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1.
Hum Mutat ; 27(7): 676-85, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16786531

ABSTRACT

Hemophilia A (HA) is an X-linked hereditary bleeding disorder defined by a qualitative and/or quantitative factor VIII (FVIII) deficiency. The molecular diagnosis of HA is challenging because of the high number of different causative mutations that are distributed throughout the large F8 gene. The putative role of the novel mutations, especially missense mutations, may be difficult to interpret as causing HA. We identified 95 novel mutations out of 180 different mutations responsible for HA in 515 patients from 406 unrelated families followed up at a single hemophilia treatment center of the Bicêtre university hospital (Assistance Publique-Hôpitaux de Paris [AP-HP], Le Kremlin-Bicêtre). These 95 novel mutations comprised 55 missense mutations, 12 nonsense mutations, 11 splice site mutations, and 17 small insertions/deletions. We therefore developed a mutation analysis based on a body of proof that combines the familial segregation of the mutation, the resulting biological and clinical HA phenotype, and the molecular consequences of the amino acid (AA) substitution. For the latter, we studied the putative biochemical modifications: its conservation status with cross-species FVIII and homologous proteins, its putative location in known FVIII functional regions, and its spatial position in the available FVIII 3D structures. The usefulness of such a strategy in interpreting the causality of novel F8 mutations is emphasized.


Subject(s)
DNA Mutational Analysis/methods , Factor VIII/genetics , Hemophilia A/genetics , Mutation , Factor VIII/chemistry , Hemophilia A/diagnosis , Humans , Male , Protein Structure, Tertiary , Sequence Analysis, Protein
2.
Br J Haematol ; 131(1): 109-17, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16173970

ABSTRACT

The development of neutralising antibodies to factor VIII (FVIII) is a major complication of haemophilia A (HA) therapy. We aimed to construct an individual risk profile for the development of inhibitors in HA and started by screening for the causative mutation in our HA patient population. A total of 109 patients and 28 carriers were screened. The analysis revealed 38 different mutations in the FVIII gene, of which 13 have not been described on the Haemophilia A Mutation, Search, Test and Resource Site (HAMSTeRS). Twenty-five mutations have been reported previously and all except two had a similar phenotype to what has been described. Three novel mutations were associated with severe HA: one non-missense mutation, a small insertion in the A2 domain, and two missense mutations, a H256R mutation in the A1 domain and a L2025P substitution in the C1 domain. One novel mutation, Y156C, was associated with moderate HA. Nine novel mutations caused mild HA. The P130R, D167E and V278M mutations are located in the A1 domain. R439C, Y511H, A544G and Q645H in the A2 domain, L1758F in the A3 domain and a S2157R mutation in the C1 domain. In conclusion, the genotypic profile of our HA population was not different from others described and is suitable to study inhibitor formation.


Subject(s)
Factor VIII/genetics , Hemophilia A/genetics , Mutation , Adolescent , Adult , Aged , Aged, 80 and over , Blood Coagulation Factor Inhibitors/analysis , Child , Child, Preschool , Cohort Studies , DNA Mutational Analysis , Exons , Factor VIII/immunology , Factor VIII/therapeutic use , Female , Genotype , Haplotypes , Hemophilia A/blood , Heterozygote , Humans , Infant , Male , Middle Aged , Phenotype , Protein Structure, Tertiary , Risk Assessment
3.
Blood ; 103(1): 155-7, 2004 Jan 01.
Article in English | MEDLINE | ID: mdl-12969981

ABSTRACT

The C2 domain of factor VIII (FVIII) mediates FVIII binding to von Willebrand factor (VWF) and phospholipids (PLs), thereby determining the stability and the activity of FVIII. A deletion of Ala2201 (Del2201) was identified in the FVIII C2 domain of 2 unrelated patients with mild hemophilia A (FVIII:C 11%-33%). This mutation prevents FVIII binding to a human monoclonal antibody recognizing the C2 domain and inhibiting FVIII binding to VWF and phospholipids. By comparison to healthy FVIII, Del2201 FVIII had a significantly reduced binding to VWF, which likely contributes to reduced FVIII levels in plasma. Del2201 FVIII interaction with phospholipids was evaluated in an FXa generation assay, using various concentrations of synthetic phospholipid vesicles mimicking an activated platelet surface. At the lowest phospholipid concentration allowing FXa generation, Del2201 FVIII activity was reduced 3-fold. This is the first report of a mutation altering FVIII binding to phospholipids and occurring in patients with hemophilia A.


