Clinical relevance of genetic variants in the von Willebrand factor according to in-silico methods.

Clinical interpretation of genetic variants in the context of the patient's phenotype is a time-consuming and costly process. In-silico analysis using in-silico prediction tools, and molecular modeling have been developed to predict the influence of genetic variants on the quality and/or quantity of the resulting translated protein, and in this way, to alert clinicians of disease likelihood in the absence of previous evidence. Our objectives were to evaluate the success rate of the in-silico analysis in predicting the disease-causing variants as pathogenic and the single-nucleotide variants as neutral, and to establish the reliability of in-silico analysis for determining pathogenicity or neutrality of von Willebrand factor gene-associated genetic variants. Using in-silico analysis, we studied pathogenicity in 31 disease-causing variants, and neutrality in 61 single-nucleotide variants from patients previously diagnosed as type 2 von Willebrand disease. Disease-causing variants and non-synonymous single-nucleotide variants were explored by in-silico tools that analyze the amino acidic sequence. Intronic and synonymous single-nucleotide variants were analyzed by in-silico methods that evaluate the nucleotidic sequence. We found a consistent agreement between predictions achieved by in-silico prediction tools and molecular modeling, both for defining the pathogenicity of disease-causing variants and the neutrality of single-nucleotide variants. Based on our results, the in-silico analysis would help to define the pathogenicity or neutrality in novel genetic variants observed in patients with clinical and laboratory phenotypes suggestive of von Willebrand disease.

From the Discovery of ADAMTS13 to Current Understanding of Its Role in Health and Disease.

ADAMTS13 (a disintegrin-like metalloprotease domain with thrombospondin type 1 motif, member 13) is a protease of crucial importance in the regulation of the size of von Willebrand factor multimers. Very low ADAMTS13 activity levels result in thrombotic thrombocytopenic purpura, a rare and life-threatening disease. The mechanisms involved can either be acquired (immune-mediated thrombotic thrombocytopenic purpura [iTTP]) or congenital (cTTP, Upshaw-Schulman syndrome) caused by the autosomal recessive inheritance of disease-causing variants (DCVs) located along the ADAMTS13 gene, which is located in chromosome 9q34. Apart from its role in TTP, and as a regulator of microthrombosis, ADAMTS13 has begun to be identified as a prognostic and/or diagnostic marker of other diseases, such as those related to inflammatory processes, liver damage, metastasis of malignancies, sepsis, and different disorders related to angiogenesis. Since its first description almost 100 years ago, the improvement of laboratory tests and the description of novel DCVs along the ADAMTS13 gene have contributed to a better and faster diagnosis of patients under critical conditions. The ability of ADAMTS13 to dissolve platelet aggregates in vitro and its antithrombotic properties makes recombinant human ADAMTS13 treatment a potential therapeutic approach targeting not only patients with cTTP but also other medical conditions.

Type 2A and 2M von Willebrand Disease: Differences in Phenotypic Parameters According to the Affected Domain by Disease-Causing Variants and Assessment of Pathophysiological Mechanisms.

Type 2A and 2M von Willebrand disease (VWD) broadly show similar phenotypic parameters, but involve different pathophysiological mechanisms. This report presents the clinical and laboratory profiles of type 2A and type 2M patients genotypically diagnosed at one large center. Higher bleeding score values and a higher incidence of major bleeding episodes were observed in type 2A compared with type 2M, potentially reflective of the absence of large and intermediate von Willebrand factor (VWF) multimers in 2A. In type 2A, most of disease-causing variants (DCVs) appeared to be responsible for increased VWF clearance and DCV clustered in the VWF-A1 domain resulted in more severe clinical profiles. In type 2M, DCV in the VWF-A1 domain showed different laboratory patterns, related to either reduced synthesis or shortened VWF survival, and DCV in the VWF-A2 domain showed patterns related mainly to shortened survival. VWF-type 1 collagen binding/Ag (C1B/Ag) showed different patterns according to DCV location: in type 2A VWD, C1B/Ag was much lower when DCVs were located in the VWF-A2 domain. In type 2M with DCV in the VWF-A1domain, C1B/Ag was normal, but with DCV in the VWF-A2 domain, C1B/Ag was low. The higher frequency of major bleeding in VWD 2M patients with DCV in the VWF-A2 domain than that with DCV in the VWF-A1 domain could be a summative effect of abnormal C1B/Ag, on top of the reduced VWF-GPIb binding. In silico modeling suggests that DCV impairing the VWF-A2 domain somehow modulates collagen binding to the VWF-A3 domain. Concomitant normal FVIII:C/Ag and VWFpp/Ag, mainly in type 2M VWD, suggest that other nonidentified pathophysiological mechanisms, neither related to synthesis/retention nor survival of VWF, would be responsible for the presenting phenotype.

