Fifth Åland Island conference on von Willebrand disease.

The fifth Åland Island meeting on von Willebrand disease (VWD) was held on the Åland Islands, Finland, from 22 to 24 September 2016-90 years after the first case of VWD was diagnosed in a patient from the Åland Islands in 1926. This meeting brought together experts in the field of VWD to share knowledge and expertise on current trends and challenges in VWD. Topics included the storage and release of von Willebrand factor (VWF), epidemiology and diagnostics in VWD, treatment of VWD, angiogenesis and VWF inhibitors.

Molecular diagnosis of von Willebrand disease.

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.

Genetic analysis of bleeding disorders.

Molecular genetic analysis of inherited bleeding disorders has been practised for over 30 years. Technological changes have enabled advances, from analyses using extragenic linked markers to next-generation DNA sequencing and microarray analysis. Two approaches for genetic analysis are described, each suiting their environment. The Christian Medical Centre in Vellore, India, uses conformation-sensitive gel electrophoresis mutation screening of multiplexed PCR products to identify candidate mutations, followed by Sanger sequencing confirmation of variants identified. Specific analyses for F8 intron 1 and 22 inversions are also undertaken. The MyLifeOurFuture US project between the American Thrombosis and Hemostasis Network, the National Hemophilia Foundation, Bloodworks Northwest and Biogen uses molecular inversion probes (MIP) to capture target exons, splice sites plus 5' and 3' sequences and to detect F8 intron 1 and 22 inversions. This allows screening for all F8 and F9 variants in one sequencing run of multiple samples (196 or 392). Sequence variants identified are subsequently confirmed by a diagnostic laboratory. After having identified variants in genes of interest through these processes, a systematic procedure determining their likely pathogenicity should be applied. Several scientific societies have prepared guidelines. Systematic analysis of the available evidence facilitates reproducible scoring of likely pathogenicity. Documentation of frequency in population databases of variant prevalence and in locus-specific mutation databases can provide initial information on likely pathogenicity. Whereas null mutations are often pathogenic, missense and splice site variants often require in silico analyses to predict likely pathogenicity and using an accepted suite of tools can help standardize their documentation.

Hemophilia B: molecular pathogenesis and mutation analysis.

Hemophilia B is an X-chromosome-linked inherited bleeding disorder primarily affecting males, but those carrier females with reduced factor IX activity (FIX:C) levels may also experience some bleeding. Genetic analysis has been undertaken for hemophilia B since the mid-1980s, through linkage analysis to track inheritance of an affected allele, and to enable determination of the familial mutation. Mutation analysis using PCR and Sanger sequencing along with dosage analysis for detection of large deletions/duplications enables mutation detection in > 97% of patients with hemophilia B. The risk of the development of inhibitory antibodies, which are reported in ~ 2% of patients with hemophilia B, can be predicted, especially in patients with large deletions, and these individuals are also at risk of anaphylaxis, and nephrotic syndrome if they receive immune tolerance induction. Inhibitors also occur in patients with nonsense mutations, occasionally in patients with small insertions/deletions or splice mutations, and rarely in patients with missense mutations (p.Gln237Lys and p.Gln241His). Hemophilia B results from several different mechanisms, and those associated with hemophilia B Leyden, ribosome readthrough of nonsense mutations and apparently 'silent' changes that do not alter amino acid coding are explored. Large databases of genetic variants in healthy individuals and patients with a range of disorders, including hemophilia B, are yielding useful information on sequence variant frequency to help establish possible variant pathogenicity, and a growing range of algorithms are available to help predict pathogenicity for previously unreported variants.

Genomics of bleeding disorders.

Molecular genetic tools are widely applied in inherited bleeding disorders. New genes involved in haemorrhagic disorders have been identified by genome wide linkage analysis on families with a specific phenotype. LMNA1 or MCFD in combined FV/FVIII-deficiency and VKORC1 in vitamin K coagulation factor deficiency type 2 are two examples. Identification of the causative gene mutation has become standard for most bleeding disorders. Knowledge of the causative mutation allows genetic counselling in affected families and most importantly adds to the pathophysiological understanding of phenotypes. Haemophilia A represents a model as the F8 gene mutation predicts the risk of developing an inhibitor and more recently also the bleeding phenotype. In this review novel genetic diagnostic strategies for bleeding disorders are outlined and inhibitor formation is presented as an example for clinical relevant phenotype/genotype correlation studies.

Genetic testing in bleeding disorders.

The aim of molecular genetic analysis in families with haemophilia is to identify the causative mutation in an affected male as this provides valuable information for the patient and his relatives. For the patient, mutation identification may highlight inhibitor development risk or discrepancy between different factor VIII assays. For female relatives, knowledge of the familial mutation can facilitate carrier status determination and prenatal diagnosis. Recent advances in understanding mutations responsible for haemophilia and methods for their detection are presented. For reporting of such mutations, participation in external quality assessment ensures that essential patient and mutation details are routinely included and that pertinent information is incorporated in the interpretation.

