Von Willebrand Factor Locus Research Articles

A rat model of severe VWD by elimination of the VWF gene using CRISPR/Cas9

BackgroundVon Willebrand Disease (VWD) is the most common inherited bleeding disorder, caused by quantitative and qualitative changes in von Willebrand factor (VWF). The biology of VWD, studied in canine, porcine, and murine models, differ in species‐specific biology of VWF and the amenability to experimental manipulations such as phlebotomy. The factor VIII (FVIII) levels in these models are higher than in humans with type 3 VWD, suggesting functional differences between FVIII and VWF.ObjectivesTo develop a VWF knock out (VWF–/–) rat by excision of all 52 exons of the VWF locus. MethodsThe entire VWF gene was eliminated in Sprague‐Dawley (Crl:SD) rats via CRISPR/Cas9‐mediated gene editing. VWF antigen (VWF:Ag), VWF propeptide, and VWF collagen IV binding (VWF:CB4) levels were determined by ELISA assays and FVIII chromogenic activity (FVIII:C) levels by chromogenic FVIII assays. Lateral tail veins were transected to measure bleeding time. VWF–/– rats were infused with FVIII–/– rat platelet poor plasma (PPP) to determine response of plasma FVIII. ResultsBreeding of VWF ± rats yielded VWF–/– offspring at normal Mendelian ratios. VWF:Ag, VWF propeptide, VWF:CB4, and FVIII:C plasma levels were undetectable in VWF–/– rats. VWF–/– rats bled longer and more than VWF+/– and VWF+/+ rats when challenged. Transfusion of FVIII‐deficient platelet‐poor plasma induced a rapid rise in endogenous FVIII:C in VWF–/– rats. ConclusionThis rat model of severe VWD due to elimination of the entire VWF gene recapitulates the severe secondary deficiency of FVIII seen in human type 3 VWD and facilitates the study of VWF and FVIII and their interactions.

Genetic determinants of VWF clearance and FVIII binding modify FVIII pharmacokinetics in pediatric hemophilia A patients

Factor VIII (FVIII) pharmacokinetic (PK) properties show high interpatient variability in hemophilia A patients. Although previous studies have determined that age, body mass index, von Willebrand factor antigen (VWF:Ag) levels, and ABO blood group status can influence FVIII PK, they do not account for all observed variability. In this study, we aim to describe the genetic determinants that modify the FVIII PK profile in a population of 43 pediatric hemophilia A patients. We observed that VWF:Ag and VWF propeptide (VWFpp)/VWF:Ag, but not VWFpp, were associated with FVIII half-life. VWFpp/VWF:Ag negatively correlated with FVIII half-life in patients with non-O blood type, but no correlation was observed for type O patients, suggesting that von Willebrand factor (VWF) half-life, as modified by the ABO blood group, is a strong regulator of FVIII PK. The FVIII-binding activity of VWF positively correlated with FVIII half-life, and the rare or low-frequency nonsynonymous VWF variants p.(Arg826Lys) and p.(Arg852Glu) were identified in patients with reduced VWF:FVIIIB but not VWF:Ag. Common variants at the VWF, CLEC4M, and STAB2 loci, which have been previously associated with plasma levels of VWF and FVIII, were associated with the FVIII PK profile. Together, these studies characterize the mechanistic basis by which VWF clearance and ABO glycosylation modify FVIII PK in a pediatric population. Moreover, this study is the first to identify non-VWF and non-ABO variants that modify FVIII PK in pediatric hemophilia A patients.

Genome‐wide studies of von Willebrand factor propeptide identify loci contributing to variation in propeptide levels and von Willebrand factor clearance