Variantes genéticas frecuentes del factor von Willebrand: su influencia en el laboratorio y la clínica / Frequent genetic variants of von Willebrand factor: its influence in the laboratory and clinic

El factor von Willebrand (VWF) es una glucoproteína altamente polimórfica. Se describen aquí diferentes variantes genéticas asintomáticas altamente frecuentes, sus influencias sobre los estudios fenotípicos, en los niveles plasmáticos del mismo, y por consiguiente en diferentes entidades clínicas. Se detallan también variaciones en la frecuencia alélica según las etnias analizadas. El objetivo de este trabajo fue alertar sobre la necesidad de conocer la frecuencia de los polimorfismos en la población normal para evitar posibles conclusiones erróneas al momento del hallazgo de cambios no previamente reportados en la literatura científica.

The von Willebrand factor (VWF) is a highly polymorphic glycoprotein. Several frequent asymptomatic genetic variants, their influences on phenotypic studies, on the plasma levels of VWF, and therefore in different clinical entities are described here. Variations in allele frequency in different ethnic groups analyzed are also detailed. The aim of this study was to highlight the need to know the frequency of polymorphisms in the normal population to avoid possible erroneous conclusions at the time of finding genetic variants not previously reported in the scientific literature.

O fator von Willebrand (VWF) é uma glicoproteína altamente polimórfica. Diversas variantes genéticas assintomáticas muito frequentes são descritas aqui, suas influências em estudos fenotípicos, nos níveis plasmáticos de VWF e, portanto, em diferentes entidades clínicas. Variações na frequência alélica também são detalhadas segundo diferentes grupos étnicos analisados. O objetivo desse trabalho é alertar sobre a necessidade de conhecer a frequência dos polimorfismos na população normal, a fim de evitar possíveis conclusões errôneas no momento de encontrar variações genéticas não relatadas anteriormente na literatura científica.

Combined effects of two mutations in von Willebrand disease 2M phenotype.

BACKGROUND: Type 2M von Willebrand disease (VWD2M) is usually characterized by VWF:RCo/VWF:Ag<0.6 and normal multimeric profile; desmopressin (DDAVP) challenge test commonly shows poor response of VWF:RCo. OBJECTIVE: We describe the bleeding tendency and the laboratory phenotype in a patient carrying two heterozygous mutations affecting VWF-A1 domain and VWF-A2 domain. SUBJECTS/METHODS: A 12-year-old patient (O blood group) with severe hemorrhagic tendency was phenotypically and genotypically analyzed; his parents were also studied. RESULTS: The proband showed decrease FVIII:C, VWF:RCo/VWF:Ag, and VWF:CB6/VWF:Ag ratios, but normal platelet count, VWF:CB1/VWF:Ag ratio, VWFpp and multimeric pattern, suggesting a VWD2M phenotype. The DDAVP challenge test, compared to controls (VWD2M patients with mutations in VWF-A1 domain), showed lower increase of FVIII:C and VWF:Ag than in heterozygous, but very similar to homozygous control. Two mutations were found in heterozygous and trans presentation: p.Pro1648fs*45 and a novel missense mutation, p.Arg1426Cys. The mother was p.Arg1426Cys heterozygous carrier, with few clinical symptoms. The father was asymptomatic, with no mutations. The p.Pro1648fs*45 was considered an apparent de novo mutation; proband's AS-PCR revealed mosaicism in the paternal allele. According to the predicted models, p.Arg1426Cys would not be affecting the binding of GPIbα to A1 domain, whereas p.Pro1648fs*45 seems to modify the folding of A2 domain, and in this way, it would affect the binding to GPIbα and type VI collagen. We believe that the combination of these two heterozygous mutations, in a child with O blood group, could result in a defective phenotype enhancer.

PT-VWD posing diagnostic and therapeutic challenges - small case series.

Despite the increased worldwide awareness, over the last decade, of the platelet-type von Willebrand Disease (PT-VWD), many uncertainties remain around this rare platelet bleeding disorder. This report aims to correctly identify and study the phenotype of new patients and highlights the diagnostic and therapeutic challenges this disease remains to pose. We describe four PT-VWD cases confirmed by genetic analysis in which either the diagnosis and/or the treatment posed challenge. We provide the details of the clinical presentation, laboratory analysis, and the treatment and the responses in each case. We show that in addition to type 2B VWD, PT-VWD can be misdiagnosed as idiopathic thrombocytopenic purpura, neonatal alloimmune thrombocytopenia, and unexplained gestational thrombocytopenia. The disease can be diagnosed as early as 1 year of age and with phenotypically normal parents. Bleeding in some patients can be managed successfully using Humate P and DDAVP combined with tranexamic acid with no significant thrombocytopenia. We provide for the first time an evidence of an efficient response to rFVIIa in PT-VWD. Anaphylactic reaction to VWF preparations may be related to PT-VWD and the development of HLA antibodies is not uncommon. Progressive thrombocytopenia with normal VWF levels can be seen with PT-VWD and the platelet count was normalized at 2.5 weeks postpartum in one case. We conclude that these studies represent a record of clinical observations/interventions that help improve diagnoses/management of PT-VWD, highlight the variations in age and clinical presentations, laboratory diagnostic approaches, the importance of genetic testing for accurate diagnosis and consideration of therapeutic alternatives.

