von Willebrand's disease: a report from a meeting in the Åland islands.

von Willebrand's disease (VWD) is probably the most common bleeding disorder, with some studies indicating that up to 1% of the population may have the condition. Over recent years interest in VWD has fallen compared to that of haemophilia, partly the result of focus on blood-borne diseases such as HIV and hepatitis. Now the time has come to revisit VWD, and in view of this some 60 international physicians with clinical and scientific interest in VWD met over 4 days in 2010 in the Åland islands to discuss state-of-the-art issues in the disease. The Åland islands are where Erik von Willebrand had first observed a bleeding disorder in a number of members of a family from Föglö, and 2010 was also the 140th anniversary of his birth. This report summarizes the main papers presented at the symposium; topics ranged from genetics and biochemistry through to classification of VWD, pharmacokinetics and laboratory assays used in the diagnosis of the disease, inhibitors, treatment guidelines in different age groups including the elderly who often have comorbid conditions that present challenges, and prophylaxis. Other topics included managing surgeries in patients with VWD and the role of FVIII in VWF replacement, a controversial subject.

Heterogeneity of the factor IX locus in nine hemophilia B inhibitor patients.

DNA from nine hemophilia B patients who produce anti-factor IX inhibitors (antibodies), including two brothers, was analyzed by the Southern blotting method and hybridization with factor IX cDNA, intragenomic, and 3'-flanking probes. Two inhibitor patients were shown to have total deletions of the factor IX gene. Two other inhibitor patients, the brothers, were shown to have a presumably identical complex rearrangement of the factor IX gene involving two separate deletions. The first deletion is of approximately 5.0 kb and removes exon e. The second deletion is between 9 and 29 kb and removes exons g and h but leaves exon f intact. An abnormal Taq I fragment at one end of the deletion junctions acted as a marker for the inheritance of hemophilia B in the patients' family. Five other inhibitor patients have a structurally intact factor IX gene as detected by this method. Our studies indicate that whereas large structural factor IX gene defects predispose hemophilia B patients to developing an anti-factor IX inhibitor, the development of an inhibitor can be associated with other defects of the factor IX gene.

Type 1 von Willebrand disease.

Since its first description in 1926, the precise nature and indeed significance of von Willebrand factor (VWD) in the area of human bleeding has been unsure and often controversial. The recognition of VWD as a distinct entity in blood and the cloning of the von Willebrand factor (VWF) gene in the 1980s encouraged both phenotypic and genotypic studies, culminating in 1994 with the recognition, by the VWF subcommittee of the Scientific and Standardization Committee (SSC) of International Society of Thrombosis and Haemostasy (ISTH), of three types of VWD, characterized by severe plasma VWF deficiency (type 3), functionally deficient plasma VWF (type 2) and reduced (below normal) levels of plasma VWF, which is functionally essentially normal (type 1; 70% of all cases). Since then, whereas gene analysis has recognized VWF gene (VWF) mutations in most individuals with type 3 and type 2 disease, the latter mutations correlating well with recognized functional domains within the VWF protein, few mutations have been reported in cases with type 1 VWD. This led to speculation that other factors, particularly ABO blood group, may be primarily responsible for the majority of such patients, perhaps combined with a generic bleeding tendency throughout the normal population. Recent large studies in Europe and Canada have considerably clarified this situation, revealing that the majority of type 1 VWD is associated with mutations within VWF. The role of these mutations in the aetiology of the disease opens up new approaches to the study of the diagnosis and treatment of the condition. Conversely, the lack of a change in the VWF gene in many recruited families will lead to enhanced efforts to identify non-VWF gene causes both at the genetic and epigenetic level.

Impact of plasma von Willebrand factor levels in the diagnosis of type 1 von Willebrand disease: results from a multicenter European study (MCMDM-1VWD).

