An investigation of the von Willebrand factor genotype in UK patients diagnosed to have type 1 von Willebrand disease.
ABSTRACT 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. These included R1205H (3614G > A) VWD Vicenza, P1648fsX45 (4944delT), D141G (422A > G) and three splice site mutations: 3108 + 5G > A, 7437 + 1G > A and 3379 + 1G > A. The Y1584C (4751A > G) polymorphism was present in eight additional families. No significant VWF gene mutation or polymorphism was identified in 15 of the 32 type 1VWD index cases (47%). Haplotype studies were performed using a panel of VWF polymorphisms to investigate the segregation in families of VWD phenotype with the VWF gene. In 13 of the 32 families it was likely that VWD segregated with the VWF gene. In eight families (25%) VWD clearly did not segregate with the VWF gene. We suggest that mutation screening of the VWF gene has limited general utility in genetic diagnostic and family studies in type 1 VWD. If genetic studies are performed, the incomplete penetrance and variable expressivity of type 1 VWD must be taken into account. Unless linkage of VWD phenotype with the VWF gene can be clearly demonstrated, the results of any genetic family studies should be interpreted with caution.
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ABSTRACT: BACKGROUND: In von Willebrand factor (VWF) the effect of mutations potentially affecting mRNA processing or splicing is less predictable than that of other mutations (e.g. nonsense or missense substitutions). Bioinformatic tools can provide a valuable means to determine the consequences of potential splice site mutations (PSSM), but functional studies are mandatory to elucidate the true effect of the variation detected. OBJECTIVES, PATIENTS AND METHODS: After identification of PSSM in VWD patients, we began a systematic study of their in vivo effect in RNA extracted from the patients' platelets and leukocytes. RESULTS AND CONCLUSIONS: Thirteen pairs of primers were designed for full amplification of VWF mRNA by RT-PCR that, after sequencing of aberrant products, enabled elucidation of the PSSM consequences for mRNA processing. This procedure was used to study seven different PSSM identified in four patients demonstrating diverse molecular mechanisms such as exon skipping (c.533-2A>G and c.8155+3G>C) and the activation of a cryptic splice site (c.7730-1G>C). No visible effect was evident for c.1533+15G>A and c.5170+10C>T and the consequence of c.[546G>A;7082-2A>G] was hidden by nonsense-mediated mRNA decay (NMD). Results were compared with in silico predictions of four splice-site analysis tools. We demonstrate selective degradation of VWF mRNA bearing PSSM by NMD for several mutations, which suggests that NMD represents a general mechanism for truncating mutations in VWF. Furthermore, because NMD efficiency varies between cell types, use of RNA from both platelets and leukocytes for in vivo study of VWF PSSM offers complementary results, particularly in cases in which NMD occurs in the allele carrying the mutation.Journal of Thrombosis and Haemostasis 01/2011; 9(4):679-88. · 6.08 Impact Factor
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ABSTRACT: Von Willebrand disease (VWD) is an autosomally inherited bleeding disorder caused by a deficiency and/or abnormality of von Willebrand factor (VWF). VWF is a multimeric adhesive protein that plays an important role in primary hemostasis by promoting platelet adhesion to the subendothelium at sites of vascular injury and platelet-platelet interactions at high-shear rate conditions. Furthermore, VWF is the carrier of factor VIII, thus indirectly contributing to the coagulation process. Most cases have a partial quantitative deficiency of VWF (type 1 VWD) with variable bleeding tendency, whereas qualitative variants (type 2 VWD), due to a dysfunctional VWF, are clinically more homogeneous and account for approximately 20-30% of cases. Type 3 VWD is rare and these patients have moderate-to-severe bleeding diathesis, display a recessive pattern of inheritance and virtual absence of VWF. The diagnosis of VWD may be difficult, especially in type 1 disease, since the laboratory phenotype of the disorder is very heterogeneous and confounded by the influence on VWF levels by factors outside the VWF gene (e.g., blood group). An array of tests are usually required to characterize the several types of the disorder in order to predict the best treatment modality. Desmopressin is the treatment of choice for most patients with type 1 VWD because it corrects the the dual defects of hemostasis, that is, abnormal coagulation expressed by low levels of factor VIII and abnormal platelet adhesion expressed by the reduction of VWF.Expert Review of Hematology 02/2011; 4(1):95-106. · 2.38 Impact Factor
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ABSTRACT: von Willebrand disease (VWD) is the most common autosomally inherited bleeding disorder. The disease represents a range of quantitative and qualitative pathologies of the adhesive glycoprotein von Willebrand factor (VWF). The pathogenic mechanisms responsible for the type 2 qualitative variants of VWF are now well characterized, with most mutations representing missense substitutions influencing VWF multimer structure and interactions with platelet GPIbα and collagen and with factor VIII. The molecular pathology of type 3 VWD has been similarly well characterized, with an array of different mutation types producing either a null phenotype or the production of VWF that is not secreted. In contrast, the pathogenetic mechanisms responsible for type 1 VWD remain only partially resolved. In the hemostasis laboratory, the measurement of VWF:Ag and VWF:RCo are key components in the diagnostic algorithm for VWD, although the introduction of direct GPIbα-binding assays may become the functional assay of choice. Molecular genetic testing can provide additional benefit, but its utility is currently limited to type 2 and 3 VWD. The treatment of bleeding in VWD involves the use of desmopressin and plasma-derived VWF concentrates and a variety of adjunctive agents. Finally, a new recombinant VWF concentrate has just completed clinical trial evaluation and has demonstrated excellent hemostatic efficacy and safety.Hematology 01/2013; 2013:254-60. · 1.49 Impact Factor