Essentials Variants at ABO, von Willebrand Factor (VWF) and 2q12 contribute to the variation in plasma in VWF. We performed a genome-wide association study of plasma VWF propeptide in 3,238 individuals. ABO, VWF and 2q12 loci had weak or no association or linkage with plasma VWFpp levels. VWF associated variants at ABO, VWF and 2q12 loci primarily affect VWF clearance rates. Background Previous studies identified common variants at the ABO and VWF loci and unknown variants in a chromosome 2q12 linkage interval that contributed to the variation in plasma von Willebrand factor (VWF) levels. Whereas the association with ABO haplotypes can be explained by differential VWF clearance, little is known about the mechanisms underlying the association with VWF single-nucleotide polymorphisms (SNPs) or with variants in the chromosome 2 linkage interval. VWF propeptide (VWFpp) and mature VWF are encoded by the VWF gene and secreted at the same rate, but have different plasma half-lives. Therefore, comparison of VWFpp and VWF association signals can be used to assess whether the variants are primarily affecting synthesis/secretion or clearance. Methods We measured plasma VWFpp levels and performed genome-wide linkage and association studies in 3238 young and healthy individuals for whom VWF levels had been analyzed previously. Results and conclusions Common variants in an intergenic region on chromosome 7q11 were associated with VWFpp levels. We found that ABO serotype-specific SNPs were associated with VWFpp levels in the same direction as for VWF, but with a much lower effect size. Neither the association at VWF nor the linkage on chromosome 2 previously reported for VWF was observed for VWFpp. Taken together, these results suggest that the major genetic factors affecting plasma VWF levels, i.e. variants at ABO, VWF and a locus on chromosome 2, operate primarily through their effects on VWF clearance.

The molecular characterization of von Willebrand disease: good in parts

Since the cloning of the gene that encodes von Willebrand factor (VWF), 27years ago, significant progress has been made in our understanding of the molecular basis of the most common inherited bleeding disorder, von Willebrand disease (VWD). The molecular pathology of this condition represents a range of genetic mechanisms, some of which are now very well characterized, and others that are still under investigation. In general, our knowledge of the molecular basis of type 2 and 3 VWD is now well advanced, and in some instances this information is being used to enhance clinical management. In contrast, our understanding of the molecular pathogenesis of the most common form of VWD, type 1 disease, is still at an early stage, with preliminary evidence that this phenotype involves a complex interplay between environmental factors and the influence of genetic variability both within and outside of the VWF locus.

Von Willebrand disease: Clinical and laboratory lessons learned from the large von Willebrand disease studies

During the past 25 years, our knowledge concerning the pathogenesis, diagnostic strategies, and treatment of von Willebrand disease (VWD) has increased significantly. Following the immunological differentiation of factor VIII (FVIII) and von Willebrand factor (VWF) in the 1970s and the cloning of the FVIII and VWF genes in the mid-1980s, substantial progress has been made in our understanding of this, the most common inherited bleeding disorder. We now recognize that VWD represents a range of genetic diseases all with the clinical endpoint of increased mucocutaneous bleeding. The molecular pathology of Type 2 and 3 VWD is now comprehensively documented and involves rare sequence variants at the VWF locus. In contrast, the genetic causation of Type 1 disease remains incompletely defined and in many cases appears to involve genetic determinants in addition to or instead of VWF. The diagnostic triad of a personal history of excessive mucocutaneous bleeding, laboratory tests for VWF that are consistent with VWD, and a family history of the condition remain the keystone to VWD identification. In the laboratory, measurement of VWF antigen and function continue to be the most important diagnostic studies, and while our understanding of the molecular genetic pathology of VWD has advanced considerably in the past decade, genetic testing as a component of diagnosis is limited to certain distinct subtypes of the disorder. Treatment of VWD has been relatively unchanged for the past decade and continues to involve either stimulation of the release of intrinsic VWF with desmopressin or the infusion of VWF concentrates.

Polymorphic variation within the VWF gene contributes to the failure to detect mutations in patients historically diagnosed with type 1 von Willebrand disease from the MCMDM-1VWD cohort

Polymorphic variation within the VWF gene contributes to the failure to detect mutations in patients historically diagnosed with type 1 von Willebrand disease from the MCMDM-1VWD cohort

Phenotypic Identification of Platelet-Type von Willebrand Disease and Its Discrimination from Type 2B von Willebrand Disease: A Question of 2B or Not 2B? A Story of Nonidentical Twins? Or Two Sides of a Multidenominational or Multifaceted Primary-Hemostasis Coin?

