A. M. Cumming

Development of resistance to activated protein C during pregnancy

Summary. We measured activated protein C (APC) anticoagulant activity in 20 healthy women at 14‐20, 28 and 36 weeks gestation, and at 1 d post‐partum. Significant reductions in the mean APC sensitivity ratio (APC‐SR) were observed at all stages of pregnancy studied compared with the mean APC‐SR obtained for baseline measurements carried out at > 8 weeks post‐partum. APC resistance was seen in 8/19 (42%) and in 11/20 (55%) women at 14‐20 and 28 weeks gestation respectively. The development of resistance to APC may contribute to the increased risk of thrombosis during pregnancy.

The prothrombin gene G20210A variant : prevalence in a U.K. anticoagulant clinic population

We have investigated the prevalence of a recently reported genetic variation in the prothrombin gene (G20210A) in patients with an objectively confirmed history of venous thrombosis. 12/219 patients (5.5%) were found to be heterozygous carriers of the 20210 A allele. The incidence of the 20210 A allele in a group of 164 healthy controls was 1.2% (allele frequency 0.61%, 95% CI 0.08–2.19). When patients with a known alternative hereditary risk factor for venous thrombosis (factor V Leiden mutation or deficiency of antithrombin, protein C or protein S) were excluded, the G20210A variant was found to increase the risk for venous thrombosis by approximately 5‐fold (odds ratio 5.4, 95% CI 1.16–25.0). This prothrombin gene sequence variation adds further to the list of recognized genetic risk factors for thrombophilia.

The diagnosis of von Willebrand disease: a guideline from the UK Haemophilia Centre Doctors' Organization.

Summary.  von Willebrand disease (VWD) is the commonest inherited bleeding disorder. However, despite an increasing understanding of the pathophysiology of VWD, the diagnosis of VWD is frequently difficult because of uncertainty regarding the relationship between laboratory assays and function in vivo. The objective of this guideline is to provide contemporary advice on a rational approach to the diagnosis of VWD. This is the second edition of this UK Haemophilia Centre Doctors’ Organisation (UKHCDO) guideline and supersedes the previous edition which was published in 1997.

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. 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.

The Molecular Analysis of von Willebrand Disease: a Guideline from the UK Haemophilia Centre Doctors' Organisation Haemophilia Genetics Laboratory Network

Summary.  von Willebrand disease (VWD) is a common autosomally inherited bleeding disorder associated with mucosal or trauma‐related bleeding in affected individuals. VWD results from a quantitative or qualitative deficiency of von Willebrand factor (VWF), a glycoprotein that is essential for primary haemostasis and that carries and protects coagulation factor VIII (FVIII) in the circulation. Through characterization of the phenotype and identification of mutations in the VWF gene in patients with VWD, understanding of the genetics and biochemistry of VWF and VWD has advanced considerably. The importance of specific regions of VWF for its interaction with other components of the vasculature has been revealed, and this has facilitated the formal classification of VWD into three subtypes based upon quantitative (types 1 and 3) and qualitative (type 2) deficiency of VWF. The underlying genetic lesions and associated molecular pathology have been identified in many cases of the qualitative type 2 VWD variants (2A, 2B, 2M, 2N) and in the severe quantitative deficiency, type 3 VWD. However in the partial quantitative deficiency, type 1 VWD, the picture is less clear: there is a variable relationship between plasma levels of VWF and bleeding, there is incomplete penetrance and variable expressivity within affected families, the causative molecular defect is unknown in a substantial number of cases, and even in those cases where the causative mutation is known, the associated molecular pathology is not necessarily understood. This guideline aims to provide a framework for best laboratory practice for the genetic diagnosis of VWD, based upon current knowledge and understanding.

A framework for genetic service provision for haemophilia and other inherited bleeding disorders

Summary.  This framework document offers guidance to patients, doctors, nurses, laboratory scientists, funders and hospitals on the provision of clinical and laboratory genetic services for haemophilia. With recent advances in molecular laboratory techniques it is now possible to give the vast majority of individual patients and family members very reliable genetic information. To enable these genetic data to be used for both the optimal treatment of patients with inherited bleeding disorders and for appropriate reproductive decisions in carriers, there needs to be a clear and robust framework for systematically acquiring the necessary clinical, personal, family and laboratory information upon which decisions can be made. This document provides guidance on the range and standards of clinical and laboratory genetic services which should be offered to patients and their families. Included are arrangements for genetic counselling and testing (including consent and confidentially issues), management of early pregnancy, standards for laboratory genetic services, as well as advice on data storage, security and retrieval.