BACKGROUND: Presence of bleeding symptoms, inheritance and reduced von Willebrand factor (VWF) contribute to the diagnosis of type 1 von Willebrand disease (VWD). However, quantitative analysis of the importance of VWF antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo) levels in the diagnosis is lacking. OBJECTIVES: To evaluate the relative contribution of VWF measurement to the diagnosis of VWD. PATIENTS AND METHODS: From the MCMDM-1VWD study cohort, 204 subjects (considered as affected by VWD based on the enrolling Center diagnoses and the presence of linkage with the VWF locus) were compared with 1155 normal individuals. Sensitivity, specificity and diagnostic positive likelihood ratios (LR) of VWF:Ag and VWF:RCo were computed. RESULTS: ABO blood group was the variable most influencing VWF levels, but adjustment of the lower reference limit for the ABO group did not improve sensitivity and specificity of VWF:Ag or VWF:RCo. The lower reference limit (2.5th percentile) was 47 IU dL(-1) for both VWF:Ag and VWF:RCo and showed similar diagnostic performance [receiver-operator curve area: 0.962 and 0.961 for VWF:Ag and VWF:RCo, respectively; P = 0.81]. The probability of VWD was markedly increased only for values below 40 IU dL(-1) (positive LR: 95.1 for VWF:Ag), whereas intermediate values (40 to 60 IU dL(-1)) of VWF only marginally indicated the probability of VWD. CONCLUSIONS: Although the conventional 2.5 lower percentile has good sensitivity and specificity, only VWF:Ag or VWF:RCo values below 40 IU dL(-1) appear to significantly indicate the likelihood of type 1 VWD. The LR profile of VWF level could be used in a diagnostic algorithm.

A quantitative analysis of bleeding symptoms in type 1 von Willebrand disease: results from a multicenter European study (MCMDM-1 VWD).

BACKGROUND: A quantitative description of bleeding symptoms in type 1 von Willebrand disease (VWD) has never been reported. OBJECTIVES: The aim was to quantitatively evaluate the severity of bleeding symptoms in type 1 VWD and its correlation with clinical and laboratory features. PATIENTS AND METHODS: Bleeding symptoms were retrospectively recorded in a European cohort of VWD type 1 families, and for each subject a quantitative bleeding score (BS) was obtained together with phenotypic tests. RESULTS: A total of 712 subjects belonging to 144 families and 195 controls were available for analysis. The BS was higher in index cases than in affected family members (BS 9 vs. 5, P < 0.0001) and in unaffected family members than in controls (BS 0 vs. -1, P < 0.0001). There was no effect of ABO blood group. BS showed a strong significant inverse relation with either von Willebrand ristocetin cofactor (VWF:RCo), von Willebrand antigen (VWF:Ag) or factor VIII procoagulant activity (FVIII:C) measured at time of enrollment, even after adjustment for age, sex and blood group (P < 0.001 for all the four upper quintiles of BS vs. the first quintile, for either VWF:RCo, VWF:Ag or FVIII:C). Higher BS was related with increasing likelihood of VWD, and a mucocutaneous BS (computed from spontaneous, mucocutaneous symptoms) was strongly associated with bleeding after surgery or tooth extraction. CONCLUSIONS: Quantitative analysis of bleeding symptoms is potentially useful for a more accurate diagnosis of type 1 VWD and to develop guidelines for its optimal treatment.

Linkage analysis in families diagnosed with type 1 von Willebrand disease in the European study, molecular and clinical markers for the diagnosis and management of type 1 VWD.

BACKGROUND: von Willebrand disease (VWD) type 1 is a congenital bleeding disorder caused by genetic defects in the von Willebrand factor (VWF) gene and characterized by a reduction of structurally normal VWF. The diagnosis of type 1 VWD is difficult because of clinical and laboratory variability. Furthermore, inconsistency of linkage between type 1 VWD and the VWF locus has been reported. OBJECTIVES: To estimate the proportion of type 1 VWD that is linked to the VWF gene. PATIENTS AND METHODS: Type 1 VWD families and healthy control individuals were recruited. An extensive questionnaire on bleeding symptoms was completed and phenotypic tests were performed. Linkage between VWF gene haplotypes and the diagnosis of type 1 VWD, the plasma levels of VWF and the severity of bleeding symptoms was analyzed. RESULTS: Segregation analysis in 143 families diagnosed with type 1 VWD fitted a model of autosomal dominant inheritance. Linkage analysis under heterogeneity resulted in a summed lod score of 23.2 with an estimated proportion of linkage of 0.70. After exclusion of families with abnormal multimer patterns the linkage proportion was 0.46. LOD scores and linkage proportions were higher in families with more severe phenotypes and with phenotypes suggestive of qualitative VWF defects. About 40% of the total variation of VWF antigen could be attributed to the VWF gene. CONCLUSIONS: We conclude that the diagnosis of type 1 VWD is linked to the VWF gene in about 70% of families, however after exclusion of qualitative defects this is about 50%.