Phenotypic Parameters in Genotypically Selected Type 2B von Willebrand Disease Patients: A Large, Single-Center Experience Including a New Novel Mutation.

von Willebrand disease type 2B (VWD2B) expresses gain-of-function mutations that enhance binding of an individual's von Willebrand factor (VWF) to its platelet ligand, glycoprotein Ib (GPIb), and which are usually identified by increased ristocetin-induced platelet aggregation (RIPA). We describe here the phenotypic profile of 38 genotypically selected VWD2B-affected family members (AFMs) belonging to 19 unrelated families. Major bleeding was observed in 68.4% of AFMs (previous to their diagnosis and registered by lifetime interviews), with a total of 46 episodes (1.21/patient), and was found to be highly related to the individual bleeding score and presence of thrombocytopenia, but otherwise unrelated to other laboratory parameters. Excessive muco-cutaneous bleeding symptoms were often reported, the most frequent of which comprised menorrhagia, epistaxis, easy bruising, and bleeding after teeth extraction/in oral cavity. Eight unaffected family members were also studied. The prevalence of VWD2B within families was 0.826, and the penetrance of mutations was complete, making it mandatory to study entire family sets to complete diagnostic profiles. Seven heterozygous missense mutations were found, the most common being p.V1316M. In the p.R1308C group, 75% of the AFMs showed absence of RIPA at 0.5 mg/mL, 66.6% of whom had VWF:RCo < 10 IU/dL, and 50% of whom had VWF:CB < 10 IU/dL. In the p.S1310F group, none of the AFMs had VWF:RCo/VWF:Ag < 0.6 (RCo/Ag), but 100% had VWF:CB/VWF:Ag < 0.6/(CB/Ag). Patients with p.P1266L and p.R1304V were characterized as atypical VWD2B. Two de novo mutations were found in four AFMs belonging to two families. We also describe a novel mutation: p.Y1258C. Of our patients, 70.5% had O blood group. In conclusion, a normal RCo/Ag and a negative RIPA at 0.5 mg/mL do not necessarily rule out a diagnosis of VWD2B.

Inhibidores adquiridos de la coagulación: enfoque diagnóstico y casos especiales / Acquired inhibitors of blood coagulation: diagnostic perpective and especial cases / Inibidores adquiridos da coagulação: abordagem diagnóstica e casos especiais

Los inhibidores adquiridos son defectos raros. Se asocian a diferentes manifestaciones clínicas con morbimortalidad significativa. Su detección es importante para implementar el tratamiento sin demora. Hay inhibidores específicos (bloquean función), contra todos los factores de la coagulación y además VWF, o de interferencia (afectan una o varias vías de la coagulación). Los inhibidores específicos son alo-anticuerpos desarrollados en pacientes deficitarios (complicación terapéutica) o auto-anticuerpos presentes en individuos sin alteraciones previas. Hay anticuerpos específicos que pueden afectar la depuración pero no inhiben función. Inhibidores de interferencia: inmunoglobulinas u otras sustancias (heparina/heparinoides, PDF/pdf, PIVKAS, moléculas anómalas, etc.) asociadas a diferentes situaciones clínicas (asintomáticos, sangrados, trombosis y/o complicaciones obstétricas). El laboratorio es fundamental para el diagnóstico. Las pruebas globales detectan el defecto, que no corrigen por el agregado de plasma normal; se caracteriza luego el tipo de inhibidor y eventualmente se titula. Esto es complejo; hay variabilidad en los resultados y posibilidad de falsos positivos o negativos, además las pruebas no son estrictamente específicas. Los algoritmos diagnósticos son útiles, pero no contemplan la posibilidad de defectos combinados. Es crítico caracterizar al inhibidor y descartar posibles interferencias o defectos concomitantes; ello requiere aplicar las pruebas adecuadas e interpretarlas correctamente.

Acquired inhibitors are rare disorders. They are associated with different clinical behaviours and significant morbi-mortality. Detection is important in order to start treatment urgently. There are either specific inhibitors, which block function, against all coagulation factors, and VWF, or with interference effects, on one or more coagulation pathways. Specific inhibitors are either allo-antibodies developed in deficient patients, which give rise to therapeutic complication; or auto-antibodies, which are present in individuals without previous defects. There are specific antibodies that can affect clearance but which cannot block the function. Inhibitors with interference effects are immunoglobulins or other substances (heparin/heparinoids, FDP/fdp, PIVKAS, abnormal molecules, etc.) associated with different clinical settings (asymptomatic, bleeding, thrombosis and/or obstetric complications). Laboratory results are fundamental for the diagnosis. Global tests are able to detect the defect, which is not corrected by the addition of normal plasma; the type of inhibitor is then characterized and titration of the inhibitor is eventually performed. This is complex; there is variability in the results and there is likelihood of false positive or negative results; moreover, the tests are not strictly specific. Diagnostic algorithms are useful tools but they do not consider combined defects. It is a critical point to characterize the inhibitor and exclude possible interferences or concomitant defects; this demands application of the correct tests without misinterpretations.

