BACKGRUOUND Nonsense mutations, caused by premature termination codons, are relatively frequent in Haemophilia (>10%) and are considered as “null mutations”. However, they have been found also in moderate/mild Haemophiliacs and, as compared with large gene deletions, are associated with lower risk for developing inhibitors. This observation point to the presence of residual expression levels arising from “ribosome readthrough” over nonsense triplets, whose sequence context has an impact on readthrough efficiency, as we have shown in a very small cohort of Haemophilia B (HB) patients. Drugs inducing readthrough such as aminoglycosides are proposed as potential therapy. AIM To investigate secreted and functional levels of factor IX (FIX) produced by an extended panel of HB nonsense mutations. METHODS Recombinant FIX (rFIX) nonsense variants were expressed in HEK293 cells, and secreted and intracellular protein levels, as well as protein isoforms, were evaluated by ELISA and Western Blotting analysis. Residual activity was assessed by coagulant assays. RESULTS We investigated a panel of F9 mutations (R75X, L103X, R162X, W240X, R294X, R298X, Y330X, Q370X, R379X, R384X), associated to severe/moderate HB, representing the vast majority of patients with nonsense mutations in HB (324 patients out of 467, corresponding to ~70%). Appreciable levels of secreted FIX were detected for the R162X, W240X, R294X, R298X, Y330X mutants, with truncated isoforms being the large predominance. Truncated forms for Q370X, R379X and R384X were observed in cell lysates only, which suggests misfolding and intracellular retention. Noticeably, the full-length FIX form was appreciable for the R162X variant, indicating the occurrence of spontaneous readthrough, which was significantly increased by the use of aminoglycosides. Moreover, aminoglycosides promoted the synthesis of full-length FIX in the presence of the R75X, Y330X, Q370X, R379X, R384X mutations, not undergoing appreciable spontaneous readthrough. Intriguingly, we investigated the peculiar W240X nonsense mutation, caused by two different termination codons, which showed a differential impact of readthrough in restoring the full-length protein depending on the sequence context. Preliminary coagulant and functional assays revealed that only a few variants were prone to undergo efficient readthrough in terms of secretion and coagulant function. CONCLUSIONS These data demonstrate that nonsense mutations can be associated to residual FIX levels through a mechanism of “productive” readthrough. The identification of “leaky” nonsense mutations, and thus of patients with trace FIX levels and potentially “high responders” to readthrough-inducing drugs, might help diagnosis and treatment.
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|Titolo:||RESPONSIVENESS OF HEMOPHILIA B-CAUSING NONSENSE MUTATIONS TO RIBOSOME READTHROUGH-INDUCING DRUGS STRICTLY DEPENDS ON THE NUCLEOTIDE AND PROTEIN CONTEXT|
|Data di pubblicazione:||2016|
|Appare nelle tipologie:||04.1 Contributi in atti di convegno (in Rivista)|