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Variable nonsense suppression in Hemophilia A
logenetic conservation of this amino acid, or whether deleterious missense mutations have been described in this codon, are additional important factors to be consid- ered.
Under our experimental conditions, the ability of RTA to increase C-terminal (light chain) positive staining or FVIII:Ag levels did not always correlate with the ability of RTA to restore FVIII:C activity. This suggests that the amino acid incorporated at the PTC and its structural sim- ilarity with the natively encoded amino acid can be criti- cal for the restoration of FVIII:C, but not necessarily for that of FVIII:Ag. According to the PTC context rule,13 the hierarchy of RTA responsiveness is T-TGA-C>T-TAG-C> T-TAA-C; thus, a stronger RTA effect at TGA (opal) is expected in PTC originally encoding tryptophan and argi- nine. However, we found that PTC generated at triplets natively encoding arginine and fulfilling the PTC context rule led to production of a non-functional protein, although this situation can also result in a gain of func- tion, as reported in a cellular model of readthrough in hemophilia B.30 Conversely, according to the PTC context rule, a PTC generated at triplets encoding glutamine is predicted to be less responsive to RTA treatment. However, in our study, RTA allowed partial restoration of FVIII:C levels in 2 of 3 recoded FVIII mutants originally encoding glutamine (pQ462X and pQ1764X). A similar result was reported in a study of cystic fibrosis in which patients harboring the Y122X mutation had a better than expected response to RTA, even though the PTC was caused by a TAA codon, followed by a C nucleotide at position +4, which was previously reported to have the lowest RTA sensitiveness.13 However, both the readthrough effect due to the presence of cytosine at position +4, and the incorporation of the natively encod- ed tyrosine might have been facilitated by the RTA treat- ment,15 which would account for the clinical benefit observed.
Our work points out the importance of not only evalu- ating RTA-mediated protein biosynthesis, but also the functional activity of the recoded protein generated. This is particularly important in studies of the potential clinical benefit of RTA therapy and the search for safer and more
effective RTA. By taking into account the functionality of the restored protein generated by RTA, our cellular model, or other similar models, provide a method for testing the effectiveness of new RTA.31
Although we evaluated only a small number of F8BDD variants, and these results cannot be generalized, our analysis of the molecular environment of these PTC pro- vides valuable information on additional factors involved in eventual responses to RTA treatment. From these results we can conclude that: 1) responsiveness is greater in PTC with the consensus sequence T-Stop-C; 2) in TGA (opal) than in TAG (amber) PTC; and 3) it will be higher when the mutated original amino acid is tryptophan or glutamine as opposed to arginine, in which, despite the efficient intracellular reduction of truncated protein and extracellular increase of FVIII:Ag levels, the increase observed in FVIII:C activity was minimal.
In conclusion, the results obtained with our model rep- resent a proof-of-concept that RTA are capable of sup- pressing PTC in some nonsense mutations causing HA, albeit with different efficiencies and different functional consequences depending on the sequence context. This model, linked to a functional assay based on the determi- nation of FVIII:C activity, and the application of the PTC rule described here, allowed for the first time the identifi- cation of five RTA-responsive nonsense mutations in HA and may facilitate the classification of nonsense mutations reported in the HA database into categories based on the predicted levels of responsiveness to RTA, thus promoting patient selection in readthrough therapy clinical trials.32
Acknowledgments
The authors thank the participating patients and their families. We also thank Yvonne Richaud-Patin and Dr. Angel Raya (CMRB, Spain) for their help with the fibroblast isolation.
Funding
This project was supported by two grants from the Instituto de Salud Carlos III (PI11/03024 and PI11/03029) / e-Rare-2 (HEMO-iPS) that were cofinanced by the European Regional Development Fund (FEDER), and the Europe Aspire 2013 Award in Hemophilia Research.
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