title: Characterization of five associations of F8 missense mutations containing FVIII B domain mutations.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  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
  C (98.5-128.5%) and
  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
  C with values in the range 3.5-44.5% (p < 0.05). However, the p.G224R variant showed
  C and
  Ag values no significantly different from FVIII-WT.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Hospices Civils de Lyon, Hôpital Edouard Herriot, Service d'hématologie Biologique, Lyon, France.|EAM 4174 Hémostase, Inflammation et Sepsis, Université Claude Bernard Lyon1, Université de Lyon, Lyon, France.
authors.forename: Y
authors.initials: Y
authors.lastname: Jourdy
authors.affiliation: Hospices Civils de Lyon, Hôpital Edouard Herriot, Service d'hématologie Biologique, Lyon, France.|EAM 4174 Hémostase, Inflammation et Sepsis, Université Claude Bernard Lyon1, Université de Lyon, Lyon, France.
authors.forename: C
authors.initials: C
authors.lastname: Nougier
authors.affiliation: Hospices Civils de Lyon, Hôpital Edouard Herriot, Service d'hématologie Biologique, Lyon, France.|EAM 4174 Hémostase, Inflammation et Sepsis, Université Claude Bernard Lyon1, Université de Lyon, Lyon, France.
authors.forename: O
authors.initials: O
authors.lastname: Roualdes
authors.affiliation: Hospices Civils de Lyon, Hôpital Edouard Herriot, Service d'hématologie Biologique, Lyon, France.
authors.forename: M
authors.initials: M
authors.lastname: Fretigny
authors.affiliation: Hospices Civils de Lyon, Hôpital de la Croix Rousse, Service d'hématologie Biologique, Lyon, France.
authors.forename: B
authors.initials: B
authors.lastname: Durand
authors.affiliation: Hospices Civils de Lyon, Hôpital Edouard Herriot, Service d'hématologie Biologique, Lyon, France.|EAM 4174 Hémostase, Inflammation et Sepsis, Université Claude Bernard Lyon1, Université de Lyon, Lyon, France.
authors.forename: C
authors.initials: C
authors.lastname: Negrier
authors.affiliation: Hospices Civils de Lyon, Hôpital Edouard Herriot, Service d'hématologie Biologique, Lyon, France.|EAM 4174 Hémostase, Inflammation et Sepsis, Université Claude Bernard Lyon1, Université de Lyon, Lyon, France.
authors.forename: C
authors.initials: C
authors.lastname: Vinciguerra
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005838:Genotype; D006467:Hemophilia A / Q000235:genetics* / Q000473:pathology; D006801:Humans; D020125:Mutation, Missense; D010641:Phenotype; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011110:Polymorphism, Genetic; D000072417:Protein Domains; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; C078147:F8 protein, human; D005169:Factor VIII
keywords: B domain; FVIII; genotyping; haemophilia A; in vitro model; missense mutation