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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.

The Meerwein&ndash, Ponndorf&ndash, Verley (MPV) reaction is an environmentally-friendly process consisting of the reduction of a carbonyl compound through hydrogen transfer from a secondary alcohol. This work deals with MPV reduction of furfural to furfuryl alcohol on different ZrOx, MgOx, TiOx, and Mg&ndash, Ti, as well as Zr&ndash, Ti mixed systems. The solids were synthesized through the sol&ndash, gel process and subsequently calcined at 200 &deg, C. Characterization was performed using a wide range of techniques: ICP-MS, N2 adsorption-desorption isotherms, EDX, TGA-DTA, XRD, XPS, TEM, TPD of pre-adsorbed pyridine (acidity) and CO2 (basicity), DRIFT of adsorbed pyridine, and methylbutynol (MBOH) test reaction. ZrOx showed the highest conversion and selectivity values, which was attributed to the existence of acid&ndash, base pair sites (as evidenced by the MBOH test reaction), whereas the introduction of titanium resulted in the drop of both conversion and selectivity probably due to the increase in Br&ouml, nsted-type acidity. As for MgOx, it had a predominantly basic character that led to the production of the condensation product of one molecule of furfural and one molecule of acetone, and thus resulted in a lower selectivity to furfuryl alcohol. The TiOx solid was found to be mainly acidic and exhibited both Lewis and Br&ouml, nsted acid sites. The presence of the latter could account for the lower selectivity to furfuryl alcohol. All in all, these results seemed to suggest that the MPV reaction is favored on Lewis acid sites and especially on acid&ndash, base pair sites. The process was accelerated under microwave irradiation.

SummaryThe 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.Supplementary Material to this article is available online at www.thrombosis-online.com.

The correct diagnosis and classification of von Willebrand disease (VWD) is difficult because of the variability of its clinical expression and limitations of laboratory methods. However, correctly diagnosing VWD is important for therapy and genetic counselling. A survey related to the referred VWD patients in Spain revealed local diagnostic problems in at least one third of the cases of VWD. Consequently, a Spanish multicenter study was carried out in which a cohort of 556 patients from 330 families was analyzed centrally. VWD was confirmed in 480 patients. Next generation sequencing (NGS) of the whole coding von Willebrand factor gene ( VWF ) was carried out in all recruited patients, compared with the phenotype, and a final diagnosis was established. A total of 238 different VWF mutations were found: 154 were not included in the Leiden Open Variation Database (LOVD). Of the patients, 467 were found to have a VWF mutation/s. A good phenotype/genotype association was estimated in 94.6% of the cases. One hundred and eighty patients had two or more mutations. Occasionally a predominant phenotype masked the presence of a second abnormality. One hundred and sixteen patients presented with mutations that had previously been associated with an increased VWF clearance. Ristocetin induced platelet agglutination (RIPA) unavailability, central phenotypic results disagreement and difficult distinction between severe type 1 and type 3 VWD prevented a clear diagnosis in 74 patients. A local/central disagreement over diagnosis occurred in 42.3% of the whole cohort. The NGS study facilitated an appropriate classification in 60 of them. Based on these results, a proposal for a change in the VWD diagnostic paradigm, suggesting the inclusion of the analysis of VWF at the initial diagnostic process, has been made. An extension of this project to include 400 new VWD patients has begun. A diagnostic strategy has been developed, and a Spanish consensus guideline for optimal treatment of VWD was developed in Spain. Desmopressin (DDAVP) is the treatment of choice in responsive VWD patients. VWF concentrates (VWF/FVIII) are used in individuals unresponsive to DDAVP, when DDAVP is contraindicated, or in VWD types 2B and 3, and for long term management needs (e.g., post major surgery).