Update on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor.

von Willebrand disease (VWD) is a bleeding disorder caused by inherited defects in the concentration, structure, or function of von Willebrand factor (VWF). VWD is classified into three primary categories. Type 1 includes partial quantitative deficiency, type 2 includes qualitative defects, and type 3 includes virtually complete deficiency of VWF. VWD type 2 is divided into four secondary categories. Type 2A includes variants with decreased platelet adhesion caused by selective deficiency of high-molecular-weight VWF multimers. Type 2B includes variants with increased affinity for platelet glycoprotein Ib. Type 2M includes variants with markedly defective platelet adhesion despite a relatively normal size distribution of VWF multimers. Type 2N includes variants with markedly decreased affinity for factor VIII. These six categories of VWD correlate with important clinical features and therapeutic requirements. Some VWF gene mutations, alone or in combination, have complex effects and give rise to mixed VWD phenotypes. Certain VWD types, especially type 1 and type 2A, encompass several pathophysiologic mechanisms that sometimes can be distinguished by appropriate laboratory studies. The clinical significance of this heterogeneity is under investigation, which may support further subdivision of VWD type 1 or type 2A in the future.

A standard nomenclature for von Willebrand factor gene mutations and polymorphisms.

Mutations in the von Willebrand factor gene responsible for von Willebrand disease, in particular those responsible for type 2 von Willebrand disease, are being increasingly identified. The plethora of mutation screening techniques now available and their enhanced sample throughput capability is also enabling an increasing number of investigations in patients with types 1 and 3 disease. An unambiguous von Willebrand factor nucleotide and amino acid nomenclature is now essential. In this paper, we present a uniform standard nomenclature for von Willebrand factor gene mutations and polymorphisms as approved and recommended by the International Society on Thrombosis and Haemostasis Scientific and Standardisation Committee subcommittee on von Willebrand factor.

Molecular genetics and counselling in haemophilia.

Genetic analysis of the human factor VIII and IX genes has resulted in accurate carrier detection and prenatal diagnosis based on either informative DNA polymorphisms or on causative mutations. By combined use of up to 10 polymorphisms within the factor IX gene, over 90% of families are informative. Within the factor VIII gene the multiallelic CA repeats in introns 13 and 22 can be analysed by multiplex PCR resulting also in over 80% of families being informative. Mutation detection has developed with advancing technology which can be used to identify mutations in the factor IX gene in practically all patients with haemophilia B and their relatives. Although this is more technically demanding for patients with haemophilia A because of the greater complexity of the factor VIII gene, the recent description of X-chromosome inversions as a cause of severe haemophilia A in almost 50% of patients has led to the ready detection of these inversions in such families with associated precise carrier detection.

The dissociation of factor VIII by reducing agents, high salt concentration and affinity chromatography.

Incubation of a factor VIII-rich fraction of plasma with a high concentration of salt confirmed the production of both high (HMW) and low (LMW) molecular weight factor VIII clotting activity (FVIIIC) as determined by agarose gel filtration but with considerable overlap. The electrophoretic mobility of factor VIII related protein (FVIIIRP) detected by precipitating rabbit antiserum was not affected by this treatment and LMW FVIIIC devoid of FVIIIRP was apparently produced. At low concentration the reducing agent dithiothreitol (DTT) altered the electrophoretic mobility of FVIIIRP. At higher concentrations it altered both its mobility and antigenicity and an LMW FVIIIRP was produced. Contrary to the findings of other workers no LMW FVIIIC devoid of FVIIIRP was produced. In further studies factor VIII-rich plasma fraction was treated with sepharose beads to which had been coupled a non-coagulation inhibitory precipitating rabbit antibody to FVIIIRP. Both FVIIIRP and FVIIIC were taken up by the beads but after elution with 1.5 M NaCl, FVIIIC of LMW and devoid of FVIIIRP was selectively removed. Antisera raised to LMW FVIIIC produced with 1.5 M NaCl either by the gel filtration or affinity chromatography methods inhibited FVIIIC and precipitated with HMW factor VIII-rich fractions. The results were consistent with the possibility that factor VIII clotting activity and FVIIIRP exist in plasma as a non-covalently bound complex.

