Almudena Pérez-Rodríguez

Molecular and clinical profile of von Willebrand disease in Spain (PCM–EVW–ES): Proposal for a new diagnostic paradigm

The diagnosis of von Willebrand disease (VWD) remains difficult in a significant proportion of patients. A Spanish multicentre study investigated a cohort of 556 patients from 330 families who were analysed centrally. VWD was confirmed in 480. Next generation sequencing (NGS) of the whole coding VWF was carried out in all recruited patients, compared with the phenotype, and a final diagnosis established. A total of 238 different VWF mutations were found, 154 were not included in the Leiden Open Variation Database (LOVD). Of the patients, 463 were found to have VWF mutation/s. A good phenotypic/genotypic association was estimated in 96.5% of the patients. One hundred seventy-four patients had two or more mutations. Occasionally a predominant phenotype masked the presence of a second abnormality. One hundred sixteen patients presented with mutations that had previously been associated with increased von Willebrand factor (VWF) clearance. RIPA unavailability, central phenotypic results disagreement and difficult distinction between severe type 1 and type 3 VWD prevented a clear diagnosis in 70 patients. The NGS study facilitated an appropriate classification in 63 of them. The remaining seven patients presented with a VWF novel mutation pending further investigation. In five patients with a type 3 and two with a type 2A or 2B phenotype with no mutation, an acquired von Willebrand syndrome (AVWS) was suspected/confirmed. These data seem to support NGS as a first line efficient and faster paradigm in VWD diagnosis.

Acquired von Willebrand syndrome and mitral valve prosthesis leakage. A pilot study

Background: Of patients with severe aortic stenosis, 15–25% present with bleeding episodes possibly attributable to acquired von Willebrand syndrome (AVWS). AVWS associated with mitral valve prosthesis leakage has not been reported. Methods and Results: Five patients receiving appropriate oral anticoagulation showed mitral valve prosthesis leakage and bleeding episodes; all of them required hospitalization and two blood transfusions, and a von Willebrand factor (VWF) analysis was performed. Two patients with normal functioning metallic prosthesis valves were included as controls. Before surgery, after cessation of acenocumarol, the patients had prolonged activated partial thromboplastin time; four had prolonged closure time (CT) from the platelet function analyzer. Factor VIII procoagulant activity (FVIII:C), VWF ristocetin cofactor activity (VWF:RCo), and VWF collagen binding (VWF:CB) were considerably elevated, while VWF antigen (VWF:Ag) was most elevated. Disproportionate VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios were seen with the loss of large VWF multimers. Following surgery, all parameters were markedly increased and the ratios, CT, and multimeric VWF profile became normal. Conclusions: Acquired VWF qualitative alterations in mitral valve prosthesis leakage may be associated with or contribute to bleeding diathesis. AVWS should be taken into consideration in patients with mitral valve prosthesis leakage with bleeding diathesis not explained by excessive oral anticoagulation.

Inherited ADAMTS13 deficiency (Upshaw-Schulman syndrome): A short review

Congenital thrombotic thrombocytopenic purpura (TTP), also known as Upshaw-Schulman syndrome, is associated with an inherited deficiency of ADAMTS13, a von Willebrand factor-cleaving protease. It is a rare, life-threatening disorder characterized by thrombocytopenia, hemolytic anemia, neurological symptoms, renal dysfunction, and fever resulting from formation of platelet thrombi within the microvasculature. Patients have initial episodes mainly during infancy or early childhood, and are conventionally treated with fresh frozen plasma. However, a more appropriate approach based on recombinant ADAMTS13 is slated to begin shortly. Mutations throughout the ADAMTS13 have been identified in congenital TTP patients. The prevalence of this entity is probably underestimated because it is often not suspected, the clinical course is usually heterogeneous and most of the symptoms are common to other diseases. The present review summarizes our current knowledge about Upshaw-Schulman syndrome.

Autosomal dominant C1149R von Willebrand disease: phenotypic findings and their implications

The classification and even the diagnosis of von Willebrand disease continues to evolve. In this paper, the authors show how a detailed examination of difficult cases using clinical laboratory and molecular analyses can be used to reach a clinically useful conclusion. See related perspective article on page 610. Background Mutation C1149R in the von Willebrand factor (VWF) gene has been thought to cause autosomal dominant severe type 1 von Willebrand disease (VWD). Design and Methods Eight patients from three unrelated families with this mutation were included in the present study who had distinct VWF abnormalities, not described in earlier studies. Results The patients showed notably low levels of VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), VWF collagen binding (VWF:CB), and a reduced ristocetin-induced platelet aggregation (RIPA). VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios were lower than 0.7. At basal conditions, all the VWF multimers were decreased in plasma, with a clearly lower relative proportion of the high molecular weight VWF multimers (HMWM). In high-resolution agarose gels, a large decrease in the relative proportions of the satellite bands was seen. The patients had a brief good response to desmopressin (DDAVP) administration, but the released VWF half-life was shorter than normal, indicating an accelerated clearance of their VWF. Platelet VWF was abnormal. Conclusions We conclude from the results obtained in these patients for plasma phenotypic data that this mutation should be classified as a VWD type 2A (IIE). DDAVP therapy may be somewhat helpful for this mutation, at least for mild to moderate bleeding. These data provide evidence that for VWD classification factors other than basal VWF, such as DDAVP response and platelet VWF, should be considered.

