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Comparative Analysis of Residual Factor VIII Expression from Recurrent F8 Nonsense Mutations Indicates that Localization in the B-domain Favours Readthrough-mediated Protein Output

1University of Ferrara, Ferrara, Italy, 2Castelfranco Veneto Hospital, Castelfranco Veneto, Italy, 3Careggi University Hospital, Florence, Italy

Abstract Number: OC 56.2

Meeting: ISTH 2021 Congress

Theme: Hemophilia and Rare Bleeding Disorders » Hemophilia - Basic

Background: Nonsense mutations, inserting premature termination codons (PTCs), might undergo, with low frequency (<0.01%), spontaneous suppression (readthrough) with production of full-length proteins upon amino acid insertion at the PTC. This process, dictated by nucleotide/protein sequence features, might have implications for hemophilia A (HA) patients.

Aims: To investigate residual factor VIII (FVIII) expression through complementary studies in HA patients’ plasma and exploiting a sensitive in-vitro expression platform.

Methods: Detection of plasma FVIII levels (ELISA, aPTT), and expression studies (HEK293 cells) with a highly-sensitive naturally-secreted luciferase (Gaussia, GL) fused to FVIII (FVIII-GL).

Results: Plasma samples from HA patients affected by six nonsense mutations (p.R446X, p.R814X, p.K1289X, p.W1726X, p.R1985X, p.R2135X) revealed traces of FVIII. Strikingly, the two B-domain variants (p.R814X, p.K1289X) showed the highest FVIII levels, suggesting a position-dependent effect. Expression studies with the FVIII-GL variants showed that those of the B-domain produced the highest luciferase activity levels, thus supporting in vivo findings. Accordingly, the predicted readthrough-deriving amino acid changes (R446W, R814W, K1289Q/Y, W1726Y, R1985W, p.R2135W) showed a minor impact for those affecting the B-domain.
To verify further our hypothesis, the panel of F8 mutations was rationally expanded to be representative of the majority of patients with nonsense mutations (60%), including the most frequent (50% of patients) in the B-domain. Through our sensitive platform we observed that all F8 nonsense variants led to detectable luciferase activity (0.4-6%). Strikingly, when categorized in two groups (B-domain, n=21; other domains, n=26), secreted luciferase activity of B-domain variants was significantly higher (p<0.0001) as compared with variants located in the other FVIII domains.

Conclusions: Our findings for the first time indicate that nonsense mutations in the B-domain, known to tolerate missense changes as those potentially arising from readthrough, are favoured in terms of readthrough-mediated protein output, which might have pathophysiological implications for HA patients.

To cite this abstract in AMA style:

Testa MF, Lombardi S, Ferrarese M, Radossi P, Belvini D, Castaman G, Bernardi F, Pinotti M, Branchini A. Comparative Analysis of Residual Factor VIII Expression from Recurrent F8 Nonsense Mutations Indicates that Localization in the B-domain Favours Readthrough-mediated Protein Output [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/comparative-analysis-of-residual-factor-viii-expression-from-recurrent-f8-nonsense-mutations-indicates-that-localization-in-the-b-domain-favours-readthrough-mediated-protein-output/. Accessed December 10, 2023.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/comparative-analysis-of-residual-factor-viii-expression-from-recurrent-f8-nonsense-mutations-indicates-that-localization-in-the-b-domain-favours-readthrough-mediated-protein-output/