OR04 - A Novel Genetic Classification of Inhibitor Risk for F8 Nonsense Mutations Based on Immunogenic Profiling of Ribosomal Readthrough in EAHAD database

OR04

A Novel Genetic Classification of Inhibitor Risk for F8 Nonsense Mutations Based on Immunogenic Profiling of Ribosomal Readthrough in EAHAD database

M. F. Testa^1,*, F. Bernardi¹, A. Branchini¹, M. E. Mancuso², M. Pinotti¹, D. Balestra¹

¹Life Sciences and Biotechnology, University of Ferrara, Ferrara, ²IRCCS, Humanitas Research Hospital, Center for Thrombosis and Hemorrhagic diseases, Milan, Italy

 

Introduction: Among F8 mutation types, Premature Termination Codon (PTC) lead to the synthesis of truncated factor VIII (FVIII), and display a wide variation of inhibitor risk, which lowers the performance of clinical risk prediction models. Translational readthrough (RT), which overrides PTCs and incorporates predictable amino acids, produces traces of full-length FVIII that may influence the immune response and risk of inhibitor formation.

Methods: To better classify PTC susceptibility for RT and inhibitor formation, F8 genotypes and inhibitor status were investigated in 1048 patients (PTCs, n=335), recorded in the EAHAD database. The predicted RT output, based on PTCs type (UGA, Trp; UAG, Gln/Tyr, UAA, Tyr/ Gln), were tested using in silico NetMHCII tool, which calculates binding affinity between FVIII novel peptides arising from RT and MHC class II molecules allowing comparisons across different HLA alleles. Affinity of novel RT peptides was compared with that of the corresponding WT counterpart.

Results: Wild-type FVIII (WT-RT), which would re-incorporate the WT residue and reduce inhibitor development risk, was i) lower in patients with than without inhibitors (p=0.022), ii) not predicted in patients with recurrent PTCs detected in at least three inhibitor-positive cases (n=136, p=0.0001), and iii) much lower (12%) for FVIII light chain PTCs, highly associated with inhibitor development than for the heavy chain (25.2%) and B-domain (27.4%). Conversely, for PTCs without WT formation (n=297), mean differences in affinity between HLA-DR alleles and novel peptides were higher for missense variants predicted in patients with than without inhibitors (p<0.0001) and increased for PTCs present in more than one patient with inhibitor. A comprehensive in silico analysis of Single Nucleotide Variants in the F8 coding sequence, consisting of prediction of WT-RT or differential affinity of missense peptides, revealed that naturally occurring PTCs have a higher inherent potential for WT-RT (31%) compared with predictable PTCs (22%, p=0.001), suggesting an evolutionary clinical negative selection.

Discussion/Conclusion: Our data provide a new classification of PTCs based on genetic features that, based on predicted RT output and immunogenicity, could be exploited to improve estimate of individual PTC-related inhibitor risk.

Disclosure of Interest: None declared