Abstract Number: PB0829
Meeting: ISTH 2020 Congress
Background: Nonsense mutations are commonly associated with “null” genetic conditions. However, an event called “ribosome readthrough” may restore the synthesis of full-length proteins through suppression of nonsense mutations and incorporation of an amino acid. This might explain why some F8 nonsense mutations are associated with moderate haemophilia A (HA).
Aims: To evaluate full-length protein production resulting from occurrence of readthrough over a wide panel of F8 nonsense mutations.
Methods: Expression of an optimized fusion protein between factor VIII (FVIII) and a high-sensitivity luciferase in HEK293 cells and luciferase assays in media and cell lysates. Evaluation of FVIII in HA patient plasma through Western blotting and ELISA.
Results: F8 nonsense mutations were subdivided into a high-frequency (12 mutations; patient number, n>10), arising from the highly frequent CGA(arginine)>TGA change, and a low-frequency (44 mutations; patient number n=1-5) group. The latter was rationally classified into two subsets including mutations predicted to be suppressed by reinsertion of the original amino acid, and localized in the B-domain, in which potential amino acid substitutions introduced during readthrough are predicted to be tolerated. Noticeably, the selected mutations (44 out of 216) have been reported in 384/611 (63%) HA patients with nonsense mutations.
Strikingly, expression of all F8 nonsense variants led to detectable luciferase activity with a different extent (0.3-7%) that appeared to be consistent with the impact of the inserted amino acid on FVIII secretion. The selected panel includes 11 nonsense mutations, found in Italian HA patients, that in preliminary results revealed traces of FVIII protein in plasma.
Conclusions: Data from our expression platform indicate that a relevant number of F8 nonsense mutations, relatively frequent in HA, undergo readthrough and can be associated with residual protein levels. This might have relevant pathophysiological implications, and might contribute to interpret the variable susceptibility of HA patients to develop inhibitors upon replacement therapy.
To cite this abstract in AMA style:Testa MF, Lombardi S, Ferrarese M, Pinotti M, Castaman G, Radossi P, Belvini D, Bernardi F, Branchini A. Detection of Residual Factor VIII Levels Reveals the Occurrence of Readthrough Over the Majority of F8 Nonsense Mutations [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/detection-of-residual-factor-viii-levels-reveals-the-occurrence-of-readthrough-over-the-majority-of-f8-nonsense-mutations/. Accessed November 29, 2023.
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