RESUMO
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.
Assuntos
Inativação Gênica , Íntrons , Mutação de Sentido Incorreto , Splicing de RNA , Fator de von Willebrand/genética , Alelos , Sequência de Bases , Plaquetas/metabolismo , Biologia Computacional , Éxons , Feminino , Frequência do Gene , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucócitos/metabolismo , Masculino , Sítios de Splice de RNA , RNA Mensageiro/genética , Doenças de von Willebrand/genéticaRESUMO
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.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Doenças de von Willebrand/diagnóstico , Doenças de von Willebrand/genética , Fator de von Willebrand/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Espanha , Adulto JovemRESUMO
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.
Assuntos
Doenças de von Willebrand/genética , Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Espanha/epidemiologia , Doenças de von Willebrand/diagnóstico , Fator de von Willebrand/genéticaRESUMO
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.
Assuntos
Mutação , Doenças de von Willebrand/epidemiologia , Doenças de von Willebrand/genética , Fator de von Willebrand/genética , Estudos de Casos e Controles , Análise Mutacional de DNA/métodos , Feminino , Estudos de Associação Genética , Marcadores Genéticos , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Epidemiologia Molecular , Fenótipo , Valor Preditivo dos Testes , Sistema de Registros , Fatores de Risco , Espanha , Doenças de von Willebrand/diagnósticoRESUMO
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.
Assuntos
Proteínas ADAM/deficiência , Proteínas ADAM/genética , Testes Genéticos/métodos , Plasma , Polimorfismo de Nucleotídeo Único/genética , Púrpura Trombocitopênica Trombótica/congênito , Púrpura Trombocitopênica Trombótica/diagnóstico , Proteína ADAMTS13 , Predisposição Genética para Doença/genética , Humanos , Púrpura Trombocitopênica Trombótica/terapiaAssuntos
Proteínas ADAM/genética , Códon sem Sentido , Mutação da Fase de Leitura , Púrpura Trombocitopênica Trombótica/genética , Proteínas ADAM/biossíntese , Proteínas ADAM/química , Proteínas ADAM/deficiência , Proteína ADAMTS13 , Erros de Diagnóstico , Feminino , Genótipo , Células HEK293 , Heterozigoto , Humanos , Lactente , Masculino , Mutação de Sentido Incorreto , Linhagem , Estabilidade Proteica , Púrpura Trombocitopênica Idiopática/diagnóstico , Púrpura Trombocitopênica Trombótica/diagnóstico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , TransfecçãoRESUMO
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.
Assuntos
Próteses Valvulares Cardíacas , Valva Mitral/cirurgia , Doenças de von Willebrand/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticoagulantes/administração & dosagem , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Projetos PilotoRESUMO
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.
Assuntos
Plaquetas/metabolismo , Mutação , Doenças de von Willebrand/genética , Fator de von Willebrand/genética , Desamino Arginina Vasopressina/administração & dosagem , Eletroforese em Gel de Ágar , Feminino , Genes Dominantes , Humanos , Masculino , Peso Molecular , Fenótipo , Multimerização Proteica/efeitos dos fármacos , Fatores de Tempo , Doenças de von Willebrand/sangue , Doenças de von Willebrand/classificação , Fator de von Willebrand/química , Fator de von Willebrand/metabolismoRESUMO
Patients initially diagnosed with type 1 von Willebrand disease (VWD) have been reclassified as type 2 after a more exhaustive analysis in several studies. Our study's objectives were (1) to reanalyze patients that were previously diagnosed as type 1 to achieve a more accurate diagnosis and (2) to compare the von Willebrand factor (VWF) ristocetin cofactor assay (VWF:RCo) and the VWF collagen binding assay (VWF:CB) in order to evaluate the possibility of replacing the former assay with the latter in the diagnosis of VWD. Twenty-one patients from two large unrelated families and 104 normal controls were studied. VWF:Ag, VWF:RCo, FVIII coagulant activity (FVIII:C), bleeding time (BT), PFA(100), and multimeric analysis of VWF were tested. Genetic analysis by sequencing exon 28 on the VWF gene was also carried out. Patients presented lower levels of VWF:Ag and VWF:RCo, a dissociation between VWF:RCo/VWF:Ag, and the presence of all sizes of multimers in plasma VWF. The results for VWF:CB varied depending on the type of collagen used. The genetic analysis showed that the mutation R1374C is responsible for type 2M VWD. A high frequency of the R1374C mutation is observed in northwestern Spain (Galicia). Some types of 2M VWD are misdiagnosed as type 1 VWD. The VWF:CB (with type I collagen) assay was unable to discriminate defective platelet binding of the R1374C VWF. This confirms that VWF:CB cannot substitute for VWF:RCo, and both should be tested when diagnosing VWD.