Molecular diagnosis of von Willebrand disease Baronciani, L.; Goodeve, A.; Peyvandi, F.
Haemophilia,
March 2017, 2017-Mar, 2017-03-00, 20170301, Volume:
23, Issue:
2
Journal Article
Peer reviewed
Open access
The role of molecular characterization in the diagnosis of von Willebrand disease (VWD) is not essential if the patients have been extensively investigated using phenotypic analysis. On the other ...hand, if some of these phenotype assays are not available, the identification of the mutation causing the disease could be crucial for an accurate diagnosis. Nevertheless, there are several reasons for performing molecular analysis in patients phenotypically well characterized, e.g. to identify the mutation causing VWD can be useful for patients and their family members when prenatal diagnosis is required (type 3 or severe type 2). In this manuscript, we report the techniques used for the molecular characterization of suspected VWD patients. We describe the use of online von Willebrand factor database and online single nucleotide variation databases, the former to verify whether a candidate mutation has been previously identified in other VWD patients and the latter to ascertain whether a putative mutation has been reported earlier in healthy individuals. We listed the available in silico analysis tools, to determine the predicted pathogenicity of a sequence variant and to establish its possible negative effect on the normal splicing process. We also report the strategy that can be used to identify VWD type 2 patients' mutations in subjects who have been fully characterized using the phenotype assays.
Summary
von Willebrand disease (VWD) is caused by a quantitative and/or qualitative deficiency of the von Willebrand factor (VWF). The laboratory diagnosis of VWD is dependent on the measurement of ...VWF antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo). The aim of this study was to undertake a two‐centre evaluation of two new automated VWF:Ag and VWF:RCo assays systems from Instrumentation Laboratory (Bedford, USA). Using the two new analytical systems that operated with different detection principles: immunoturbidimetric (TOP500 analyser) and chemiluminescent (AcuStar analyser), VWF:Ag and VWF:RCo levels were determined in samples from 171 healthy normal subjects, 80 VWD patients (16 type 1, 58 type 2 and 6 type 3) and 7 acquired von Willebrand syndrome patients. With commercial lyophilized normal and pathological plasmas VWF: Ag and VWF:RCo assays performed on both analysers exhibited low levels of inter‐assay imprecision (AcuStar: CV% range 3.3–6.9; TOP500: CV% range 2.6–6.3). Samples from normal healthy subjects (range: VWF:Ag 44.6–173.9 IU dL−1; VWF:RCo 43.1–191.5 IU dL−1) and patients (range: VWF:Ag <0.3–115.1 IU dL−1; VWF:RCo <0.5–57.2 IU dL−1) showed a good correlation between the two VWF:Ag and VWF:RCo methods (rs = 0.92 and 0.82 respectively), with only a few inconsistent cases among the patients' samples evaluated. The chemiluminescent assays had a lower limit of detection for both VWF:Ag and VWF:RCo compared to immunoturbidimetric tests (0.3 IU dL−1 vs. 2.2 IU dL−1 and 0.5 IU dL−1 vs. 4.4 IU dL−1 respectively). The TOP500 and AcuStar VWF:Ag and VWF:RCo assays were precise and compare well between centres, making these systems suitable for the diagnosis of VWD in non‐specialized and reference laboratories.
Essentials
New VWF activity assays are increasingly used but information on their comparability is limited.
This is an ISTH SSC‐organized study (expert labs, 5 countries) to compare all available ...assays.
VWF activity by six assays correlated well with each other.
The new assays show improved characteristics ‐ minor differences are noted.
Summary
Background
Several new assays have become available to measure von Willebrand factor (VWF) activity. The new assays appear to have improved performance characteristics compared with the old reference standard, ristocetin cofactor activity (VWF:RCo), but information is limited about how they compare with VWF:RCo and each other.
Methods
The von Willebrand factor Subcommittee of the International Society for Thrombosis and Haemostasis (ISTH) Scientific and Standardization Committee (SSC) designed a collaborative study involving expert laboratories from several countries to compare available tests with each other and with VWF:RCo. Eight laboratories from five countries were provided with blinded samples from normal healthy individuals and well‐characterized clinical cases. Laboratories measured VWF activity using all tests available to them; data from six laboratories, not affected by thawing during transportation, are included in this study.