A novel deletion mutation is recurrent in von Willebrand disease types 1 and 3.

Direct sequencing of VWF genomic DNA in 21 patients with type 3 von Willebrand disease (VWD) failed to reveal a causative homozygous or compound heterozygous VWF genotype in 5 cases. Subsequent analysis of VWF mRNA led to the discovery of a deletion (c.221-977_532 + 7059del [p.Asp75_Gly178del]) of VWF in 7 of 12 white type 3 VWD patients from 6 unrelated families. This deletion of VWF exons 4 and 5 was absent in 9 patients of Asian origin. We developed a genomic DNA-based assay for the deletion, which also revealed its presence in 2 of 34 type 1 VWD families, segregating with VWD in an autosomal dominant fashion. The deletion was associated with a specific VWF haplotype, indicating a possible founder origin. Expression studies indicated markedly decreased secretion and defective multimerization of the mutant VWF protein. Further studies have found the mutation in additional type 1 VWD patients and in a family expressing both type 3 and type 1 VWD. The c.221-977_532 + 7059del mutation represents a previously unreported cause of both types 1 and 3 VWD. Screening for this mutation in other type 1 and type 3 VWD patient populations is required to elucidate further its overall contribution to VWD arising from quantitative deficiencies of VWF.

Clinical and laboratory evaluation of National Health Service factor VIII concentrate (8Y) for the treatment of von Willebrand's disease

This study was carried out to assess the efficacy of NHS 8Y concentrate in the treatment of patients with von Willebrands disease (vWD). Eight patients (two type I vWD, one type IIA vWD, two type IIB vWD, and three type III vWD) were treated on a total of 10 occasions with 8Y. Following each treatment episode there was a temporary correction of patients’bleeding time (BT) measurements. Other laboratory parameters—von Willebrand factor ristocetin cofactor activity (vWf:RiCo), vWf antigen (vWf:Ag) levels, and factor VIII coagulant activity (factor VIII:C)—were also corrected. Plasma vWf multimers temporarily reflected those present in the infused concentrate. An effective clinical response was observed in each case despite, as revealed by autoradiography and scanning densitometry of SDS‐agarose electrophoresis gels, a reduction in the concentration of the largest vWf multimers in 8Y compared with normal plasma. Overall, the clinical effectiveness of 8Y in vWD was comparable to that seen with cryoprecipitate. We conclude that NHS 8Y concentrate may be used as an alternative to cryoprecipitate for the treatment of vWD.

The prevalence of the cysteine1584 variant of von Willebrand factor is increased in type 1 von Willebrand disease: co-segregation with increased susceptibility to ADAMTS13 proteolysis but not clinical phenotype

The molecular pathogenesis of type 1 von Willebrand disease (VWD) is uncertain in most patients. We examined 30 type 1 VWD families in the UK Haemophilia Centre Doctors’ Organization study. Heterozygosity for Y/C1584 was present in eight of 30 (27%) families and 19 of 76 (25%) individuals with type 1 VWD recruited into the study. Eighteen (95%) of these 19 individuals were blood group O. C1584 did not co‐segregate with VWD in four families, and co‐segregated in one family; the results were equivocal in three families. In all families increased susceptibility of von Willebrand factor (VWF) to a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) 13 proteolysis co‐segregated with C1584 in affected and unaffected individuals. These data show that C1584, associated with blood group O, is prevalent among patients with type 1 VWD but not necessarily causative of disease and should not be used in isolation to diagnose VWD. Increased susceptibility of C1584 VWF to ADAMTS13 proteolysis may be physiologically significant and increase an individuals risk of bleeding and presenting with VWD.

Type 1 von Willebrand disease: application of emerging data to clinical practice

Summary.  There has been much recent data published on type 1 von Willebrand disease (VWD) predominantly from three multi‐centre cohort studies. These data have influenced a revision of the classification of type 1 VWD and have important implications for the management of this disorder. Patients with low von Willebrand factor (VWF) levels tend to have VWF mutations and VWD is transmitted predictably within families. In patients with VWF levels close to the lower end of the normal range, candidate mutations are found less often, ABO blood group is a more important factor and the disease has variable heritability within families. The importance of bleeding symptoms, in addition to VWF levels, in the diagnosis of type 1 VWD has been highlighted.