Novel characterization of a breakpoint in F8: an individualized approach to gene analysis when PCR and MLPA results contradict.

Haemophilia A is an X-linked bleeding disorder caused by heterogeneous mutations in the F8 gene. Two inversion hotspots in intron 22 and intron 1, as well as point mutations, small insertions and deletions in the F8 gene account for causal mutations leading to severe haemophilia A. Rarely, novel molecular mechanisms lead to a haemophilia A phenotype which cannot be completely characterized by routine molecular diagnostic methods.

Expression of 14 von Willebrand factor mutations identified in patients with type 1 von Willebrand disease from the MCMDM-1VWD study.

Background: Candidate von Willebrand factor (VWF) mutations were identified in 70% of index cases in the European study 'Molecular and Clinical Markers for the Diagnosis and Management of type 1 von Willebrand Disease'. The majority of these were missense mutations.

Objectives: To assess whether 14 representative missense mutations are the cause of the phenotype observed in the patients and to examine their mode of pathogenicity.

An investigation of the von Willebrand factor genotype in UK patients diagnosed to have type 1 von Willebrand disease.

Forty families diagnosed by UK centres to have type 1 VWD were recruited. Following review, six families were re-diagnosed to have type 2 VWD, one to have a platelet storage pool disorder, and one family was determined to be unaffected. Direct DNA sequencing of the promoter region and all exons and intronic boundaries of the VWF gene identified six mutations likely to be causative of VWD in index cases of nine of the 32 (28%) confirmed type 1 VWD families.

Phenotype and genotype of a cohort of families historically diagnosed with type 1 von Willebrand disease in the European study, Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease (MCMDM-1VWD).

Type 1 von Willebrand disease (VWD) is characterized by a personal and family history of bleeding coincident with reduced levels of normal plasma von Willebrand factor (VWF). The molecular basis of the disorder is poorly understood. The aims of this study were to determine phenotype and genotype and their relationship in patients historically diagnosed with type 1 VWD.

Distinguishing between type 2B and pseudo-von Willebrand disease and its clinical importance.

Pseudo-von Willebrand disease (p-VWD) and type 2B von Willebrand disease (VWD) have similar phenotypic parameters and clinical symptoms, but different aetiologies. Fourteen individuals from five families with a historical diagnosis of type 2B VWD but with no mutation in the von Willebrand factor gene were re-investigated for the possibility of p-VWD, using platelet aggregation in the presence of cryoprecipitate. p-VWD was confirmed by targeted DNA sequencing of the glycoprotein Ibalpha gene, identifying a heterozygous Glycine 233 Valine substitution.

Aberrant dimerization of von Willebrand factor as the result of mutations in the carboxy-terminal region: identification of 3 mutations in members of 3 different families with type 2A (phenotype IID) von Willebrand disease.

The 3' end of the VWF gene was screened in the affected members of 3 different families with type 2A (phenotype IID) von Willebrand disease (vWD). Exons 49 to 52 of the VWF gene were amplified and screened for mutations by chemical cleavage mismatch detection. Mismatched bands were detected in exon 52 of 2 patients and in exon 51 of a third patient.

A new candidate missense mutation (Leu 1657 IIe) in an apparently asymptomatic type 2A (phenotype IIA) von Willebrand disease family.

Type 2A von Willebrand disease (VWD) is mostly an autosomal dominantly inherited bleeding disorder characterised by a qualitative defect of von Willebrand factor (VWF). Mutation screening was used to screen the whole of VWF gene followed by direct sequencing to detect the mutation in a father and son diagnosed with type 2A (phenotype IIA) von Willebrand disease. A C5219 to A transversion was detected predicting Leucine to Isoleucine substitution in codon 1657.

Assembly of multimeric von Willebrand factor directs sorting of P-selectin.

We designed a model system to study the role of von Willebrand factor (vWF) in the sorting of P-selectin and the biogenesis of Weibel-Palade body (WPB)-like organelles. For that purpose, a human epithelial cell line (T24) that synthesizes P-selectin mRNA, but which is devoid of vWF mRNA synthesis and storage organelles, was transfected with full-length vWF cDNA or a deletion mutant thereof. Stable transfectants of T24 with full-length vWF cDNA revealed the generation of WPB-like organelles as demonstrated by colocalization of vWF and P-selectin with double-labeling immunofluorescence.

Characterisation of type 2N von Willebrand disease using phenotypic and molecular techniques.

von Willebrand factor (vWF) is a multimeric glycoprotein found in plasma non covalently linked to factor VIII (FVIII). Type 2N von Willebrand disease (vWD) is caused by a mutation in the vWF gene that results in vWF with a normal multimeric pattern, but with reduced binding to FVIII. We have utilised methods for the phenotypic and genotypic detection of type 2N vWD.