Class 1 CF Mutations

Since the discovery of the gene that causes Cystic Fibrosis, our knowledge of how mutations in this gene cause the varied pathophysiological manifestations of this disease has increased substantially. This knowledge has led to the possibility of new therapeutic approaches aimed at the basic defect. Class I mutations of CFTR include premature termination codons (PTCs) or stop codons. In the last 10 years there has been a concerted international effort to utilize the concept of read-through of the stop codon producing full length functioning CFTR protein. This author considers that this approach will result in clinical trials in CF patients carrying these mutations. Class I mutations include PTCs or nonsense codons. A nonsense mutation is a single point alteration in DNA that results in the inappropriate presence of a UAA, UAG, or UGA stop codon in the protein-coding region of the corresponding messenger RNA (mRNA) transcript. Such a stop codon causes premature cessation of translation, with protein truncation leading to loss of function and consequent disease. Nonsense mutations are responsible for about 10% of cystic fibrosis cases worldwide. However, in Israel, nonsense mutations are the cause of cystic fibrosis in most patients (Kerem et al., 1997). As such mutations produce little functional CFTR, these patients usually have a phenotype of CF with exocrine pancreatic insufficiency. The increased understanding of ribosomal function, the process of translation, and small molecules that change the interaction between the ribosome and mRNA have led to the identification of several agents that are capable of suppressing PTCs. This has resulted in a novel strategy to treat CF and other genetic disorders caused by PTCs by restoring full length protein. Aminoglycoside antibiotics were the first drugs demonstrated to suppress PTCs in disease-causing mutations, allowing the translation of full length proteins (Hermann, 2007). Aminoglycosides are antibacterial agents, their mode of action is interfering with normal translation via binding to the bacteria 16S rRNA. There is reduced discrimination between cognate and near-cognate tRNA hence reducing translational fidelity. Eventually, there is accumulation of truncated and non-functioning proteins resulting in bacterial cell death. Gorini and Kataja (1964) demonstrated that aminoglycosides may suppress PTCs and lead to full length translation in E. coli. Aminoglycosides may also bind to human 18S rRNA subunit reducing discrimination of near-cognate tRNAs. This interaction is less stable than in bacteria but may be sufficient to lead to an insertion of a near-cognate aminoacyl-tRNA into the ribosomal A site that is subsequently incorporated into the polypeptide chain. Howard et al. (1996) described PTC suppression by the synthetic aminoglycoside geneticin (G418) to restore function in HeLa cells expressing nonsense codons in 1996. This pivotal work was extended to four nonsense mutations of cftr who were expressed by the human airway cell line IB3-1.In this study, the commonly used aminoglycoside, gentamicin, was incubated with these cells and full length protein was produced (Bedwell et al., 1997).