Altered mRNA processing and FVIII biosynthesis/function as determinants of phenotype variability in the frequent Arg2016Trp Haemophilia A patients.

The relationship between the molecular defect, the coagulation and the clinical phenotype in Haemophilia is often unclear. While the association of the so-called “null mutations” with severe forms is well-established, that of missense mutations, frequent in Haemophilia A (HA) and largely predominant in Haemophilia B (HB), with the phenotype is elusive. Missense mutations are candidate to affect protein biosynthesis/function but might also impair pre-mRNA splicing, an emerging mechanism whose pathophysiological impact is still poorly investigated. The interplay between these mechanisms might concur to factor VIII/IX levels and to HA/HB severity. As an ideal model to investigate this issue, we propose the frequent F8 c.6046C>T mutation that has been found in severe and moderate HA patients with different degrees of bleeding tendency. The mutation is candidate to affect FVIII protein biology, and preliminary data suggest it alters F8 pre-mRNA splicing. Aim To elucidate the pathogenic mechanisms of the frequent F8 p.Arg2016Trp (c.6046C>T) mutation and define the underlying residual factor VIII levels, thus identifying determinants of phenotypic variability of Haemophilia A. Expected results By investigation at the protein and mRNA level in several p.Arg2016Trp patients and with recombinant FVIII variants we expect to define the contribution of impaired mRNA splicing and/or protein biosynthesis/function to FVIII expression. This will provide insights into a poorly known, and largely underestimated feature of missense mutations, which are frequent cause of Haemophilia and of the other rare coagulation factor disorders, and could boost the investigation of mRNA splicing patterns in patients with missense mutations to help interpretation of the related coagulation and clinical phenotypes. We believe that our findings on the frequent F8 c.6046C>T mutation could help clinicians in the patient’s diagnosis and treatment.