Método de detección de la Telangiectasia Hemorrágica Hereditaria

Método de detección de la Telangiectasia Hemorrágica Hereditaria. Método para la detección de la Telangiectasia Hemorrágica Hereditaria (HHT) que comprende el análisis in vitro de los productos de la expresión de los genes ENG o FLT1 Y ENG o ANGPT2 en muestras biológicas de pacientes y que, además, permite sub-clasificar las variantes HHT1 y HHT2 de la enfermedad. En particular, la presente invención se refiere a un kit para la detección molecular de HHT capaz de llevar a cabo la detección mencionada anteriormente.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER)A1 Solicitud de patentes con informe sobre el estado de la técnic

Mutation affecting the proximal promoter of Endoglin as the origin of hereditary hemorrhagic telangiectasia type 1

[Background] Hereditary hemorrhagic telangiectasia (HHT) is a vascular multi-organ system disorder. Its diagnostic criteria include epistaxis, telangiectases in mucocutaneous sites, arteriovenous malformations (AVMs), and familial inheritance. HHT is transmitted as an autosomal dominant condition, caused in 85% of cases by mutations in either Endoglin (ENG) or Activin receptor-like kinase (ACVRL1/ACVRL1/ALK1) genes. Pathogenic mutations have been described in exons, splice junctions and, in a few cases with ENG mutations, in the proximal promoter, which creates a new ATG start site. However, no mutations affecting transcription regulation have been described to date in HHT, and this type of mutation is rarely identified in the literature on rare diseases.[Methods] Sequencing data from a family with HHT lead to single nucleotide change, c.-58G > A. The functionality and pathogenicity of this change was analyzed by in vitro mutagenesis, quantitative PCR and Gel shift assay. Student t test was used for statistical significance.[Results] A single nucleotide change, c.-58G > A, in the proximal ENG promoter co-segregated with HHT clinical features in an HHT family. This mutation was present in the proband and in 2 other symptomatic members, whereas 2 asymptomatic relatives did not harbor the mutation. Analysis of RNA from activated monocytes from the probands and the healthy brother revealed reduced ENG mRNA expression in the HHT patient (p = 0.005). Site-directed mutagenesis of the ENG promoter resulted in a three-fold decrease in luciferase activity of the mutant c.-58A allele compared to wild type (p = 0.005). Finally, gel shift assay identified a DNA-protein specific complex.[Conclusions] The novel ENG c.-58G > A substitution in the ENG promoter co-segregates with HHT symptoms in a family and appears to affect the transcriptional regulation of the gene, resulting in reduced ENG expression. ENG c.-58G > A may therefore be a pathogenic HHT mutation leading to haploinsufficiency of Endoglin and HHT symptoms. To the best of our knowledge, this is the first report of a pathogenic mutation in HHT involving the binding site for a transcription factor in the promoter of ENG.This study has been supported by grants from Ministerio de Economia y Competitividad of Spain (SAF2011-23475 and SAF2014-52374-R) to L.M. Botella and Centro de Investigación Biomedica en Red de Enfermedades Raras (CIBERER).Peer reviewe

Screening pulmonary arteriovenous malformations in a large cohort of Spanish patients with hemorrhagic hereditary telangiectasia

