Unraveling the Influence of Common von Willebrand factor variants on von Willebrand Disease Phenotype: An Exploratory Study on the Molecular and Clinical Profile of von Willebrand Disease in Spain Cohort.

The clinical diagnosis of von Willebrand disease (VWD), particularly type 1, can be complex because several genetic and environmental factors affect von Willebrand factor (VWF) plasma levels. An estimated 60% of the phenotypic variation is attributable to hereditary factors, with the ABO blood group locus being the most influential. However, recent studies provide strong evidence that nonsynonymous single nucleotide variants (SNVs) contribute to VWF and factor VIII phenotypic variability in healthy individuals. This study aims to investigate the role of common VWF SNVs on VWD phenotype by analyzing data from 219 unrelated patients included in the "Molecular and Clinical Profile of von Willebrand Disease in Spain project." To that end, generalized linear mixed-effects regression models were fitted, and additive and epistatic analyses, and haplotype studies were performed, considering five VWD-related measures (bleeding score, VWF:Ag, VWF:RCo, factor VIII:C, and VWF:CB). According to these analyses, homozygotes: for p.Thr789Ala(C) would be expected to show 39% higher VWF:Ag levels; p.Thr1381Ala(C), 27% lower VWF:Ag levels; and p.Gln852Arg(C), 52% lower VWF:RCo levels. Homozygotes for both p.Thr789Ala(C) and p.Gln852Arg(T) were predicted to show 185% higher VWF:CB activity, and carriers of two copies of the p.Thr1381Ala(T)/p.Gln852Arg(T) haplotype would present a 100% increase in VWF:RCo activity. These results indicate a substantial effect of common VWF variation on VWD phenotype. Although additional studies are needed to determine the true magnitude of the effects of SNVs on VWF, these findings provide new evidence regarding the contribution of common variants to VWD, which should be taken into account to enhance the accuracy of the diagnosis and classification of this condition. ClinicalTrials.gov identifier: NCT02869074.

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.

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.

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.

High levels of latent antithrombin in plasma from patients with antithrombin deficiency.

Antithrombin is an anticoagulant serpin that efficiently inhibits multiple procoagulant proteases. The cost for the structural flexibility required for this function is the vulnerability to mutations that impact its folding pathway. Most conformational mutations identified in serpins cause polymerisation. Only three mutations in SERPINC1 affecting two residues have been found to favour transformation to the latent conformation of antithrombin, another hyperstable non-anticoagulant form with strong antiangiogenic activity that constitutes 3 % of plasma antithrombin in healthy subjects. The analysis of latent antithrombin in 141 unrelated patients with antithrombin deficiency carrying 89 different SERPINC1 mutations identified four cases with higher levels than that of controls: p.Pro439Thr, p.Pro461Ser, p.Met283Val, and p.His401Tyr, the last also with circulating polymers. Heating of plasma at 42ºC exacerbated the transformation to the latent conformation in p.Pro439Thr and p.Pro461Ser. The conformational effect of p.Met283Val, the mutation associated with the highest levels of latent antithrombin detected in four members of a family, was verified in a recombinant model. Antithrombin deficiency in these cases should be classified as pleiotropic based on the impaired reactivity and low heparin affinity of the variant. Despite high levels of latent antithrombin (up to 80 µg/ml in p.Met283Val carriers), no vascular defects were described in carriers of these mutations. In conclusion, our study identifies new residues involved in the structural stability of antithrombin (and potentially of all serpins). High levels of endogenous latent antithrombin seem to play a minor antiangiogenic effect. Finally, pleiotropic deficiencies may be caused by mutations inducing transformation to the latent conformation.

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.

Vitamin B12 deficiency, hyperhomocysteinemia and thrombosis: a case and control study.

This study aimed at assessing the relationship between thrombosis, hyperhomocysteinemia and vitamin B12 deficiency using a case-control study carried out in 326 patients with thrombosis (case group) and 351 patients from the same hospital (control group). Apart from the classic risk factors, a number of hematological variables were evaluated, including serum vitamin B12 (B12), red cell folate (RCF), and serum homocysteine (Hcy). An evaluation of serum methylmalonic acid (MMA) and a clinical study were carried out to investigate B12 pathology. Results of univariate analysis demonstrated decreased B12 levels in thrombosis (Student's t test, p < 0.0001). Vitamin B12 below 200 pmol/l (LB200) or below 150 pmol/l (LB150), and red cell folate below 600 nmol/l were found in 17.2, 8.6, and 2.2% of cases with thromboembolism, respectively. An increase in Hcy was detected in 86 cases with thrombosis (26.3%). An abnormality in vitamin B12 and/or renal function was found in 80% of cases with hyperHcy and thrombosis. The MMA increase demonstrated that vitamin B12 deficiency was present in these patients with low levels of vitamin B12 in serum, and the MMA levels were in concordance with Hcy levels. The clinical study revealed B12 malabsorption in most cases with LB200. Multivariate analysis showed that serum vitamin B12 (RR 0.998, CI 0.997-0.999) was moderately related to thromboembolism. The results indicated that vitamin B12 deficiency was common among patients with hyperhomocysteinemia and thrombosis. Moreover, HyperHcy was caused by vitamin B12 deficiency and/or chronic renal failure in most patients with thrombosis. As the main cause of vitamin B12 deficiency was vitamin malabsorption, parenteral vitamin B12 with or without folic acid should be administered for the treatment of this condition. However, it remains to be demonstrated whether this treatment approach prevents recurrent thromboses in patients with vitamin B12 deficiency and thrombosis, as suggested by some case reports.

