Type 2N Research Articles

Factor VIII-von Willebrand Factor Binding Defects in Autosomal Recessive von Willebrand Disease Type Normandy and in Mild Hemophilia A

This concise review is focused on genetic, molecular and clinical aspects of von Willebrand disease (VWD) type 2N and of mild hemophilia A due to mutations impairing FVIII-von Willebrand factor (VWF) interactions. Missense mutations in the VWF gene impairing the binding to FVIII do not impair the structure of VWF multimers nor the ability of VWF to aggregate platelets but causes an accelerated clearance of FVIII. Missense mutations in the FVIII gene impairing the binding to VWF are a common cause of mild/moderate hemophilia A. The implications of these observations for the treatment of patients with coagulation factor concentrates and desmopressin are discussed.

Von Willebrand disease and other bleeding disorders in women: consensus on diagnosis and management from an international expert panel

Reproductive tract bleeding in women is a naturally occurring event during menstruation and childbirth. In women with menorrhagia, however, congenital bleeding disorders historically have been underdiagnosed. This consensus is intended to allow physicians to better recognize bleeding disorders as a cause of menorrhagia and consequently offer effective disease-specific therapies.

Measurement of von Willebrand factor‐FVIII binding activity in patients with suspected von Willebrand disease type 2N: application of an ELISA‐based assay in a reference laboratory

Laboratory diagnosis of von Willebrand disease type 2N (VWD2N) is based on costly mutation analysis or in vitro measurement of the ability of plasma von Willebrand factor (VWF) to bind exogenous factor VIII (FVIII); however, the VWF-FVIII binding activity assay is complex and not widely used. Our aim was to assess the utility of the in-house VWF-FVIII binding assay in the investigation of patients with suspected VWD2N. A previously described ELISA-based FVIII binding method was simplified and adapted for the clinical laboratory use by optimizing incubation time, reagent concentrations and assay standardization. The assay was validated using samples from eight individuals with known homozygous or heterozygous VWD2N mutations, and 100 healthy adults. An additional 392 patient samples were tested, including 314 with FVIII activity <50% of normal and 78 received for routine VWF-FVIII binding activity testing. Intra- and inter-assay variations were less than 10% and 17%, respectively, and the limit of quantification was estimated as 0.12. The reference range for healthy adults was 0.73-1.42. VWF:FVIII binding activity was consistent with the genotype in subjects with available genetic data, being low in three individuals with homozygous mutation (<0.12) and intermediate in five heterozygous individuals (0.44-0.61). Screening of the 392 clinical samples identified reduced VWF:FVIII binding in 19 subjects. This assay provides accurate measurement of VWF:FVIII binding activity and successfully identifies homozygous VWD2N patients and heterozygous carriers. Use of this ELISA-based assay may help avoid the need for mutation analysis in patients with unexplained low FVIII activity.

Novel missense mutation c.2685G&gt;C (p.Q895H) in VWF gene associated with very low levels of VWF mRNA

Homozygous patients for null alleles in VWF gene show a severe von Willebrand phenotype, whereas compound heterozygous patients only show the phenotype of the expressed allele. Five members of the same family were studied. The two patients showed borderline VWF levels, a mild factor VIII (FVIII) deficiency and a decrease of the binding of VWF to exogenous FVIII. The genetic analyses of the VWF gene confirmed that the patients were compound heterozygous for c.2561G>A (R854Q) and c.2685G>C (p.Q895H) mutations. The latter, is located in the 3' extreme of exon 20, and it has not been previously described. Studies of the cDNA from platelet mRNA were performed to investigate the expression of p.H895 allele. The loss of heterozygosity at the cDNA level suggests a lack of expression of the p.H895 allele. The overall studies can explain the type 2N phenotype of the two patients, since the allele carrying the new missense mutation p.Q895H has shown a low expression of VWF gene.

Levels of quaternion algebras

The level of a ring R with 1 ≠ 0 is the smallest positive integer s such that −1 can be written as a sum of s squares in R, provided −1 is a sum of squares at all. D. W. Lewis showed that any value of type 2n or 2n + 1 can be realized as level of a quaternion algebra, and he asked whether there exist quaternion algebras whose levels are not of that form. Using function fields of quadratic forms, we construct such examples.