Os inibidores adquiridos são defeitos raros. Associam-se a diferentes manifestações clínicas com morbimortalidade significativa. Sua detecção é importante para implementar o tratamento sem demora. Há inibidores específicos (bloqueiam função), contra todos os fatores da coagulação e também VWF, ou de interferência (afetam uma ou várias vias da coagulação). Inibidores específicos: - aloanticorpos desenvolvidos em pacientes deficitários (complicação terapêutica); - autoanticorpos, em indivíduos sem alterações prévias. Existem anticorpos específicos que podem afetar a depuração, mas não inibem função. Inibidores de interferência: imunoglobulinas ou outras substâncias (heparina/heparinoides, PDF/pdf, PIVKAS, moléculas anômalas, etc.) associadas a diferentes situações clínicas (assintomáticos, sangramentos, tromboses e/ou complicações obstétricas). O laboratório é fundamental para o diagnóstico. Os testes globais detectam o defeito, que não corrige pelo acréscimo de plasma normal; caracteriza-se depois o tipo de inibidor e eventualmente é titulado. Isto é complexo; há variabilidade nos resultados e possibilidade de falsos positivos ou negativos, além disso os testes não são rigorosamente específicos. Os algoritmos diagnósticos são úteis, mas não consideram a possibilidade de defeitos combinados. É crítico caracterizar o inibidor e descartar possíveis interferências ou defeitos concomitantes; isso requer aplicar os testes adequados e interpretá-los corretamente.

Factor von Willebrand y Enfermedad de von Willebrand: nuevos enfoques diagnósticos / Von Willebrand Factor and von Willebrand disease: new approaches to diagnosis / Fator Von Willebrand e doença de von Willebrand: novas abordagens diagnósticas

El factor von Willebrand (VWF) es una glicoproteína que se sintetiza en células endoteliales y en megacariocitos. Su vida media es de ~12 horas. Está formado por multímeros de diferentes pesos moleculares, pequeños, intermedios, grandes y extragrandes. La actividad funcional reside en los multímeros grandes, y los extragrandes son trombogénicos. Promueve la adhesión plaquetaria al subendotelio, la agregación plaquetaria y transporta al FVIII en plasma, protegiéndolo de su degradación por proteasas. La enfermedad de von Willebrand es el trastorno hemorrágico más frecuente; se describen deficiencias cuantitativas (parcial: VWD1; total: VWD3) o defectos cualitativos (VWD2A, VWD2M, VWD2B y VWD2N). La expresión clínica es variable (sangrado muco-cutáneo) y su herencia autosómica, dominante o recesiva, según las variantes. Los niveles del VWF dependen de factores genéticos y no genéticos que afectan el diagnóstico y la expresión clínica. Para llegar al diagnóstico se precisan varias pruebas, algunas inespecíficas. El laboratorio comienza con pruebas orientadoras, se continúa con pruebas confirmatorias, y posteriormente pruebas para definir la variante de VWD. El diagnóstico genotípico es fundamental para lograr el diagnóstico diferencial entre VWD2B vs. PT-VWD y VWD2N vs. Hemofilia A (leve-moderada), diferenciar VWD de AVWS y discriminar variantes VWD2.

Von Willebrand factor (VWF) is a glycoprotein with essential roles in both primary and secondary hemostasis, synthesized by endothelial cells and megakaryocytes. Its half-life is ~12 hours. VWF consists in multimers of different molecular weight: small, intermediate, large and ultra large. The functional activity resides in the large multimers; the ultra large are thrombogenic. VWF promotes platelet adhesion to subendothelium, platelet aggregation and binds FVIII, protecting it from proteolysis and preserving its hemostatic function. Von Willebrand disease is the most common bleeding disorder; qualitative defects (VWD2A, VWD2M, VWD2B and VWD2N) and quantitative deficiencies (VWD1 and VWD3) are described. The clinical expression is variable (mucocutaneous bleeding); VWF levels depend on genetic and non-genetic factors affecting diagnosis and clinical expression. The inheritance can be autosomal, dominant or recessive according to the variants. To reach diagnosis, several tests are required, being some of them unspecific. The laboratory testing begins with global tests, followed by confirmatory tests and further tests to define the variant of VWD. Genotypic studies are essential to achieve the differential diagnosis between VWD2B vs. PT-VWD, VWD2N vs. Hemophilia A (mild to moderate) and differentiate VWD from AVWS and discriminate VWD2 variants.