Laboratory aspects of von Willebrand disease: test repertoire and options for activity assays and genetic analysis.

The deficiency or abnormal function of von Willebrand factor (VWF) causes von Willebrand disease (VWD), the most frequent inherited bleeding disorder. The laboratory diagnosis of VWD can be difficult as the disease is heterogeneous and an array of assays is required to describe the phenotype. Basic classification of quantitative (type 1 and 3) and qualitative (type 2) VWD variants requires determination of VWF antigenic (VWF:Ag) levels and assaying of VWF ristocetin cofactor (VWF:RCo) activity, determining the capacity of VWF to interact with the platelet GPIb-receptor. Knowing the VWF:RCo activity is essential for identifying, subtyping and monitoring VWD, but the assay is poorly standardized and many protocols do not fulfil the clinical need in all situations. This has led to the development of novel activity assays, independent of ristocetin, with enhanced assay characteristics. Results from the first independent clinical evaluations are promising, showing that they are reliable and suitable for VWD diagnosis. The qualitative type 2 VWF deficiency can be further divided into four different subtypes (A, B, M and N) using specific assays that explore other activities or the size distribution of VWF multimers. These methods are discussed herein. However, in a number of patients it may be difficult to correctly classify the VWD phenotype and genetic analysis may provide the best option to clarify the disorder, through mutation identification.

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

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

Genetics of haemostasis.

Congenital defects of platelets or plasma proteins involved in blood coagulation generally lead to bleeding disorders. In some of these disorders, patients with a severe phenotype are prone to spontaneous bleeds with critical consequences. This situation occurs more commonly in haemophilia A and haemophilia B and to a certain extent in severe forms (type 3) of von Willebrand disease. Defects in other plasma coagulation proteins and platelet factors are relatively rare, with an incidence of ≤ 1: 1-2 million. Molecular genetic studies of the human coagulation factors, especially factors VIII and IX, have contributed to a better understanding of the biology of these genetic disorders, the accurate detection of carriers and genetic counselling, and have also fostered new therapeutic strategies. This article reviews the evolution of genetics over the last five decades as a tool for bleeding disorder investigations, the recent advances in molecular techniques that have contributed to improved genetic diagnosis of this condition, and the development and utility of proficiency testing programmes and reference materials for genetic diagnosis of bleeding disorders.

Deep intronic variations may cause mild hemophilia A.

BACKGROUND: In about 10% of patients with mild hemophilia A, no candidate gene mutations are apparent after complete gene sequencing. AIM OF THE STUDY: To analyze factor VIII gene (F8) mRNA for mutations in five families with mild hemophilia A with no apparent genomic mutation and a reduced response to desmopressin. RESULTS: In four cases, mRNA studies revealed the presence of an abnormal mRNA transcript in addition to normal F8 mRNA. Sequencing of the abnormal transcripts revealed complex abnormalities, which allowed the identification of three different intronic variations (c.2113+1152delA, c.5587-93C>T and c.5999-277G>A) at the DNA level, absent from 387 normal alleles. By in silico analysis, c.2113+1152delA and c.5587-93C>T were strongly predicted to result in the generation of new splice sites with the introduction of premature termination codons, while c.5999-277G>A was predicted to generate a new protein with 30 additional amino acids. However, these predictions were not homogeneous across the different mutations and programs used. The detrimental effect of two mutations was also confirmed by in vitro expression studies. These changes were also identified in related female carriers and in other mild HA patients not included in the original study. No mRNA abnormality was identified in the remaining patient. CONCLUSIONS: Although rare, deep intronic variations may be responsible for mild hemophilia A where no other F8 mutations have been identified and may be associated with a reduced biologic response to desmopressin. F8 mRNA analysis is a useful tool for the identification of deep intronic variation not detectable by standard DNA sequencing.

F8 and F9 mutations fail to co-segregate in a family with co-incident haemophilia A and B.

Haemophilia A and B in one individual may arise from co-incident inheritance of independent mutations in the F8 and F9 genes. However, this association is rare and has been studied poorly at a genetic level. We report a male patient with abnormal bleeding and reduced factor VIII:C (26 IU dL(-1)) and factor IX:C (35 IU dL(-1)). This index case harboured a F8 c.979C>G transversion (predictive of p.Leu327Val) and a F9 c.845A>G transition (predictive of p.His282Arg) which have been previously associated with mild haemophilia A and B, respectively. Identical F8 and F9 mutations were identified in the mother and maternal grandmother. However, an affected maternal uncle showed only the F8 c.979C>G mutation, indicating haemophilia A alone. The sister of the index case was heterozygous only for F9 c.845A>G, indicating carriership of haemophilia B alone. The non-Mendelian inheritance of F8 c.979C>G and F9 c.845A>G in this kindred is consistent with recombination between F8 and F9 and illustrates the large recombination distance between these loci. Recognition of this phenomenon was essential for accurate genetic counselling in this kindred.

