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

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

Mice lacking endoglin in macrophages show an impaired immune response

24 p.-9 fig.-1 tab. Ojeda Fernández, Luisa et al.Endoglin is an auxiliary receptor for members of the TGF-β superfamily and plays an important role in the homeostasis of the vessel wall. Mutations in endoglin gene (ENG) or in the closely related TGF-β receptor type I ACVRL1/ALK1 are responsible for a rare dominant vascular dysplasia, the Hereditary Hemorrhagic Telangiectasia (HHT), or Rendu-OslerWeber syndrome. Endoglin is also expressed in human macrophages, but its role in macrophage function remains unknown. In this work, we show that endoglin expression is triggered during the monocyte-macrophage differentiation process, both in vitro and during the in vivo differentiation of blood monocytes recruited to foci of inflammation in wild-type C57BL/6 mice. To analyze the role of endoglin in macrophages in vivo, an endoglin myeloid lineage specific knock-out mouse line (Engfl/flLysMCre) was generated. These mice show a predisposition to develop spontaneous infections by opportunistic bacteria. Engfl/flLysMCre mice also display increased survival following LPS-induced peritonitis, suggesting a delayed immune response. Phagocytic activity is impaired in peritoneal macrophages, altering one of the main functions of macrophages which contributes to the initiation of the immune response. We also observed altered expression of TGF-β1 target genes in endoglin deficient peritoneal macrophages. Overall, the altered immune activity of endoglin deficient macrophages could help to explain the higher rate of infectious diseases seen in HHT1 patients.This work was funded by: Ministerio de Economía y Competitividad of Spain (SAF2011-23475 to LMB; SAF2013-43421-R and SAF2010- 19222 to CB.Peer reviewe

Research on potential biomarkers in hereditary hemorrhagic telangiectasia

9 p.-4 fig.Hereditary hemorrhagic telangiectasia (HHT) is a genetically heterogeneous disorder, involving mutations in two predominant genes known as Endoglin (ENG; HHT1) and activin receptor-like kinase 1 (ACVRL1/ALK1; HHT2), as well as in some less frequent genes, such as MADH4/SMAD4 (JP-HHT) or BMP9/GDF2 (HHT5). The diagnosis of HHT patients currently remains at the clinical level, according to the “Curaçao criteria,” whereas the molecular diagnosis is used to confirm or rule out suspected HHT cases, especially when a well characterized index case is present in the family or in an isolated population. Unfortunately, many suspected patients do not present a clear HHT diagnosis or do not show pathogenic mutations in HHT genes, prompting the need to investigate additional biomarkers of the disease. Here, several HHT biomarkers and novel methodological approaches developed during the last years will be reviewed. On one hand, products detected in plasma or serum samples: soluble proteins (vascular endothelial growth factor, transforming growth factor b1, soluble endoglin, angiopoietin-2)and microRNA variants (miR-27a, miR-205, miR-210). On the other hand, differential HHT gene expression fingerprinting, next generation sequencing of a panel of genes involved in HHT, and infrared spectroscopy combined with artificial neural network patterns will also be reviewed. All these biomarkers might help to improve and refine HHT diagnosis by distinguishing from the non-HHT population.This study has been supported by grants from Ministerio de Economia y Competitividad of Spain(SAF2011-23475toLMB;SAF2013-43421-R andSAF2010-19222toCB)and Centro de Investigación Biomedica en Red de Enfermedades Raras (CIBERER).CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain,supported by FEDER funds.Peer reviewe

Propranolol as an antiangiogenic candidate for the therapy of hereditary haemorrhagic telangiectasia

28 p.-9 fig.The β-blocker propranolol, originally designed for cardiological indications (angina, cardiac arrhythmias and high blood pressure), is nowadays, considered the most efficient drug for the treatment in infantile haemangiomas (IH), a vascular tumour that affects 5–10% of all infants. However, its potential therapeutic benefits in other vascular anomalies remain to be explored. In the present work we have assessed the impact of propranolol in endothelial cell cultures to test if this drug could be used in the vascular disease hereditary haemorrhagic telangiectasia (HHT). This rare disease is the result of abnormal angiogenesis with epistaxis, mucocutaneous and gastrointestinal telangiectases, as well as arteriovenous malformations in several organs, as clinical manifestations. Mutations in Endoglin (ENG) and ACVLR1 (ALK1) genes, lead to HHT1 and HHT2, respectively. Endoglin and ALK1 are involved in the TGF-β1 signalling pathway and play a critical role for the proper development of the blood vessels. As HHT is due to a deregulation of key angiogenic factors, inhibitors of angiogenesis have been used to normalise the nasal vasculature eliminating epistaxis derived from telangiectases. Thus, the antiangiogenic properties of propranolol were tested in endothelial cells. The drug was able to decrease cellular migration and tube formation, concomitantly with reduced RNA and protein levels of ENG and ALK1. Moreover, the drug showed apoptotic effects which could explain cell death in IH. Interestingly, propranolol showed some profibrinolytic activity, decreasing PAI-1 levels. These results suggest that local administration of propranolol in the nose mucosa to control epistaxis might be a potential therapeutic approach in HHT.Virginia Albiñana was supported by fellowships from Fundación Ramón Areces and Real e Ilustre Colegio de Farmaceúticos de Sevilla.Peer reviewe