Fundamento y objetivo Caracterizar los defectos moleculares y estudiar su distribucion en los portadores de betatalasemia de Lanzarote. Pacientes y metodo El estudio molecular del gen beta de la globina se llevo a cabo con la tecnica de reaccion en cadena de la polimerasa (PCR) en tiempo real (RT-PCR LyghtCycler, Roche), PCR con amplificacion de alelos especificos (PCR-ARMS) y secuenciacion automatica del ADN del gen beta de la globina. Resultados Se incluyo en el estudio a 243 portadores diagnosticados de betatalasemia heterocigota entre julio de 1991 y febrero de 2007. La RT-PCR identifico la lesion molecular en el 81% de los cromosomas [113 codon CD 39 (C → T); 41 IVS-1-nt-110 (G → A), 25 IVS 1-nt-1 (G → A) y 19 IVS 1-nt-6 (T → C)]. Las 12 alteraciones moleculares restantes incluyeron la delecion 619 bp (7,8%) y las mutaciones -28 (A → G), IVS1-nt-2 (T → G), CD 41/42 (-TTCT), CD 8/9 (+G), CD 51 (-C), CD 22 (G → T), CD 24 (T → A), CD 67 (-TG), ademas de una nueva mutacion CD20/21-TGGA. Conclusiones La distribucion de las mutaciones de la betatalasemia heterocigota en Lanzarote es similar a la descrita en la zona mediterranea. El aumento de los flujos migratorios a la comunidad canaria puede explicar la presencia de mutaciones no descritas antes en nuestra comunidad.

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.

The aim of this study was to determine the molecular defects of heterozygous beta thalassaemia and to ascertain their distribution in Lanzarote.Molecular characterization was achieved by real time polymerase chain reaction (RT-PCR LightCycler, Roche), PCR-ARMS (PCR-amplification reaction mutations system) and DNA sequencing on an automated DNA sequencer.Two hundred forty-three heterozygous beta thalassaemia carriers were included between July 1991 and February 2007. RT-PCR detected the molecular defect in 81% of the beta thalassaemia chromosomes analyzed [113 codon CD 39 (C --T); 41 IVS-1-nt-110 (G --A), 25 IVS 1-nt-1 (G --A) and 19 IVS 1-nt-6 (T --C)]. The remaining 12 molecular defects included the deletion 619 bp (7.8%) and the mutations -28 (A --G), IVS1-nt-2 (T --G), CD 41/42 (-TTCT), CD 8/9 (+G), CD 51 (-C), CD 22 (G --T) and CD 24 (T --A), CD 67 (-TG) and the novel mutation CD 20/21-TGGA.The distribution of the mutations is similar to that found in the Mediterranean area. The increasing migratory flow received in the Canary Islands may explain the emergence of new mutations not reported before in our area.

Although an increased level of serum soluble transferrin receptor (sTfR) have been found in both heterozygous β-thalassaemia patients with iron deficiency and in those with more severe genotype (β0), it is not a useful marker of iron deficiency status associated to β-thalassaemia. The aim of this study was to analyse the use of two biochemical parameters (sTfR and sTfR/log of ferritin ratio) to determine the iron status and to evaluate the degree of erythropoietic activity in a group of 221 β-thalassaemic heterozigotes patients (155 β0 and 66 β+). Serum ferritin and transferrin saturation index were measured in order to establish the iron status. Of the whole group, 51 patients were iron defficient (βthal-ID) while the remaining 170 were iron sufficient (βthal-IS). Based on the combination of β-thalassaemia genotype and iron status, patients were classified into four subgroups: β0thalassaemia and iron-sufficient (β0thal-IS) (n=124); β0thalassaemia and iron-deficient (β0thal-ID) (n=31); β+thalassaemia and iron-sufficient (β+thal-IS) (n=46); β+thalassaemia and iron-deficient (β+thal-ID) (n=20). 258 healthy and 56 iron-deficient individuals were used as controls. All the haematological parameters were measured by using analyzer Coulter® GEN-S™. Haemoglobins A2 (Hb A2) and F (HbF) were analysed by high performance liquid chromatography and molecular analysis was performed by real-time PCR and direct sequencing techniques. Chemical, inmunoturbidimetrical and nephelometric methods were used to measure iron status as well as sTfR. Comparison of haemalogical and biochemical parameters between subgroups was performed by using the t-student test and correlation analysis was calculated by using least-squares regression model. Mean sTfR level obtained was 2.63 ± 0.8 mg/dL and 2.57 ± 1.1 mg/dL in βthal-ID and βthal-IS patients respectively (p=0.783). Soluble transferrin receptor showed a positive correlation with HbA2, HbF and reticulocyte count values in βthal-IS patients (r=0.208 [p