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Recessive inheritance of von Willebrand's disease type I
Abstract
The inheritance of type I von Willebrand's disease is thought to be autosomally dominant. The laboratory profile may, however, vary between affected people, even within a single family. There is also a large variation in the severity of clinical symptoms. To see if there is an association between the von Willebrand factor genotype, the laboratory profile, and the severity of the clinical symptoms we did a genetic analysis of four families with type I von Willebrand's disease. The proband of each family proved to be a compound heterozygote for defects in the von Willebrand factor gene. Simple heterozygotes in these families were either symptomless or only mildly affected. One of the identified mutations, which was shared by the probands of three of the four families, may have a carrier prevalence of 1:50 in the general population. These results suggest that the inheritance of von Willebrand's disease is often recessive rather than dominant and so have important implications for diagnosis and genetic counselling.
... Type 2 accounts for about 20% o f all VWD with type 2A the most common subtype (15% o f all VWD) and type 2B accounting for 5% of VWD cases . Other variants o f VWD are rare with only a few case reports, although the prevalence o f type 2N may be substantial (Eikenboom 1993). Recently, type 2M and 2M Vicenza have been reported with a higher frequency than previously thought (Zieger 1997, Schneppenheim 2000 ...
... In several mutation reports it has been shown that an increased ratio o f FVIII to VWF suggests the presence o f a carrier o f a null allele (gene deletions, ffameshift mutations, nonsense mutations and defects o f mRNA expression) (Peake 1990, Standen 1990, Eikenboom 1993, Schenppenheim 1994, Zhang 1995, Eikenboom 1998). The 'carriers' have usually normal FVIII and lower VWF levels in plasma (mean value 22-46 lU/dl) and the FVIII:C/VWF:Ag ratio is above 1.6 (usually above 2) (Blomback 1992 Some individuals with type 1 VWD have been shown to be compound heterozygous with a different mutation on each allele (mutation 1 /mutation 2) ). ...
... In general one mutation gives rise to a null allele (with a defective expression at the RNA level) and the other mutation is usually a missense mutation, such as a type 2N mutation (Eikenboom 1993, Zhang 1993. In these situations the FVIII:C/VWF:Ag ratio is less than 1, between 0.4-0.7. ...
Von Willebrand disease (VWD) has been the subject of numerous studies and debates regarding diagnosis, classification and treatment. My research involved prospective and retrospective studies of patients with VWD registered at the Royal Free Hospital London. During the evaluation of the diagnosis of VWD we found that the von Willebrand factor ristocetin cofactor assay was superior to the ELISA assays. The study of new tests, such as the collagen binding assay and the platelet function analyser were shown to have an adjunctive role in the characterisation of patients with VWD. Another study investigated the proposed criteria for defining type 1 VWD (into 'definite' and 'possible' categories adjusted for the ABO blood group) and we showed that these criteria were too stringent as they left many individuals with bleeding histories unclassified. In another study we demonstrated that a group of patients previously diagnosed with type 1 VWD were misclassified as they were type 2 VWD as shown by phenotypes, genotypes and molecular modelling of the identified mutations. Furthermore, using molecular diagnosis and modelling a 2A/2B genotype was found in a kindred with type 2A VWD and thrombocytopaenia. The role of desmopressin is well established in mild forms of type 1 VWD but there is little data on its role in severe type 1 and type 2 VWD. Our research showed that 30% of these patients can also benefit from desmopressin therapy. There are currently no guidelines on the management of elective surgery in patients with VWD and this thesis reviewed the treatment with desmopressin and clotting factor concentrates in these patients. In conclusion, this thesis brings new data for the clarification of the diagnosis of VWD, it offers new perspectives into the complex classification and, finally, it investigates the optimal therapeutic options for patients with VWD.
... Missense mutations in the FVIII binding domain at the amino-terminal portion of VWF are responsible for type 2N [7]. TheR854Q mutation is the most frequent and has found in about 2% of the Dutch population [8]. This mutation may cause symptoms only in homozygous or compound heterozygous states. ...
... Despite its high prevalence, the precise genetic cause of type 1 VWD is still elusive in most cases, especially those with a mild phenotype. Many type 1 VWD cases might be compound heterozygotes, producing an apparent dominant transmission [8] or alternatively, the mutated allele is negatively influenced by the effects of gene(s) outside the VWF gene and by other non-genetic factors contributing to the expression of a bleeding phenotype. In rare cases it is caused by frame shifts, nonsense mutations, or deletions that overlap those identified in type 3 VWD [2]. ...
... The variable penetrance and severity of type 1 VWD may indeed be explained in some cases by the inheritance of two different VWD alleles [8,10]. Coinheritance of R854Q mutation with a null mutation (for example, R2535X) increases the severity of bleeding within a given family [8]. ...
Von Willebrand disease is the most frequent inherited bleeding disorder and is due to a deficiency or abnormality of von Willebrand factor. Despite the reported association of vWF gene mutations, the comprehensive computational analysis of coding, noncoding and regulatory SNPs, and have functional impacts on protein level. In this study, we performed a comprehensive analysis of the functional and structural impact of non-synonymous (nsSNP) that are deleterious to vWF structure and function. A sequence homology-based approach was adopted for screening nsSNPs in vWF, including seven different in silico prediction algorithms; SIFT, PolyPhen-2, I-Mutant 3.0, MUpo , SNP&GO, PhD-SNP and PANTHER. Moreover, Protein structural analysis was done by modeling of amino acid substitutions using Project Hope and chimera. Our results demonstrate that 155nsSNPs in the vWF sgene was deleterious. 18 deleterious and high-risk nsSNPs were identified in vWF gene. 16 mutations of these predicted to have decrease effective stability of the protein and two were predicted to have increased effective stability of the protein. Two mutations couldn't predicted by Project HOPE software, so Chimera was used instead to determine the position of wild type residue and replaced with the new amino acid. We hope our results will provide useful information that needed to help researchers to do further study.
... For the VWD patient analysis, missense variations clustered in exon 28 usually associate with VWD types 2A, 2M, or 2B [21]. Patient Af1VW presents variations c.3835G > A and c.4133C > T. This patient is a compound heterozygote for defects in the VWF as found in the Eikenboom et al. study (1993) [46]. Since the variation is within the domains of the VWF that are involved in binding to the platelet membrane glycoprotein Ib as shown in Figure 9. ...
... The mutations may interfere with the ristocetin-induced VWF binding to platelets. This variation was found in patients with VWD type I diagnosis [46]. Variation c.4133C > T was found in a cohort study, where it was identified as type 1 VWD [47]. ...
Inherited bleeding disorders (IBDs) are the most frequent congenital diseases in the Colombian population; three of them are hemophilia A (HA), hemophilia B (HB), and von Willebrand Disease (VWD). Currently, diagnosis relies on multiple clinical laboratory assays to assign a phenotype. Due to the lack of accessibility to these tests, patients can receive an incomplete diagnosis. In these cases, genetic studies reinforce the clinical diagnosis. The present study characterized the molecular genetic basis of 11 HA, three HB, and five VWD patients by sequencing the F8, F9, or the VWF gene. Twelve variations were found in HA patients, four in HB patients, and 19 in WVD patients. From these variations a total of 25 novel variations were found. Disease-causing variations were used as positive controls for validation of the high-resolution melting (HRM) variant-scanning technique. This approach is a low-cost genetic diagnostic method proposed to be incorporated in developing countries. For the data analysis, we developed an accessible open-source code in Python that improves HRM data analysis with better sensitivity of 95% and without bias when using different HRM equipment and software. Analysis of amplicons with a length greater than 300 bp can be performed by implementing an analysis by denaturation domains.
... Mutations included nonsense mutations, stop mutations and recombination between VWF gene and pseudo-gene. The presence of mutations associated with type 2N VWD, defective binding of FVm to VWF, have been shown to occur in patients with type 1 VWD (Eikenboom et al. 1993). ...
... The inheritance of effectively a null allele that results in low level of VWF mRNA level from one allele and a mutation associated with a factor VH[ binding defect has been previously described (Peerlink et al. 1992;Eikenboom et al. 1993 ...
Von Willebrand disease (VWD), the most common inherited bleeding disorder, is caused by either qualitative or quantitative abnormalities of the circulating glycoprotein Von Willebrand Factor (VWF). This thesis describes an approach to the study of mutations causing VWD by analysis of the essential coding region of VWF. This was performed by the amplification of regions of VWF derived from mRNA and genomic DNA. By use of this approach mutations were identified in a series of patients with VWD, and the inheritance of causative mutations demonstrated in affected families. Molecular analysis demonstrated that a number of the patients have qualitative rather than the quantitative abnormalities of VWF originally diagnosed. In addition to the identification of causative mutations in VWD, this thesis describes the modelling of the three VWF-A domains and the location of known mutation sites within the models. This provided an explanation for the various effects of the mutations on VWF structure and function. Important residues involved in ligand binding to the VWF-A1 domains were identified and a mechanism proposed for the action of mutations causing an upregulation in binding of VWF to platelets, and other mutations causing downregulation. VWF is a large protein that interacts with a variety of ligands and exhibits multiple functions. In many cases of VWD the action of mutations cannot be simply explained by knowledge of the sequence alteration. This thesis demonstrates that a variety of approaches are required for integrated molecular understanding of VWD and VWF.
... The frequency might be largely underestimated due to the lack of transcript analysis. Patients with heterozygous genotypes for these variants are frequently associated with mild forms of type 1 VWD or low VWF level and maybe phenotypically silent (Eikenboom et al., 1993). However, when splicing variants are in a homozygous state or in a compound heterozygous state with another pathogenic variant, they could result in type 3 VWD and severe bleeding. ...
Approximately 10% of von Willebrand factor (VWF) gene variants are suspected to disrupt mRNA processing, the number of which might be underestimated due to the lack of transcript assays. In present study, we provided a detailed strategy to evaluate the effects of nine putative splice site variants (PSSVs) of VWF on mRNA processing as well as protein properties and establish their genotype-phenotype relationships. Eight of nine PSSVs affected VWF splicing: c.322A>T, c.1534-13_1551delinsCA and c.8116-2del caused exon skipping; c.221-2A>C, c.323+1G>T and c.2547-13T>A resulted in the activation of cryptic splice sites; c.2684A>G led to exon skipping and activation of a cryptic splice site; c.2968-14A>G created a new splice site. The remaining c.5171-9del was likely benign. The efficiency of nonsense mediated mRNA decay (NMD) was much higher in platelets compared to leukocytes, impairing the identification of aberrant transcripts in 4 of 8 PSSVs. The nonsense variant c.322A>T partially impaired mRNA processing, leaking a small amount of correct transcripts with c.322T (p.Arg108*), while the missense variant c.2684A>G totally disrupted normal splicing of VWF, rather than produced mutant protein with the substitution of Gln895Arg. The results of this study would certainly added novel insights into the molecular events behind von Willebrand disease.
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... Along this line, 25% of synonymous mutations positioned at exon-intron boundaries result in altered splicing, which, in itself, can cause disease, modify the severity of the disease phenotype, or be linked with disease susceptibility. 8 In VWF, heterozygotes for PSSM may be associated with mild forms of VWD type 1 or be phenotypically silent, but when two such mutations are found in different alleles, the phenotype is associated with VWD type 3. 9 Almost all related studies of splicing effects have examined peripheral blood platelets, as VWF is exclusively expressed in these cells and endothelial cells. 10,11 Platelets are anucleated, but they contain small amounts of translationally active megakaryocytic mRNA. ...