CLN5 in heterozygosis may protect against the development of tumors in a VHL patient

© The Author(s).Von Hippel-Lindau syndrome (VHL) is a rare disease of dominant inheritance that increases susceptibility to tumor development, with a complete penetrance at the age of 60. In this report, we present the unprecedented case of a VHL carrier who remains healthy at 72. Under the course of this study, it was discovered that this patient carries a mutation for a second rare disease, Neuronal Ceroid Lipofuscinosis (NCL or CNL). We hypothesize that the CLN mutation she carries offers a protective effect, preventing tumor development in the cells potentially suffering a VHL second hit mutation. To test this hypothesis, we ran a series of molecular experiments and confirmed that cell viability of primary endothelial cells decreases upon CLN5 silencing. Our results further elucidate the cell biology implications of two rare diseases interacting.Funding was provided by the projects SAF2014–52374-R and SAF2017–83351-R from the Ministry of Economy and Competitivity to LMB. The group is part of the CIBER Spanish consortium for Rare Diseases, CIBERER, unit 707, and was also financially supported by the Spanish VHL patient AlliancePeer reviewe

Immunosuppressor FK506 increases endoglin and activin receptor-like kinase 1 expression and modulates transforming growth factor-β1 signaling in endothelial cells

11 páginas, 6 figuras -- PAGS nros. 833-843Hereditary hemorrhagic telangiectasia (HHT), or Rendu-Osler-Weber syndrome, is an autosomal-dominant vascular disease. The clinical manifestations are epistaxis, mucocutaneous and gastrointestinal telangiectases, and arteriovenous malformations in internal organs. Patients show severe epistaxis, and/or gastrointestinal bleeding, both of which notably interfere with their quality of life. There are two predominant types of HHT caused by mutations in endoglin (ENG) and ACVRL1/activin receptor-like kinase 1 (ALK1) genes, named HHT1 and HHT2, respectively. ENG and ALK1 code for proteins involved in the transforming growth factor (TGF)-β1 signaling pathway, and it is widely accepted that HHT pathogenicity results from haploinsufficiency. No cure for HHT has been found, so identification of drugs able to increase the expression of these genes is essential when proposing new therapies. We report the efficacy of tacrolimus (FK506) in increasing ENG and ALK1 expression. The rationale comes from a case report of a patient with HHT who received a liver transplantation after hepatic failure due to a liver arteriovenous malformation. The liver was transplanted, and the immunosuppressor FK506 was used to prevent the rejection. After the first month of FK506 treatment, the internal and external telangiectases, epistaxes, and anemia disappeared. Here, we find that the immunosuppressor FK506 increases the protein and mRNA expression of ENG and ALK1 in cultured endothelial cells and enhances the TGF-β1/ALK1 signaling pathway and endothelial cell functions like tubulogenesis and migration. These results suggest that the mechanism of action of FK506 involves a partial correction of endoglin and ALK1 haploinsufficiency and may therefore be an interesting drug for use in patients with HHT who undergo transplantationThis work was supported by the Ministerio de Ciencia e Innovacion [Grants SAF2008-01218, SAF2007-61827, and SAF2010-19222]. The therapeutical in vitro assays in HHT were supported by Fundacion Ramón Areces of Spain (Rare and Emergent Diseases) and a Fundacion Ramon Areces fellowship (to V.A.)Peer reviewe

Review of Pharmacological Strategies with Repurposed Drugs for Hereditary Hemorrhagic Telangiectasia Related Bleeding