39 p.-1 fig.-7 tab.Background and objectives Because of the serious nature of potential complications, screening for pulmonary arteriovenous malformations is required in patients with hereditary hemorrhagic telangiectasia. The aim of this study was to evaluate the utility of contrast echocardiography and compare the performance of two contrast agents: agitated saline and Gelofusine.Material and methods Two hundred and five patients screened for PAVMs using TTCE and computed tomography (CT) performed with an interval of less than 180 days. Contrast echocardiography studies were graded on a 4-point semiquantitative scale based on the amount of microbubbles seen in left heart chambers.Results Positive TTCE findings were seen in 137 (66.8%) patients, whereas CT confirmed PAVMs in 59 (43.1%). Two of 67 grade 1 patients; 18 of 42 grade 2; 17 of 22 grade 3 and all grade 4 had PAVMs on CT. Embolotherapy was feasible in 38.9% patients in grade 2 and 82.3% and 95.2% in grades 3–4. No patients in grade 1 were embolized. The mean cardiac cycle in which bubbles were first seen in the left heart in patients without and with PAVMs on CT was 6.1 and 3.9 (p < 0.0001). Compared to saline, Gelofusine produced an overall increase in grade.Conclusions No grade 1 patients had treatable PAVMs. There is a need for improvement in the selection of patients for CT in grade 2, where less than half have PAVMs on CT. The cardiac cycle may help to differentiate between patients with and without PAVMs. Gelofusine was not better than saline for PAVM screening.This study has been supported by grants from Instituto de Salud Carlos III (ISCIII; PI11/0246 to JAP), FEDER (to JAP), Ministerio de Economía y Competitividad of Spain (SAF2011-23475 to LMB and SAF2013-43421-R to CB), and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; ISCIIICB06/ 07/0038 to CB).Peer reviewe

Characterization of chicken endoglin, a member of the zona pellucida family of proteins, and its tissue expression

Endoglin is a TGF-β co-receptor expressed in endothelial cells, where it plays a crucial role in angiogenesis, cardiovascular development and vascular remodeling. In humans, mutations in the endoglin gene give rise to Hereditary Hemorrhagic Telangiectasia type 1 (HHT1), an autosomal dominant disorder associated with vascular lesions in skin, mucosa and internal organs. So far, endoglin cDNA has been sequenced in several species from mammals, amphibians and birds. While in mammals the characterization of endoglin protein expression and function is well documented, little is known about the protein homologue in birds. In silico analysis by multiple sequences alignment showed a low homology score of 30-33 between the full length chicken endoglin protein and several mammalian homologues. However, a high homology score (80-85) was observed with the cytoplasmic and transmembrane regions and the overall structure of the zona pellucida (ZP) and orphan domains of the extracellular region appear to be conserved. Transient expression of chicken endoglin allowed the identification of a 180-kDa disulfide linked homodimer similar to the mammalian homologues. To further characterize its tissue expression, the novel specific monoclonal antibody (mAb) 7H5A8 was generated against chicken endoglin transfectant cells. The mAb 7H5A8 specifically recognized chicken endoglin by western blot, immunoprecipitation, immunofluorescence flow cytometry as well as immunofluorescence microscopy assays and displayed a positive staining of the endothelium in veins and arteries from frozen tissue sections of lung and bursa of Fabricius. These results may help to further understand the endoglin expression in vertebrates. © 2011 Elsevier B.V.Ministerio de Ciencia e Innovación of Spain (SAF2010-19222 to CB and BFU2010-19144 to CC); Genoma España (MEICA); Centro de Investigación Biomédica en Red de Enfermedades Raras; Fondo de Investigaciones Sanitarias; European Union (PI081813)Peer Reviewe

Propranolol reduces viability and induces apoptosis in hemangioblastoma cells from von Hippel-Lindau patients