Large studies in von Willebrand disease patients, including Spanish and Portuguese registries,
led to identification of >250 different mutations. It is a challenge to determine the pathogenic
effect of potential splice site mutations on VWF mRNA. This study aimed to elucidate the true
effects of 18 mutations on VWF mRNA processing, investigate the contribution of nextgeneration
sequencing to in vivo mRNA study in von Willebrand disease, and compare the
findings with in silico prediction. RNA extracted from patient platelets and leukocytes was
amplified by RT-PCR and sequenced using Sanger and next generation sequencing techniques.
Eight mutations affected VWF splicing: c.1533+1G>A, c.5664+2T>C and c.546G>A (p.=)
prompted exon skipping; c.3223-7_3236dup and c.7082-2A>G resulted in activation of cryptic
sites; c.3379+1G>A and c.7473G>A (p.=) demonstrated both molecular pathogenic
mechanisms simultaneously; and the p.Cys370Tyr missense mutation generated two aberrant
transcripts. Of note, the complete effect of 3 mutations was provided by next generation
sequencing alone because of low expression of the aberrant transcripts. In the remaining 10
mutations, no effect was elucidated in the experiments. However, the differential findings
obtained in platelets and leukocytes provided substantial evidence that 4 of these would have
an effect on VWF levels. In this first report using next generation sequencing technology to
unravel the effects of VWF mutations on splicing, the technique yielded valuable information.
Our data bring to light the importance of studying the effect of synonymous and missense
mutations on VWF splicing to improve the current knowledge of the molecular mechanisms
behind von Willebrand disease.
... Since the D′ domain contains several disulfide bonds important in N-terminal multimerization of VWF, several type 2N mutations are associated with defective multimerization as well [101][102][103]. One of the most common type 2N mutation is the p.Arg854Gln mutation that, because of the high frequency, has a high prevalence of co-inheritance with other types of VWD [104]. ...
Von Willebrand disease (VWD) is a bleeding disorder that is mainly caused by mutations in the multimeric protein von Willebrand factor (VWF). These mutations may lead to deficiencies in plasma VWF or dysfunctional VWF. VWF is a heterogeneous protein and over the past three decades, hundreds of VWF mutations have been identified. In this review we have organized all reported mutations, spanning a timeline from the late eighties until early 2017. This resulted in an overview of 750 unique mutations that are divided over the VWD types 1, 2A, 2B, 2M, 2N and 3. For many of these mutations the disease-causing effects have been characterized in vitro through expression studies, ex vivo by analysis of patient-derived endothelial cells, as well as in animal or (bio)physical models. Here we describe the mechanisms associated with the VWF mutations per VWD type.
... Strictly speaking, only the first of these three groups should be considered true cases of type 1 VWD, but flexible criteria are commonly used to define type 1 VWD. We can also distinguish between severe and mild cases of type 1 VWD: the former have more markedly reduced VWF levels, often to below 10 U/dL, while the latter have levels ranging between 20 and 60 U/dL [25]. The severe cases can be diagnosed definitively as type 1 VWD, and they usually have forms characterized by a dominant inheritance. ...
... Similarly, the presence of the mutation R854Q, which in the homozygous or compound heterozygous situation results in type 2N VWD, 40 was excepted because it has previously been shown to occur in up to 1% of the healthy population. 41 We believe that the spectrum of VWD phenotype and profile of mutations seen in this cohort indicate those that may be expected when routinely diagnosed type 1 VWD patients are investigated for VWF gene mutations. For personal use only. ...
... In such cases, the ratio between VWF levels measured either in terms of the antigen (VWF:Ag) or function (measured as ristocetin cofactor activity, VWF:RCo, a test that mimics the physiological interaction between VWF and platelet GpIb) is approximately equal to unity, and the multimeric pattern is usually normal or exhibits minor abnormalities that are not usually detectable using standard methodologies [8]. In more complex cases of type 1 VWD, variable penetrance and expressivity within the same family is due to compound heterozygosity, such as the co-inheritance of a missense mutation with a null mutation [9]. In other cases, increased clearance of the secreted mutant multimers by still unknown mechanisms has been found. ...
Recent studies have suggested a unifying pathophysiological concept to explain the underlying defects of von Willebrand factor (VWF) causing von Willebrand disease (VWD) and have highlighted the relevance of simple VWF related activities in producing a useful diagnosis. A standardized bleeding history condensed into a final bleeding score and few widely available laboratory tests, such as VWF ristocetin cofactor activity, VWF antigen and factor VIII (FVIII), in the index case and in his/her relatives are of critical importance. Ristocetininduced platelet aggregation (RIPA) should also be tested. Trial with desmopressin should be carried out in patients, except those with virtual absence of VWF or with increased RIPA. Desmopressin should be used in all responsive patients as first choice. Substitutive treatment with VWF/FVIII containing products should be used in unresponsive patients, in those with heightened response to desmopressin or in those undergoing interventions requiring good hemostasis for more than 3–5 days. Special consideration should be deserved to the treatment of menorrhagia and parturition.
... Studies conducted in healthy Dutch and Welsh populations have indicated that the type 2N VWF defect (mainly the R854Q mutation) may be found in up to 1% of the general population (14)(15)(16). It remains to be seen whether this might be explained by a founder effect. ...
Background. The impaired capacity of von Willebrand factor (VWF) to carry factor VIII (FVIII) is identified as type 2N von Willebrand disease (VWD). R854Q is the most common type 2N mutation, and almost the only one identified in Italy. Design and methods. This study aimed to ascertain whether R854Q mutations in an Italian cohort of type 2N VWD patients originated from single or recurrent events. Thirteen unrelated Italian families were investigated, analyzing the VWF gene haplotype associated with the R854Q mutation. Results. A common haplotype emerged in all the families, extending from SNPs rs2166902 to rs216293 over 48.2 kb and including five intragenic markers. This haplotype is infrequent in the healthy Italian population (17% vs 100%, p<0.0001) and each genetic marker within said haplotype is similarly rare. These data strongly suggest a founder effect, supporting the existence of a single R854Q mutation event in our cohort of type 2N VWD patients. Using the DMLE+ software and the Bengtsson and Thomson mathematical method, it was estimated that the R854Q mutation occurred from 10,000 to 40,000 years ago, which is consistent with the short dimension of the haplotype shared by our patients. Conclusions. Together with the fact that the R854Q mutation seems to be limited to the Caucasian populations, these findings suggest that a single mutational event took place after human populations moved from Africa towards Europe.
... Consistent with this observation, screening of a large number of normal individuals demonstrated that the Arg89Gln mutation is a common polymorphism in the general population whereas Arg91Gln was only observed in the patient and none of the control samples. This particular mutation is found in over 50% of type 2 N patients and may be particularly common in some populations [30]. This mutation, along with most of the other reported type 2 N mutations, does not result in complete disruption of FVIII binding and in homozygous patients is associated with residual levels of FVIII in the range of 5%. ...
von Willebrand disease is the most common inherited bleeding disorder in humans, with the general population prevalence estimated to be as high as 1% in some studies. This condition exhibits extensive heterogeneity with over 20 distinct subtypes distinguished based on subtle clinical and laboratory differences in presentation. Recent research laboratory advances have shed considerable new light on the molecular basis of this disorder. Specific mutations within the von Willebrand factor gene have been identified in many of the qualitative variants of von Willebrand disease, providing important new insight into the structure and function of this central clotting protein. However, the complex genetic factors determining the clinical severity of type 1 von Willebrand disease, the most common variant, still remain largely unknown and are the subject of current investigation.
... The phenotype may vary considerably depending on the mutations involved. The most severe but rare mutation has been reported to be p.E787K [59] with an associated FVIII activity as low as 1%, and the mildest mutation which qualifies as a polymorphism due to its allele frequency of > 0.01 in the normal Western European population is p.R854Q [59,65]. ...
von Willebrand disease (VWD), the most common inherited bleeding disorder in humans, is characterised by a prolonged bleeding time due to quantitative and/or functional deficits of von Willebrand factor (VWF), a huge multimeric protein. Given the large size and complexity of the protein, the many functions of VWF, for example, binding to collagen, to platelet GPIb, and to FVIII, the localisation of these binding sites in different VWF domains, as well as the dependence on a high molecular weight multimer structure for proper function, VWF is prone to quantitative and very heterogeneous structural and functional defects. Comprehensive clinical and laboratory phenotypic description of patients with VWD in correlation to the genotype has considerably increased our knowledge on this disorder and the physiology and pathophysiology of VWF. This article focuses on the phenotype/genotype relationship in VWD and the context of VWD types and subtypes with particular VWF domains.
... In the French INSERM network study, 47% of unrelated patients with type 2N VWD are homozygous for this mutation and 43% are compound heterozygous with this mutation on one allele [17]. The R854Q mutation was found in 0.92% of the normal population of the Netherlands [18] and 0.31% in Euro-Brazilians [19]. ...
Although von Willebrand disease (VWD) is now well-described, many facets of diagnosis and management continue to be debated. The diagnosis of type 1 disease can be difficult but recent genetic analyses help to distinguish many factors which can influence von Willebrand factor (VWF) levels and bleeding phenotype. Type 2 disease (functional abnormalities) includes a particularly interesting group of disorders with faulty binding between VWF and FVIIIC (Normandy) where treatment methods need careful consideration. Type 3 VWD is the most severe form of VWD and a new international study is underway to examine the use of prophylaxis.
Introduction
With the increase in life expectancy of haemophilia patients (PWH), the risk of osteoporosis increases, but there is little research on whether haemophilia is the cause of osteoporosis.
Aim
To conduct systematically review whether bone mineral density (BMD) in PWH decreased and the factors affecting BMD.
Methods
Two authors independently searched databases and reviewed citations from relevant articles, selecting studies published in any language and performed in humans before March 2023. Eligibility criteria were observational studies in PWH, with BMD as at least one outcome other than osteoporosis or bone loss, and analyses in a group of PWH and healthy controls.
Results
Twelve studies were ultimately identified, consisting of 1210 individuals (534 PWH and 676 healthy controls), compared with the control group, BMD in PWH decreased by 0.13 g/cm ² [95% confidence interval (CI) ‐0.18 to ‐0.08, I ² = 89%]. No evidence of publication bias was detected. There was no evidence that age, BMI, level of physical activity, the types of haemophilia, haemophilia severity, a blood‐borne virus (HCV) and treatment modality predicted the BMD in PWH.
Conclusion
The results indicate that BMD in PWH is lower than in healthy controls. Therefore, we strongly recommend PWH early measurement of BMD to prevent osteoporosis.
Neonatal hematology is a fast-growing field, and the majority of sick neonates will develop hematological problems. This is an essential guide to the pathogenesis, diagnosis and management of hematologic problems in the neonate. Guidance is practical, including blood test interpretation, advice on transfusions and reference ranges for hematological values. Chapters have been thoroughly revised according to the latest advances in the field for this updated third edition. Topics discussed include erythrocyte disorders, platelet disorders, leukocyte disorders, immunologic disorders and hemostatic disorders. Coverage of oncological issues has been expanded to two separate chapters on leukemia and solid tumors, making information more easily accessible. Approaches to identifying the cause of anemia in a neonate are explained, with detailed algorithms provided to aid clinicians in practice. Covering an important hematologic niche with an ever increasing amount of specialized knowledge, this book is a valuable resource for hematologists, neonatologists and pediatricians.
