Mutation analysis in F9 gene of 17 families with haemophilia B from Iran.

Seventeen haemophilia B families from Iran were investigated to determine the causative mutation. All the essential regions of the F9 gene were initially screened by conformational sensitive gel electrophoresis and exons with band shift were sequenced. Seven of the 15 mutations identified in these families were novel mutations. The mutations were authenticated in nine families as other affected members or heterozygous female carriers were available for verification.

Site and type of mutations in the factor VIII gene in patients and carriers of haemophilia A.

Haemophilia A is an X-linked bleeding disorder caused by reduced or absent FVIII (FVIII) protein caused by mutations in the FVIII gene. We have used Southern blotting and chemical mismatch analysis (CMA) to identify the mutations causing haemophilia A in 59 local or referred patients or carriers of haemophilia A. Southern blot analysis of 87 families with FVIII : C < 5% identified 31 as positive for the intron 22 inversion. Analysis of 19 of the inversion-negative families and a further nine families with mild or moderate haemophilia A by CMA resulted in the identification of a heterogeneous spectrum of mutations in the FVIII gene comprising 21 single base-pair substitutions and nine deletions. Seventeen of the base-pair substitutions are missense, two nonsense, and two are splice-site mutations. Two patients were found to have compound mutations with two mutations identified on a single X chromosome. Six of the point mutations and six of the deletions have not been reported previously in the haemophilia A mutation database. Unusually, a missense mutation, as well as deletion and splice-site mutations, was found to be associated with exon-skipping events.

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. Using direct DNA sequencing, a heterozygous G8562A transition leading to a Cys2008Tyr substitution was found in all the patients in family 1, and a T8561A transversion leading to a Cys2008Ser substitution was found in both patients from family 2. In a patient from a third family, an 8-base deletion from nucleotide 8437 to 8444 was identified in exon 51. The 2 mutations in exon 52 were reproduced by in vitro site-directed mutagenesis of full-length von Willebrand factor (vWF) cDNA and transiently expressed in COS-7 cells. The corresponding recombinant VWFs for these 2 mutations exhibited the typical aberrant vWF:Ag multimer pattern seen in the plasma of the patients. These 3 mutations demonstrate the importance of other carboxy-terminal cysteines in addition to the reported Cys2010 residue, in the normal dimerization of vWF, and their essential role in the assembly of normal multimeric vWF. (Blood. 2001;98:674-680)

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. This novel missense mutation which was also identified by MboI restriction enzyme analysis, was found in both patient and his father but not in any other unaffected family member or 50 unrelated normal individuals. This substitution was reproduced by in vitro site directed mutagenesis of full-length VWF cDNA and transiently expressed in COS-7 cells. The corresponding recombinant VWF protein exhibited the full spectrum of VWF multimers, suggesting that the abnormal multimer seen in the patient results from increased proteolysis.

Identification of type 2 von Willebrand disease in previously diagnosed type 1 patients: a reappraisal using phenotypes, genotypes and molecular modelling.

In order to investigate the possibility that qualitative type 2 defects in von Willebrand factor (VWF) occurred in patients previously diagnosed with quantitative type 1 von Willebrand disease (VWD), the phenotypes and genotypes were reanalysed in 30 patients who exhibited discrepant VWF activity/VWF:Ag ratios of less than 0.7. The capacity of VWF to bind to glycoprotein Ib (GpIb) was reassessed using the ristocetin co-factor activity (VWF:RiCo) assay compared to an in-house and a commercial ELISA assay (based on a mAb directed against the GpIb binding site on VWF). This was supplemented by multimeric analysis and the amplification and sequencing of a 936 bp fragment of exon 28 of the VWF gene with the aim of identifying mutations in the A1 domain. On reappraisal, using the VWF:RiCo assay all patients demonstrated a disproportionately reduced VWF:RiCo/VWF:Ag ratio, indicative of a qualitative defect, while abnormal ratios were detected in only seven kindreds using the in-house ELISA assay and in only one kindred with the commercial ELISA assay. Eight single amino acid substitutions were found in nine kindreds, four of which were novel candidate VWF mutations and four previously described in association with type 2 VWD. In agreement with the phenotype, the novel VWF mutations were located in the VWF-A1 crystal structure at positions that corresponded to potential type 2M defects. This study underlines the difficulties of correct diagnosis of the subtype of VWD and emphasises the importance of using sensitive phenotypic assays, the relevance of the VWF:RiCo/ VWF:Ag ratio, multimeric analysis and molecular modelling analysis.