Gel filtration patterns of factor VIII coagulant antigen and factor VIII related antigen in normal and von Willebrand's disease.

Gel filtration (sepharose 2B CL) patterns of factor VIII coagulant antigen (VIIIC:Ag) and factor VIII related antigen (VIIIR:Ag) were obtained using normal plasma and plasma from patients with von Willebrand's disease. The latter group consisted of five individuals with normal mobility of factor VIIIR:Ag on cross-immunoelectrophoresis (Type I) and five others with abnormal (increased) mobility (Type II). Results showed that the elution of VIIIC:Ag was delayed in those subjects whose ratio of VIIIR:Ag to VIIIC:Ag was reduced. It has previously been reported that VIIIR:Ag exerts a stabilizing influence on the coagulant activity of factor VIII (VIII:C); our data suggests that when VIIIR:Ag is deficient, abnormal (low molecular weight) forms of VIIIC:Ag circulate.

A single base pair deletion in the promoter region of the factor IX gene is associated with haemophilia B.

Patients with the haemophilia B Leyden phenotype show a distinct pattern of factor IX expression characterized by a post-pubertal increase in FIX levels and the remission of clinical symptoms in adult life. This phenotype has previously been linked to single base mutations within transcription factor binding sites in a region of approximately 40 bp around the major start point of transcription of the FIX gene. Here we report a novel mutation in this region within the transcription factor C/EBP binding site at +1 to +18. The mutation is a single base pair deletion from a triplet of thymine residues at +6 to +8. We show that the extent to which this mutation disrupts the binding of C/EBP to its binding site is less marked than the disruption caused by the +13 A-->G mutation of FIX Norwich (1). This correlates with age-matched phenotypic data we have available for the patient reported here and that of FIX Norwich.

Relationship between factor VIII mutation type and inhibitor development in a cohort of previously untreated patients treated with recombinant factor VIII (Recombinate). Recombinate PUP Study Group.

A cohort of 79 previously untreated patients (PUPs) with moderate-severe haemophilia A (baseline Factor VIII < or =2%) were enrolled in a study to evaluate the safety, efficacy and immunogenicity of recombinant factor VIII (r-FVIII, Recombinate). Blood samples were obtained retrospectively from a total 55 PUPs who were investigated for the spectrum of FVIII gene mutations responsible for their haemophilia. FVIII gene inversion mutations were found in 27 (49%) patients. Two patients had partial gene deletions. The remaining 26 patients were then screened for mutations in the FVIII gene coding region using conformation sensitive gel electrophoresis. Point mutations were identified in 22 (85%) of the patients and 14 of these mutations were novel. Study subjects were monitored for the development of FVIII inhibitors throughout the study. A total of 23 of the 73 evaluable subjects (including one subject with a low inhibitor titer at baseline) demonstrated an inhibitor on one or more occasions; 11 (15%) were persistent. Inhibitors were detected in patients with partial gene deletions and inversions and in three of eight patients with missense mutations. No inhibitors were found in 11 patients with small insertions or deletions resulting in an alteration of the protein translation reading frame (frameshift mutations). The results corroborate the observation that mutation type is an important determinant of the propensity to develop inhibitory anti-FVIII antibody.

The application of a monoclonal antibody to factor VIII related antigen (VIIIRAg) in immunoradiometric assays for factor VIII.