Diagnosis and treatment of von Willebrand disease: new perspectives and nuances

In 1926, Erik von Willebrand, a Finnish internist andacademic, evaluated and described a 5-year-old girlwith extreme bleeding and bruising due to what hadbeen designated the A˚landic haemorrhagic diseaseby inhabitants of this archipelago island in the Gulfof Bothnia. Hjordis, the propositus, was the ninth of12 children born to a pedigree in which four femalesiblings had already died before the age of 4 withuncontrolled haemorrhage and in which 23 of 66family members, predominantly females, had expe-rienced significant bleeding and bruising complica-tions [1]. In fact, Hjordis herself eventually died atthe age of 13 during her fourth menstrual cycle.Professor von Willebrand mistakenly concluded thatthis bleeding diathesis was an unusual form ofhaemophilia and decided to label the new diseaseas pseudo-haemophilia to differentiate it from thesex-linked recessive haemophilia A. Little did vonWillebrand realize that this disease, eventually tobecomeeponymous[vonWillebranddisease(VWD)],would become the most commonly diagnosed con-genital bleeding disorder, with a prevalence rangingbetween 1 per 10 000 individuals to 1.3% [2]. Type1 VWD is the most common subtype, representingup to 75–80% of all cases while the subtype 3 VWDoccurs in approximately 1 per million population inthe United States and Europe [2]. Determination ofthe exact prevalence of VWD is hindered somewhatby the heterogeneity of clinical and diagnosticlaboratory features. Even Professor von Willebrandappreciated the challenges of diagnosing VWD as hecollaborated with Professor Rudolf Ju¨gens at theBerlin University to examine patients blood sampleswith a newly invented kapilla¨rtrombometer appa-ratus [3]. Although they were technically mistakenwhen they attributed the bleeding manifestations ofVWD to a platelet defect, their observations wereremarkably prescient since it was not until early1970s that the existence of a specific von Willebrandfactor (VWF) glycoprotein separate from factor VIIIwas finally appreciated and demonstrated to supportplatelet adhesion to the subendothelial matrix ofdamaged blood vessels.Over the last 81 years since von Willebranddescribed the bleeding disorder, there have beennumerous attempts and approaches to refine thediagnosis of the disease so that appropriate andefficacious treatment can be delivered. The clinicalphenotype of VWD includes, in part, information onwhether bleeding is spontaneous or related to surgi-cal or physical trauma; family history and inheri-tance pattern; menstrual history in women; age ofonset of bleeding (to distinguish between acquired vs.inherited coagulation disorders); and sites of bleed-ing(mucocutaneousvs.visceral,intra-articular,intra-muscular, or soft tissue locations). Subsequentlaboratory testing is necessary to exclude or confirmthe diagnosis and to further classify the subtype ofVWD. This article will focus on selected vagariesassociated with the ability to utilize clinical pheno-typic information gathered through the history andphysical examination to predict the presence ofVWD. The clinical diagnosis of VWD must be based,however, on more than physician suspicion andintuition. Thus, the need for confirmatory testing inthe laboratory. The frailties of conventional labora-tory testing for diagnosing VWD and the use of anin vitro surrogate of the bleeding time will also be

Novel VWF Sequence Variations Identified in Normal Controls and Index Cases Enrolled in the TS Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD).

