The aminoglycoside geneticin permits translational readthrough of the CTNS W138X nonsense mutation in fibroblasts from patients with nephropathic cystinosis
Cystinosis is an ultrarare disorder caused by mutations of the cystinosin (CTNS) gene, encoding a cystine-selective efflux channel in the lysosomes of all cells of the body. Oral therapy with cysteamine reduces intralysosomal cystine accumulation and slows organ deterioration but cannot reverse renal Fanconi syndrome nor prevent the eventual need for renal transplantation. A definitive therapeutic remains elusive. About 15% of cystinosis patients worldwide carry one or more nonsense mutations that halt translation of the CTNS protein. Aminoglycosides such as geneticin (G418) can bind to the mammalian ribosome, relax translational fidelity, and permit readthrough of premature termination codons to produce full-length protein.
To ascertain whether aminoglycosides permit readthrough of the most common CTNS nonsense mutation, W138X, we studied the effect of G418 on patient fibroblasts.
G418 treatment induced translational readthrough of CTNSW138X constructs transfected into HEK293 cells and expression of full-length endogenous CTNS protein in homozygous W138X fibroblasts.
Reduction in intracellular cystine indicates that the CTNS protein produced is functional as a cystine transporter. Interestingly, similar effects were seen even in W138X compound heterozygotes. These studies establish proof-of-principle for the potential of aminoglycosides to treat cystinosis and possibly other monogenic diseases caused by nonsense mutations.
KeywordsCystinosis Aminoglycoside Geneticin Nonsense mutation Translational readthrough
EJB participated in the experimental design; performed the cell culture, qPCR, cell transfection, and intracellular half-cystine measurements; analyzed the results; and participated in the manuscript preparation.
LLC participated in the experimental design; performed the cell culture, qPCR, Western immunoblotting, plasmid construction, and intracellular half-cystine measurements; analyzed the results; and participated in the manuscript preparation.
REK participated in the experimental design.
JHS performed the cell transfection.
RL performed the cell culture.
DMI participated in the experimental design.
PG participated in the experimental design, data analysis, and manuscript preparation.
This work was supported by operating grants from the Cystinosis Research Foundation, the Kidney Foundation of Canada, and the Canadian Institutes of Health Research and an infrastructure grant from the Fonds de Recherche en Santé du Québec to the Research Institute of the McGill University Health Centre. Emma Brasell was the recipient of a graduate studentship award from the Cystinosis Research Foundation. Dr. Paul Goodyer is the recipient of a James McGill Research Chair.
Compliance with ethical standards
All primary cell lines used in this article were subject to IRB approval from the RI-MUHC REB (protocol: 2018-2922).
Conflict of interest
The authors declare that they have no conflicts of interest.
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