Diagnosis and management of von Willebrand disease in Spain.

The correct diagnosis and classification of Von Willebrand disease (VWD) is important for therapy and genetic counseling but is made difficult due to the variability of its clinical expression and limitations of laboratory methods. A national registry of VWD patients has been initiated in Spain. The results of a concise survey on the diagnosis of VWD show the frequency of VWD is fivefold greater in Spain than that expected from epidemiological studies in other European countries; this may result from overdiagnosis and/or a higher prevalence of VWD. These results clearly reinforce the need for the Spanish VWD registry. A consensus guideline for optimal treatment of VWD is being elaborated in Spain. Desmopressin (DDAVP) is the choice of treatment in responsive VWD patients. Von Willebrand factor concentrates (VWF/factor VIII) are used in individuals nonresponsive to DDAVP, when DDAVP is contraindicated, or in VWD types 2B and 3.

Molecular and clinical profile of von Willebrand disease in Spain (PCM-EVW-ES): Comprehensive genetic analysis by next-generation sequencing of 480 patients

Molecular diagnosis of patients with von Willebrand disease is pending in most populations due to the complexity and high cost of conventional molecular analyses. The need for molecular and clinical characterization of von Willebrand disease in Spain prompted the creation of a multicenter project (PCM-EVW-ES) that resulted in the largest prospective cohort study of patients with all types of von Willebrand disease. Molecular analysis of relevant regions of the VWF, including intronic and promoter regions, was achieved in the 556 individuals recruited via the development of a simple, innovative, relatively low-cost protocol based on microfluidic technology and next-generation sequencing. A total of 704 variants (237 different) were identified along VWF, 155 of which had not been previously recorded in the international mutation database. The potential pathogenic effect of these variants was assessed by in silico analysis. Furthermore, four short tandem repeats were analyzed in order to evaluate the ancestral origin of recurrent mutations. The outcome of genetic analysis allowed for the reclassification of 110 patients, identification of 37 asymptomatic carriers (important for genetic counseling) and re-inclusion of 43 patients previously excluded by phenotyping results. In total, 480 patients were definitively diagnosed. Candidate mutations were identified in all patients except 13 type 1 von Willebrand disease, yielding a high genotype-phenotype correlation. Our data reinforce the capital importance and usefulness of genetics in von Willebrand disease diagnostics. The progressive implementation of molecular study as the first-line test for routine diagnosis of this condition will lead to increasingly more personalized and effective care for this patient population.

Role of multimeric analysis of von Willebrand factor (VWF) in von Willebrand disease (VWD) diagnosis: Lessons from the PCM-EVW-ES Spanish project

The multimeric analysis (MA) of plasma von Willebrand factor (VWF) evaluates structural integrity and helps in the diagnosis of von Willebrand disease (VWD). This assay is a matter of controversy, being considered by some investigators cumbersome and only slightly informative. The centralised study ‘Molecular and Clinical Profile of von Willebrand Disease in Spain (PCM-EVW-ES)’ has been carried out by including the phenotypic assessment and the genetic analysis by next generation sequencing (NGS) of the VWF gene (VWF). The aim of the present study was to evaluate the role of MA to the diagnosis of these patients and their potential discrepancies. Two hundred and seventy out of 480 patients centrally diagnosed with VWD had normal multimers, 168 had abnormal multimers and 42 a total absence of multimers. VWF MA was of great significance in the diagnosis of 83 patients (17.3%), it was also of help in the diagnosis achieved in 365 additional patients (76%) and was not informative in 32 cases (6.7%). With regard to discrepancies, 110 out of 480 (23%) patients centrally diagnosed with VWD presented some kind of discordance between VWF:RCo/VWF:Ag and/or VWF:CB/VWF:Ag ratios, multimeric study and/or genetic results. The VWF MA was key in the presence of novel mutations as well as in cases with phenotypic discrepancies. A comparison between the contribution of MA and VWF:CB showed a clearly higher contribution of the former in the diagnostic process. These data seem to reinforce the relevance of the VWF MA in VWD diagnosis, despite all its limitations.

Unraveling the effect of silent, intronic and missense mutations on VWF splicing: contribution of next generation sequencing in the study of mRNA.

Large studies in von Willebrand disease patients, including Spanish and Portuguese registries, led to the 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 next-generation 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.7437G>A) demonstrated both molecular pathogenic mechanisms simultaneously; and the p.Cys370Tyr missense mutation generated two aberrant transcripts. Of note, the complete effect of three 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 four 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. clinicaltrials.gov identifier:02869074.