O fator de von Willebrand (vWF) é uma glicoproteína sintetizada em células endoteliais e em megacariócitos. Sua vida média é de ~12 horas. É constituído por multímeros de pesos moleculares diferentes, pequenos, intermediários, grandes e extragrandes. A atividade funcional reside nos multímeros grandes, sendo os extragrandes, trombogênicos. Promove adesão das plaquetas ao subendotélio, a agregação plaquetária e transporta o FVIII em plasma, protegendo-o de sua degradação. A doença de von Willebrand é o distúrbio hemorrágico mais frequente; são descritas deficiências quantitativas (parcial: VWD1; total: VWD3) ou defeitos qualitativos (VWD2A, VWD2M, VWD2B e VWD2N). A expressão clínica é variável, (sangramento mucocutâneo), e sua herança autossômica dominante ou recessiva de acordo com as variantes. Os níveis de vWF dependem de fatores genéticos e não-genéticos que afetam o diagnóstico e a expressão clínica. Para fazer o diagnóstico, vários testes são necessários, alguns inespecíficos. O laboratório começa com testes orientadores, continua com testes de confirmação e, mais tarde, com testes para definir a variante de VWD. O diagnóstico genotípico é essencial para alcançar o diagnóstico diferencial entre VWD2B vs. PT-VWD e VWD2N vs. Hemofilia A (leve a moderada), diferenciar VWD de AVWS, discriminar variantes VWD2.

Inhibidor lúpico en situaciones clínicas diferentes al síndrome antifosfolipídico / Lupus inhibitor in clinical situations other than antiphospholipid syndrome

El inhibidor lúpico (IL) es uno de los criterios de laboratorio para Síndrome Antifosfolipídico (SAF); sin embargo, puede detectarse en individuos asintomáticos o estar asociado a otras situaciones clínicas. Presentamos un análisis retrospectivo de 1000 exámenes consecutivos para IL (TTPA, DRVVT) de los cuales 249 casos no presentaban criterios clínicos de SAF. Aplicando los criterios SSC-ISTH, hallamos IL+ en 27,30% (205/751) y 43,37% (108/249) de los casos con y sin criterios clínicos de SAF respectivamente; analizándose en estos últimos casos las características clínicas y de laboratorio. Contexto clínico de casos IL+ sin SAF: 18,52% asintomáticos, 34,26% síntomas de sangrado y 47,22% otras manifestaciones. Otras alteraciones de laboratorio en casos IL+ sin SAF, con síntomas de sangrado: detectamos alteraciones plaquetarias, descenso de VWF:RCo y/o VWF:Ag, disminución de FVIII, FV, FVII, FXI o fibrinógeno e hiperfibrinolisis en el 54,05% de los casos. El análisis mostró detección de IL+ en un número importante de estudios (108/1000) sin criterios SAF. Los casos con IL+ y sangrado representan un desafío particular, al requerir evaluar otros posibles defectos subyacentes, que pudiesen justificar el comportamiento clínico. La detección e identificación de defectos combinados requiriere de un análisis minucioso, a fin de alcanzar un diagnóstico correcto, esencial para tomar decisiones terapéuticas adecuadas. (AU)

Despite lupus anticoagulant (LA) is one of the laboratory criteria for antiphospholipid syndrome (APS), it can be present in asymptomatic subjects or it can be associated with other clinical settings. We present a retrospective analysis of 1000 consecutive LA assays (APTT, DRVVT), 249 of them were performed in patients without clinical criteria for APS. According to ISTH criteria, positive LA was found in 27.30% (205/751) and 43.37% (108/249) of cases with or without APS criteria respectively; in the last group, the analysis of clinical background and laboratory characteristics was done. Clinical background of LA+ cases without APS: 18.52% asymptomatic, 34.26% bleeding symptoms and 47.22% other clinical settings. Other abnormal laboratory tests in LA+ cases without APS and bleeding symptoms: platelet dysfunction; low VWF:RCo and/or VWF:Ag; decrease of FVIII, FV, FVII, FXI or fibrinogen and hyperfibrinolysis were found in the 54.05% of the cases. The analysis showed positive LA in an important number of cases (108/1000) without criteria of APS. Those LA+ cases with bleeding symptoms represent a particular challenge because other possible underlying defects have to be analysed in order to explain the clinical behaviour. The detection and identifications of combined defects required a careful analysis in order to achieve accurate diagnosis, essential for therapeutic decisions. (AU)

Identificación por nuestro grupo de una nueva mutación en el gen GP1BA (P.W246L) asociada al fenotipo PT-VWD. Sexta mutación reportada internacionalmente / Identification by our group of a novel mutation in the GP1BA gene (P.W246L) responsible for PT-VWD. The 6th mutation internationally reported