[Background] Von Hippel-Lindau (VHL) disease is a rare oncological disease with an incidence of 1:36,000, and is characterized by the growth of different types of tumors: hemangioblastomas in the central nervous system (CNS) and retina, renal carcinoma, pheochromocytomas, pancreatic serous cystadenoma, and endolymphatic sac tumors. These tumors do not express VHL protein (pVHL). pVHL ubiquitinates hypoxia inducible factor (HIF) protein for degradation by the proteasome; in the absence of VHL, HIF translocates to the nucleus to activate the expression of its target genes. Targeting VHL-derived tumors with drugs that have reduced side effects is urgent to avoid repeat CNS surgeries. Recent reports have shown that propranolol, a β-blocker used for the treatment of hypertension and other cardiac and neurological diseases, is the best option for infantile hemangioma (IH). Propranolol could be an efficient treatment to control hemangioblastoma growth in VHL disease because of its antiangiogenic effects demonstrated in IH and the hypothetical impact on HIF levels.[Methods] HeLa 9X (HRE) hypoxia responsive element cell line and primary hemangioblastoma-derived cells were subjected to propranolol treatment and cell viability and apoptosis were evaluated. HIF1-α and Hif-2α expression after propranolol treatment was analyzed by western blotting. Quantitative PCR was performed to study the mRNA expression of HIF target genes. Vascular endothelial growth factor (VEGF) was measured in culture supernatants by immunoassay.[Results] Propranolol downregulated HIF-dependent transcription in HeLa 9XHRE cells. Under hypoxic conditions, propranolol decreased the expression of HIF target genes in hemangioblastoma cells, which stopped proliferating and died following long-term treatment. These results suggests that propranolol treatment promoted reduced HIF protein expression and corresponding downregulation of HIF target genes, and inhibited cell proliferation in parallel with induction of cell death by apoptosis.[Conclusions] Our results suggest that propranolol could reduce the growth of HIF-dependent tumors and may thus be a promising treatment to delay surgery in VHL patients.This work was supported by grants from Ministerio de Economia y Competitividad SAF2011-23475 and from Alianza VHL Spain& Fundación Iberdrola to LMB. Virginia Albiñana was supported by Alianza VHL Spain, Fundación Iberdrola and Fundación Divina Pastora.Peer reviewe

MMP-12, Secreted by Pro-Inflammatory Macrophages, Targets Endoglin in Human Macrophages and Endothelial Cells

Upon inflammation, monocyte-derived macrophages (MF) infiltrate blood vessels to regulate several processes involved in vascular pathophysiology. However, little is known about the mediators involved. Macrophage polarization is crucial for a fast and e cient initial response (GM-MF) and a good resolution (M-MF) of the inflammatory process. The functional activity of polarized MF is exerted mainly through their secretome, which can target other cell types, including endothelial cells. Endoglin (CD105) is a cell surface receptor expressed by endothelial cells and MF that is markedly upregulated in inflammation and critically involved in angiogenesis. In addition, a soluble form of endoglin with anti-angiogenic activity has been described in inflammation-associated pathologies. The aim of this work was to identify components of the MF secretome involved in the shedding of soluble endoglin. We find that the GM-MF secretome contains metalloprotease 12 (MMP-12), a GM-MF specific marker that may account for the anti-angiogenic activity of the GM-MF secretome. Cell surface endoglin is present in both GM-MF and M-MF, but soluble endoglin is only detected in GM-MF culture supernatants. Moreover, MMP-12 is responsible for the shedding of soluble endoglin in vitro and in vivo by targeting membrane-bound endoglin in both MF and endothelial cells. These data demonstrate a direct correlation between GM-MF polarization, MMP-12, and soluble endoglin expression and function. By targeting endothelial cells, MMP-12 may represent a novel mediator involved in vascular homeostasis.Ministerio de Ciencia, Innovación y Universidades of Spain (SAF2013-43421-R to C.B.; SAF2017-83785-R and SAF2014-23801 to A.L.C.)Consejo Superior de Investigaciones Cientificas (201920E022 to C.B.)Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; ISCIII-CB06/07/0038 to C.B.)Czech Republic Specific University Research (SVV-260414 to P.N.)CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER fundsM.A. was funded with a fellowship from Ministerio de Ciencia e Innovación (BES-2008-003888)M.V. was supported by a short-term mobility fellowship from the European Erasmus Programm