Del Culturalismo al Ciudadanismo / From Culturalism to Citizenship

El artículo plantea el debate entre las miradas culturalista y ciudadanista en al ámbito de los procesos de integración entre la población inmigrante y la autóctona. Se parte de un análisis de la concepción esencialista del término ‘cultura’, señalando algunos de sus déficits y algunos de los riesgos que supone el manejo de esa concepción; lo cual permitirá entender la cultura como algo más que las costumbres o la esencia de un pueblo. Asimismo se exponen -a la luz del continuo ético universalismo-relativismo- los riesgos de manejar una idea de integración centrada en la cultura, y se ofrece una propuesta de carácter ciudadanista que pretende superar el enfoque culturalista al considerar la integración como multidireccional, procesual y encaminada hacia la equiparación de derechos entre todas las personas (AU)

This article places the immigrants’ integration issue within the culture-citizenship debate. In order to understand culture as something else than people’s given customs or "essence", the text begins by analyzing the essentialist notion of the term ‘culture’, pointing out some of its deficiencies and risks involved in dealing with such a notion. Drawing on the ethical relativism-universalism continuum, the authors also discuss the drawbacks they see in a culture-centred integration, suggesting instead a citizenship approach based on a multidirectional and dynamic notion -aimed at achieving equal rights for everyone involved, immigrant and native populations alike (AU)

SIDA y discriminación: percepción de un grupo de adolescentes y personas que viven con el virus de inmunudeficiencia humana / AIDS and discrimination: perception of a group of adolescents and persons who live with the human immune-deficiency virus

La infección por el virus de inmunudeficiencia humana desde sus comienzos ha constituido una fuente de preocupación para la humanidad. Surgió acompañada de una serie de estigmas que en cierta medida dañan la integridad personal de los que la padecen.Objetivo: Comprobar el grado de conocimientos que poseen los adolescentes sobre el VIH y su percepción acerca de la enfermedad y el trato a los que la padecen. Métodos: Estudio descriptivo realizado en Cienfuegos, en el que fueron encuestados 120 adolescentes y 29 personas que viven con virus de inmunudeficiencia humana, y durante el cual fueron impartidas conferencias acerca de la enfermedad.Resultados y Conclusiones: Los 120 encuestados plantearon que conocen qué es el síndrome de inmunudeficiencia adquirida y han recibido información sobre la enfermedad. La mayoría identifica a la sangre, las secreciones vaginales, semen, el compartir agujas y jeringas contaminadas como principales vías de transmisión, consideran que tener una pareja estable es el mejor modo de protegerse. El 50,8 porciento piensa que deben ser informados los nombres de las personas que viven con VIH/SIDA y al 15 porciento les preocupa compartir su vida laboral o escolar con ellos, mientras 26 porciento consideran que estas personas deben vivir aisladas en un sanatorio. Por otra parte 16 personas que viven con virus de inmunudeficiencia humana, plantearon que alguna vez se han sentido discriminados y 11 consideran que al solicitar ayuda médica han sido atendidos con verdaderas muestras de temor. Tres sintieron el rechazo de su familia y 7 plantearon que los amigos y la comunidad se fueron alejando poco a poco de ellos(AU)

Morbimortalidad del paciente quirúrgico en sala de terapia intensiva / Morbility of the surgery patient at the intensive care unit

Con el objetivo de determinar el comportamiento de la morbimortalidad del paciente quirúrgico en la unidad de cuidados intensivos del Hospital Clínico Quirúrgico Docente Amalia Simoni de la ciudad de Camagüey, se realizó un estudio descriptivo transversal desde enero de 2002 a diciembre de 2003. El universo estuvo representado por los pacientes quirúrgicos admitidos en el servicio en este período. La causa de muerte que más se presentó fue el shock séptico y la insuficiencia renal aguda fue la complicación más frecuente; se observó que a menor tiempo de estadía postoperatoria antes del ingreso mayor sobrevida. Un por ciento de los pacientes reintervenidos tuvieron un desenlace fatal, la dehiscencia de sutura fue la causa fundamental(AU)

Morbimortalidad del paciente quirúrgico en sala de terapia intensiva / Morbility of the surgery patient at the intensive care unit

Con el objetivo de determinar el comportamiento de la morbimortalidad del paciente quirúrgico en la unidad de cuidados intensivos del Hospital Clínico Quirúrgico Docente Amalia Simoni de la ciudad de Camagüey, se realizó un estudio descriptivo transversal desde enero de 2002 a diciembre de 2003. El universo estuvo representado por los pacientes quirúrgicos admitidos en el servicio en este período. La causa de muerte que más se presentó fue el shock séptico y la insuficiencia renal aguda fue la complicación más frecuente; se observó que a menor tiempo de estadía postoperatoria antes del ingreso mayor sobrevida. Un por ciento de los pacientes reintervenidos tuvieron un desenlace fatal, la dehiscencia de sutura fue la causa fundamental