La enfermedad de von Willebrand tipo plaquetario (PT-VWD) y tipo 2B (2B-VWD) son trastornos hemorrágicos raros, caracterizados por agregación plaquetaria a bajas concentraciones de ristocetina (RIPA). El diagnóstico diferencial no es fácil y representa un desafío. Hasta el presente, sólo se habían reportado cinco mutaciones en el gen GP1BA relacionadas con este desorden. Describimos aquí la sexta mutación relacionada con PT-VWD, en un paciente con sintomatología hemorrágica severa, macro-trombocitopenia, leve agregación plaquetaria espontánea, RIPA positivo a 0,3 y 0,4 mg/mL, VWF:RCo/VWF: Ag<0,2 y estudios discriminatorios positivos para PT-VWD. VWFpp/VWF: Ag resultó normal a diferencia del 2B-VWD que en algunas oportunidades resulta afectado. El exón 28 del gen VWF del paciente y su madre no reveló mutaciones. Identificamos una sustitución G>T en el nucleótido 3805 en el gen GP1BA del paciente, resultando en un cambio de Trp a Leu en el residuo 246 (p.W246L), en la región de la GPIBa que une al VWF. Esta mutación no se identificó en su madre ni en 100 controles sanos. Es considerada como dañina por análisis in sílico. Consideramos que esta sustitución es responsable del fenotipo PT-VWD del paciente. Dada la ausencia de la misma en los 100 normales estudiados, no se considera un polimorfismo

Platelet-type von Willebrand disease (PT-VWD) and type 2B von Willebrand disease (2B-VWD) are rare bleeding disorders characterized by increased ristocetin-induced platelet aggregation (RIPA) at low concentrations. Diagnosis of either condition is not easy and the differential diagnosis is especially challenging. Five mutations in the GP1BA gene related to PT-VWD and near 50 patients are currently reported worldwide. We herein describe a patient with severe bleeding symptoms, macro thrombocytopenia, mild spontaneous platelet aggregation, positive RIPA at 0.3 and 0.4 mg/mL, VWF: RCo/VWF: Ag <0.2, normal VWFpp/VWF: Ag ratio, and RIPA mixing tests and cryoprecipitate challenge positive for PT-VWD. GP1BA gene was studied in the patient, his mother, and 100 healthy control subjects. We identified a substitution G>T at nucleotide 3805 in the patient's GP1BA gene, resulting in a Trp to Leu amino acid change at residue 246 (p.W246L), within the VWF binding region. This mutation was absent in his unaffected mother and also in the 100 controls, and was predicted as damaging by in silico analysis. The residue is located in a strongly conserved position in the phylogenetic tree. These findings argue in favor of considering this substitution does not represent a polymorphism, and is therefore responsible for the PT-VWD phenotype of the patient

Identification of p.W246L as a novel mutation in the GP1BA gene responsible for platelet-type von Willebrand disease.

Platelet-type von Willebrand disease (PT-VWD) and type 2B von Willebrand disease (2B-VWD) are rare bleeding disorders characterized by increased ristocetin-induced platelet aggregation (RIPA) at low concentrations of ristocetin. Diagnosis of either condition is not easy and the differential diagnosis between the two entities is especially challenging as evidenced by high levels of misdiagnosis of both conditions, but particularly PT-VWD. Five mutations in the GP1BA gene related to PT-VWD and less than 50 patients are currently reported worldwide. We herein describe a patient with severe bleeding symptoms, macrothrombocytopenia, mild spontaneous platelet aggregation, positive RIPA at 0.3 and 0.4 mg/mL, von Willebrand factor ristocetin cofactor (VWF:RCo) to antigen (VWF:Ag) < 0.2, normal VWF propeptide/VWF:Ag ratio, and RIPA mixing tests and cryoprecipitate challenge positive for PT-VWD. GP1BA gene was studied in the patient, in his mother, and in 100 healthy control subjects. We identified a heterozygous substitution G > T located at nucleotide 3805 in the g.DNA of the patient's GP1BA gene, resulting in a Trp to Leu amino acid change at residue 246 (p.W246L). This mutation was absent in his unaffected mother and also in the 100 controls, and was predicted as damaging by in silico analysis. The residue W246 is located within the VWF-binding region and exists in a strongly conserved position in the phylogenetic tree, which is expected to be unable to tolerate substitutions without changing its functional characteristics. These findings argue strongly in favor of the view that this substitution does not represent a polymorphism and is therefore responsible for the PT-VWD phenotype of the patient.

Diagnosis and management of von Willebrand disease in a single institution of Argentina.