Results
All tests correlated well with VWF:RCo activity (r‐values ranged from 0.963 to 0.989). Slightly steeper regression lines for VWF:Ab and VWF:GPIbM were clinically insignificant. The new assays showed improved performance characteristics. Of the commercially available assays, the VWF:GPIbR using the AcuStar system was the most sensitive and could reliably detect VWF activity below 1 IU dL−1. The lower limit of the measuring interval for the VWF:GPIbM and the VWF:GPIbR assays was in the 3–4 and 3–6 IU dL−1 range, respectively. Inter‐laboratory variation was also improved for most new assays.
Conclusion
All VWF activity assays correlated well with each other and the VWF:RCo assay. The slight differences in characteristics found in the COMPASS‐VWF study will assist the VWF community in interpreting and comparing activity results.
Introduction
Laboratory diagnosis of von Willebrand disease (VWD) is made by the measurement of von Willebrand factor (VWF) protein level and its activities. Current VWF activity tests include ...ristocetin cofactor and collagen binding (VWF:CB) assays.
Aim
We have undertaken an evaluation of a new fully automated VWF:CB assay relative to an established enzyme‐linked immunosorbent assay (ELISA) method.
Methods
The two analytical systems operate with different detection principles: a chemiluminescent method performed on ACL AcuStar Analyzer (the former) and a colorimetric ELISA by Asserachrom Stago (the latter) (type III collagen from human placenta). The HemosIL AcuStar VWF:CB assay is a chemiluminescent 2‐step immunoassay that uses magnetic particles coated with a type III collagen triple‐helical peptide. VWF:CB levels were determined in 50 healthy subjects and 100 VWD patients (22 type 1, 73 type 2 and 5 type 3).
Results
Eleven VWD samples reported VWF:CB values below the lower detection limit of one or both methods. The new method showed a good correlation with the ELISA method (r > .9, mean bias 3.85 IU/dL) in both healthy and VWD samples. One of 150 samples gave inconsistent results using the two assays, leading to an uncertain diagnosis of VWD type 1 (ELISA method) or type 2 MCB (fully automated method).
Conclusion
The new assay is rapid and simple to use, with its ready‐to‐use reagent cartridges. This VWF:CB assay, in addition to the measurement of VWF:Ag and VWF:RCo made on the same platform, gives additional information for the diagnosis of VWD in both nonspecialized and reference laboratories.
Introduction
We characterized five patients affected with von Willebrand disease (VWD) carrying the p.Arg1379Cys mutation. One was diagnosed as VWD type 1 and four as type 2M. The 2M patients also ...have the variant p.Ala1377Val in cis with p.Arg1379Cys.
Aim
To evaluate the role of p.Ala1377Val and p.Arg1379Cys von Willebrand factor (VWF) variants to explain patients' phenotype.
Methods
Conventional phenotype tests were used to evaluate patients' plasma and platelets. Direct sequence analysis of exon 28 was carried out. The allele frequency of p.Ala1377Val was evaluated using online database. pcDNA3.1‐VWF‐WT and mutant (A1377V, R1379C and A1377V‐R1379C) expression vectors were transiently transfected in HEK293 cells. The capacity of WT and mutant recombinant (r)VWF (along with patients' plasma VWF) to bind glycoprotein Ibα (GpIbα) were evaluated, using two ELISA assays. One with a wild‐type (WT) recombinant (r)GpIbα at increasing ristocetin concentrations (from 0 to 1.50 mg mL−1) and the other with a gain‐of‐function mutant rGpIbα (VWF:GPIbM).
Results
The substitution c.4130C>T (p.Ala1377Val) was reported as rare variant in online databases. At 0.25 mg mL−1 of ristocetin, WT, A1377V and R1379C showed 6, 7.5 and 12‐fold increased binding to rGpIbα, respectively. A1377V‐R1379C rVWF showed no increased binding to rGpIbα at the same ristocetin concentration and reached the highest binding, of only 3‐fold increased, at 1.50 mg mL−1 of ristocetin. The VWF:GPIbM showed strongly reduced values for the A1377V‐R1379C rVWF and the 2M patients' plasma.