The aim of this thesis is to investigate the obstetric and gynaecological problems and their management in women with inherited bleeding disorders, as well as the role of such disorders in obstetric and gynaecological haemorrhage. The uptake of prenatal diagnosis and termination of an affected pregnancy is low in carriers of haemophilia. Fetal gender determination has important implications in the management of labour in carriers who do not wish to have specific prenatal diagnosis. The attitude of women towards reproductive choices is influenced by ethnic and cultural issues and family experience with the disease. Haemostatic response to pregnancy is variable in different types and subtypes of inherited bleeding disorders and in the same patient in different pregnancies. Haemorrhagic complications are confined to post-abortal and post-partum period. The incidence of primary and secondary post-partum haemorrhage was 22% and 11% in carriers of haemophilia, 18.5% and 20% in vWD and 16% and 24% in FXI deficient women, respectively. Women with low factor levels (<50 iu/dl) and no prophylactic treatment for labour and puerperium are especially at risk. There are great inter- and intra-individual variations in coagulation markers in women due to different physiological conditions including age, ethnicity, blood group and hormonal changes during different phases of the menstrual cycle. Women with inherited bleeding disorders suffer from heavy and prolonged menstruation which adversely affects their quality of life. Objectively confirmed menorrhagia is significantly higher in these women (67%) compared with the control group (29%). On the other hand, undiagnosed inherited bleeding disorders can be the underlying cause in a significant proportion (17%) of women presenting with unexplained menorrhagia. The DDAVP nasal spray was shown not to be superior to placebo in the treatment of menorrhagia. Increased awareness among clinicians responsible for women's health of these disorders and their morbidity and the availability of management guidelines are essential for optimal care and improvement of the quality of life of these patients.
Introduction
von Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays an important role in haemostasis. von Willebrand disease (VWD) is an inherited heterogeneous bleeding disorder caused by either a quantitative or qualitative defect of VWF. Type 3 VWD, the most severe form of the disease, leads to complete quantitative VWF deficiency.
Aim
The present study aims to investigate the molecular pathogenesis of type 3 VWD patients from Southern Brazil.
Methods
The VWF gene was sequenced in 26 cases clinically diagnosed with type 3 VWD by next‐generation sequencing using Ion Torrent PGM.
Results
In 25 patients, we were able to identify both disease‐causing variants. We identified 72 different variants: 31 intronic and 41 exonic. Five novel variants were found: c.6976+5G>T; c.6885_6886insC; c.3378C>T (p.Cys1126); c.3346_3347insCCA; and c.2503G>T (p.Glu835*). Variants p.Pro2063Ser and p.Arg324* co‐segregated in 17 patients, 15 of them in homozygosity.
Conclusion
Our results may contribute to the discussion on whether the variant p.Pro2063Ser is pathogenic or not. Finally, the presence of a common haplotype in patients bearing these two variants suggests a founder effect for this variant in our region.
Platelet adhesion to collagen-coated surfaces in whole blood under flow conditions is mediated by both von Willebrand factor (vWF)-dependent recruitment of the platelet glycoprotein Ib-IX receptor complex and collagen interaction with the integrin 2β1. In type 1 von Willebrand disease (vWD), platelet adhesive functions are impaired due to the decrease in vWF levels in plasma and platelets. There are at least three alleles of the human 2 gene, distinguishable by a cluster of silent or noncoding sequence differences within a segment of the gene. Two alleles, associated with low receptor density can be distinguished by nucleotide 807C, while the third allele associated with high receptor density, expresses nucleotide 807T. Gene frequencies of these alleles in a normal population (n = 167) are 0.58 for 807C and 0.42 for 807T. We measured the frequencies of these alleles in symptomatic patients with five types of vWD (type 1, n = 78; type 2A, n = 25, type 2B, n = 14; type 2M, n = 10; and type 3, n = 20). Compared with the normal group, no significant difference in allele frequencies was observed among individuals with types 2A, 2B, 2M, or 3 vWD. However, the frequency of the 807C allele, associated with low collagen receptor density, among type 1 vWD patients (807C = .71; 807T = .29) was significantly higher than that of the normal population (P = .007). Also, in patients with vWD type 1 and borderline to normal ristocetin-cofactor (vWF:RCo) activity values, collagen receptor density correlates inversely with closure time in a high shear stress system (platelet function analyzer [PFA-100]). We propose that low platelet 2β1 density results in less efficient primary platelet adhesion and may result in increased tendency to bleed, as evidenced by the high frequency of this polymorphism in patients with type 1 vWD compared with normal individuals. In addition, this may account for the variability between patients with similar levels of vWF antigen, but strikingly different bleeding histories.
Background:
von Willebrand factor (VWF) and factor VIII (FVIII) circulate as a non-covalent complex with VWF serving as the carrier for FVIII. VWF indirectly influences secondary hemostasis by stabilizing FVIII and transporting it to the site of primary hemostasis. Type 2N von Willebrand disease involves impaired binding of VWF to FVIII, resulting in decreased plasma levels of FVIII.
Objectives:
In these studies, we characterize the impact of three type 2N VWD variants (R763A, R854Q, R816W) on VWF secretion, FVIII stabilization, and thrombus formation in a murine model.
Methods:
Type 2N VWD mice were generated by hydrodynamic injections of mutant murine VWF cDNAs and the influence of these variants on VWF secretion and FVIII binding was evaluated. In vivo hemostasis and the dynamics of thrombus formation and embolization were assessed using a murine tail vein transection hemostasis model and an intravital thrombosis model in the cremaster arterioles.
Results:
Type 2N VWD variants were associated with decreased VWF secretion using cell and animal-based models. FVIII-binding to type 2N variants was impaired in vitro and was variably stabilized in vivo by expressed or infused 2N variant VWF protein. Both transgenic type 2N VWD and FVIII KO mice demonstrated impaired thrombus formation associated with decreased thrombus stability.
Conclusions:
The type 2N VWD phenotype can be recapitulated in a murine model and is associated with both quantitative and qualitative VWF deficiencies and impaired thrombus formation. Patients with type 2N VWD may have normal primary hemostasis formation but decreased thrombus stability related to ineffective secondary hemostasis. This article is protected by copyright. All rights reserved.
Von Willebrand disease (VWD) is a common autosomal inherited bleeding disorder caused by quantitative or qualitative defects of von Willebrand factor (VWF), a multiadhesive protein which binds platelets to exposed subendothelium and carries factor VIII (FVIII) in circulation. Clinical manifestations are mainly represented by mucous membrane and soft tissue bleeding and their severity and frequency usually correlate with the degree of VWF and FVIII reduction. The diagnosis requires an array of tests, but in mild cases the advantage of pursuing a definite diagnosis is not always straightforward. The correction of the dual defect of hemostasis (abnormal/ reduced VWF and the concomitant deficiency of FVIII) is required for an appropriate treatment. Desmopressin is the treatment of choice for type 1 VWD patients with FVIIII and VWF levels ≥ 10 U/dL responsive to a test infusion with the compound. VWF/FVIII concentrates are needed when desmopressin is ineffective (mainly type 2 and 3 VWD).
Interaction between von Willebrand factor (VWF) and platelet GPIbα is required for primary haemostasis. Lack or loss-of-function in the ligand-receptor pair results in bleeding complications. Paradoxically, gain-of-function mutations in VWF or GPIbα also results in bleeding complications as observed in type 2B von Willebrand disease (VWD) and platelet-type- (PT-) VWD, respectively. A similar phenotype is observed with increased ristocetin-induced platelet agglutination and disappearance of the highest molecular weight multimers of VWF. We evaluated a patient with a bleeding disorder and a biological presentation compatible with type 2B VWD. VWF and platelet functional assays, sequencing of the VWF and GPIBA genes, and expression studies in HEK cells were performed. Sequencing of the VWF gene in the propositus revealed a heterozygous p.Pro1266Leu mutation previously found in type 2B VWD Malmö/New York. These variants are characterised by a mild phenotype and a normal VWF multimer composition suggesting presence of a second mutation in our propositus. Sequencing of the GP1BA gene revealed a heterozygous c.765G>A substitution changing Met at position 255 of GPIbα to Ile. This new mutation is located in the β-switch domain where five other gain-of-function mutations have been reported in PT-VWD. Expression of GPIbα Ile255 in HEK GPIb-IX cells resulted in enhanced VWF binding compared to wild-type, similar to known PT-VWD mutations (p.Val249, p.Ser249 and p.Val255) indicating that it contributes to the propositus defects. This first report associating PT- with type 2B VWD illustrates the importance of combining biological assays with genetic testing to better understand the clinical phenotype.
von Willebrand disease (VWD) is caused by quantitative or qualitative defects of von Willebrand factor (VWF), associated with mucocutaneous bleeding symptoms. VWD is classified into three major groups and four subcategories. The classification is intended to be simple, to rely on widely available laboratory tests, and to correlate with important clinical characteristics. It is meant to facilitate the diagnosis, treatment, and counseling of patients with VWD. The International Society on Thrombosis and Haemostasis/Scientific and Standardization Committee (ISTH/SSC) reviewed the classification and published update information on the diagnosis and treatment of VWD in 2006 based on the most recent pathophysiology data for VWF. Notably, Vicenza type has been classified into type 1 from type 2M. In epidemiology, there may be small differences of frequency in VWD subcategories (type 2) between Europe and lapan. For treatment, factor VIII/VWF concentrate is commonly employed in Japan, although DDAVP has been the first choice for VWD in Europe.
Historically discovered in 1926 by Erik von Willebrand studying bleeding patients living on an island of the Åland archipelago, von Willebrand disease (vWD) is now recognised as the most common inherited bleeding disorder. It defines a heterogeneous group of patients with quantitative or qualitative defects of the von Willebrand factor (vWF) [1] which is a large glycoprotein present in plasma, subendothelium and the alpha granules of platelets. Synthesized exclusively by both endothelial cells and megakaryocytes, vWF plays a dual role in haemostasis. It mediates platelet adhesion to the subendothelium at the site of vessel injury and its circulating form carries and stabilizes factor VIII (FVIII), [2].
Platelet von Willebrand Factor (vWF) was studied in normal donors (n = 10) and in patients with von Willebrand disease (vWD) type I (n = 12). The vWF antigen (Ag) and the functional parameter, measured as the vWF collagen binding activity (CBA), were determined by parallel quantitative ELISAs. In the platelets of normal donors the concentrations (mean±SD) of Ag and CBA were 0.40 ±0.18 U/109 platelets and 0.44 ± 0.24 U/109 platelets, respectively, with a CBA to Ag ratio of 1.06 ±0.17. There was no correlation between the plasma concentrations of Ag and CBA with those in platelets either in normal donors or in patients with vWD type I. On the basis of their concentrations of Ag and CBA in platelets, patients could be subdivided into two groups: (1) platelet normal, i.e. those with a normal Ag and CBA content of 0.35±0.18 U/109 platelets and 0.42±0.21 U/109 platelets, respectively, and (2) platelet low/discordant, i.e. those with a low Ag and CBA content of 0.15±0.06U/109 platelets and 0.07±0.03 U/109 platelets, respectively. Patients with “platelet low/discordant” vWF content had an increased bleeding tendency when compared to those patients with “platelet normal”, confirming the relevance of platelet vWF in haemostasis.
Das Von-Willebrand-Syndrom (VWS) ist die häufigste angeborene Gerinnungsstörung im Kindesalter, obgleich bezüglich der Prävalenz der unterschiedlichen Subtypen keine exakten Daten existieren. Diese defizitäre Beschreibung kann auf die große Variabilität der Symptomatik des VWS zurückgeführt werden. Die Prävalenz des VWS bewegt sich im Bereich von 5 bis 10 000 pro 1 Million Menschen in Abhängigkeit der Schwere der Erkrankung [1]. Unter den verschiedenen Typen des VWS ist der Typ I mit einer Prävalenz von 1–2 auf 100 Personen als häufigste Form beschrieben [3].
von Willebrand disease (VWD) is an autosomal inherited bleeding disorder related to quantitative and/or qualitative abnormalities of the von Willebrand factor (VWF). Many different types have been described based on phenotypic characteristics of the VWF protein and the VWF genotype. The revised and updated classification issued by the International Society on Thrombosis and Haemostasis (ISTH) VWF Subcommittee is the one that is currently accepted. VWD is not restricted to mutations within the VWF gene. There are two major levels of classification: primary—1, 2, and 3; and secondary—A, B, M, and N. Additional “tertiary” information that is not reflected in the defined types of VWD can be appended in parentheses. Such information may include a place name that indicates a remarkable phenotype (e.g., Vicenza), the patient's mutation using standard nomenclature, or a VWF multimer pattern that suggests a specific disease mechanism (e.g., IIA, IIC, IID, or IIE). A potential redundancy in type 2M VWD has been examined.
