Aminoglycoside-Mediated Rescue of a Disease-Causing Nonsense Mutation in the V2 Vasopressin Receptor Gene In Vitro and In Vivo

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Title: Aminoglycoside-Mediated Rescue of a Disease-Causing Nonsense Mutation in the V2 Vasopressin Receptor Gene In Vitro and In Vivo
Authors: Sangkuhl, Katrin; Schulz, Angela; Rompler, Holger; Yun, June; Wess, Jurgen; Schoneberg, Torsten
Publisher: Human Molecular Genetics
Date Published: May 01, 2004
Reference Number: 645
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Many human diseases are caused by inactivating mutations in specific G-protein-coupled receptors (GPCRs). In about 10% of these cases, a premature stop codon leads to the generation of a truncated, functionally inactive receptor protein. In this study, we tested the hypothesis that such GPCR mutations can be functionally rescued in vitro and in vivo by treatment with aminoglycoside antibiotics, which are known for their ability to suppress premature termination codons. As a model system, we studied a mutant V2 vasopressin receptor (AVPR2) containing the inactivating E242X nonsense mutation which mimics human X-linked nephrogenic diabetes insipidus (XNDI) when introduced into mice via gene targeting techniques. Studies with cultured mammalian cells expressing the E242X mutant receptor showed that G418 (geneticin) was by far the most potent aminoglycoside antibiotic capable of suppressing the E242X nonsense codon. Strikingly, G418 treatment increased AVP-mediated cAMP responses in cultured kidney collecting duct cells prepared from E242X mutant mice in vitro, and significantly improved the urine-concentrating ability of E242X mutant mice in vivo. This is the first study demonstrating that G418 (aminoglycosides) can ameliorate the clinical symptoms of a disease-causing premature stop codon in a member of the GPCR superfamily.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

Each gene carries the code for the formation of a specific protein, which is a specific chain of amino acid residues. Genes are made up of codons, which are sets of three adjacent bases on a single strand of DNA and RNA. Each codon carries the code for a specific amino acid that is to become part of the protein the gene codes for. Each gene has 64 different codons, 61 of which encode a specific amino acid, and three of which signal the chain of amino acids that is the protein when to stop developing. The latter are called terminator codons.

There is a type of gene mutation called a nonsense mutation. It signals the protein synthesizing process to stop adding amino acids to the developing protein before the protein is complete. This results in a truncated protein that is unable to perform the function for which it was intended. 18 different nonsense mutations of the vasopressin 2 receptor (V2R) gene have been identified that result in congenital X-linked NDI.

There is a class of aminoglycoside antibiotics that are known for their ability to suppress premature termination codons. Sangkuhl, et al., tested one such antibiotic called G418 on the V2R mutation, E242X, which is a nonsense mutation that produces a truncated, functionally inactive V2R protein. The researchers hypothesized that G418 could functionally rescue E242X, that is, enable it to perform its function of being able to bind with the hormone, arginine vasopressin (AVP). When AVP binds with V2R, it initiates a molecular sequence that allows the kidney to reabsorb body water and concentrate urine. The researchers studied the effectiveness of G418 on E242X in different types of cell cultures and in mice bred to bear the E242X V2R mutation.

Their research results were striking. G418 was able to suppress the E242X stop mutation in two different mammalian cell cultures either stably or temporarily expressing E242X. G418 allowed the cells to respond to AVP as indicated in the rise of cAMP levels, part of the molecular sequence that leads to urine concentration. Further, by suppressing the E242X stop codon, the G418 allowed the mutated gene to produce V2Rs of the proper length. However, these full length V2Rs were abnormally shaped. The result being only a small percentage of these V2Rs were able to get from the cell interior to the cell membrane, a place they must be to bind with AVP. Even though only a small percentage of these V2Rs reached the cell membrane, they resulted in a robust cAMP response, thus indicating the possibility of therapeutic benefit for NDI patients bearing the E242X mutation.

When the research team tested G418’s effect on living mice carrying the E242X mutation, they found a profound increase in AVP induced cAMP formation. The researchers speculated that G418 enabled the generation of small numbers of full length AVPR2s. Also, G418 led to an approximately 50% increase in the urine concentrating abilities of the mice.

These findings indicate that further research into G418’s therapeutic potential would be useful as it holds both challenges and promise for those XNDI patients with the E242X mutation.