Von Willebrand disease (VWD) is caused by quantitative (types 1 and 3) and qualitative (type 2) defects in von Willebrand factor (VWF). The TS Zimmerman Program for the Molecular and Clinical Biology of VWD is a multinational Program Project established to further the study of VWD in the United States and to contrast these studies with the studies initiated previously in the EU and Canada. As one of the components of this study we sought further insight into the clinical expression and penetrance of established types of VWD by performing full gene DNA sequence analysis in VWD patients and normal controls. This report is an interim report of the first 50 index cases and 113 normal individuals recruited into this study. Twenty four of these index cases were found to have known mutations, four of which had a second new mutation, and 11 cases had 1 or 2 new mutations. In cases where mutations were identified, 46% of the identified mutations were new mutations that have not been reported in the Sheffield VWF Mutation Database. In 15 patients, no mutations were identified in the coding region, although analysis of the non-coding regions is still in progress. Five of the mutations were deletions, insertions, or nonsense mutations that have clear functional consequences. The other 12 mutations were missense mutations. Since VWF polymorphisms are not well characterized in all exons, we have also completed studies of the first 113 normal control individuals in our study. These are individuals without a bleeding history and in whom full VWF laboratory testing and VWF sequencing was also undertaken. Since some estimates in the EU and Canadian studies have determined the prevalence of VWF mutations varies by the severity of type 1 VWD patients, we wanted to determine the frequency of VWF variation in a normal population and determine if sequence variations correlate with VWF levels. There were three linked common polymorphims identified in normal African Americans that are discussed elsewhere and are not included in this present analysis. We found 19 new sequence variations in the normal control group of which three (2900G>A, 6554G>A, 7997C>T) were found individually in 4–6% of the normal control samples. In addition, in 12 normal control samples we identified 6 sequence variations that were previously reported as VWF mutations. Four were reported as type 1 mutations (2220 G>A, 3686T>G, 3692A>C, 6859C>T) and two as type 2N mutations (2451T>A, 2771G>A). The 2220G>A and 2451T>A mutations were seen in 6 normal controls (5%) and 5 of these 6 normal controls had both mutations. In another normal control, both 3686T>G and 3692A>C were identified. Although the reported prevalence of VWD is 1% or greater, the frequency of these mutations in our normal controls is higher than expected (as high as 5%). In our normal control group, the mean VWF:Ag concentration in the patients with polymorphisms/mutations did not differ from the normal control group as a whole and did not cluster on the lower end of the normal range. Thus, the data on our normal individuals suggest that VWF gene variation is considerable and that many mutations and polymorphisms remain to be identified. Differentiation of those that affect the diagnosis of VWD and/or hemorrhagic risk continues to be difficult.

Acquired von Willebrand Syndrome in Cardiac Patients

p ON WILLEBRAND DISEASE (VWD) was first described by Erik von Willebrand in 1926. It is caused by quantitative nd/or qualitative defects of the von Willebrand factor (VWF). WF is a multimeric plasma protein composed of 250 kilodalon subunits and a multimer fraction that shows a wide range in ize from 500 kilodaltons to 10 million daltons. High–moleclar-weight (HMW) VWF multimers mediate platelet adhesion longside vascular injury by binding to connective tissue and to latelets. VWF also binds and stabilizes coagulation factor FVIII.1 herefore, patients’ bleeding symptoms may refer to platelet dysunction and/or deficiency of FVIII. VWD is classified as 3 main ypes: type 1 (VWF moderately reduced), type 3 (VWF absent), nd type 2 (VWF antigen in normal range, but qualitative defect). ype 2 VWD is divided into subtypes, with type 2A showing ecreased platelet adhesion resulting from the deficiency of mulimer fractions, type 2B with increased affinity for platelet glycorotein Ib, type 2M with reduced platelet adhesion despite relaively normal multimer fraction, and type 2N with decreased ffinity for FVIII and low FVIII levels.1 Inherited VWD is the ost frequent bleeding disorder, showing an autosomal dominant r recessive inheritance pattern in most cases, and the majority of atients exhibit mild-to-moderate mucocutaneous bleeding sympoms.2 In contrast, acquired von Willebrand syndrome (AVWS) is rare nd occurs in patients without a bleeding history. It is summarized nder VWS type 1 if associated with hypothyroidism or Wilm’s umor, or more commonly as VWS type 2A because of a lack of r decrease in the HMW VWF paralleled by a decrease in plateletependent functions.3 AVWS is associated with several underlyng diseases (eg, lymphoproliferative disorders, myeloproliferative isorders, neoplasias, hypothyroidism, thrombocythemia, immuologic diseases, and cardiovascular diseases4); with drugs like iprofloxacin, griseofulvin, valproic acid, and hydroxyethyl starch; nd with various pathogenic mechanisms including developing utoantibodies to VWF, adsorption of VWF onto tumor cells or ctivated platelets, increased proteolysis, and mechanical destrucion of HMW VWF multimers under high shear stress.5 It is well known that neither routine coagulation tests nor hromboelastometric measurements enable exact diagnosis of VWS. Testing for AVWS includes patient (lifelong mucocutaeous, postoperative bleeding) and family bleeding history, creening procedures (eg, platelet count, partial thromboplastin ime, concentration of FVIII [FVIII:C], platelet function analyzer PFA-100; Dade Behring, Marburg, Germany]), and confirming ests (eg, VWF antigen [VWF:Ag], VWF ristocetin cofactor acivity [VWF:RCo], VWF collagen-binding activity [VWF:CB], istocetin-induced platelet aggregation [RIPA], and analysis of WF multimers by gel electrophoresis).3 All these tests are time onsuming, and several are available only in specialized laboral