Neonatal hematology is a fast-growing field, and hematologic problems occur in the majority of sick neonates. Focusing on clinical issues and problem-solving, this is a fully revised and updated revision of a successful practical guide to the pathogenesis, recognition and management of hematologic problems in the neonate. The second edition begins with chapters on the history of neonatal hematology, hematopoiesis, and the immunologic system. Subsequent sections are devoted to erythrocyte disorders, platelet disorders, leucocyte disorders, immunologic disorders and hemostatic disorders. New to this edition are an expanded coverage of neonatal oncology, cord blood utilization, neonatal screening, prenatal diagnosis and hyperbilirubinemia. Written by practising physicians specializing in pediatric hematology, neonatology, immunology, pediatric infectious disease and transfusion medicine, this is an essential text for pediatric hematologists, NICU specialists, neonatologists and neonatal nurse practitioners.
Introduction Hemostatic abnormalities can be considered as either congenital or acquired and can be classified as hemorrhagic or thromboembolic disorders. In newborns, the clinical presentation, diagnosis, and management of hemorrhagic and thromboembolic disorders differ from those in older infants and children, likely reflecting profound differences of the hemostatic system at birth. While severe congenital hemostatic defects usually present in the first hours to days of life with distinct symptoms in otherwise well newborns, acquired hemostatic disorders usually present in sick newborns with a variety of presentations and distinct etiologies that differ from older children and adults. In newborns, the diagnosis of hemostatic abnormalities based upon plasma concentrations of components of the hemostatic system requires age-appropriate reference ranges, because plasma concentrations of several procoagulant and inhibitor proteins are physiologically decreased at birth. The aim of this chapter is to discuss the clinical presentation, diagnosis, and management of the most common congenital and acquired hemostatic disorders in newborns. Hemorrhagic disorders Congenital hemorrhagic disorders General information Hemostatic proteins For congenital deficiencies of components of the hemostatic system, both a severe and a milder form occur. Severe congenital deficiencies of prothrombin, factor V (FV), factor VII (FVII), factor VIII (FVIII), factor IX (FIX), factor X (FX), factor Ⅺ (FXI), fibrinogen, factor XIII (FXIII) and alpha2-antiplasmin (α2AP) all can present with bleeding in the first days of life [1–4]. Mild congenital deficiencies of these proteins usually do not cause bleeding at birth in otherwise healthy full-term newborns. © Cambridge University Press 2005 and Cambridge University Press, 2009.
The diagnosis of von Willebrand's disease is becoming complex as more is understood about the disease. Clinical information and laboratory data are necessary for the diagnosis because of the overlap of normal and abnormal laboratory values. A complete evaluation including von Willebrand factor multimers, ristocetin-induced platelet aggregation, factor VIII activity level, and a template bleeding time is necessary to correctly classify the patient so that optimal treatment may be given.
GeneticsProteomicsPhysiologyClassification of von Willebrand diseaseClinical manifestationsLaboratory diagnosis of von Willebrand diseaseManagement of patients with von Willebrand diseaseConclusions
Further reading
Fibrinogen, coagulation factor VII (FVII), factor VIII (FVIII), and its carrier von Willebrand factor (vWF) play key roles in hemostasis. Previously identified common variants explain only a small fraction of the trait heritabilities and additional variation may be explained by associations with rarer variants with larger effects. The aim of this study was to identify low-frequency (minor allele frequency [MAF] ≥0.01 and <0.05) and rare (MAF <0.01) variants that influence plasma concentrations of these 4 hemostatic factors by meta-analyzing exome chip data from up to 76,000 participants of 4 ancestries. We identified 12 novel associations of low-frequency (n=2) and rare (n=10) variants across the fibrinogen, FVII, FVIII, and vWF traits that were independent of previously identified associations. Novel loci were found within previously reported genes and had effect sizes much larger than and independent of previously identified common variants. In addition, associations at KCNT1, HID1, and KATNB1 identify new candidate genes related to hemostasis for follow-up replication and functional genomic analysis. Newly identified low-frequency and rare-variant associations accounted for modest amounts of trait variance and therefore are unlikely to increase predicted trait heritability but provide new information to understanding individual variation in hemostasis pathways.
Copyright © 2015 American Society of Hematology.
A simplified phenotypic classification of Von Willebrand disease is proposed that is based on differences in pathophysiology. Quantitative defects are divided into partial deficiency (type 1) and severe deficiency (type 3). Qualitative defects (type 2) are divided into four subcategories. Type 2A refers to variants with decreased platelet‐dependent function associated with the loss of high‐molecular weight Von Willeband factor multimers. Type 2B refers to variants with increased affinity for platelet glycoprotein Ib. Type 2M refers to qualitatively abnormal variants with decreased platelet‐dependent function not associated with the loss of high‐molecular weight multimers. Type 2N refers to variants with decreased affinity for factor VIII. When recognized, mixed phenotypes caused by compound heterozygosity are indicated by separate classification of each allele. Standard amino acid and nucleotide numbering schemes are recommended for the description of mutations.
von Willebrand disease (vWD) is a bleeding disorder caused by quantitative or qualitative defects of von Willebrand factor (vWF). vWF is synthesized by endothelial cells and megakaryocytes and circulates in plasma as a multimeric high molecular weight glycoprotein. vWF plays a major role in the early phases of ostasis by promoting platelet‐vessel wall and plateletplatelet interactions under high shear conditions. It is also the carrier of coagulation factor VIII (FVIII) in plasma. A deficiency of vWF results in impairment of both primary and secondary phases of ostasis. Therefore, patients with vWD manifest bleeding symptoms that are typical of defects of primary ostasis (mucocutaneous haemorrhages) but, in case of severe deficiency of vWF, there are also haemarthroses and haematomas, which are typical of those seen with coagulation defects. Several types and subtypes of vWD have been described with a high degree of heterogeneity. The diagnosis is based on measurements of plasma and platelet vWF, the ability of vWF to interact with its platelet receptor and the analysis of the multimeric composition of vWF. Due to the heterogeneity of vWF defects, a correct diagnosis of types and subtypes may be sometimes difficult but is very important for an appropriate treatment of patients with vWD.
This article has no abstract; the first 100 words appear below.
Presentation of Case A six-year-old boy was admitted to the hospital because of a painful hip. The child had been well until 11 days earlier, when an upper respiratory tract infection developed. Two days later, he fell while playing and noticed mild pain in the right hip. During the night, the pain increased, and the next day a physician prescribed ibuprofen. Radiographs of the hip showed no abnormalities. On the following day, the child was admitted to another hospital because of more severe pain and low-grade fever. The urea nitrogen, creatinine, glucose, and electrolyte levels were normal. Other laboratory findings . . .
* Director, Boston Hemophilia Center, Brigham and Women's Hospital and Children's Hospital, Boston; assistant professor of medicine, Harvard Medical School.
Classical hemophilia A (HA) is characterized by absence or decrease in factor VIII activity. F8 is an essential cofactor of F9 to activate F10. Most known genetic mutations that lead to HA phenotype can be traced to the F8 gene itself. However, in some cases, mutations in chaperon proteins, such as LMNA1 and MCFD2, cause a decrease in secretion of both F5 and F8 resulting in a combined F5/F8 deficiency. Moreover, mutations in the domain of von Willebrand factor (VWF) that interact with F8 cause a HA-like syndrome known as type 2N (Normandy) von Willebrand disease (VWD-2N). Still, in a minority of HA cases, no mutations could be attributed to any of the genes known to be involved in the F8 pathway. In this article we will give an overview of these cases and outline future efforts needed to identify the molecular defects in such patients.
Among the three principal types of vWD, type 2 includes all qualitative abnormalities of vWF, and type 3 is the recessive form with total or near total absence. Type 1 with a purely quantitative reduction of vWF is considered to be the most common. As opposed to types 2 and 3, the molecular genetic background of type 1 has not been detected except in a few cases. We found that almost 25% of our patients diagnosed as type 1 vWD several years ago had the substitutions R611C och R552C recently labelled as type 2 mutations based on in vitro and/or clinical studies. The laboratory characteristics and the good response to DDVP, however, make them difficult to distinguish from type 1. Obviously, these substitutions confer only a minor and clinically insignificant functional deficiency of the vWF when present in heterozygous form. Based on this experience we question the clinical usefulness of the present vWD classification.
Mutation analysis in von Willebrand disease (VWD) is often undertaken to help clarify diagnosis of the disorder. This includes, but is not limited to, differential diagnosis between type 2N VWD and hemophilia A and between type 2B VWD and platelet-type VWD. The correct diagnosis is important for decisions on the most appropriate treatment modality and for appropriate genetic counseling of at-risk family members. This chapter reviews the mutations that contribute to VWD and the nature and location of mutation in each type of VWD. It then examines how diagnostic laboratories can use this knowledge in their genetic analysis strategies and in interpreting the pathogenesis of the candidate mutations identified.
Patients presenting with a low FVIII:C and with normal VWF levels are usually presumed to have hemophilia (males) or be carriers for hemophilia (females). Some of these patients may instead have VWD:2N. Such patients if misdiagnosed are likely to suffer from insufficiently treated bleeds. We report 2 males and 1 female who presented with a low FVIII:C (1–21%) and minimally reduced/normal VWF and were assumed to have, or be a carrier for, hemophilia A. Eventually all were found to have VWD:2N. Prior to the correct diagnosis the males had been treated with rFVIII with poor responses and ultimately adverse clinical consequences. Pediatr Blood Cancer 2011; 57: 1081–1083. © 2011 Wiley-Liss, Inc.
von Willebrand disease (vWD) is a bleeding disorder caused by quantitative (type 1 and 3) or qualitative (type 2) defects of von Willebrand factor (vWF). The molecular basis of type 2And 3 vWD are now known and those of type 1 vWD are being understood. Phenotypic diagnosis is based on the measurements of plasma and platelet vWF, of the ability of vWF to interact with platelet receptors and the analysis of the multimeric structure of vWF. Due to the heterogeneity of vWF defects and the variables that interfere with vWF levels, a correct diagnosis of types and subtypes may sometimes be difficult but is very important for therapy. The aim of treatment is to correct the dual defects of haemostasis, i.e. abnormal intrinsic coagulation expressed by low levels of factor VIII (FVIII) and abnormal platelet adhesion. Desmopressin is the treatment of choice in patients with type 1 vWD, who account for approximately 70% of cases, because it corrects FVIII–vWF levels and the prolonged bleeding time (BT) in the majority of these patients. In type 3 and in severe forms of type 1 and 2 vWD patients, desmopressin is not effective and it is necessary to resort to plasma concentrates containing FVIII and vWF. Treated with virucidal methods, these concentrates are effective and safe, but they cannot always correct BT defect. Platelet concentrates or desmopressin can be used as adjunctive treatments when poor correction of BT after plasma concentrate treatment is associated with continued bleeding.