Subject(s)
Factor VIII/chemistry , Factor VIII/genetics , Hemophilia A/blood , Hemophilia A/genetics , Alanine/chemistry , Antibodies, Monoclonal , Case-Control Studies , Epitopes/chemistry , Epitopes/genetics , Factor VIII/immunology , Factor VIII/physiology , Humans , In Vitro Techniques , Models, Molecular , Phospholipids/blood , Protein Binding , Protein Structure, Tertiary , Sequence Deletion , von Willebrand Factor/metabolism
4.
Br J Haematol ; 120(4): 643-51, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12588351

ABSTRACT

We report the identification of a new mutation in exon 28 of the von Willebrand factor (VWF) gene in two related patients with type 2M von Willebrand disease (VWD). The molecular abnormality changes the Ser 1285 to Phe within the A1 loop of VWF. The S1285F mutation was reproduced by site-directed mutagenesis on the full-length VWF cDNA. The mutated recombinant VWF (rVWF), F1285rVWF, and the hybrid, S/F1285rVWF, were expressed in COS-7 cells. F1285rVWF exhibited a slight decrease of high-molecular-weight multimers and markedly reduced ristocetin- or botrocetin-induced binding of VWF to platelets in association with a decreased binding to botrocetin. The hybrid S/F1285rVWF showed a normal multimeric profile and bound to platelets in a similar way to the patients' plasma VWF, in the presence of ristocetin or botrocetin. Thus, the new S1285F mutation within the A1 loop was responsible for the type 2M VWD observed in these patients, and was involved in the binding of VWF to botrocetin and to platelet glycoprotein Ib (GPIb). Three anti-VWF monoclonal antibodies, with conformational epitopes within the A1 loop but distinct GPIb binding inhibitory properties, showed a different interaction with F1285rVWF. These results indicate that the S1285F substitution alters the folding of the A1 loop and prevents the correct exposure of the VWF binding sites to botrocetin and GPIb.


Subject(s)
Crotalid Venoms/metabolism , Mutation , Platelet Glycoprotein GPIb-IX Complex/metabolism , von Willebrand Diseases/genetics , von Willebrand Factor/genetics , Adult , Animals , Binding Sites/genetics , Blood Platelets/metabolism , COS Cells , Electrophoresis, Polyacrylamide Gel , Humans , Male , Protein Binding/genetics , Protein Folding , Protein Structure, Tertiary , Recombinant Proteins/metabolism , Ristocetin/metabolism , von Willebrand Factor/chemistry , von Willebrand Factor/metabolism
5.
Br J Haematol ; 119(2): 390-2, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12406074

ABSTRACT

In this study, we reinvestigated a 20-year-old woman, the first cousin of two brothers with severe haemophilia A. This patient was previously assumed to be a carrier of haemophilia A due to her FVIII deficiency. We identified a novel FVIII gene mutation in the family and demonstrated that the FVIII deficiency in this female patient did not result from this gene mutation, but was linked to molecular defects in the von Willebrand factor gene.


Subject(s)
Hemophilia A/genetics , Mutation, Missense , von Willebrand Diseases/genetics , von Willebrand Factor/genetics , Adult , Dosage Compensation, Genetic , Factor VIII/metabolism , Female , Heterozygote , Humans , Male , Pedigree , Protein Binding , von Willebrand Factor/metabolism
6.
Blood ; 100(8): 3034-6, 2002 Oct 15.
Article in English | MEDLINE | ID: mdl-12351418

ABSTRACT

This study describes the genetic mechanisms responsible for the de novo occurrence of severe and mild hemophilia A in monozygotic twin females. Both twins were found to carry a previously known factor VIII mutation (Tyr16Cys) in the heterozygous state which most probably arose in the paternal germ line. Both twins showed concordant skewing of X inactivation toward the maternally derived normal X chromosome, the most severely affected twin exhibiting a higher percentage of inactivation of the normal X chromosome. The degree of skewing of X inactivation closely correlated with both the coagulation parameters and the clinical phenotype of the twins. Since these twins were monochorionic, such results suggest that the twinning event in this case has occurred after the onset of the X-inactivation period.


Subject(s)
Chromosomes, Human, X/genetics , Hemophilia A/genetics , Amino Acid Substitution , Chromosome Mapping , Factor VIII/genetics , Female , Genetic Predisposition to Disease , Humans , Male , Mutation , Pedigree , Sex Chromosome Aberrations , Twins, Monozygotic
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