Translational readthrough at F8 nonsense variants in the factor VIII B domain contributes to residual expression and lowers inhibitor association

In hemophilia A (HA), F8 nonsense variants, and particularly those affecting the large factor VIII (FVIII) B domain that is dispensable for coagulant activity, display lower association with replacement therapy-related anti-FVIII inhibitory antibodies as retrieved from multiple international databases. Since null genetic conditions favour inhibitor development, we hypothesized that translational readthrough over premature termination codons (PTCs) may contribute to immune tolerance by producing full-length (FL) proteins through the insertion of amino acid subset(s). To quantitatively evaluate in vitro the readthrough output, we developed a very sensitive luciferase-based system to detect very low FL-FVIII synthesis from a wide panel (n=45; ~60% patients with PTCs) of F8 nonsense variants. PTCs not associated with inhibitor displayed higher readthrough-driven expression levels than inhibitor-associated PTCs, a novel observation. Particularly, higher levels were detected for B-domain variants (n=20) than for variants in other domains (n=25). Studies on plasma from six HA patients with PTCs, integrated by expression of the corresponding nonsense and readthrough-deriving missense variants, consistently revealed higher FVIII levels for B-domain variants. Only one B-domain PTC (Arg814*) was found among the highly represented PTCs not sporadically associated with inhibitors, but with the lowest proportion of inhibitor cases (four out of 57). These original findings into HA molecular genetics, and particularly into genotype-phenotype relationships related with disease treatment, demonstrate that B-domain features favour PTC readthrough output. This provides a potential molecular mechanism contributing to differential PTC-associated inhibitor occurrence, with translational implications for a novel, experimentally based classification of F8 nonsense variants.