Von Willebrand disease (VWD) is an autosomally inherited bleeding disorder caused by a deficiency and/or abnormality of von Willebrand factor (VWF), a multimeric adhesive protein which plays an important role in primary hemostasis. VWF interacts with glycoprotein Ib on platelet surface thus promoting platelet adhesion to the subendothelium at sites of vascular injury and platelet-platelet interactions at high-shear rate conditions. Moreover, VWF is the carrier of factor VIII (FVIII), thus indirectly contributing to coagulation process. As a consequence, also FVIII is usually low in VWD. There are 3 types of VWD: most cases have a partial quantitative deficiency of VWF (Type 1 VWD) with variable bleeding tendency, while only a minority has a virtually complete reduction (Type 3), with moderate to severe bleeding tendency. Qualitative variants (type 2 VWD), due to a dysfunctional VWF, are clinically more homogeneous and account for about 20-30% of cases. The diagnosis of VWD may be difficult, especially in type 1, since the laboratory phenotype of the disorder is greatly heterogeneous and confounded by the influence on VWF levels by factors outside the VWF gene (e.g., blood group). The molecular bases of VWD are being unraveled in most cases and have provided useful information especially in those patients with partial or total gene deletion, associated with the occurrence of inhibitors and with allergic reactions upon treatement. The aim of treatment in VWD is to correct the dual defects of hemostasis (i.e. low levels of FVIII and low VWF-dependent platelet activities) Desmopressin (DDAVP) is the treatment of choice in patients with type 1 VWD because it corrects these abnormalities in the majority of cases. In type 3 and in severe forms of type 1 and 2 VWD, DDAVP is not effective and plasma virally-inactivated concentrates containing FVIII and VWF are the mainstay of treatment.
Type 2N von Willebrand's disease (VWD) is characterized by a factor VIII (FVIII) deficiency and a low FVIII/VWF ratio related to a markedly decreased affinity of von Willebrand factor (VWF) to FVIII. Type 2N VWD is diagnosed using assays allowing the measurement of plasma VWF capacity to bind FVIII (VWF:FVIIIB). These assays, crucial in order to distinguish type 2N VWD patients from mild haemophiliacs A and haemophilia A carriers, remain exclusively homemade and limited to laboratories possessing a high level of expertise in VWD. We evaluated the first commercial ELISA (Asserachrom® VWF:FVIIIB; Stago) comparated to a reference method in a multicentric study involving 205 subjects: 60 healthy volunteers, 37 haemophiliacs A, 17 haemophilia A carriers, 37 patients with type 2N VWD, 9 heterozygous carriers for a 2N mutation and 45 patients with miscellaneous other types of VWD (all previously characterized). A diluted plasma sample adjusted to 10 IU dL(-1) of VWF:Ag was incubated with a rabbit antihuman VWF polyclonal antibody. After removing the endogenous FVIII, recombinant FVIII (rFVIII) was added and bound rFVIII was quantified using a peroxydase-conjugated mouse antihuman FVIII monoclonal antibody. The intra-assay and inter-assay reproducibility was satisfactory. In all subgroups, both methods were well correlated. All type 2N VWD patients exhibited a markedly decreased VWF:FVIIIB (lower than 15%) and all heterozygous 2N carriers had a moderately decreased VWF:FVIIIB (between 30% and 65%). All controls (healthy subjects, haemophiliacs A and haemophilia A carriers) had a normal VWF:FVIIIB (higher than 80%) except one healthy volunteer and three haemophiliacs who exhibited a moderately decreased VWF:FVIIIB suggesting a heterozygous status for a 2N mutation. In conclusion, the Asserachrom® VWF:FVIIIB is easy to perform, standardized and accurate for type 2N VWD diagnosis with a 100% sensitivity and specificity.
We have previously demonstrated within intron 40 of the von Willebrand factor (vWF) gene a region of ATCT repeats that was shown to vary in length between two different DNA clones from unrelated individuals. The polymerase chain reaction (PCR) was used to examine the variability in length of this variable number tandem repeat (VNTR) in 53 normal individuals, using primers to DNA sequence flanking the repeat region. Overall, eight different length allelic bands were seen. These were individually sequenced and shown to contain from 6 to 14 ATCT repeats (a nine-repeat band was not seen). Seventy-five percent of individuals were shown to be heterozygous for this vWF.VNTR, and family studies showed Mendelian inheritance with allelic frequencies from 1% (vWF.VNTR [8] and vWF.VNTR [14]) to 39% (vWF.VNTR [7]). In the family of a patient with type III severe von Willebrand disease (vWD), vWF.VNTR results mirrored the phenotypic data and results with previously reported intragenic vWF restriction fragment length polymorphisms (RFLP). The patient was shown to be a compound heterozygote. In a family with a child with severe type III vWD, prenatal diagnosis by vWF.VNTR analysis on DNA obtained by chorionic villus sampling at 10 weeks gestation during a subsequent pregnancy indicated a severely affected fetus. This diagnosis was confirmed by fetal blood sampling at 18 weeks.
To evaluate the prevalence of von Willebrand's disease (vWd) we carried out an epidemiological investigation among school children of the Veneto region in northern Italy. A total of 1,218 of 1,281 possible children participated in the study. They were 11 to 14 years of age, and all attended secondary schools in two distinct small areas, 70 km apart, between which there is no social contact. A blood sample was taken from each subject for determination of the blood group and von Willebrand factor (vWf) level (measured as ristocetin cofactor and expressed in IU/dL after calibration of the internal pool against an international standard), and the parents were given a questionnaire concerning hemorrhagic symptoms in the members of the family in the last three generations. Separate normal ranges were calculated for blood group O and non-O subjects (1,166 children and 289 adults) with a nonparametric method because the distribution curves of the reference values did not fit the gaussian distribution. Diagnoses of vWd were considered only for children who had low vWf levels and were members of a family with a convincing bleeding history (case of "probable vWd"). A final diagnosis was assigned if, in addition to these criteria, at least one other family member on the side with hemorrhagic history had a low vWf level. Of the 1,218 children examined, ten were classified as having vWd (0.82%). Taking into account the 90% confidence interval for the lower limit of the normal range, this figure could range from 7 (0.57%) to 14 (1.15%). All these subjects were mildly to moderately affected and presented features of heterozygous classic vWd (type I). Affected subjects were distributed evenly in the two areas examined. Our results suggest that the prevalence of vWd might be much higher than previously reported and that a different screening approach might be of use for patients with mild bleeding diathesis.
In order to determine the phenotypic variability within families transmitting classic von Willebrand's disease (vWd), a single cross-sectional examination was made of two large seven-generation vWd kindred living in the Carolinas. A total of 181 subjects were examined at least once. Fifty-eight were classified as 'affected,' 24 in the smaller kindred of 418 persons and 34 (2 of whom were homozygous) in the larger kindred of 750 persons. Detailed histories were taken, and bleeding time (BT), coagulant factor VII (VIII:C), factor-VIII-related antigen (VIIIR:Ag), and Willebrand factor (VIIIR:WF) were determined on all subjects. Affected persons were present in several generations in each kindred, and the affected state (defined as having a value outside the normal range in one or more of the four tests in the direction expected in vWd) was transmitted by both sexes to both sexes. Segregational analysis showed the pattern of inheritance to be consistent with an autosomal dominant mode, and both kindred appeared to be transmitting classic vWd. The abnormal gene was found to have a highly variable expression, 11 of the 16 possible combinations of normal or abnormal results for BT, VIII:C, VIIIR:Ag, and VIIIR:WF being observed among the affected persons. Penetrance of the abnormal gene was incomplete, 11 of the 26 presumably heterozygous transmitters having all F-VIII-related activities within the normal range. Two years after the initial study, a subsample of one kindred was reexamined twice, 5 mo apart. The phenotype of the affected state was found to be highly stable over time; 77% of those restudied were assigned the same classification as originally. Among the 5 of 22 who were phenotypically reclassified on second or third test, no changes in genotypic designation were required, since their genotypes could be correctly determined from genetic data.
von Willebrand disease (vWD), the most common inherited bleeding disorder in humans, results from abnormalities in the plasma clotting protein von Willebrand factor (vWF). Severe (type III) vWD is autosomal recessive in inheritance and is associated with extremely low or undetectable vWF levels. We report a method designed to distinguish mRNA expression from the two vWF alleles by PCR analysis of peripheral blood platelet RNA using DNA sequence polymorphisms located within exons of the vWF gene. This approach was applied to a severe-vWD pedigree in which three of eight siblings are affected and the parents and additional siblings are clinically normal. Each parent was shown to carry a vWF allele that is silent at the mRNA level. Family members inheriting both abnormal alleles are affected with severe vWD, whereas individuals with only one abnormal allele are asymptomatic. The maternal and paternal silent alleles are identical at two coding sequence polymorphisms as well as an intron 40 variable number tandem repeat, suggesting a possible common origin. Given the frequencies of the two exon polymorphisms reported here, this analysis should be applicable to approximately 70% of type I and type III vWD patients. This comparative DNA and RNA PCR-restriction fragment length polymorphism approach may also prove useful in identifying defects at the level of gene expression associated with other genetic disorders.
von Willebrand factor (vWF) and factor VIII (FVIII) circulate in plasma as a noncovalently linked protein complex. The FVIII/vWF interaction is required for the stabilization of procoagulant FVIII activity. Recently, we reported a new variant of von Willebrand disease (vWD) tentatively named "Normandy," characterized by plasma vWF that appears to be structurally and functionally normal except that it does not bind FVIII. Three patients from one family were found to be homozygous for a C----T transition at codon 816 converting Arg 53 to Trp in the mature vWF subunit. To firmly establish a causal relationship between this missense mutation and vWD Normandy phenotype, we have characterized the corresponding recombinant mutant vWF(R53W). Expressed in COS-7 cells or CHO cell lines, normal vWF and vWF(R53W) were processed and formed multimers with equal efficiency. However, vWF(R53W) exhibited the same defect in FVIII binding as did plasma vWF from patients with vWD Normandy, confirming that this mutation is responsible for the vWD Normandy phenotype. These results illustrate the importance of Arg 53 of the mature vWF subunit for the binding of FVIII to vWF, and identify an amino acid residue within a disulfide loop not previously known to be involved in this interaction.
von Willebrand disease Normandy (vWD Normandy) is a recently described phenotype in which a mutant von Willebrand factor (vWF) appears structurally and functionally normal except that it does not bind to blood coagulation factor VIII. This interaction is required for normal survival of factor VIII in the circulation; consequently, vWD Normandy can present as apparent hemophilia A but with autosomal recessive rather than X chromosome-linked inheritance. A vWF missense mutation, Thr28----Met, was identified in the propositus in or near the factor VIII binding site. The corresponding mutant recombinant vWF(T28M) formed normal multimers and had normal ristocetin cofactor activity. However, vWF(T28M) exhibited the same defect in factor VIII binding as natural vWF Normandy, confirming that this mutation causes the vWD Normandy phenotype. The distinction between hemophilia A and vWD Normandy is clinically important and should be considered in families affected by apparent mild hemophilia A that fail to show strict X chromosome-linked inheritance and, particularly, in potential female carriers with low factor VIII levels attributed to extreme lyonization.
von Willebrand factor (vWF) plays a central role in blood coagulation, mediating the adhesion of the initial platelet plug to the subendothelium, and serving as the carrier for factor VIII (FVIII) in the circulation. In previous studies, we have mapped the epitope for an anti-vWF monoclonal antibody which inhibits the interaction between FVIII and vWF to a region spanning Thr78 to Thr96 of the mature protein (Bahou, W.F., Ginsburg, D., Sikkink, R., Litwiller, R., and Fass, D. N. (1989) J. Clin. Invest. 84, 56-61). We now report the identification of a mutation within this region of vWF that results in decreased FVIII binding. Sequence analysis of polymerase chain reaction amplified platelet vWF mRNA from a von Willebrand disease (vWD) patient with a disproportionately low FVIII level identified a single nucleotide substitution (G----A), resulting in the conversion of Arg91----Gln. Recombinant vWF carrying this substitution showed decreased binding to FVIII compared with wild-type vWF or vWF carrying a polymorphic substitution in the same region (Arg89----Gln). These observations suggest a critical role for Arg91 in the interaction of vWF with FVIII and identify the molecular mechanism for a variant of vWD associated with unusually low FVIII levels.
von Willebrand factor (vWf) is a multimeric plasma glycoprotein that functions in hemostasis as the initiator of platelet adhesion to damaged blood vessels and as the carrier of Factor VIII (FVIII). Montgomery et al. (Montgomery, R.R., Hathaway, W.E., Johnson, J., Jacobsen, L., and Muntean, W. (1982) Blood 60, 201-207) reported a variant of von Willebrand disease characterized by the abnormal interaction between FVIII and a defective vWf. To identify the molecular basis of this abnormal interaction, we isolated platelet RNA from members of one of the affected families and determined the nucleotide sequence of the FVIII-binding domain encoded by the vWf mRNA. A single G to A transition at nucleotide 2561 was linked with disease expression and results in the substitution of Gln for Arg91 in mature vWf. A restriction fragment containing this mutation was introduced into a full-length vWf expression vector, and both wild type and mutant vWf were expressed in COS-7 cells. In a solid-phase binding assay, expressed vWf was captured with anti-vWf monoclonal antibody AVW1 and then incubated with 6.25-400 milliunits of recombinant FVIII. After washing, vWf-bound FVIII activity was determined with a chromogenic assay. Mutant vWf showed reduced binding of FVIII compared with wild type, suggesting that the substitution of Gln for Arg91 is the likely basis for the abnormal vWf/FVIII interaction in this von Willebrand disease variant.
