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. (24th February 2015)

• Record Type: Journal Article
• Authors: Roualdes, O.; Nougier, C.; Fretigny, M.; Talagrand, E.; Durand, B.; Negrier, C.; Vinciguerra, C.
• Is Part Of: Haemophilia. Volume 21:Number 3(2015:May)
• Page Start: e202
• Page End: e209
• Publication Date: 2015-02-24
• Subjects: Hemophilia -- Periodicals; 616.1572005
• DOI: 10.1111/hae.12651
• Languages: English
• ISSNs: 1351-8216

Abstract

<abstract abstract-type="main" id="hae12651-abs-0001"> <title>Summary</title> <p>This study aims to determine the way to predict the haemophilia A (HA) carrier status and the potential severity in six females with low FVIII:C levels (<0.50 IU mL<sup>−1</sup>), <italic>F8</italic> gene variations and without family history of HA. Except p.Ser577Tyr, <italic>F8</italic> 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 (FVIII:C) and antigen (FVIII:Ag) were assayed <italic>in vitro</italic>. <italic>In silico</italic> analysis showed the probably damaging effect of all substitutions and the full conservation of the residues across mammalian species, except for p.Leu2032Pro. The <italic>in vitro</italic> variant expression model showed abnormal intra and/or extracellular FVIII:C and FVIII: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 <italic>in vitro</italic>. <italic>In vitro</italic> results suggest that mutations p.Ser577Tyr and p.Ala315dup could led to a severe HA in men. This study demonstrates the ability of this <italic>in vitro</italic> cellular expression model to contribute to the diagnosis strategy for female suspected of being HA carrier, without HA family history and with a novel <italic>F8</italic> gene variation and to provide new criteria for the genetic counselling.</p> </abstract>