A two site solid phase immunoradiometric assay (IRMA) for VIIIRAg has been developed using a monoclonal antibody as both the solid phase ligand and the radiolabelled antibody. A range of normal plasmas and von Willebrand's disease (vWd) plasmas has been assayed for VIIIRAg by this method and compared with VIIIRAg values measured by an IRMA using polyclonal antibodies and by immunoelectrophoresis and with ristocetin cofactor activity (VIIIRiCoF). It was found that the monoclonal IRMA correlated well with the polyclonal IRMA and the immunoelectrophoretic assay, but the correlation with the VIIIRiCoF assay was relatively poor, in spite of the strong inhibitory effect of the antibody on VIIIRiCoF activity.

Major structural defects in the antithrombin gene in four families with type I antithrombin deficiency--partial/complete deletions and rearrangement of the antithrombin gene.

The molecular basis of quantitative antithrombin deficiency was investigated in four families predicted to have major antithrombin gene rearrangements. A 1,442 bp deletion and insertion of the sequence 5'T(n = 38-40)GAGACG was characterised in one case. Sequence surrounding the breakpoints contained two perfect, and one imperfect, inverted repeats which may have mediated formation of a stem loop structure on one strand during DNA replication potentiating the deletion. A 9,219 bp deletion spanning introns 2 to 5 was identified in a second family. The identical 6 bp sequence was upstream of each breakpoint and the 5' breakpoint was located in a sequence of the Alu 3 repeat predicted to be susceptible to strand breakage during transcription. This may have promoted misalignment, and deletion, of one of the repeats and the intervening DNA. A novel 1.8 kb antithrombin gene fragment was present in DNA digests from affected members of the third family suggesting a partial antithrombin gene duplication event while in the remaining family, evidence supporting a complete gene deletion was obtained.

A novel mutation in intron K of the PROS1 gene causes aberrant RNA splicing and is a common cause of protein S deficiency in a UK thrombophilia cohort.

In the course of investigating the molecular basis of protein S deficiency in 31 index cases with thrombophilia, we identified seven kindred where the underlying defect was a novel A to G transition 9 bp upstream of exon 12 in intron K of the PROS1 gene. In all but one case, the mutation caused type I deficiency. One individual had type III deficiency. While ectopic transcript analysis using the BstXI dimorphism in exon 15 failed to detect a transcript from the mutated allele, analysis of transcripts spanning exons 11 and 12 revealed a minor mRNA species. Sequencing confirmed the mutation created a new RNA acceptor site introducing 8 nucleotides of intronic sequence into the mature mRNA. Haplotype analysis of the defective PROS1 alleles in six families revealed the same haplotype in all affected individuals suggesting the presence of a common ancestor. Six of the fourteen relatives with the mutation experienced at least one venous thrombotic event strongly supporting the association of the mutation with venous thrombosis.

A common splice site mutation is shared by two families with different type 2N von Willebrand disease mutations.

Using an ELISA-based method to detect type 2N von Willebrand disease (VWD), we found two individuals with absent FVIII binding. Direct sequencing of the FVIII binding region of the von Willebrand factor (VWF) gene showed that one individual had an R854Q substitution whilst the other had a T791M substitution. The very low FVIII binding and the VWF:Ag levels in both individuals suggested a second defect on the other VWF allele. Conformation sensitive gel electrophoresis of polymerase chain reaction amplified DNA was used to detect an additional change in the VWF gene of each patient. Direct sequencing confirmed a previously unreported G to A transition in the donor splice site in intron 25 of both individuals which should result in a null allele. This was confirmed by mRNA analysis. These two individuals therefore have compound heterozygous VWD in which the only expressed allele has a type 2N mutation. In our population, such compound heterozygosity appears to be a significant cause of type 2N VWD.

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. The binding of FVIII to vWF in 69 patients, 36 with type 1 vWD, 32 with mild haemophilia A and one possible haemophilia A carrier with low FVIII levels was studied. Of these, six were found to have reduced binding (five type 1 vWD, one possible haemophilia A carrier). DNA was extracted from these patients and exons 18-23 of the vWF gene encoding the FVIII binding region of vWF were analysed. After direct sequencing and chemical cleavage mismatch detection, a Thr28Met mutation was detected in two unrelated individuals, one of whom appears to be a compound heterozygote for the mutation and a null allele. No mutations were found in the region of the vWF gene encoding the FVIII binding region of vWF in the other four patients.