Congenital thrombophilia and thrombosis: a study in a single centre.

AIM: To identify the incidence of congenital thrombophilia in a cohort of children presenting with symptomatic thromboembolism. METHOD: A review of children with thromboembolism investigated for thrombophilia over a 12 month period. SUBJECTS: Thirty children with thromboembolic episodes and 16 of their family members. MEASUREMENTS AND DATA COLLECTION: Data were collected on age at diagnosis, underlying diagnosis, site of thrombosis, associated precipitating factors, occurrence of other thromboembolic events, and family history. Investigations included measurement of protein C activity, total and free protein S antigen, antithrombin III activity, screening for factor V Leiden and prothrombin 20210A, urinary homocysteine estimation, and a screen for lupus anticoagulant. RESULTS: Twenty seven of 30 patients had one or more risk factors present at the time of thromboembolism. Eighty three per cent had acquired precipitating factors present, and 43% had underlying congenital thrombophilia. CONCLUSIONS: There was a high incidence of congenital thrombophilia in this group of patients with symptomatic thromboembolism. These findings emphasise the importance of such defects in the pathogenesis of childhood thrombosis, and suggest that full thrombophilia investigations should be performed on all children presenting with thromboembolic disease.

Use of Intron 40 VNTR I in vWD Gene Tracking.

Common alleles or polymorphisms form the basis of human diversity, and some of these polymorphisms closely linked on the same chromosome with a defective gene have been used for gene tracking in many genetic disorders. The success of any linkage analysis also relies on the nature of the polymorphisms used, and the most useful are those that can have a large numbers of alleles.

Multimeric analysis of von Willebrand factor.

Von Willebrand Factor (vWF) in normal plasma is composed of a series of high molecular multimers, ranging in size from 8×10(5) to over 15×10(6) Daltons (1). The multimeric structure of vWF was first investigated by two-dimensional crossed immunoelectrophoresis (2D-IEP). In 1974, Kernoff et al. (2) used this method, and since then multimer sizing of von Willebrand Factor Antigen (vWF∶Ag) in this form and other variations have been used in the diagnosis and classification of von Willebrand's disease (vWD).

Independent occurrence of the novel Arg2163 to His mutation in the factor VIII gene in three unrelated families with haemophila A with different phenotypes. Mutations in brief no. 126. Online.

Using chemical mismatch analysis or denaturing gradient gel electrophoresis followed by nucleotide sequencing, we have identified the same G6545A mutation leading to an Arg2163 His subsitution in the factor VIII gene of three haemophiliacs from unrelated families. One of the affected individuals has severe haemophilia, while the other two are moderately severe. While we cannot exclude the possibility that these differences in phenotype arise from differences in VIII:C assay methods, other studies have also identified different clinical phenotypes in individuals with the same mutations, and suggested that they may arise from extragenic factors that affect or modify gene expression or protein function. The G6545A mutation occurs at a CG dinucleotide which is a known mutation hotspot, and which may explain the independent occurrence in unrelated families.

De novo mutation causing sporadic type 2A von Willebrancd's disease: report of three cases.

Chemical mismatch detection has been used to screen selectively part of the A2 domain of exon 28 of the von Willebrand factor gene of three unrelated patients with apparently sporadic type 2A von Willebrand disease (vWD) and their parents and siblings. Mismatches have been defined by DNA sequencing and mutations authenticated by restriction enzyme analysis. While a mutation was identified in all three patients, no evidence of mutation could be found in their asymptomatic/un-affected parents or siblings, proving the disease to be truly sporadic in these cases. Of these, two with severe clinical bleeding had a serine 743 to leucine substitution while the third patient with clinically less severe bleeding had an arginine 834 to tryptophan substitution. The possible genetic mechanisms for sporadic type 2A vWD in these families are discussed.

Congenital microangiopathic haemolytic anemia: a variant of thrombotic thrombocytopenic purpura?

We describe a child with recurrent microangiopathic haemolytic anemia (MAHA) and thrombocytopenia (TCP) who presented on the first day of life. Remission was maintained only by regular infusions of fresh frozen plasma (FFP). This case shows similarities with others described in the literature as examples of thrombotic thrombocytopenic purpura (TTP), although the absence of renal or neurological involvement distinguishes this disorder from classical TTP.