Von Willebrand disease (VWD) is a bleeding disorder with variable clinical expression. In this article we describe types, clinical features, genetic testing when needed, genotype/phenotype relationships, and the response to desmopressin (DDAVP) testing, according to our experience. Our findings are possible type 1, 69.6%; type 1, 13.5%; severe type 1, 0 .35%; type 3, 0.55%; type 2A, 9.5%; probable 2B, 0.6%; type 2M, 2.5%; and probable type 2N, 3.4%. The most frequent symptoms are ecchymoses-hematomas and epistaxis, and, in females >over 13 years also menorrhagia. In pregnant patients, assessment of laboratory parameters in months 7 and 8 is recommended to plan the need for prophylaxis at term. DDAVP merits to be considered as the first-choice therapy, including pregnant women and children, and no patient showed significant unwanted effects. Because this is a safe, effective, and affordable therapy, we hope to encourage clinicians, mainly pediatricians and obstetricians, to a wider use of DDAVP, especially in developing countries. We also report two patients with prophylactic treatment.

Hemorragias mayores asociadas a cirugía en pacientes con enfermedad de Von Willebrand leve / Major haemorrhages associated with surgery in patients with mild von Willebrand disease

Introducción: Los pacientes con enfermedad de von Willebrand frecuentemente sangran frente a desafíos hemostáticos. Objetivos: Nuestro propósito fue identificar marcadores predictivos de hemorragia mayor en cirugías en pacientes con las variantes tipo 1 y posible tipo 1 de la enfermedad. Material y Métodos: Se registraron las hemorragias mayores en cirugías anteriores al diagnóstico y los parámetros de laboratorio en 311 pacientes. Éstos se agruparon de acuerdo con la ausencia (grupo A) o presencia (grupo B) de hemorragia mayor en cirugías. Resultados: Presentaron hemorragia mayor el 26 por ciento de los pacientes y 17,5 por ciento de las cirugías. No hubo diferencias en el porcentaje de pacientes tipo 1 (32,6 por ciento) y posible tipo 1 (24,8 por ciento) que tuvieron hemorragia mayor. Tampoco se observaron diferencias en la prevalencia del grupo sanguíneo O, edad, género, historia familiar y niveles de FVIII y VW entre los grupos A y B. La hemorragia post exodoncia fue el antecedente clínico más frecuente (P<0,000; RR=2,11; IC 95 por ciento = 1,3-3,5) y podría definir riesgo de hemorragias mayores. El bleeding score y el número de sitios de sangrado no resultaron predictivos de hemorragias mayores. Las cesáreas (24,6 por ciento) y adenoamigdalectomías (22,3 por ciento) fueron las cirugías con mayor frecuencia de hemorragias mayores. Conclusión: Los pacientes con VWD tipo 1 y posible tipo 1 mostraron similar incidencia de hemorragia mayor en cirugías. Los niveles de FVIII y VWF, el tiempo de sangría, historia familiar y grupo sanguíneo no resultaron efectivos como marcadores predictivos de hemorragia mayor. Sin embargo, el antecedente de sangrado post exodoncia y el tipo de cirugía a llevar a cabo (cesáreas y adenoamigdalectomías) en pacientes con enfermedad de von Willebrand tipo 1 y posible tipo 1 parecen importantes como marcadores de riesgo.

Introduction: Patients with von Willebrand disease frequently bleed under haemostatic challenges. Objectives: The aim of our study was to identify predictive markers of perioperative major haemorrhage in type 1 and possible type 1 patients von Willebrand disease. Material and Methods: We recorded perioperative bleeding complications previous to diagnosis and laboratory parameters in 311 patients. They were grouped according to the absence (group A) or presence (group B) of perioperative major haemorrhage. Results: Twenty-six per cent of patients and 17.5 per cent of surgical procedures presented major haemorrhage. There was no difference neither between percentages of type 1 (32.6 per cent) and possible type 1 patients (24.8 per cent) who had major haemorrhage nor in the prevalence of O blood group, age, gender, family history and levels of FVIII and VWF, between group A and B. A history of bleeding after tooth extraction was the most frequent clinical feature (P<0.000; RR=2.11; CI 95 per cent = 1.3-3.5) observed in patients with major haemorrhage, and could defi ne risk factor. The bleeding score and the number of bleeding sites were not predictors of major haemorrhages. Caesarean sections (24.6 per cent) and adeno-tonsillectomies (22.3 per cent) showed the highest frequency of major haemorrhage. Conclusion: Type 1 and possible type 1 VWD patients showed similar incidence of perioperative major haemorrhage. The levels of FVIII and VWF, the bleeding time, blood group and family history did not prove to be effective as predictive markers of major haemorrhage. However, the personal history of bleeding after tooth extraction and the type of surgery (caesarean section and adeno-tonsillectomies) in patients with either type 1 or possible type 1 von Willebrand disease shown to be important in determining risk.

Biological and clinical response to desmopressin (DDAVP) in a retrospective cohort study of children with low von Willebrand factor levels and bleeding history.