Identifying Carriers of Type 2N von Willebrand Disease

The defective FVIII carrier function of von Willebrand factor (VWF) identifies type 2N von Willebrand disease (VWD), a variant with a pattern resembling hemophilia A. Type 2N characterization is based on the evaluation of the capacity of VWF to bind exogenous FVIII (VWF:FVIIIB). Here we report on a retrospective evaluation of hemostatic laboratory parameters most useful in detecting type 2N carriers. The diagnostic capacity of aPTT, FVIII, VWF:Ag, FVIII/VWF:Ag ratio, VWF:FVIIIB and VWF:FVIIIB/VWF:Ag ratio was evaluated in 21 type 2N VWD carriers. Twenty subjects were heterozygous for the R854Q mutation, one was heterozygous for the R760C missense mutation, which interferes with cleavage of the VWF propeptide. We found that prolongation of aPTT and decrease in FVIII and FVIII/VWF:Ag ratio were not frequent findings in type 2N carriers. The same was true for VWF:FVIIIB which was not always abnormal. On the contrary, VWF:FVIIIB/VWF:Ag ratio was always defective and its values were not related with FVIII and FVIII/VWF:Ag ratio or influenced by plasma VWF concentration. Given these results, we attribute the greatest significance to VWF:FVIIIB/VWF:Ag ratio in the diagnosis of type 2N defects, and only search for type 2N mutations, to validate the diagnosis, if the ratio proves abnormal.

Von Willebrand disease—Phenotype versus genotype: Deficiency versus disease

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

The water buffalo: evolutionary, clinical and molecular cytogenetics

Although buffalo population is about 1/10 of that of cattle, buffaloes interest a larger human population, especially in the east countries. For this reason, this species is of great economic importance. Two main species of buffalo are found in the world: the Asiatic (water) buffalo (Bubalus bubalis) and the African buffalo (Syncerus caffer). These two different species have both two different sub-species differing in diploid number but interbreeding within the same genus. The water buffalo, especially the river type (2n=50), is the most important one and a summary of the most important cytogenetic findings obtained until now in this species is reported in this paper.

Intravenous DDAVP and Factor VIII-von Willebrand Factor Concentrate for the Treatment and Prophylaxis of Bleedings in Patients With von Willebrand Disease Type 1, 2 and 3

The current standard set of von Willebrand factor (VWF) parameters used to differentiate type 1 from type 2 VWD include bleeding times (BTs), factor VIII coagulant activity (FVIII:C), VWF antigen (VWF:Ag), VWF ristocetine cofactor activity (VWF:RCo), VWF collagen binding activity (VWF:CB), ristocetine induced platelet aggregation (RIPA), and analysis of VWF multimers in low and high resolution agarose gels and the response to DDAVP. The BTs and RIPA are normal in asymptomatic carriers of a mutant VWF allele, in dominant type 1, and in recessive type 2N VWD, and this category has a normal response of VWF parameters to DDAVP. The response of FVIII:C is compromised in type 2N VWD. The BTs and RIPA are usually normal in type Vicenza and mild type 2A VWD, and these two VWD variants show a transiently good response of BT and VWF parameters followed by short in vivo half life times of VWF parameters. The BTS are strongly prolonged and RIPA typically absent in recessive severe type 1 and 3 VWD, in dominant type 2A and in recessive type 2C (very likely also 2D) VWD and consequently associated with low or absent platelet VWF, and no or poor response of VWF parameters to DDAVP. The BTs are prolonged and RIPA increased in dominant type 2B VWD, that is featured by normal platelet VWF and a poor response of BT and functional VWF to DDAVP. The BTs are prolonged and RIPA decreased in dominant type 2A and 2U, that all have low VWF platelet, very low VWF:RCo values as compared to VWF:Ag, and a poor response of functional VWF to DDAVP. VWD type 2M is featured by the presence of all VWF multimers in a low resolution agarose gel, normal or slightly prolonged BT, decreased RIPA, a poor response of VWF:RCo and a good response of FVIII and VWF:CB to DDAVP and therefore clearly in between dominant type 1 and 2U. The existing recommendations for prophylaxis and treatment of bleedings in type 2 VWD patients with FVIII/VWF concentrates are mainly derived from pharmocokinetic studies in type 3 VWD patients. FVIII/VWF concentrates should be characterised by labelling with FVIII:C, VWF:RCo, VWF:CB and VWF multimeric pattern to determine their safety and efficacy in prospective management studies. As the bleeding tendency is moderate in type 2 and severe in type 3 VWD and the FVIII:C levels are near normal in type 2 and very low in type 3 VWD patients. Proper recommendations of FVIII/VWF concentrates using VWF:RCo unit dosing for the prophylaxis and treatment of bleeding episodes are proposed and has to be stratified for the severity of bleeding, the type of surgery either minor or major and for type 2 and type 3 VWD as well.