Platelet-type von Willebrand disease (PT-VWD) and type 2B von Willebrand disease (2B-VWD) have different etiologies although both present with a similar clinical bleeding and basic laboratory phenotype. Both PT-VWD and 2B-VWD represent gain-of-function mutations that lead to enhanced binding between plasma von Willebrand factor (VWF) and its platelet ligand, glycoprotein Ib alpha (GP1BA). However, 2B-VWD results from a functionally abnormal VWF molecule arising from mutations in the VWF gene, whereas PT-VWD is caused by hyperresponsive platelets resulting from mutations in the platelet GP1BA gene. A definitive diagnosis of PT-VWD versus 2B-VWD is critical for treatment decisions (as differential therapies might be respectively required) and also for family counseling. However, laboratory discrimination of PT-VWD versus 2B-VWD is problematic because simple phenotypic testing will not permit their differentiation, and the more complex testing approaches that might permit their differentiation are rarely applied, or are perhaps poorly applied. Although differential identification of PT-VWD versus 2B-VWD can most definitively be achieved by identifying the gene defect at either the VWF or GP1BA loci, such tests are not commonly available, not always successful, and even if available and successful might not be readily available to serve time-critical treatment decisions. Accordingly, simple laboratory tools to enable discrimination of the two disorders would be a valuable addition to the test repertoire of the hemostasis laboratory. This article provides a review of PT-VWD-related literature and an overview of a phenotypic laboratory test process that should enable the effective identification of PT-VWD and its discrimination from 2B-VWD.

Platelet-Type von Willebrand Disease and Type 2B von Willebrand Disease: A Story of Nonidentical Twins when Two Different Genetic Abnormalities Evolve into Similar Phenotypes

Platelet-type von Willebrand disease (PT-VWD, or pseudo-VWD) and type 2B VWD share a common bleeding phenotype with different etiologies. Both PT-VWD and type 2B VWD represent an enhanced binding between the plasma von Willebrand factor (VWF) to its platelet ligand, glycoprotein Ib alpha ( GP1BA). However, type 2B VWD results from a functionally abnormal VWF molecule, whereas PT-VWD is caused by hyperresponsive platelets due to defects in the platelet GP1BA gene. The laboratory discrimination between the two disorders can be a challenge because simple phenotypic testing will not differentially identify the disorders, and the more complex testing approaches are often poorly applied. Definitive diagnosis is critical for treatment decisions and can be most definitively achieved by identifying the gene defect at either the VWF or GP1BA loci. A systematic international molecular genetic study would be helpful to address the question of whether PT-VWD is being misdiagnosed as type 2B VWD. Such a study can be facilitated by an international online database/disease registry to enhance international awareness about this otherwise long-recognized diagnostic dilemma.

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

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. To evaluate the relative contribution of VWF measurement to the diagnosis of VWD. 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. 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. 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.

Von Willebrand disease—Phenotype versus genotype: Deficiency versus disease

Von Willebrand disease (VWD) is classified into three primary subtypes; type 1 includes partial quantitative deficiency, type 2 (A, B, M and N) includes qualitative defects and type 3 includes virtually complete deficiency of von Willebrand factor (VWF). 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 normal sized VWF multimers. Finally, type 2N includes variants with markedly decreased affinity for factor VIII (FVIII). Studies have begun to address the association between particular genetic variants of VWF and the disease phenotype. For example, the recessive type 3 disease appears to be due to a variety of mutations that result in a null phenotype, while type 2N disease results from a group of recessive missense mutations that interfere with FVIII binding. Recently, the molecular pathogenesis of type 1 VWD has begun to be characterised revealing a diverse spectrum of genetic changes. In two large studies, patients with type 1 VWD were found to be heterozygous for a variety of missense, splice site and transcriptional mutations. In some patients, more than one variant sequence was present, and in a significant number of individuals no changes were evident in the VWF gene. These results suggest that the molecular correlates for type 1 VWD are complex and, in addition to a wide array of changes at the VWF locus, are likely to involve mutations in other genes.

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.

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. To estimate the proportion of type 1 VWD that is linked to the VWF gene. 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. 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. 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%.