Southern blotting was performed with cDNA probes for the human von Willebrand factor (vWF) gene on six patients with severe type III von Willebrand's disease (vWD). A partial deletion in the 3' end of the vWF gene was demonstrated in one individual whose parents were related and who had an alloantibody inhibitor to vWF. A resulting novel 2.0-kilobase (kb) EcoRI fragment was used for carrier detection within the patient's family, and seven carriers of this recessive trait were identified. Of the six tested, five had normal or only slightly reduced levels of vWF antigen, but with generally higher levels of factor VIII. The sixth carrier had moderately severe vWD and it is proposed that this patient is heterozygous for the defective vWF gene and a second recessive vWF defect. The novel 2.0-kb EcoRI restriction fragment was cloned and sequenced, and compared with that of the corresponding normal 4.2-kb EcoRI fragment that includes exons 41 and 42 of the vWF gene. A deletion of 2,320 base pairs (bp) which included exon 42, was identified and a novel 182-bp insert was found between the breakpoints. This insert was detected by polymerase chain reaction amplification both in the patient's DNA and in his carrier relatives.
We have previously demonstrated within intron 40 of the von Willebrand factor (vWF) gene a region of ATCT repeats that was shown to vary in length between two different DNA clones from unrelated individuals. The polymerase chain reaction (PCR) was used to examine the variability in length of this variable number tandem repeat (VNTR) in 53 normal individuals, using primers to DNA sequence flanking the repeat region. Overall, eight different length allelic bands were seen. These were individually sequenced and shown to contain from 6 to 14 ATCT repeats (a nine-repeat band was not seen). Seventy-five percent of individuals were shown to be heterozygous for this vWF.VNTR, and family studies showed Mendelian inheritance with allelic frequencies from 1% (vWF.VNTR [8] and vWF.VNTR [14]) to 39% (vWF.VNTR [7]). In the family of a patient with type III severe von Willebrand disease (vWD), vWF.VNTR results mirrored the phenotypic data and results with previously reported intragenic vWF restriction fragment length polymorphisms (RFLP). The patient was shown to be a compound heterozygote. In a family with a child with severe type III vWD, prenatal diagnosis by vWF.VNTR analysis on DNA obtained by chorionic villus sampling at 10 weeks gestation during a subsequent pregnancy indicated a severely affected fetus. This diagnosis was confirmed by fetal blood sampling at 18 weeks.
von Willebrand factor is a multifunctional adhesive protein of plasma, platelets, and endothelial cells that mediates a crucial interaction for normal hemostasis and thrombus formation by binding to platelet membrane glycoprotein Ib. We provide here evidence that this function involves two limited noncontiguous regions of the molecule, each contained within 15 amino acid residues, separated in the linear sequence by 205 residues, and maintained in close spatial proximity in the folded molecule by disulfide bonding. Definition of this chemical structure clarifies a fundamental mechanism of platelet adhesion to thrombogenic surfaces and sets the bases for obtaining synthetic replicas that may be used to modulate platelet function.
von Willebrand factor is a large multimeric plasma protein composed of identical subunits which contain four types of repeated domains. von Willebrand factor is essential for normal hemostasis, and deficiency of von Willebrand factor is the most common inherited bleeding disorder of man. Four human genomic DNA cosmid libraries and one bacteriophage lambda library were screened with von Willebrand factor cDNA probes. Twenty positive overlapping clones were characterized that span the entire von Willebrand factor gene. A high-resolution restriction map was constructed for approximately 75% of the locus and a total of approximately 33.8 kilobases was sequenced on both strands including all intron-exon boundaries. The gene is approximately 178 kilobases in length and contains 52 exons. The exons vary from 40 to 1379 base pairs in length, and the introns vary from 97 base pairs to approximately 19.9 kilobases in length. The signal peptide and propeptide (von Willebrand antigen II) of von Willebrand factor are encoded by 17 exons in approximately 80 kilobases of DNA while the mature subunit of von Willebrand factor and 3' noncoding region are encoded by 35 exons in the remaining approximately 100 kilobases of the gene. A number of repetitive sequences were identified including 14 Alu repeats and a approximately 670-base pair TCTA simple repeat in intron 40 that is polymorphic. Regions of the gene that encode homologous domains have similar structures, supporting a model for their origin by gene segment duplication.
We report three members of a family who had reduced levels of plasma von Willebrand factor (vWF) and increased ristocetin-induced platelet aggregation (RIPA) (aggregation of platelet-rich plasma with ristocetin at a concentration of 0.45 mg/mL), as previously reported in type IIB and pseudo-von Willebrand's disease (vWD). However, in contrast to the latter two disorders in which the larger vWF multimers are absent in plasma, the entire range of vWF multimers was observed in the patients' plasma after sodium dodecyl sulfate-agarose gel electrophoresis, and all vWF multimers (including the largest) were present in the same proportion as in normal plasma and type I vWD. Thus, despite increased RIPA, the levels and multimeric pattern of vWF in this family's plasma were indistinguishable from those in type I vWD in which RIPA is usually decreased. Addition of ristocetin to the patients' platelet-rich plasma resulted in the removal of vWF (and, more selectively, of the large multimers) at lower concentrations of ristocetin than normal, as in type IIB and pseudo-vWD. The defect in the patients was localized to their vWF, which had an enhanced capacity for aggregating washed normal platelets in the presence of low concentrations of ristocetin and for aggregating pseudo-vWD platelets (in the absence of ristocetin). Both glycoproteins (GP) Ib and IIb-IIIa were involved in the enhanced aggregation response. RIPA (at low ristocetin concentrations) in the patients' platelet-rich plasma was abolished by a monoclonal antibody (AP1) to GPIb and was markedly reduced by monoclonal antibodies (10E5 and LJP9) that block adenosine diphosphate and thrombin-induced binding of vWF and fibrinogen to GPIIb-IIIa but was unaffected by an antibody (LJP5) that only blocks vWF binding. Partial inhibition of the initial aggregation slope (and complete inhibition of second phase aggregation) was achieved with creatine phosphate/creatine phosphokinase. EDTA blocked second-phase aggregation but was without effect on the initial slope. The findings in this family combine some features of both type I vWD (normal pattern of vWF multimers in plasma) and type IIB vWD (increased RIPA) and further demonstrate the increasing complexity of the structure-function relationships in vWD.
von Willebrand factor (vWF) and factor VIII (FVIII) circulate in plasma as a noncovalently linked protein complex. The FVIII/vWF interaction is required for the stabilization of procoagulant FVIII activity. Recently, we reported a new variant of von Willebrand disease (vWD) tentatively named “Normandy,” characterized by plasma vWF that appears to be structurally and functionally normal except that it does not bind FVIII. Three patients from one family were found to be homozygous for a C----T transition at codon 816 converting Arg 53 to Trp in the mature vWF subunit. To firmly establish a causal relationship between this missense mutation and vWD Normandy phenotype, we have characterized the corresponding recombinant mutant vWF(R53W). Expressed in COS-7 cells or CHO cell lines, normal vWF and vWF(R53W) were processed and formed multimers with equal efficiency. However, vWF(R53W) exhibited the same defect in FVIII binding as did plasma vWF from patients with vWD Normandy, confirming that this mutation is responsible for the vWD Normandy phenotype. These results illustrate the importance of Arg 53 of the mature vWF subunit for the binding of FVIII to vWF, and identify an amino acid residue within a disulfide loop not previously known to be involved in this interaction.
To evaluate the prevalence of von Willebrand's disease (vWd) we carried out an epidemiological investigation among school children of the Veneto region in northern Italy. A total of 1,218 of 1,281 possible children participated in the study. They were 11 to 14 years of age, and all attended secondary schools in two distinct small areas, 70 km apart, between which there is no social contact. A blood sample was taken from each subject for determination of the blood group and von Willebrand factor (vWf) level (measured as ristocetin cofactor and expressed in IU/dL after calibration of the internal pool against an international standard), and the parents were given a questionnaire concerning hemorrhagic symptoms in the members of the family in the last three generations. Separate normal ranges were calculated for blood group O and non-O subjects (1,166 children and 289 adults) with a nonparametric method because the distribution curves of the reference values did not fit the gaussian distribution. Diagnoses of vWd were considered only for children who had low vWf levels and were members of a family with a convincing bleeding history (case of “probable vWd”). A final diagnosis was assigned if, in addition to these criteria, at least one other family member on the side with hemorrhagic history had a low vWf level. Of the 1,218 children examined, ten were classified as having vWd (0.82%). Taking into account the 90% confidence interval for the lower limit of the normal range, this figure could range from 7 (0.57%) to 14 (1.15%). All these subjects were mildly to moderately affected and presented features of heterozygous classic vWd (type I). Affected subjects were distributed evenly in the two areas examined. Our results suggest that the prevalence of vWd might be much higher than previously reported and that a different screening approach might be of use for patients with mild bleeding diathesis.
Southern blotting was performed with cDNA probes for the human von Willebrand factor (vWF) gene on six patients with severe type III von Willebrand's disease (vWD). A partial deletion in the 3′ end of the vWF gene was demonstrated in one individual whose parents were related and who had an alloantibody inhibitor to vWF. A resulting novel 2.0- kilobase (kb) EcoRI fragment was used for carrier detection within the patient's family, and seven carriers of this recessive trait were identified. Of the six tested, five had normal or only slightly reduced levels of vWF antigen, but with generally higher levels of factor VIII. The sixth carrier had moderately severe vWD and it is proposed that this patient is heterozygous for the defective vWF gene and a second recessive vWF defect. The novel 2.0-kb EcoRI restriction fragment was cloned and sequenced, and compared with that of the corresponding normal 4.2-kb EcoRI fragment that includes exons 41 and 42 of the vWF gene. A deletion of 2,320 base pairs (bp) which included exon 42, was identified and a novel 182-bp insert was found between the breakpoints. This insert was detected by polymerase chain reaction amplification both in the patient's DNA and in his carrier relatives.