The diagnosis and management of von Willebrand disease (VWD) in paediatrics is challenging. Our aim was to review patient's characteristics related to biological and clinical response to DDAVP in children with low von Willebrand factor (VWF) levels and bleeding history from a single institution. We included a retrospective cohort of 221 children (median age 11 years; 137 females): 27 type 1 (VWF levels within 15-30 IU dL-1) and 194 possible type 1 (VWF levels within 31-49 IU dL-1). The DDAVP infusion-test was performed in 214/221 children, 93.4% of whom showed good response. Patients with type 1 were at higher risk of DDAVP-test failure: 9/26 (34.6%) vs. 18/188 (9.6%) with possible type 1 (RR 3.44, 1.75-6.79; p= 0.002, Fisher's exact test). In 68 children, the clinical response to DDAVP was evaluated 87 times: i) to stop bleeding: menorrhagia (13), mucocutaneous (12), haemarthrosis (1); and ii) to prevent surgical bleeding: adenotonsillectomy (17), major (15) and minor surgery (10); and dental procedures (19). No major adverse events or bleeding were observed. The treatment was effective with one single dose of DDAVP in almost all patients, without antifibrinolytic or local therapy, except in a girl with severe haemorrhage during menarche who required replacement therapy. In conclusion, patients with VWD type 1 were at higher risk of no response to DDAVP infusion-test. In this series, one dose of DDAVP proved effective and safe for children with VWD. Since this is a safe, effective and affordable therapy, we consider that a wider use should be promoted, especially in developing countries.

Uso de desmopresina durante el embarazo en enfermedad de Von Willebrand / Desmopressin use during pregnancy in Von Willebrand diseases

La enfermedad de Von Willebrand es el desorden hemorragíparo más frecuente. Las mujeres constituyen una población particularmente sintomática debido al desafío hemostático de las menstruaciones y el parto. Nosotros revisamos las historias médicas de 54 mujeres con niveles disminuidos de factor von Willebrand (VWF) e historia de sangrado, quienes hubieran usado desmopresina durante el embarazo. No se observaron efectos adversos ni en las mujeres ni en los recién nacidos, incluso en los 5 expuestos a la medicación en el primer trimestre de gestación. No se observaron complicaciones locales asociadas a la colocación del catéter epidural. La DDAVP fue efectiva para prevenir el sangrado posparto. La desmopresina merece ser considerada como la primera elección de tratamiento; en aquellas pacientes con bajo niveles de VWF presentan complicaciones hemorrágicas, incluyendo mujeres embarazadas. Aunque el sangrado posparto aparece en una pequeña proporción de mujeres con VWD, no hay un modo apropiado de identificar quiénes van a sangrar. El uso de profilaxis con DDAVP debería ser tenido en cuenta como una alternativa segura y efectiva.

The von Willebrand disease (VWD) is the most frequent hemorrhagic disorder. Women with VWD are more symptomatic than men because the challenged of menses and delivery. We reviewed the records of 54 women with a low plasmatic VWF level and bleeding history, who had used desmopressin during pregnancy. No adverse effects were observed in mothers or newborns, incluiding those exposed to the drug during the first trimester. No local complication of epidural placement was observed. DDAVP was effective to prevent post-partum hemorrhage. DDAVP merits to be considered as the first choice of therapy, when patients with a previous or current low plasmatic VWF level present bleeding complications, including pregnant women. Although post-partum bleeding will appear in a small proportion of VWD women, there is no accurate way to identify who is going to bleed. The use of DDAVP should be regarded as a highly valuable option.

Major haemorrhage related to surgery in patients with type 1 and possible type 1 von Willebrand disease.

Patients with von Willebrand disease (VWD) frequently bleed under a challenge. The aim of our study was to identify predictive markers of perioperative major haemorrhage in type 1 (VWF:RCo = 15-30 IU dl(-1)) and possible type 1 (VWF:RCo = 31-49 IU dl(-1)) VWD patients. We recorded perioperative bleeding complications previous to diagnosis and laboratory parameters in 311 patients with 498 surgical procedures. The patients were grouped according to the absence (A) or presence (B) of perioperative major haemorrhages. Eighty-one patients (26%) and 87 surgical procedures (17.5%) presented major haemorrhages associated with surgeries. There was no difference between the percentage of type 1 and possible type 1 VWD patients who had major haemorrhages (32.6% and 24.8% respectively; p = ns). No difference in the prevalence of O blood group, age, gender, positive family history and laboratory test results (FVIII and VWF) was observed, independent of the haemorrhagic tendency. Bleeding after tooth extraction was the most frequent clinical feature observed in patients with perioperative major haemorrhages. The bleeding score and the number of bleeding sites (> or = 3) were not predictors of major haemorrhage associated with surgery. Caesarean section and adenotonsillectomy showed the highest frequency of major haemorrhages (24.6% and 22.3%, respectively). In conclusion, type 1 and possible type 1 VWD patients showed similar incidence of perioperative major haemorrhages. Laboratory tests and positive family history did not prove to be effective at predicting major haemorrhages in patients that had either type 1 or possible type 1 VWD. The history of bleeding after tooth extraction could define risk factors of major haemorrhage.