Conclusion
Our study showed that the presence of both p.Ala1377Val and p.Arg1379Cys mutations (synergistic effect) abolishes the binding of rVWF to rGpIbα, explaining patients' 2M phenotype.
Summary
Background
Diagnosis of von Willebrand disease (VWD) type 2 usually relies on the discrepancy between the von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) and VWF antigen ...(VWF:Ag). Type 2B patients can be discriminated from other qualitative VWD variants by using ristocetin‐induced platelet agglutination (RIPA) test. The major limitation of RIPA is the requirement of fresh blood sample.
Objectives
In this study, we evaluated the VWF gain‐of‐function mutant GPIb binding (VWF:GPIbM) and VWF:RCo assays to investigate whether the VWF:GPIbM/VWF:RCo ratio was able to identify the type 2B variant among an heterogeneous VWD population, previously characterized following the ISTH‐SSC guidelines.
Patients/methods
Seventy‐six VWD patients and 31 healthy subjects were evaluated by using VWF:Ag, VWF:RCo, and VWF:GPIbM assays.
Results
The mean (minimum–maximum values) VWF:GPIbM/VWF:RCo ratio was higher in type 2B patients (2.53, 0.84–6.11) than in healthy controls (1.05, 0.87–1.34), type 1 (0.85, 0.51–1.15), 2A (1.20, 0.36–2.82), and 2M (1.07, 0.91–1.38) (P < 0.0001). Type 2B variants were divided into four groups (A, B, C, and D) according to their different multimeric patterns. The mean value of the VWF:GPIbM/VWF:RCo ratio in the four groups showed an increasing trend from group A (1.08) to D (3.69), proportional to the loss of high molecular weight multimers. Among 32 type 2B patients, previously diagnosed with RIPA, 8 (mainly with a type I New York/Malmö phenotype) were not confirmed using the VWF:GPIbM/VWF:RCo ratio.
Conclusions
Whenever the RIPA test is not feasible, the VWF:GPIbM/VWF:RCo ratio might help to identify severe type 2B VWD patients.
Summary
Background
In individuals with borderline von Willebrand factor (VWF) plasma levels, second‐level tests are required to confirm or exclude von Willebrand disease (VWD). These tests are ...time‐consuming and expensive.
Objective
To assess which parameters can predict VWD diagnosis in individuals with borderline VWF levels (30–60 IU dL−1).
Methods
Nine hundred and fifty individuals with bleeding episodes or abnormal coagulation test results were investigated with first‐level tests (blood count, prothrombin time, activated partial thromboplastin time, blood clotting factor VIII, VWF ristocetin cofactor activity VWF:RCo, and VWF antigen), and 93 (62 females and 31 males; median age, 28 years; interquartile range 15–44) had borderline VWF:RCo levels. All underwent second‐level investigations to confirm or exclude VWD. A multivariable logistic regression model was fitted with sex, age, bleeding score, family history, VWF:RCo and ABO blood group as predictors, and used to predict VWD diagnosis.
Results
Forty‐five of the 93 individuals (48%) had VWD (84% type 1). A negative linear relationship between VWF:RCo levels and risk of VWD diagnosis was present, and was particularly evident with blood group non‐O adjusted odds ratio 7.00 (95% confidence interval CI 1.48–33.11) for every 5 IU dL−1 decrease in VWF:RCo. The other variable clearly associated with VWD diagnosis was female sex (adjusted odds ratio 5.76 95% CI 1.47–22.53). The area under the receiver operating characteristic curve of the full logistic model was 0.89 (95% CI 0.82–0.95).
Conclusions
In individuals with borderline VWF, the two strongest predictors of VWD diagnosis are low VWF:RCo levels (particularly in those with blood group non‐O) and female sex. This predictive model has a promising discriminative ability to identify patients with borderline VWF levels who are likely to have VWD.
PDF
