Identification of 58 Mutations (26 Novel) in 94 of 109 Symptomatic Spanish Probands with Protein C Deficiency.

Presently, no data on the molecular basis of hereditary protein C (PC) deficiency in Spain is available. We analyzed the PC gene (PROC) in 109 patients with symptomatic PC deficiency and in 342 relatives by sequencing the 9 PROC exons and their flanking intron regions. In 93 probands, we found 58 different mutations (26 novel). Thirty-seven consisted of a nucleotide change, mainly missense mutations, 1 was a 6-nucleotide insertion causing the duplication of 2 amino acids, and 4 were deletions of 1, 3, 4, and 16 nucleotides. Nine mutations caused type II deficiencies, with the presence of normal antigen levels but reduced anticoagulant activity. Using a PC level of 70% as lowest normal limit, we found no mutations in 16 probands and 25 relatives with PC levels ≤ 70%. On the contrary, 4 probands and 12 relatives with PC levels > 70% carried the mutation identified in the proband. The spectrum of recurrent mutations in Spain is different from that found in the Netherlands, where the most frequent mutations were p.Gln174* and p.Arg272Cys, and is more similar to that found in France, where the most frequent were p.Arg220Gln and p.Pro210Leu. In our study, p.Val339Met (9 families), p.Tyr166Cys (7), p.Arg220Gln (6), and p.Glu58Lys (5) were the most prevalent. This study confirms the considerable heterogeneity of the genetic abnormality in PC deficiencies, and allowed genetic counseling to those individuals whose PC levels were close to the lower limit of the normal reference range.

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.

Novel mutations in RASGRP2, which encodes CalDAG-GEFI, abrogate Rap1 activation, causing platelet dysfunction.

In addition to mutations in ITG2B or ITGB3 genes that cause defective αIIbß3 expression and/or function in Glanzmann's thrombasthenia patients, platelet dysfunction can be a result of genetic variability in proteins that mediate inside-out activation of αIIbß3 The RASGRP2 gene is strongly expressed in platelets and neutrophils, where its encoded protein CalDAG-GEFI facilitates the activation of Rap1 and subsequent activation of integrins. We used next-generation sequencing (NGS) and whole-exome sequencing (WES) to identify 2 novel function-disrupting mutations in RASGRP2 that account for bleeding diathesis and platelet dysfunction in 2 unrelated families. By using a panel of 71 genes, we identified a homozygous change (c.1142C>T) in exon 10 of RASGRP2 in a 9-year-old child of Chinese origin (family 1). This variant led to a p.Ser381Phe substitution in the CDC25 catalytic domain of CalDAG-GEFI. In 2 Spanish siblings from family 2, WES identified a nonsense homozygous variation (c.337C>T) (p.Arg113X) in exon 5 of RASGRP2 CalDAG-GEFI expression was markedly reduced in platelets from all patients, and by using a novel in vitro assay, we found that the nucleotide exchange activity was dramatically reduced in CalDAG-GEFI p.Ser381Phe. Platelets from homozygous patients exhibited agonist-specific defects in αIIbß3 integrin activation and aggregation. In contrast, α- and δ-granule secretion, platelet spreading, and clot retraction were not markedly affected. Integrin activation in the patients' neutrophils was also impaired. These patients are the first cases of a CalDAG-GEFI deficiency due to homozygous RASGRP2 mutations that are linked to defects in both leukocyte and platelet integrin activation.

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.

[Follow-up of pulmonary thromboembolism]. / Seguimiento de la tromboembolia pulmonar.

The aims of follow-up of pulmonary thromboembolism (PTE) are to avoid recurrence and possible sequels, such as pulmonary hypertension and postthrombotic syndrome of the lower limbs. Recurrences are reduced by anticoagulant therapy. In most PTE triggered by a transitory risk factor, without additional risk factors, the duration of oral anticoagulant therapy (OAT) is well established. However, in at least half of all cases, the triggering factors are not clear, the risk of recurrence is higher, and the duration of OAT has not been well-defined. Consequently, the factors that increase the risk of recurrence should be identified and monitored. These factors include cancer, some thrombophilias, and recurrent PTE or deep veinous thrombosis (DVT). In the last few years, idiopathic etiology, residual venous thrombosis, and other factors such as persistent right ventricular dysfunction, have also been demonstrated to be markers of recurrence. In some patients, D-dimers also seem to predict the risk of recurrence. Finally, the duration of OAT will be defined by periodically weighing the risk of recurrence against hemorrhagic risk in each individual patient. Current evidence on the balance of risks indicates a tendency toward indefinite anticoagulation, especially in idiopathic PTE. Moreover, functional monitoring through echocardiography, at least in the first 2 years, is essential to detect pulmonary hypertension associated with chronic pulmonary thromboembolism.

