Suppression of “leaky” nonsense mutations by ribosome readthrough accounts for residual factor IX levels in Haemophilia B patients

INTRODUCTION Nonsense mutations, relatively frequent in Haemophilia (>10%), are considered “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, 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 residual levels of factor IX (FIX) produced by an extended panel of HB nonsense mutations. METHODS Expression of nonsense FIX variants in HEK293 cells, evaluation of intracellular and secreted FIX levels (ELISA, Western Blotting, fluorogenic functional assays). RESULTS We investigated nine F9 mutations (R75X, L103X, R162X, R294X, R298X, Y330X, Q370X, R379X, R384X) associated to severe/moderate HB. Appreciable levels of secreted FIX were detected for the R162X, R294X, R298X, Y330X mutants, with a large predominance of truncated forms. Truncated forms for Q370X, R379X and R384X were observed in cell lysates only, which suggests misfolding. Noticeably, the full-length FIX form was appreciable for the R162X, R294X and R298X and indicated 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. Preliminary functional assays revealed a residual activity for the R162X mutant. 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.