© 2020 by the authors.The diagnosis of hereditary hemorrhagic telangiectasia (HHT) is based on the Curaçao criteria: epistaxis, telangiectases, arteriovenous malformations in internal organs, and family history. Genetically speaking, more than 90% of HHT patients show mutations in ENG or ACVRL1/ALK1 genes, both belonging to the TGF-β/BMP9 signaling pathway. Despite clear knowledge of the symptoms and genes of the disease, we still lack a definite cure for HHT, having just palliative measures and pharmacological trials. Among the former, two strategies are: intervention at “ground zero” to minimize by iron and blood transfusions in order to counteract anemia. Among the later, along the last 15 years, three different strategies have been tested: (1) To favor coagulation with antifibrinolytic agents (tranexamic acid); (2) to increase transcription of ENG and ALK1 with specific estrogen-receptor modulators (bazedoxifene or raloxifene), antioxidants (N-acetylcysteine, resveratrol), or immunosuppressants (tacrolimus); and (3) to impair the abnormal angiogenic process with antibodies (bevacizumab) or blocking drugs like etamsylate, and propranolol. This manuscript reviews the main strategies and sums up the clinical trials developed with drugs alleviating HHT.This research was funded by Ministry of Economy and Competitivity, grant number SAF2017-83351-R to L.M.B.Peer reviewe

Blockade of β2-Adrenergic Receptor Reduces Inflammation and Oxidative Stress in Clear Cell Renal Cell Carcinoma

Von Hippel-Lindau (VHL) syndrome is a rare inherited cancer disease where the lack of VHL protein triggers the development of multisystemic tumors such us retinal hemangioblastomas (HBs), CNS-HBs, and clear cell renal cell carcinoma (ccRCC). Since standard therapies in VHL have shown limited response, leaving surgery as the only possible treatment, targeting of the β2-adrenergic receptor (ADRB2) has shown therapeutic antitumor benefits on VHL-retinal HBs (clinical trial), VHL-CNS HBs, and VHL-ccRCC (in vitro and in vivo). In the present study, we wanted to look deep into the effects of the ADRB2 blockers propranolol and ICI-118,551 on two main aspects of cancer progression: (i) the changes on the inflammatory response of ccRCC cells; and (ii) the modulation on the Warburg effect (glycolytic metabolism), concretely, on the expression of genes involved in the cell reactive oxygen species (ROS) balance and levels. Accordingly, in vitro studies with primary VHL-ccRCC and 786-O cells measuring ROS levels, ROS-expression of detoxifying enzymes, and the expression of p65/NF-κB targets by RT-PCR were carried out. Furthermore, histological analyses of ccRCC samples from heterotopic mouse xenografts were performed. The obtained results show that ADRB2 blockade in ccRCC cells reduces the level of oxidative stress and stabilizes the inflammatory response. Thus, these data further support the idea of targeting ADRB2 as a promising strategy for the treatment of VHL and other non-VHL tumors

Copy number variations in endoglin locus: mapping of large deletions in Spanish families with hereditary hemorrhagic telangiectasia type 1

Abstract Background The hereditary hemorrhagic telangiectasia syndrome (HHT), also known as the Rendu–Osler-Weber syndrome is a multiorganic vascular disorder inherited as an autosomal dominant trait. Diagnostic clinical criteria include: epistaxis, telangiectases in mucocutaneous and gastrointestinal sites, arteriovenous malformations (AVMs) most commonly found in pulmonary, hepatic and cerebral circulations, and familial inheritance. HHT is transmitted in 90% of the cases as an autosomal dominant condition due to mutations in either endoglin (ENG), or activin receptor-like kinase 1 (ACVRL1/ALK1) genes (HHT type 1 and 2, respectively). Methods We have carried out a genetic analysis of four independent Spanish families with HHT clinical criteria, which has permitted the identification of new large deletions in ENG. These mutations were first detected using the MLPA technique and subsequently, the deletion breakpoints were mapped using a customized copy number variation (CNV) microarray. The array was designed to cover the ENG gene and surrounding areas. Results All tested families carried large deletions ranging from 3-kb to 100-kb, involving the ENG gene promoter, several ENG exons, and the two downstream genes FGSH and CDK9. Interestingly, common breakpoints coincident with Alu repetitive sequences were found among these families. Conclusions The systematic hybridization of DNA from HHT families, with deletions or duplications, to custom designed microarrays, could allow the mapping of breakpoints, coincident with repetitive Alu sequences that might act as “hot spots” in the development of chromosomal anomalies.This work was supported by grants from Ministerio de Economia y Competitividad (SAF2008-01218 and SAF2011-23475 to LMB; and SAF2010-19222 to CB), CIBERER (Intramural 11-707/112.02) and Fundación Ramón Areces (FRA; Rare and Emergent Diseases to LMB) of Spain. Virginia Albiñana was supported by FRA. Maria L. Ojeda-Fernandez is recipient of a CIBERER contract. CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII), Spain.Peer Reviewe