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

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, 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.