The variability of laboratory findings in von Willebrand's disease (vWd) was evaluated by performing serial studies of bleeding time (BT), factor VIII coagulant activity (VIII:C), factor-VIII-related antigen (VIIIR:Ag) and ristocetin cofactor (VIIIR:Rcof) in 50 individuals from 25 families with this disorder. The types of results were characterized from 1 to 16 based on the possible combinations of findings using these four tests. The only patients observed to have consistently abnormal results of all four tests were three individuals with homozygous vWd. Individuals with autosomal dominant vWd were found to have a variety of results and all 16 possible types were observed. Although a consistent pattern was present within some families, others with equivalent history of bleeding demonstrated widely variable types of results. The results within some families, others with equivalent history of bleeding demonstrated widely variable types of results. The results of serial studies of the same tests in 10 normal individuals indicated relative stability, with nearly all values within the usual range of normal, but some independent variation of factor-VIII-related activities was observed. These studies indicate that: (1) the results of BT, VIII:C, VIIIR:Ag, and VIIIR:Rcof vary considerably from time to time in many individuals with vWd, (2) a classification of “variants” of vWd based solely on such studies may be inappropriate, particularly if the tests are not repeated, and (3) repeated testing may be required to establish the diagnosis of vWd in some individuals.
We report three members of a family who had reduced levels of plasma von Willebrand factor (vWF) and increased ristocetin-induced platelet aggregation (RIPA) (aggregation of platelet-rich plasma with ristocetin at a concentration of 0.45 mg/mL), as previously reported in type IIB and pseudo-von Willebrand's disease (vWD). However, in contrast to the latter two disorders in which the larger vWF multimers are absent in plasma, the entire range of vWF multimers was observed in the patients' plasma after sodium dodecyl sulfate-agarose gel electrophoresis, and all vWF multimers (including the largest) were present in the same proportion as in normal plasma and type I vWD. Thus, despite increased RIPA, the levels and multimeric pattern of vWF in this family's plasma were indistinguishable from those in type I vWD in which RIPA is usually decreased. Addition of ristocetin to the patients' platelet- rich plasma resulted in the removal of vWF (and, more selectively, of the large multimers) at lower concentrations of ristocetin than normal, as in type IIB and pseudo-vWD. The defect in the patients was localized to their vWF, which had an enhanced capacity for aggregating washed normal platelets in the presence of low concentrations of ristocetin and for aggregating pseudo-vWD platelets (in the absence of ristocetin). Both glycoproteins (GP) Ib and IIb-IIIa were involved in the enhanced aggregation response. RIPA (at low ristocetin concentrations) in the patients' platelet-rich plasma was abolished by a monoclonal antibody (AP1) to GPIb and was markedly reduced by monoclonal antibodies (10E5 and LJP9) that block adenosine diphosphate and thrombin-induced binding of vWF and fibrinogen to GPIIb-IIIa but was unaffected by an antibody (LJP5) that only blocks vWF binding. Partial inhibition of the initial aggregation slope (and complete inhibition of second phase aggregation) was achieved with creatine phosphate/creatine phosphokinase. EDTA blocked second-phase aggregation but was without effect on the initial slope. The findings in this family combine some features of both type I vWD (normal pattern of vWF multimers in plasma) and type IIB vWD (increased RIPA) and further demonstrate the increasing complexity of the structure-function relationships in vWD.
The size of human factor VIII-related protein in plasma has been determined by sodium dodecyl sulfate (SDS) glyoxyl agarose electrophoresis. The protein was immobilized after the electrophoresis by coupling it to the modified agarose, and it was identified by autoradiography using purified rabbit anti-factor VIII-related antigen (VIIR:Ag). A series of multimeric forms was identified with Mr of 0.85- 12 x 10(6). The distribution of VIIR:Ag multimers was the same in heparin and citrate anticoagulated plasmas and in serum, and the pattern was the same after freezing as in plasma kept at 37 degrees C from the time of venipuncture until the electrophoresis was complete. These observations indicate that VIIR:Ag circulates in normal plasma as a population of very large multimers and that the size distribution is not an artifact induced by purification methods, freezing, or calcium chelation.
In each of two kindreds with von Willebrand's disease patients the pattern of inheritance exhibited both ‘recessive’ and ‘dominant’ features. It is suggested that this phenomenon may be associated with the fact that the factor VIII related antigen/Willebrand factor exists in normal plasma as a series of homologous oligomers of which those with the highest molecular weight exhibit the most potent platelet-related biological activities. The variable inheritance patterns in some von Willebrand's disease kindreds may thus be due to interaction between maternally and paternally derived protomers or related biosynthetic pathways so that various series of oligomers are produced in the offspring.
The von Willebrand factor (vWF) genes of nine unrelated, severe, type III von Willebrand's disease (vWD) patients (six of Dutch origin) and four unrelated Dutch type I vWD patients were screened for mutations in exons that contain CGA codons (Arg), which are liable to mutation to TGA stop codons. The nine exons of the vWF gene (3, 8, 9, 10, 28, 31, 32, 43 and 45) that contain all the CGA codons (11 in total) of the vWF cDNA were amplified by the polymerase chain reaction and screened for mutations by single-strand conformation polymorphism analysis, restriction enzyme - and/or nucleotide sequence analysis. Three of the severe vWD patients were found to be heterozygous for a nonsense mutation: CGA Arg 2535-->TGA Stop. Three other severe vWD patients were homozygous for a single nucleotide substitution, AAC Asn 2546-->TAC Tyr. The transcription of these mutated alleles was tested by cDNA dependent amplification of platelet RNA. The level of transcription product was strongly reduced for either mutant allele.
We describe a patient with a lifelong bleeding disorder previously classified as von Willebrand's disease (vWD) type I. The factor VIII (FVIII) level in this patient was disproportionately low and we showed that this was due to a decreased factor VIII binding capacity of her vWF. To characterize the molecular defect in this type of vWD, a cDNA-dependent polymerase chain reaction (PCR) amplification was performed using platelet RNA as a template. Direct sequencing of the amplified fragment, which encodes for the FVIII-binding domain, showed a single nucleotide change in exon 20 at codon 854, resulting in the substitution of CAG glutamine (Gln) for CGG arginine (Arg). At the level of the cDNA only the mutated sequence was found, whereas at genomic DNA level the patient was heterozygous for this mutation. This patient is therefore a compound heterozygote for a point mutation resulting in a FVIII-binding defect and a vWF allele with low transcript levels.
In community-wide immunisation programmes against childhood infections there is a conflict between the interests of the individual (vaccine safety and efficacy) and the interests of the community (vaccine uptake and level of herd immunity). Studies suggesting that the complication rate is greater with the high efficacy Urabe Am 9 mumps vaccine than with the lower efficacy Jeryl Lynn vaccine, have led to concern about whether the higher efficacy mumps vaccine should be introduced or retained in nationwide mass immunisation programmes. We describe the use of a mathematical model to assess benefits and risks to both individual and community, and illustrate this method by reference to immunisation programmes based on these vaccines. On the basis of current epidemiological data on viral transmission and vaccine coverage in England and Wales, data on vaccine-associated and infection-associated complication rates, and vaccine efficacies estimated from clinical trials, our analyses suggest there is little to choose between the two vaccines, but that overall performance depends on the level of vaccine coverage in a defined population. In community-based programmes, the greater apparent safety of the Jeryl Lynn vaccine (fewer vaccine-induced complications) is offset by the greater apparent efficacy of the Urabe Am 9 vaccine (fewer complications due to natural infection). The findings suggest that it may not always be in the interests of the community to use the vaccine with the lowest complication rate.
Plasma von Willebrand factor (vWf) is a multi-domain multimerized glycoprotein which has a dual role in haemostasis: it promotes platelet adhesion to subendothelium and is the carrier of blood coagulation factor VIII (FVIII). We previously characterized a functional defect of vWf, limited to its ability to bind FVIII, in two families whose affected members have the same phenotype that mimics mild haemophilia A and was tentatively named von Willebrand's disease (vWD) 'Normandy'. A homozygous point mutation C----T converting Thr 28 to Met in mature vWf subunit was identified in one of these patients who was born of third-cousin parents. In the present studies we report two unrelated new cases of vWD 'Normandy' and characterize, using the analysis of the vWf gene intron 40 region containing a variable number of tandem repeats, the recessive inheritance of the disease in two affected families without known consanguinity. Exons 18-24 of the vWf gene encoding for the first 311 amino acids of mature vWf subunit were amplified by the polymerase chain reaction method and sequenced. Two new missense mutations, both corresponding to a C----T transition and predicting respectively an Arg 53----Trp and an Arg 91----Gln substitution, were characterized. The three patients from family 1 were homozygous for the first-mentioned mutation while the patient from family 3 was homozygous for the second. The patient from family 2 was found a compound heterozygote for the two mutations. None of the two point mutations reported, both destroying a MspI restriction site, could be detected in DNA from 50 normal controls screened by restriction endonuclease analysis. Our data show that different mutations may be found in patients with the 'Normandy' phenotype. The mutations characterized so far are all localized on the N-terminal region of mature vWf subunit, within or near the major FVIII binding domain, and some of them occur within the epitope of monoclonal antibodies inhibiting the vWf/FVIII interaction. These observations suggest a causal relationship between these mutations and the vWD 'Normandy' phenotype.
Besides having a large number of restriction fragment length polymorphisms (RFLP) the von Willebrand factor (vWF) gene contains several sequence polymorphisms in the coding regions. Eight nucleotide substitutions have been reported in two or more independent cDNA clones. Four of them give rise to amino acid substitutions, two of which are in the mature vWF subunit (at positions 26 and 709). We have investigated a previously suggested putative alanine-threonine polymorphism at position 618 of the mature subunit in normal subjects and patients with various types of von Willebrand's disease (vWD). the codon for amino acid 618 is located in exon 28, which encodes several important vWF functional domains. We amplified the whole exon 28 and parts of it by polymerase chain reaction (PCR) and distinguished gene from pseudogene sequences. The alanine----threonine (G----A) substitution was studied with restriction enzyme cleavage of the products, since it creates a new HphI site. Moreover, in two individuals we confirmed the polymorphism by cDNA sequencing. In 23 normals the frequencies of the h- (Ala) and the h+ (Thr) alleles were 0.50/0.50. In eight patients with type III vWD from seven different families, the h- allele was present in 13 of 16 genes, but whether this signifies a common mutation in some of the patients is not known. In types I and II, both alleles were present in roughly similar proportions. Owing to the high frequency of heterozygosity, the polymorphism should prove useful as an aid in genetic counselling.
We have previously identified a microsatellite variable number tandem repeat region of the nucleotide sequence ATCT within intron 40 of the von Willebrand factor (vWF) gene. By polymerase chain reaction (PCR) amplification of this region, eight major alleles have been demonstrated in the South Wales population, with an overall heterozygosity rate of 75%. Direct sequencing has shown that the alleles correspond to lengths of between six and 14 ATCT repeats. In the present study we describe the use of this variable repeat sequence and previously reported restriction fragment length polymorphisms (RFLP) to study inheritance patterns in families with type I, IIA and severe type III von Willebrand's disease (vWD). The results confirm that analysis of this precisely localized intragenic locus provides a highly informative marker for gene tracking studies in the major forms of vWD.