Enseñanzas Artísticas en la Universidad de Alicante

La implantación de las titulaciones de grado adaptadas al Espacio Europeo de Educación Superior -E.E.E.S.- comporta establecer un diseño curricular conforme al sistema de créditos ECTS –European Transfer System-. Esto implica un seguimiento de la totalidad de las actividades y tareas que el estudiante debe realizar para adquirir las competencias y objetivos de la asignatura en el tiempo establecido. En este contexto, las opiniones de los alumnos constituyen una importante fuente de información para validar la guía docente de Música en la Educación Primaria, dentro de la titulación de grado de Maestro de Primaria. Asimismo, ayuda a detectar los aspectos donde los discentes encuentran mayores dificultades a la hora de superar la materia en cuestión. Para alcanzar los objetivos propuestos el equipo investigador ha adaptado un cuestionario de corte cuantitativo realizado por Castejón (2005). Los datos obtenidos a través del campus virtual de la universidad han sido tratados con el paquete informático SPSS mediante procedimientos descriptivos

A Novel Splicing Mutation in the ACVRL1/ALK1 Gene as a Cause of HHT2

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare disorder of vascular development. Common manifestations include epistaxis, telangiectasias and arteriovenous malformations in multiple organs. Different deletions or nonsense mutations have been described in the ENG (HHT1) or ACVRL1/ALK1 (HHT2) genes, all affecting endothelial homeostasis. A novel mutation in ACVRL1/ALK1 has been identified in a Peruvian family with a clinical history compatible to HHT. Subsequently, 23 DNA samples from oral exchanges (buccal swaps) of the immediate family members were analyzed together with their clinical histories. A routine cDNA PCR followed by comparative DNA sequencing between the founder and another healthy family member showed the presence of the aforementioned specific mutation. The single mutation detected (c.525 + 1G > T) affects the consensus splice junction immediately after exon 4, provokes anomalous splicing and leads to the inclusion of intron IV between exons 4 and 5 in the ACVRL1/ALK1 mRNA and, therefore, to ALK1 haploinsufficiency. Complete sequencing determined that 10 of the 25 family members analyzed were affected by the same mutation. Notably, the approach described in this report could be used as a diagnostic technique, easily incorporated in clinical practice in developing countries and easily extrapolated to other patients carrying such a mutation

Mutation study of Spanish patients with hereditary hemorrhagic telangiectasia and expression analysis of Endoglin and ALK1

10 páginas, 2 figuras, 1 tabla -- PAGS nro. 295Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant and age-dependent vascular disorder originated by mutations in Endoglin (ENG) or activin receptor-like kinase-1 (ALK1, ACVRL1) genes. The first large series HHT analysis in Spanish population has identified mutations in 17 unrelated families. Ten different mutations in ALK1 and six in ENG genes were found. Six unrelated families had a mutation in ENG gene, four representing new mutations, p.Y258fs, pV323fs, p.F279fs (c.834_837del CTTC), and p.F279fsdupC. Eleven unrelated families harboured mutations in ALK1; ten were new mutations identified as p.H328P, p.R145fs, p.G68C, p.A377T, p.H297R, p.M376T, p.C36Y, p.H328P, p.T82del and p.R47P. Overall, ALK1 mutations (HHT2) were predominant over ENG mutations (HHT1), in agreement with data reported for other Mediterranean countries (France, Italy), but at variance with Northern Europe or North America. Endoglin expression in HHT1 or HHT2 activated monocytes and blood outgrowth endothelial cells (BOECs) from older patients was well below the theoretical 50% level expected from the HHT1 haploinsufficiency model, suggesting that the pathogenic endoglin haploinsufficiency leading to the HHT phenotype is age-dependent. Interestingly, ALK1 protein levels of HHT BOECs in some missense ALK1 mutants were similar to controls. In vitro expression of these ALK1 constructs suggests that, in addition to the haploinsufficiency model, certain ALK1 mutants may inhibit the function of the wild type alleleAuthors are indebted to Drs. Michelle Letarte and Ursula Cymerman for suggestions on methods of HHT patient sequencing, Dr. Kohei Miyazono for ALK1 constructs, Carmen Langa for technical assistance, Prof. Ginevra Guanti for hosting in her lab to A.F-L. and L.M.B., and to all the volunteers and HHT patients for their collaboration. A.F-L is a predoctoral fellow of I3P Program from Ministerio de Educación y Ciencia, SpainPeer reviewe