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

O Roualdes; C Nougier; M Fretigny; E Talagrand; B Durand; C Negrier; C Vinciguerra

doi:10.1111/hae.12651

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

Haemophilia. 2015 May;21(3):e202-e209. doi: 10.1111/hae.12651. Epub 2015 Feb 24.

Authors

O Roualdes^{1

2}, C Nougier^{1

2}, M Fretigny¹, E Talagrand¹, B Durand³, C Negrier^{1

2}, C Vinciguerra^{1

2}

Affiliations

¹ Hospices Civils de Lyon, Hôpital Edouard Herriot, Service d'Hématologie Biologique, Lyon.
² EAM 4174 Hémostase, Inflammation et Sepsis, Université Claude Bernard Lyon 1, Lyon.
³ Hospices Civils de Lyon, Hôpital de la Croix Rousse, Laboratoire d'Hématologie Biologique, Lyon.

PMID: 25708597
DOI: 10.1111/hae.12651

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

Fviii: 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 (

Fviii: C) and antigen (

Fviii: 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

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 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.

Keywords: carrier detection; factor VIII; gene expression; genetic counselling; haemophilia A; in vitro model.

MeSH terms

Animals
Blood Coagulation Tests
COS Cells
Cell Line
Chlorocebus aethiops
Factor VIII / genetics*
Factor VIII / immunology
Factor VIII / metabolism
Female
Gene Expression*
Hemophilia A / blood
Hemophilia A / diagnosis*
Hemophilia A / genetics*
Heterozygote*
Humans
In Vitro Techniques
Male
Mutation*
Mutation, Missense
Phenotype

Substances

Factor VIII