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

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

Sequencing of the factor 8(F8) coding regions in 10 Turkish hemophilia A patients reveals three novel pathological mutations, and one rediagnosis of von Willebrand's disease type 2N

The most common cause for severe cases of hemophilia A is the homologous recombination involving intron 22 and related sequences outside the F8 gene. F8 coding regions of the gene including the exon/intron junctions were sequenced in 10 Turkish hemophilia A patients all of whom have been typed negative for intron 22 inversion and who did not have a detectable change by DGGE analysis. Pathological changes including two novel deletions (c. 205del CT and c. 3699del ACAT), one novel missense mutation (9546A) and two recurrent missense mutations were observed in five patients. The c. 2110C > T is another novel pathological change affecting exonic splicing enhancer site in two patients. One of the remaining three patients had a recurrent vWD type 2N mutation in the F8 binding site of the vWF (C788R). The S1269S polymorphism (c. 3864A > C) detected phenotype. Conclusively, sequencing of the promoter and the coding regions of 10 hemophilia A patients contributes four novel pathological mutations to the F8 mutations list and reveals a rediagnosis of hemophilia A but is still not sufficient to confirm hemophilia A phenotype in two patients.

Type 2N von Willebrand disease due to compound heterozygosity for R854Q and a novel R763G mutation at the cleavage site of von Willebrand factor propeptide

Type 2N von Willebrand disease (VWD) is characterized by a markedly decreased affinity of von Willebrand factor (VWF) for factorVIII (FVIII) and is caused by mutations in the D' or D3 domain of mature VWF. We now report a French patient with an atypical 2N VWD phenotype associating FVIII deficiency with plasmaVWF unable to bind FVIII (undetectableVWF:FVIIIB) but with an abnormal multimeric profile. This patient is heterozygous for both the frequent R854Q type 2NVWD mutation and a novel R763G mutation at the cleavage site between VWF propeptide and mature VWF. Four children of the patient displayed moderately decreased VWF:FVIIIB of plasma VWF and were heterozygous for either the R763G or the R854Q mutation. Children with the R763G mutation displayed the same abnormal multimeric profile as their father. Recombinant VWF (rVWF) expression studies performed in COS-7 cells showed that the R763G mutation subtly affects its multimeric profile and dramatically impairs its FVIII binding function. Furthermore, the characteristics of hybrid G763/Q854 rVWF resulting from cotransfection experiments were in agreement with the type 2N VWD diagnosis of the patient. We conclude that R763G is a new type 2N VWD mutation located in the VWF propeptide which alters the proteolytic processing of VWF and consequently its binding to FVIII.

Secondary Chromosome Associations in &lt;i&gt;Ocimum&lt;/i&gt; spp.

Secondary association of chromosomes at metaphase I was noted in 61.94%, 60.00%, 50.38% and 21.74% meiocytes of Ocimum tenuiflorum–green type (2n=36), O. gratissimum (2n=40), O. canum (2n=26) and O. kilimandscharicum (2n=76) respectively. Secondary polyploidy has been attributed as the possible cause of secondary association of chromosomes and statistical analysis of cytological data revealed that the basic chromosome number in the species is 12 (x=6 for O. canum) with probable origin from x=6 through polyploidy.

