Immunogenic implications of translational readthrough on the inhibitor risk associated with F8 nonsense mutations in Hemophilia A

Among F8 mutation types, main determinants of inhibitor development in hemophilia A, nonsense mutations (premature termination codons, PTC) display wide variation in the associated risk. Translational readthrough (Rdthr) at PTCs, which produces traces of full-length factor VIII (FVIII) including missense and wild-type molecules, may influence the immune response and risk of inhibitor formation. To better classify PTC susceptibility in relation to inhibitor occurrence, F8 genotypes, inhibitor status and Rdthr characteristics were investigated in 335 PTCs (1048 patients) recorded in the European Association for Haemophilia and Allied Disorders (EAHAD) database. Rdthr producing wild-type FVIII (WT-Rdthr), hypothesized to minimize inhibitor development, was not predicted in patients with recurrent PTCs detected in at least three inhibitor-positive cases (n=136, p=0.0001). These PTCs, previously expressed in a wide panel of FVIII-luciferase recombinants, showed lower readthrough output. In addition, WT-Rdthr was lower for light-chain PTCs, highly associated with inhibitor occurrence. We also estimated lower WT-Rdthr for PTCs predictable by single nucleotide variations (n=662) than in those observed in the EAHAD database. In silico mean differences in affinity of HLA-DR alleles for FVIII peptides and their missense counterparts, potentially arising from Rdthr of PTCs without WT formation (n=297), were higher for missense variants predicted in patients with inhibitors than without (p<0.0001). The difference increased for PTCs present in more than one patient with inhibitor. We provide a new classification of PTCs, based on predicted Rdthr output and immunogenicity, that could be exploited to improve estimate of individual PTC-related inhibitor risk, and particularly in HLA genotyped patients.