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.

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.

Thrombotic microangiopathies: First report of 294 cases from a single institution experience in Argentina.

Introduction: Introduction: Thrombotic microangiopathies (TMAs) are rare disorders associated with fatal outcomes if left uncared for. However, healthcare problems in developing countries tend to limit medical assistance to patients. Methods: Methods: We prospectively studied an Argentine cohort of 294 consecutive patients from 2013 to 2016. Patients' subcategory classification relied on clinical symptoms and presence or absence of trigger events associated with TMA. Results: Main suspected disorders were the primary TMAs known as thrombotic thrombocytopenic purpura (TTP) (n = 72/294, 24%) and atypical haemolytic uraemic syndrome (aHUS) (n = 94/294, 32%). In acute phase, demographic parameters for acquired TTP (aTTP) (n = 28) and aHUS (n = 47) showed that both groups were characterised by a young median age (37 and 25 years, respectively) and female predominance (60% and 86%). Median of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 activity was significantly lower in aTTP than in aHUS group (1.4% vs 83%) and was associated with a more severe thrombocytopenia (15 × 109 vs 53 × 109/L). Creatinine (Cr) and urea (Ur) were significantly increased in aHUS compared to aTTP subjects (Cr: 3.7 vs 0.7 mg/dL, Ur: 118 vs 33 mg/dL). Gastrointestinal and neurological symptoms were more frequent in aHUS and aTTP, respectively. Conclusion: The first description of a TMA cohort in Argentina revealed similar clinical presentations to those of other countries.

The effect of fludarabine on interferon-gamma production by lymphoid cells from healthy donors and patients with B-cell chronic lymphocytic leukemia.

Fludarabine treatment in patients with B-cell chronic lymphocytic leukemia (B-CLL) can trigger or exacerbate the development of autoimmune hemolytic anemia (AHA) through a currently ill-defined mechanism. We here show that exposure of peripheral blood lymphocytes from healthy donors and B-CLL patients to fludarabine increases in vitro production of interferon-gamma, a key cytokine in the pathogenesis of AHA.

Antiphospholipid antibodies impact the protein C (PC) pathway behavior.

Antiphospholipid antibodies may interfere with the PC pathway, displaying a resistance to the activated PC (resistant phenotype). This effect was evaluated by the APCR and the ProCG systems in 36 lupus anticoagulant samples, yielding abnormal results in 47% of APCR(original), 17% of APCR(modified), and 22% of ProCG test. ProCG values correlated with APCR(original) but not with APCR(modified). Most of lupus anticoagulants affecting the PC pathway showed abnormal APCR(original) results but not abnormal ProCG values. The different behavior between both systems may be due to the heterogeneity of the antibodies or could be attributed to the fact that, in the ProCG, a PC activator is added, while the APCR employs already activated exogenous PC.