Resolvable Maximum Packings with Quadruples

Let V be a finite set of v elements. A packing of the pairs of V by k-subsets is a family F of k-subsets of V, called blocks, such that each pair in V occurs in at most one member of F. For fixed v and k, the packing problem is to determine the number of blocks in any maximum packing. A maximum packing is resolvable if we can partition the blocks into classes (called parallel classes) such that every element is contained in precisely one block of each class. A resolvable maximum packing of the pairs of V by k-subsets is denoted by RP(v, k). It is well known that an RP(v, 4) is equivalent to a resolvable group divisible design (RGDD) with block 4 and group size h, where h = 1, 2 or 3. The existence of 4-RGDDs with group-type hn for h = 1 or 3 has been solved except for (h, n) = (3, 4) (for which no such design exists) and possibly for (h, n) ∈{(3, 88), (3,124)}. In this paper, we first complete the case for h = 3 by direct constructions. Then, we start the investigation for the existence of 4-RGDDs of type 2n. We shall show that the necessary conditions for the existence of a 4-RGDD of type 2n, namely, n≥4 and n ≡4 (mod 6) are also sufficient with 2 definite exceptions (n = 4, 10) and 18 possible exceptions with n = 346 being the largest. As a consequence, we have proved that there exists an RP(v, 4) for v≡0 (mod 4) with 3 exceptions (v = 8, 12 or 20) and 18 possible exceptions.

Two novel mutations, Q1053H and C1060R, located in the D3 domain of von Willebrand factor, are responsible for decreased FVIII‐binding capacity

In type 2N von Willebrand disease (VWD), von Willebrand factor (VWF) is characterized by a markedly decreased affinity for Factor VIII (FVIII), and the mutations responsible are essentially located in the D' domain of VWF. We report the identification, in seven unrelated French families, of two novel type 2N VWD mutations, Q1053H and C1060R (Gln290His and Cys297Arg in mature VWF sequence), in exon 24 of the VWF gene. These missense mutations have been identified in the heterozygous, homozygous or hemizygous states. Using site-directed mutagenesis and transient expression in COS-7 cells, we showed that both mutations, although located in the D3 domain of VWF, outside the tryptic fragment containing the FVIII domain, dramatically decrease the binding of VWF to FVIII. In contrast, the R924Q substitution, which was identified in a patient who was heterozygous for C1060R, was shown to be a polymorphism.

Identification of von Willebrand disease type 2N (Normandy) in Australia: a cross-laboratory investigation using different methods.

We report on a cross-laboratory study of type 2N von Willebrand disease (vWD). We tested 101 selected plasma samples for factor VIII and factor VIII binding activity of von Willebrand factor (vWF). Of these plasma samples, 31 were cotested by 2 specialist centers using different detection procedures for vWF-factor VIII binding: there was good agreement between results obtained by chromogenic assay and enzyme-linked immunosorbent assay. In total, 8 patients with type 2N vWD were identified. The 2-stage factor VIII assay detected a deficiency of factor VIII relative to vWF antigen in all 8 patients; the 1-stage factor VIII assay detected a relative deficiency in only 3 patients. Four patients were homozygous for the most common type 2N mutation (R854Q), 3 patients were presumed to be compound heterozygotes, and in 1 patient no type 2N mutations were identified. In this study of patients from 5 specialist centers in Australia, type 2N vWD was found in 5 families. The 2-stage factor VIII assay was more useful as a screening test than the 1-stage assay, and both vWF-factor VIII binding assays were equally effective.

Large experience with a factor VIII binding assay of plasma von Willebrand factor using commercial reagents.

The present diagnostic assay for type 2N von Willebrand disease (VWD) is based on the in vitro measurement of the capacity of plasma von Willebrand factor (VWF) to bind exogeneous factor VIII (VWF:FVIIIB). We report a method using only commercially available reagents that is easy to perform. This method has been validated in a cohort of 144 patients with FVIII/VWF ratios < 0.6 using a plasma control mixture representative of intermediate VWF:FVIIIB. In total, 15 patients were diagnosed with markedly decreased VWF:FVIIIB and five patients were shown to have moderately decreased VWF:FVIIIB. Specific type 2N mutations were identified in all these patients.