Characterization of five associations of F8 missense mutations containing FVIII B domain mutations

Introduction Haemophilia A (HA) is a bleeding disorder due to an absence or a reduced activity of coagulation factor VIII (FVIII) caused by mutations in F8 gene. Missense mutations represent approximately 45% of the reported molecular defects in HA. However, only few missense mutations in FVIII B domain have been described. Aim The aim of this study was to characterize five genetic variations (three novels and two previously reported) localized in the FVIII B domain. In all cases, an additional missense variation located outside the FVIII B domain was found. We investigated each of these variations separately and in combination too for their contribution to HA phenotype. Methods F8 variants were transiently expressed in COS-1 cells. Media and cell lysates were collected after 72 h. Then, FVIII activity, secretion and thermostability were analysed and compared to FVIII wild-type. Results The 5 FVIII B domain variants showed normal FVIII:C (98.5–128.5%) and FVIII:Ag (97.7–154%). No synergistic effect was observed between the B domain variant and their associated mutations. In contrast, the variants located outside the B domain, p.V682L, p.S714L, p.V592D and p.C573F revealed significantly decrease of FVIII:C with values in the range 3.5–44.5% (p < 0.05). However, the p.G224R variant showed FVIII:C and FVIII:Ag values no significantly different from FVIII-WT. Conclusion The FVIII B domain variants, p.D963N, p.S806T, p.G873D, p.H998Q and p.Q1225R may be considered as polymorphism or non-pathologic mutations. In five patients, clinical phenotype could be explained by the additional causative missense mutation. For the p.G224T variant further splicing studies are necessary to determine its pathogenicity.