Provisional criteria for the diagnosis of VWD type 1: on behalf of the ISTH SSC Subcommittee on von Willebrand Factor

Provisional criteria for the diagnosis of VWD type 1: on behalf of the ISTH SSC Subcommittee on von Willebrand Factor

Linkage analysis of factor VIII and von Willebrand factor loci as quantitative trait loci

Elevated factor (F)VIII levels contribute to venous thrombotic risk. FVIII levels are determined to a large extent by levels of von Willebrand factor (VWF), its carrier protein which protects FVIII against proteolysis. VWF levels are largely dependent on ABO blood group. Subjects with blood group non-O have higher VWF and FVIII levels than individuals with blood group O. Apart from ABO blood group no genetic determinants of high FVIII levels have been identified, whereas clustering of FVIII levels has been reported within families even after adjustment for ABO blood group and VWF levels. We investigated the FVIII and VWF loci as possible quantitative trait loci (QTL) influencing FVIII and VWF levels. Two sequence repeats in the FVIII gene and three repeats in the VWF gene were typed in 52 FV Leiden families. Multipoint sib-pair linkage analysis was performed with the MAPMAKER/SIBS program. FVIII levels adjusted for VWF levels and age, and VWF levels adjusted for ABO blood group and age, were used for this linkage analysis. No linkage of FVIII levels to the FVIII locus was found, whereas we found evidence that the VWF locus contains a QTL for VWF levels [maximum likelihood no dominance variance lod score = 0.70 (P = 0.04) and non-parametric Z-score = 1.92 (P = 0.03)]. About 20% of the total variation in VWF levels may be attributed to this VWF locus.

Significant linkage and non‐linkage of type 1 von Willebrand disease to the von Willebrand factor gene

Significant linkage of types 2A and 2B von Willebrand disease (VWD) to the von Willebrand factor (VWF) gene have been reported, as well as mutations in the VWF gene. However, data for the partial quantitative variant are less consistent. An inconsistency of association between the type 1 VWD phenotype and genotype has been reported recently. We undertook linkage analysis of 12 families with definite or possible type 1 VWD patients. One family with classic type 1 VWD had a high lod score (Z = 5.28, theta = 0.00). A total lod score of 10.68 was obtained for the four families with fully penetrant disease. In two families linkage was rejected, while three families did not show conclusive evidence of linkage. This study corroborates ABO blood group influence, especially in patients with mild deficiencies and/or incomplete penetrance. Indirect genetic analysis may be an option for diagnosing asymptomatic or presymptomatic type 1 VWD carriers, particularly in families showing higher penetrance. The study indicates defects of the VWF locus are to be expected in more than half of the families studied. However, as defects at different loci may be the cause of this phenotype, the results of the segregation analyses should be interpreted with caution, especially in studies involving small families, or mild expressions of the disorder or incomplete penetrance.

Inconsistency of Association between Type 1 von Willebrand Disease Phenotype and Genotype in Families Identified in an Epidemiological Investigation

In a previous epidemiological investigation among schoolchildren of Northern Italy, a conservative 1% prevalence of type 1 von Willebrand disease (VWD) was found. Diagnosis was based on a positive family history and low von Willebrand factor (VWF) ristocetin cofactor activity. To investigate whether the type 1 VWD phenotype as detected by our original methodology cosegregates with one or more specific alleles of the VWF gene, we have performed genotype analysis in affected subjects and their family members. Eleven of the 14 subjects previously identified as having VWD, all with mild personal bleeding symptoms, agreed to participate in the genetic study. Remarkably, the laboratory measurements of the previous investigation were completely confirmed in 10 of the 11 subjects. Clear cosegregation of the VWD type 1 and a specific VWF allele was observed in one family and was likely in the family of two other pro-bands. In three additional propositi and their families a possible association of the phenotype with a VWF allele was found. No association was observed in the remaining five subjects and their families. During 13-year follow-up few additional bleeding episodes were recorded among investigated subjects, most often occurring in the one family manifesting clear cosegregation. The results of this study illustrate that a personal and family bleeding history and persistently low VWF ristocetin cofactor activity, fitting the usual criteria for type 1 VWD, may not cosegregate with genetic markers at the VWF gene locus. Thus the prevalence of VWD defined as a disorder involving the VWF locus might be overestimated in population study. However, phenotypic diagnosis still remains fundamental to identify patients at risk of bleeding. Further research should clarify whether in families with more severe clinical and laboratory phenotype a clear association with markers of VWF is found.