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

MicroRNA-223 is a novel negative regulator of HSP90B1 in CLL

Background MicroRNAs are known to inhibit gene expression by binding to the 3′UTR of the target transcript. Downregulation of miR-223 has been recently reported to have prognostic significance in CLL. However, there is no evidence of the pathogenetic mechanism of this miRNA in CLL patients. Methods By applying next-generation sequencing techniques we have detected a common polymorphism (rs2307842), in 24% of CLL patients, which disrupts the binding site for miR-223 in HSP90B1 3′UTR. We investigated whether miR-223 directly targets HSP90B1 through luciferase assays and ectopic expression of miR-223. Quantitative real-time polymerase chain reaction and western blot were used to determine HSP90B1 expression in CLL patients. The relationship between rs2307842 status,HSP90B1 expression and clinico-biological data were assessed. Results HSP90B1 is a direct target for miR-223 by interaction with the putative miR-223 binding site. The analysis in paired samples (CD19+ fraction cell and non-CD19+ fraction cell) showed that the presence of rs2307842 and IGHV unmutated genes determined HSP90B1 overexpression in B lymphocytes from CLL patients. These results were confirmed at the protein level by western blot. Of note, HSP90B1 overexpression was independently predictive of shorter time to the first therapy in CLL patients. By contrast, the presence of rs2307842 was not related to the outcome. Conclusions HSP90B1 is a direct target gene of miR-223. Our results provide a plausible explanation of why CLL patients harboring miR-223 downregulation are associated with a poor outcome, pointing out HSP90B1 as a new pathogenic mechanism in CLL and a promising therapeutic target. Keywords Chronic lymphocytic leukemia MicroRNAs Next-generation sequencingEuropean Commision (EC). Funding FP7/SP1/HEALTH. Project Code: 30624

Núcleo Novatores: una plataforma en Internet para la gestión del sistema regional de ciencia y tecnología de Castilla y León

Presentación del proyecto NOVATORES de gestión de información científica en Castilla y León[ES]En este trabajo se describe la experiencia de implantación del Núcleo Novatores, un sistema de información y gestión de actividades científicas y tecnológicas cuyas fuentes son las instituciones, empresas o grupos sociales que financian, promueven o demandan las actividades de Investigación, Desarrollo e Innovación (I+D+i). El proyecto se enmarca dentro del Sistema Regional de Información en Ciencia y Tecnología de Castilla y León, promovido por la Junta de Castilla y León a través de la Dirección General de Universidades e Investigación y por la Fundación General de la Universidad de SalamancaJunta de Castilla y Leó

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

Molecular characterization of chronic lymphocytic leukemia patients with a high number of losses in 13q14.

BACKGROUND: Patients with chronic lymphocytic leukemia and 13q deletion as their only FISH abnormality could have a different outcome depending on the number of cells displaying this aberration. Thus, cases with a high number of 13q- cells (13q-H) had both shorter overall survival and time to first therapy. The goal of the study was to analyze the genetic profile of 13q-H patients. DESIGN AND METHODS: A total of 102 samples were studied, 32 of which served as a validation cohort and five were healthy donors. RESULTS: Chronic lymphocytic leukemia patients with higher percentages of 13q- cells (>80%) showed a different level of gene expression as compared to patients with lower percentages (<80%, 13q-L). This deregulation affected genes involved in apoptosis and proliferation (BCR and NFkB signaling), leading to increased proliferation and decreased apoptosis in 13q-H patients. Deregulation of several microRNAs, such as miR-15a, miR-155, miR-29a and miR-223, was also observed in these patients. In addition, our study also suggests that the gene expression pattern of 13q-H cases could be similar to the patients with 11q- or 17p-. CONCLUSIONS: This study provides new evidence regarding the heterogeneity of 13q deletion in chronic lymphocytic leukemia patients, showing that apoptosis, proliferation as well as miRNA regulation are involved in cases with higher percentages of 13q- cells

Quantitative RT-PCR validation for miR-15a, miR-29a, miR-155 and miR-223 in independent CLL patients.

<p>Relative expression of miR-15a, miR-29a, miR-155 and miR-223 [represented as arbitrary units (a.u.)] was evaluated by individual TaqMan miRNA assays performed in duplicate and normalized to RNU43 (2^−dCt). Box plots indicate the median value (horizontal line) and the 25^th–75^th percentile range (box) while whiskers showing the maximum and minimum values. Values outside this range are shown as outliers (open circles). <i>P</i>-values were determined by the Mann-Whitney U test. In every case, miRNAs downregulated in 13q-H CLL patients relative to 13q-L patients were also found to be downregulated by quantitative RT-PCR. Similar observations were made for miR-155, which was upregulated in 13q-H patients. All comparisons were statistically significant (<i>P</i><0.05).</p

miRNAs significantly deregulated between 13q- CLL subgroups (patients with 80% or more of cells with 13q deletion and patients with less than 80% 13q cells).

<p>Upregulation or downregulation refers to 13q-H relative to13q-L CLL patients.</p><p>miRNA: microRNA.</p>*<p>deregulation shared with 17p/11q CLL patients.</p