCK cells were transfected with the vectors, and the read-through was induced using an aminoglycoside derivative, G418. Cellular expression of the target protein was monitored via immunohistochemistry. While cells transfected with the mutant CLCNKB failed to express ClC-Kb, G418 treatment of the cells induced the full-length protein expression, which was localized to the basolateral plasma membranes. It is demonstrated that the W610X mutation in CLCNKB can be a good candidate for trial of translational read-through induction as a therapeutic modality.

Keyword

Bartter Syndrome; CLCNKB Gene; Nonsense Codon; Translational Read-Through Induction

MeSH Terms

Animals
Bartter Syndrome/genetics/*pathology
Chloride Channels/analysis/genetics/*metabolism
Cloning, Molecular
Codon, Nonsense
Dogs
Humans
Immunohistochemistry
Madin Darby Canine Kidney Cells
Microscopy, Confocal
Mutagenesis, Site-Directed
Recombinant Fusion Proteins/analysis/biosynthesis/genetics
Transfection
Chloride Channels
Codon, Nonsense
Recombinant Fusion Proteins