title: Usefulness of an in vitro cellular expression model for haemophilia A carrier diagnosis: illustration with five novel mutations in the F8 gene in women with isolated factor VIII:C deficiency.
abstract: This study aims to determine the way to predict the haemophilia A (HA) carrier status and the potential severity in six females with low
  C levels (<0.50 IU mL(-1) ), F8 gene variations and without family history of HA. Except p.Ser577Tyr, F8 gene variations that we reported have never been described (p.Leu107His, p.Pro521Leu, p.Val682Leu, p.Leu2032Pro, p.Ala315dup). Prediction of their potential causal impact was studied by two strategies: bioinformatics approaches and site-directed mutagenesis followed by FVIII cellular expression into COS-1 cell. FVIII clotting assay (
  C) and antigen (
  Ag) were assayed in vitro. In silico analysis showed the probably damaging effect of all substitutions and the full conservation of the residues across mammalian species, except for p.Leu2032Pro. The in vitro variant expression model showed abnormal intra and/or extracellular
  C and
  Ag levels for five mutations, which suggest their causality in HA and provide informations about the involved mechanism. We suspect a defect in synthesis and secretion for p.Leu107His, p.Ala315dup and p.Pro521Leu. The mutation p.Val682Leu only affects the FVIII function while p.Ser577Tyr alters function and synthesis. The variant p.Leu2032Pro is probably a polymorphism because no alteration of the FVIII protein expression was observed in vitro. In vitro results suggest that mutations p.Ser577Tyr and p.Ala315dup could led to a severe HA in men. This study demonstrates the ability of this in vitro cellular expression model to contribute to the diagnosis strategy for female suspected of being HA carrier, without HA family history and with a novel F8 gene variation and to provide new criteria for the genetic counselling.
authors.affiliation: Hospices Civils de Lyon, Hôpital Edouard Herriot, Service d'Hématologie Biologique, Lyon.|EAM 4174 Hémostase, Inflammation et Sepsis, Université Claude Bernard Lyon 1, Lyon.
authors.forename: O
authors.initials: O
authors.lastname: Roualdes
authors.affiliation: Hospices Civils de Lyon, Hôpital Edouard Herriot, Service d'Hématologie Biologique, Lyon.|EAM 4174 Hémostase, Inflammation et Sepsis, Université Claude Bernard Lyon 1, Lyon.
authors.forename: C
authors.initials: C
authors.lastname: Nougier
authors.affiliation: Hospices Civils de Lyon, Hôpital Edouard Herriot, Service d'Hématologie Biologique, Lyon.
authors.forename: M
authors.initials: M
authors.lastname: Fretigny
authors.affiliation: Hospices Civils de Lyon, Hôpital Edouard Herriot, Service d'Hématologie Biologique, Lyon.
authors.forename: E
authors.initials: E
authors.lastname: Talagrand
authors.affiliation: Hospices Civils de Lyon, Hôpital de la Croix Rousse, Laboratoire d'Hématologie Biologique, Lyon.
authors.forename: B
authors.initials: B
authors.lastname: Durand
authors.affiliation: Hospices Civils de Lyon, Hôpital Edouard Herriot, Service d'Hématologie Biologique, Lyon.|EAM 4174 Hémostase, Inflammation et Sepsis, Université Claude Bernard Lyon 1, Lyon.
authors.forename: C
authors.initials: C
authors.lastname: Negrier
authors.affiliation: Hospices Civils de Lyon, Hôpital Edouard Herriot, Service d'Hématologie Biologique, Lyon.|EAM 4174 Hémostase, Inflammation et Sepsis, Université Claude Bernard Lyon 1, Lyon.
authors.forename: C
authors.initials: C
authors.lastname: Vinciguerra
mesh_terms: D000818:Animals; D001780:Blood Coagulation Tests; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D005169:Factor VIII / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006467:Hemophilia A / Q000097:blood / Q000175:diagnosis* / Q000235:genetics*; D006579:Heterozygote; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D009154:Mutation; D020125:Mutation, Missense; D010641:Phenotype
chemical_list: D005169:Factor VIII
keywords: carrier detection; factor VIII; gene expression; genetic counselling; haemophilia A; in vitro model