Seguimiento de la tromboembolia pulmonar / Follow-up of pulmonary thromboembolism

Los objetivos del seguimiento de la tromboembolia pulmonar (TEP) son evitar recidivas y secuelas, como hipertensión arterial pulmonar y síndrome postrombótico de extremidades inferiores, cuando se acompaña de trombosis venosa profunda (TVP). En la mayoría de las TEP desencadenadas por un factor de riesgo transitorio, sin otros añadidos, la duración del tratamiento anticoagulante oral (TAO) está establecida en 3-6 meses. En las TEP idiopáticas, las circunstancias desencadenantes no son claras, el riesgo de recidiva es más elevado, y la duración del TAO no está bien definida, con una tendencia progresiva hacia la anticoagulación a largo plazo. La duración se determinará por un balance periódico individualizado entre riesgo de recidiva y riesgo hemorrágico. Es por ello que se debe conocer y monitorizar los factores que aumentan el riesgo de recidiva. En los últimos años se ha demostrado que, además del cáncer, algunas trombofilias y TEP o TVP de repetición, también son marcadores de recidiva la trombosis venosa residual, así como la persistencia de disfunción del ventrículo derecho. Asimismo, el dímero D parece tener un papel como predictor de riesgo de recidiva. Por otra parte, también es imprescindible la monitorización funcional, al menos los primeros 2 años, mediante ecocardiografía, para detectar hipertensión pulmonar asociada a tromboembolia pulmonar crónica, lo que implicaría mantener el TAO indefinidamente

The aims of follow-up of pulmonary thromboembolism (PTE) are to avoid recurrence and possible sequels, such as pulmonary hypertension and postthrombotic syndrome of the lower limbs. Recurrences are reduced by anticoagulant therapy. In most PTE triggered by atransitory risk factor, without additional risk factors, the duration of oral anticoagulant therapy (OAT) is well established. However, in at least half of all cases, the triggering factors are not clear, the risk ofrecurrence is higher, and the duration of OAT has not been well-defined. Consequently, the factors that increase the risk of recurrence should be identified and monitored. These factors include cancer, some thrombophilias, and recurrent PTE or deep veinous thrombosis (DVT). In the last few years, idiopathic etiology, residual venous thrombosis, and other factors such as persistent right ventricular dysfunction,have also been demonstrated to be markers of recurrence.In some patients, D-dimers also seem to predict the risk of recurrence. Finally, the duration of OAT will be defined by periodically weighing the risk of recurrence against hemorrhagic risk in each individualpatient. Current evidence on the balance of risks indicates a tendency toward indefinite anticoagulation, especially in idiopathic PTE. Moreover, functional monitoring through echocardiography, at least in the first 2 years, is essential to detect pulmonary hypertension associated with chronic pulmonary thromboembolism

Consensus opinion for the selection and use of therapeutic products for the treatment of haemophilia in Spain.

The period between isolation of HIV in the early 1980s and the development of effective viral inactivation procedures able to eradicate the virus from the blood supply was long and unfortunately many recipients of blood-derived products became infected; this translated into a devastating impact on their quality of life, quality of care as well as on their life expectancy. Some years later, hepatitis C virus infection was identified as another known blood-borne disease complicating the treatment of haemophilia. Nowadays, the potential threat of emerging new pathogens has stressed the need to provide effective but primarily safe products with regard to infectious agents, as well as to regularly update therapeutic guidelines for haemophilia. The aim of the present publication was to review some of the crucial aspects related to the choice of haemostatic concentrates for the treatment of haemophilia and other inherited bleeding disorders, to analyse the current situation in the United States, Canada and European Union countries and to report the most relevant aspects of the Spanish consensus opinion of haemophilia-treating doctors for the use of therapeutic products for haemophilia recently issued. Essentially, it suggests that a gradual switch to recombinant concentrates may be a beneficial decision for patients with haemophilia and for the National Health Service.