Structural analysis of the von Willebrand factor gene located on chromosome 12 is complicated by the presence of a partial unprocessed pseudogene on chromosome 22q11-13. The structures of the von Willebrand factor pseudogene and corresponding segment of the gene were determined, and methods were developed for the rapid differentiation of von Willebrand factor gene and pseudogene sequences. The pseudogene is 21-29 kilobases in length and corresponds to 12 exons (exons 23-34) of the von Willebrand factor gene. Approximately 21 kilobases of the gene and pseudogene were sequenced, including the 5' boundary of the pseudogene. The 3' boundary of the pseudogene lies within an 8-kb region corresponding to intron 34 of the gene. The presence of splice site and nonsense mutations suggests that the pseudogene cannot yield functional transcripts. The pseudogene has diverged approximately 3.1% in nucleotide sequence from the gene. This suggests a recent evolutionary origin approximately 19-29 million years ago, near the time of divergence of humans and apes from monkeys. Several repetitive sequences were identified, including 4 Alu, one Line-1, and several short simple sequence repeats. Several of these simple repeats differ in length between the gene and pseudogene and provide useful markers for distinguishing these loci. Sequence differences between the gene and pseudogene were exploited to design oligonucleotide primers for use in the polymerase chain reaction to selectivity amplify sequences corresponding to exons 23-34 from either the von Willebrand factor gene or the pseudogene. This method is useful for the analysis of gene defects in patients with von Willebrand disease, without interference from homologous sequences in the pseudogene.
In 1989 a nationwide surveillance of neurologic complications after the administration of mumps vaccine was conducted in Japan, based on the notification of cases and the testing of mumps viruses isolated from cerebrospinal fluid for their relatedness to the vaccine by nucleotide sequence analysis. Among 630,157 recipients of measles-mumps-rubella trivalent (MMR) vaccine containing the Urabe Am9 mumps vaccine, there were at least 311 meningitis cases suspected to be vaccine-related. In 96 of these 311 cases, mumps virus related to the vaccine was isolated from cerebrospinal fluid. The unusually high incidence may have been partly a result of the adverse media publicity of the problem at the time of surveillance. We analyzed clinical features of 165 and 27 laboratory-confirmed mumps vaccine-related meningitis cases that occurred among the recipients of MMR and monovalent mumps vaccines, respectively, during a 1-year period after the introduction of MMR vaccine. The incidence of vaccine-related meningitis was similar among the recipients of MMR and monovalent Urabe Am9 mumps vaccines. Meningitis was generally mild and there were no sequelae from the illness. The complication was more frequent among male than among female children.
A survey of untoward reactions, especially central nervous system reactions, after the administration of a newly introduced measles, mumps and rubella (MMR) vaccine in Gunma Prefecture, Japan, was initiated soon after 4 patients were hospitalized for aseptic meningitis. Thirty-five, 6 and 2 children developed meningitis, convulsive disorders and parotitis, respectively, within 2 months after MMR vaccination during the 8-month period extending from April to November, 1989. The time lag between MMR vaccination and meningitis ranged from 14 to 28 days in the 35 cases of meningitis. Mumps virus, isolated from the cerebrospinal fluid in 13 patients with aseptic meningitis, was characterized by determination of the nucleotide sequences of the P gene as mumps vaccine strain. The incidence of aseptic meningitis with positive mumps vaccine virus was estimated to be 0.11% (0.3% as a whole) during the 8 months from April to November and increased to 0.3% (0.7% as a whole) in September and October. We conclude that the incidence of aseptic meningitis after MMR vaccination seems to be higher than that reported previously.
Parts of the F gene from 16 mumps viruses derived from vaccines and clinical isolates were amplified using the polymerase chain reaction and their nucleotide sequences were determined. Over a region of 111 nucleotides, eight regions of variability were detected with a maximum of six (5.4%) changes occurring between any two virus strains. The Jeryl Lynn and Urabe vaccine strains were clearly different from each other and from wild virus isolated from cases of non-vaccine-associated mumps. In contrast, viruses isolated from the cerebrospinal fluid and throat in cases of meningitis and parotitis following vaccination with the Urabe strain were identical to this strain. We conclude that the vaccine was the source of these infections.
The COVER scheme, a method for the rapid evaluation of vaccine coverage in England and Wales, is described. The primary aim of the scheme is to improve cover by providing health district vaccination programme coordinators with relevant timely information. Quarterly data were obtained from, analysed and promptly fed back to, 126 health districts on cohorts of children who had recently attained the target ages for receiving the selected sentinel vaccines; 18 months for third diphtheria and third pertussis and 2 years for measles. Although the data suggested that vaccination cover is improving, national performance still falls well short of 90%, the 1990 target set by the World Health Organisation for countries in Europe.
In 1982 a two dose regimen was introduced in Sweden for the combined vaccination against measles, mumps, and rubella of children aged 18 months and 12 years. Since 1977 about half of the preschool children were vaccinated against measles annually, and since 1974 about 80% of 12 year old girls were vaccinated against rubella. During the period 1982 to 1985 90-93% of the eligible age cohorts of 18 month old children and 88-91% of the 12 year old children were immunised with the new combined vaccine. A study in 1982 of about 140 18 month old children who were nearly all seronegative before vaccination showed that 96%, 92%, and 99% seroconverted against measles, mumps, and rubella, respectively. A second study was carried out in 1983 of 247 12 year old children, of whom 11% lacked antibodies to measles, 27% to mumps, and 45% to rubella. This showed seroconversion in 82% and 80% against measles and mumps, respectively, and all children seroconverted against rubella. In the latest study in 1985 of 496 12 year olds 9% and 13% were seronegative against measles and mumps before vaccination, and 41% against rubella. Of these, 88% seroconverted to measles and 80% to mumps, and all converted to rubella when sera were tested by the haemolysis in gel method. After a neutralisation test against measles as well all children showed immunity to the disease. A low incidence of measles and declining figures for mumps and rubella were reported in 1984 to 1986. An outbreak of rubella during 1985 affected mainly boys in age cohorts in which only the girls had been vaccinated during the 1970s.
Before the introduction of measles, mumps, and rubella vaccine a survey was carried out to measure antibody prevalence to the three viruses by age. A total of 8716 samples of serum collected by five public health laboratories in different parts of England during 1986-7 were tested. Despite the current measles vaccination programme 60% of children aged 1-2 years did not have measles antibody and over 80% did not have antibodies to mumps and rubella. In the 3-4 year age group 17% of the children were susceptible to measles, 55% to mumps, and 73% to rubella. The results suggest that vaccinating children early in the second year of life will be necessary to eliminate the three diseases. The survey provides baseline data for continuing surveillance of the immediate and long term effects of the new vaccination strategy.
Progress has occurred in the past several years in the understanding of the structure and function of von Willebrand factor (vWF). This multimeric glycoprotein exhibits a dual role, that of mediating platelet adhesion and aggregation onto thrombogenic surfaces, and that of functioning as carrier in plasma for the factor VIII procoagulant protein. New insights into the nature of the several functional domains of vWF have led to the identification of the regions of the molecule that interact with factor VIII, heparin, the glycoprotein lb of platelets, and collagen. Alterations of vWF are the cause of von Willebrand disease (vWD), a congenital bleeding disorder. In the majority of patients, the plasma levels of vWF are decreased, but there is no demonstrable structural or functional alteration of the protein. In other patients, however, the structure of vWF is abnormal. This review summarizes the current knowledge on vWF and vWD.
Methods to identify disease risk factors from a series of cases are considered. These include methods that compare risk factor levels among diagnostic categories and methods that relate risk factor levels to age at diagnosis, with a single diagnostic category. Statistical aspects considered include modelling assumptions, parameter identifiability, hypothesis-testing efficiency, assumptions concerning unsampled diagnostic categories and requirements for risk factor data and confounding factor data. It is argued that methods to identify risk factors using data on a single diagnostic category involve such strong assumptions that they have limited usefulness. Analyses that compare risk factor levels among diagnostic categories, on the other hand, should continue to play an important role in epidemiologic research, though there are important limitations in relation to analyses involving disease-free controls.
The size of human factor VIII-related protein in plasma has been determined by sodium dodecyl sulfate (SDS) glyoxyl agarose electrophoresis. The protein was immobilized after the electrophoresis by coupling it to the modified agarose, and it was identified by autoradiography using purified rabbit anti-factor VIII-related antigen (VIIR:Ag). A series of multimeric forms was identified with Mr of 0.85-12 x 10(6). The distribution of VIIR:Ag multimers was the same in heparin and citrate anticoagulated plasmas and in serum, and the pattern was the same after freezing as in plasma kept at 37 degrees C from the time of venipuncture until the electrophoresis was complete. These observations indicate that VIIR:Ag circulates in normal plasma as a population of very large multimers and that the size distribution is not an artifact induced by purification methods, freezing, or calcium chelation.
The variability of laboratory findings in von Willebrand's disease (vWd) was evaluated by performing serial studies of bleeding time (BT), factor VIII coagulant activity (VIII:C), factor-VIII-related antigen (VIIIR:Ag) and ristocetin cofactor (VIIIR:Rcof) in 50 individuals from 25 families with this disorder. The types of results were characterized from 1 to 16 based on the possible combinations of findings using these four tests. The only patients observed to have consistently abnormal results of all four tests were three individuals with homozygous vWd. Individuals with autosomal dominant vWd were found to have a variety of results and all 16 possible types were observed. Although a consistent pattern was present within some families, others with equivalent history of bleeding demonstrated widely variable types of results. The results within some families, others with equivalent history of bleeding demonstrated widely variable types of results. The results of serial studies of the same tests in 10 normal individuals indicated relative stability, with nearly all values within the usual range of normal, but some independent variation of factor-VIII-related activities was observed. These studies indicate that: (1) the results of BT, VIII:C, VIIIR:Ag, and VIIIR:Rcof vary considerably from time to time in many individuals with vWd, (2) a classification of "variants" of vWd based solely on such studies may be inappropriate, particularly if the tests are not repeated, and (3) repeated testing may be required to establish the diagnosis of vWd in some individuals.
The value of studying factors of haemostasis and thrombosis in patients with coronary artery disease is established. The endothelial lesion and evolution of the thrombus play key roles in acute coronary syndromes and coronary angioplasty. The von Willebrand factor (VWF) is known for its participation in primary haemostasis. Deficits of this factor lead to a haemorrhagic syndrome, von Willebrand's disease. This glycoprotein is mainly synthesised by the endothelial cells. Its polymeric composition allows identification of two types of multimeres. The high molecular weight, active multimeres are liberated from the endothelium after stimulation by thrombin. Low molecular weight multimeres are less active and are secreted continuously. The VWF promotes platelet adhesion and facilitates platelet aggregation. Experimental pig models with VWF deficiency show that this factor is essential for the constitution of an occlusive thrombus. Several physiopathological mechanisms interact to increase VWF concentrations during thrombosis: the endothelial lesion, adrenergic stimulation, acute phase reaction. Increased VWF concentrations have been reported in many clinical situations. The results are most demonstrative in coronary artery disease. The VWF is abnormally high from the time of hospital admission in patients with acute myocardial infarction and continues to increase up to the 5th day before falling, without returning to normal values, at the 15th day. It is a sensitive though not specific late diagnostic marker of myocardial infarction. Increased VWF concentrations are not proportional to the severity of coronary atherosclerosis. They are, however, related to the infarct size, to the inflammatory reaction and to the prothrombotic phase.(ABSTRACT TRUNCATED AT 250 WORDS)
Die mumps-meningitis-ein Mumps-komplikation?
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ADDRESSES: Department of Haematology, Haemostasis and Thrombosis Research Centre, University Hospital Leiden, Netherlands (J. C J Eikenboom, MD, P. H. Reitsma, PhD, E. Briet, MD) and Centre for Thrombosis and Vascular Research, University of Leuven, Belgium (K. M. J. Peerlinck, MD). Correspondence to Dr Jeroen C. J. Eikenboom, Department of Haematology, Building 1: C2-R, Haemostasis and Thrombosis Research Centre, University Hospital Leiden, PO Box 9600, 2300 RC Leiden, Netherlands REFERENCES
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