Validation of a rapid test (VWF-LIA) for the quantitative determination of von Willebrand factor antigen in type 1 von Willebrand disease diagnosis within the European multicenter study MCMDM-1VWD.

BACKGROUND: Accurate measurement of von Willebrand factor (VWF) is a critical requirement for the diagnosis of von Willebrand disease (VWD). AIM OF THE STUDY: To evaluate the diagnostic efficiency of a rapid quantitative test for the measurement of VWF antigen (VWF:Ag) in type 1 VWD. PATIENTS AND METHODS: VWF:Ag was measured with an ELISA in a robotic instrument, as a reference method, and with a fully automated latex-immunoassay (LIA) on an ACL 9000 analyser in 1,716 subjects enrolled within the Molecular and Clinical Markers for Diagnosis and Management of Type 1 von Willebrand Disease (MCMDM-1VWD) Study. Among these subjects, 1,049 were healthy controls, 281 healthy family members and 386 affected members from 127 European families with type 1 VWD. RESULTS: The assay linearity range was 10-125 IU/dL for LIA (R2=0.99) and 5-133 IU/dL for ELISA (R2=0.99). The inter-assay CV for low VWF levels (approximately 30 IU/dL) was 2% for the LIA test and 8.7 % for ELISA. The sensitivity for detection of type 1 VWD affected members was 86% and the specificity 91% for LIA, 87% and 90% for ELISA. A receiver-operator (ROC) analysis disclosed only a marginal difference between the two tests, LIA having a slightly greater area under the curve (0.94 vs. 0.93, p=0.03). CONCLUSION: VWF:Ag LIA compared well to standard ELISA in this large population of patients and controls, showing better CV. However the lower detection limit for the VWF:Ag LIA compared to the VWF:Ag ELISA means that the LIA assay is less good at discriminating between type 3 VWD and moderate type 1 VWD.

Haemophilia A and von Willebrand's disease.

SUMMARY: Deficient or defective coagulation factor VIII (FVIII) and von Willebrand factor (VWF) can cause bleeding through congenital deficiency or acquired inhibitory antibodies. Recent studies on type 1 von Willebrand's disease (VWD), the most common form of the disease, have begun to explain its pathogenesis. Missense mutations of varying penetrance throughout VWF are the predominant mutation type. Other mutation types also contribute while about one-third of patients have no mutation identified. Enhanced clearance and intracellular retention contribute to pathogenic mechanisms. Chromogenic substrate (CS) methods to determine FVIII coagulant activity have several advantages over one-stage methods, which include minimal influence by variable levels of plasma components, notably lupus anticoagulant. Direct proportionality between FVIII activity and FXa generation results in high resolution at all FVIII levels, rendering the CS method suitable for measuring both high and low levels of FVIII activity. FVIII inhibitors in patients with inherited or acquired haemophilia A present several challenges in their detection and accurate quantification. The Nijmegen method, a modification of the Bethesda assay is recommended for inhibitor analysis by the International Society on Thrombosis and Haemostasis. Understanding potential confounding factors including heparin and residual FVIII in test plasma, plus optimal standardization can reduce assay coefficient of variation to 10-20%.These areas are all explored within this article.

von Willebrand factor variant p.Arg924Gln marks an allele associated with reduced von Willebrand factor and factor VIII levels.

BACKGROUND: von Willebrand factor (VWF) variant c.2771G>A; p.R924Q has been described as a benign polymorphism or a possible marker for a null allele and been associated with mild bleeding phenotypes. It was identified in several patients in recent type 1 von Willebrand disease (VWD) studies. OBJECTIVES: To determine whether the p.R924Q allele contributes to reduced VWF levels and type 1 VWD. METHODS: One thousand one hundred and fifteen healthy controls and 148 index cases from the MCMDM-1VWD study were genotyped for c.2771G>A; VWF and FVIII levels were analyzed in ABO blood group stratified individuals and the p.R924Q variant was expressed in 293 EBNA cells. RESULTS: c.2771G>A was present in six index cases, five of whom had a second VWF variant which probably contributed to the phenotype. A common core haplotype identified in families, which included the rare G allele of c.5843-8C>G, was present in the majority of 35 c.2771G>A heterozygous controls. c.2771G>A contributed about 10% variance in VWF and FVIII levels in controls and 35% variance when co-inherited with blood group O. Recombinant p.R924Q VWF had no effect on in vitro expression and heterozygous family members had normal VWF-FVIII binding and normal clearance of VWF and FVIII. CONCLUSIONS: The allele bearing c.2771A leads to reductions in VWF and FVIII levels particularly in combination with blood group O. Its inheritance alone may be insufficient for VWD diagnosis, but it appears to be associated with a further VWF level reduction in individuals with a second VWF mutation and it contributes to population variance in VWF and FVIII levels.