Statistical Analysis of Readthrough Levels for Nonsense Mutations in Mammalian Cells Reveals a Major Determinant of Response to Gentamicin

The efficiency of translation termination depends on the nature of the stop codon and the surrounding nucleotides. Some molecules, such as aminoglycoside antibiotics (gentamicin), decrease termination efficiency and are currently being evaluated for diseases caused by premature termination codons. However, the readthrough response to treatment is highly variable and little is known about the rules governing readthrough level and response to aminoglycosides. In this study, we carried out in-depth statistical analysis on a very large set of nonsense mutations to decipher the elements of nucleotide context responsible for modulating readthrough levels and gentamicin response. We quantified readthrough for 66 sequences containing a stop codon, in the presence and absence of gentamicin, in cultured mammalian cells. We demonstrated that the efficiency of readthrough after treatment is determined by the complex interplay between the stop codon and a larger sequence context. There was a strong positive correlation between basal and induced readthrough levels, and a weak negative correlation between basal readthrough level and gentamicin response (i.e. the factor of increase from basal to induced readthrough levels). The identity of the stop codon did not affect the response to gentamicin treatment. In agreement with a previous report, we confirm that the presence of a cytosine in +4 position promotes higher basal and gentamicin-induced readthrough than other nucleotides. We highlight for the first time that the presence of a uracil residue immediately upstream from the stop codon is a major determinant of the response to gentamicin. Moreover, this effect was mediated by the nucleotide itself, rather than by the amino-acid or tRNA corresponding to the −1 codon. Finally, we point out that a uracil at this position associated with a cytosine at +4 results in an optimal gentamicin-induced readthrough, which is the therapeutically relevant variable

Readthrough of Premature Termination Codons in the Adenomatous Polyposis Coli Gene Restores Its Biological Activity in Human Cancer Cells

The APC tumor suppressor gene is frequently mutated in human colorectal cancer, with nonsense mutations accounting for 30% of all mutations in this gene. Reintroduction of the WT APC gene into cancer cells generally reduces tumorigenicity or induces apoptosis. In this study, we explored the possibility of using drugs to induce premature termination codon (PTC) readthrough (aminoglycosides, negamycin), as a means of reactivating endogenous APC. By quantifying the readthrough of 11 nonsense mutations in APC, we were able to identify those giving the highest levels of readthrough after treatment. For these mutations, we demonstrated that aminoglycoside or negamycin treatment led to a recovery of the biological activity of APC in cancer cell lines, and showed that the level of APC activity was proportional to the level of induced readthrough. These findings show that treatment with readthrough inducers should be considered as a potential strategy for treating cancers caused by nonsense mutations APC gene. They also provide a rational basis for identifying mutations responsive to readthrough inducers

Ex Vivo Treatment with a Novel Synthetic Aminoglycoside NB54 in Primary Fibroblasts from Rett Syndrome Patients Suppresses MECP2 Nonsense Mutations

BACKGROUND: Nonsense mutations in the X-linked methyl CpG-binding protein 2 (MECP2) comprise a significant proportion of causative MECP2 mutations in Rett syndrome (RTT). Naturally occurring aminoglycosides, such as gentamicin, have been shown to enable partial suppression of nonsense mutations related to several human genetic disorders, however, their clinical applicability has been compromised by parallel findings of severe toxic effects. Recently developed synthetic NB aminoglycosides have demonstrated significantly improved effects compared to gentamicin evident in substantially higher suppression and reduced acute toxicity in vitro. RESULTS: We performed comparative study of suppression effects of the novel NB54 and gentamicin on three MECP2 nonsense mutations (R294X, R270X and R168X) common in RTT, using ex vivo treatment of primary fibroblasts from RTT patients harboring these mutations and testing for the C-terminal containing full-length MeCP2. We observed that NB54 induces dose-dependent suppression of MECP2 nonsense mutations more efficiently than gentamicin, which was evident at concentrations as low as 50 µg/ml. NB54 read-through activity was mutation specific, with maximal full-length MeCP2 recovery in R168X (38%), R270X (27%) and R294X (18%). In addition, the recovered MeCP2 was translocated to the cell nucleus and moreover led to parallel increase in one of the most important MeCP2 downstream effectors, the brain derived neurotrophic factor (BDNF). CONCLUSION: Our findings suggest that NB54 may induce restoration of the potentially functional MeCP2 in primary RTT fibroblasts and encourage further studies of NB54 and other rationally designed aminoglycoside derivatives as potential therapeutic agents for nonsense MECP2 mutations in RTT

PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR-G542X nonsense allele in a CF mouse model

Nonsense mutations inactivate gene function and are the underlying cause of a large percentage of the individual cases of many genetic disorders. PTC124 is an orally bioavailable compound that promotes readthrough of premature translation termination codons, suggesting that it may have the potential to treat genetic diseases caused by nonsense mutations. Using a mouse model for cystic fibrosis (CF), we show that s.c. injection or oral administration of PTC124 to Cftr−/− mice expressing a human CFTR-G542X transgene suppressed the G542X nonsense mutation and restored a significant amount of human (h)CFTR protein and function. Translational readthrough of the premature stop codon was demonstrated in this mouse model in two ways. First, immunofluorescence staining showed that PTC124 treatment resulted in the appearance of hCFTR protein at the apical surface of intestinal glands in Cftr−/− hCFTR-G542X mice. In addition, functional assays demonstrated that PTC124 treatment restored 24–29% of the average cAMP-stimulated transepithelial chloride currents observed in wild-type mice. These results indicate that PTC124 can effectively suppress the hCFTR-G542X nonsense mutation in vivo. In light of its oral bioavailability, safety toxicology profile in animal studies, and efficacy with other nonsense alleles, PTC124 has the potential to be an important therapeutic agent for the treatment of inherited diseases caused by nonsense mutations