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1.  Hereditary haemorrhagic telangiectasia: a questionnaire based study to delineate the different phenotypes caused by endoglin and ALK1 mutations 
Journal of Medical Genetics  2003;40(8):585-590.
Background: Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant vascular dysplasia characterised by mucocutaneous telangiectasis, epistaxis, gastrointestinal haemorrhage, and arteriovenous malformations in the lung and brain. Causative mutations for HHT have been identified in two genes, endoglin and ALK1, which encode proteins involved in serine-threonine kinase signalling in the endothelial cell.
Methods: A number of people affected with HHT had completed a postal questionnaire as part of an international study to delineate the HHT phenotype. We identified questionnaires completed by subjects in whom we had identified a mutation in endoglin or ALK1. Further questionnaires were sent to families with known mutations. Data were only included from questionnaires returned by people known to carry disease causing mutations.
Results: Questionnaires were completed by 83 subjects with known mutations. Of these, 49 had endoglin mutations (HHT1) and 34 had ALK1 mutations (HHT2). Subjects with HHT1 reported an earlier onset of epistaxis (p=0.01) and telangiectasis (p=0.0001) than those with HHT2. Pulmonary arteriovenous malformations were only reported in the endoglin mutation group in our study (p<0.001).
Conclusions: Our questionnaire based study provides evidence that the HHT phenotype caused by mutations in endoglin (HHT1) is distinct from, and more severe than, HHT caused by mutations in ALK1 (HHT2). This has significant implications for diagnosis, screening, and treatment in the two different forms of HHT, as well as for understanding the pathogenesis of the disease.
PMCID: PMC1735540  PMID: 12920067
2.  Clinical and genetic analyses of three Korean families with hereditary hemorrhagic telangiectasia 
BMC Medical Genetics  2011;12:130.
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal-dominant vascular disorder, characterized by recurrent epistaxis, mucocutaneous telangiectases, and arteriovenous malformations (AVMs) in various visceral organs. Endoglin (ENG) and activin receptor-like kinase 1 (ACVRL1; ALK1), receptors for transforming growth factor-β (TGF-β) superfamily, have been identified as the principal HHT-causing genes.
Three unrelated Korean HHT patients and their asymptomatic as well as symptomatic family members were genetically diagnosed by sequencing whole exons and their flanking regions of ENG and ACVRL1. Functionality of an aberrant translation start codon, which is created by a substitution mutation at the 5'-untranslated region (UTR) of ENG found in a HHT family, was tested by transient in vitro transfection assay. Decay of the mutant transcripts was also assessed by allele-specific expression analysis.
Two ENG and one ACVRL1 mutations were identified: a known ENG mutation (c.360+1G > A; p.Gly74_Tyr120del); a novel ENG mutation (c.1-127C > T); and a novel ACVRL1 mutation (c.252_253insC; p.Val85fsX168). We further validated that the 5'-UTR ENG mutation prevents translation of ENG from the biological translation initiation site of the mutant allele, and leads to degradation of the mutant transcripts.
This is the first experimental demonstration that a 5'-UTR mutation can prevent translation of ENG among HHT patients, and further supports the previous notion that haploinsufficiency is the primary mechanism of HHT1. Our data also underscore the importance of including exons encoding 5' UTR for HHT mutation screening.
PMCID: PMC3202234  PMID: 21967607
3.  Hereditary haemorrhagic telangiectasia: current views on genetics and mechanisms of disease 
Journal of Medical Genetics  2006;43(2):97-110.
Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterised by epistaxis, telangiectases, and multiorgan vascular dysplasia. The two major types of disease, HHT1 and HHT2, are caused by mutations in the ENG (endoglin) and ACVRL1 genes, respectively. The corresponding endoglin and ALK‐1 proteins are specific endothelial receptors of the transforming growth factor β superfamily essential for maintaining vascular integrity. Many mutations have been identified in ENG and ACVRL1 genes and support the haploinsufficiency model for HHT. Two more genes have recently been implicated in HHT: MADH4 mutated in a combined syndrome of juvenile polyposis and HHT (JPHT), and an unidentified HHT3 gene linked to chromosome 5. Current knowledge on the genetics of HHT is summarised, including the pathways that link the genes responsible for HHT and the potential mechanisms underlying the pathogenesis of the disease.
PMCID: PMC2603035  PMID: 15879500
ACVRL1; ALK‐1; ENG; hereditary haemorrhagic telangiectasia; vascular disorders
4.  Molecular and functional analysis identifies ALK-1 as the predominant cause of pulmonary hypertension related to hereditary haemorrhagic telangiectasia 
Journal of Medical Genetics  2003;40(12):865-871.
Background: Mutations of the transforming growth factor ß (TGFß) receptor components ENDOGLIN and ALK-1 cause the autosomal dominant vascular disorder hereditary haemorrhagic telangiectasia (HHT). Heterozygous mutations of the type II receptor BMPR2 underlie familial primary pulmonary hypertension.
Objective: To investigate kindreds presenting with both pulmonary hypertension and HHT.
Methods: Probands and families were identified by specialist pulmonary hypertension centres in five countries. DNA sequence analysis of ALK-1, ENDOGLIN, and BMPR2 was undertaken. Cellular localisation was investigated by heterologous overexpression of mutant constructs in both BAEC and HeLa cells. The impact of a novel sequence variant was assessed through comparative analysis and computer modelling.
Results: Molecular analysis of 11 probands identified eight missense mutations of ALK-1, one of which was observed in two families. Mutations were located within exons 5 to 10 of the ALK-1 gene. The majority of ALK-1 mutant constructs appeared to be retained within the cell cytoplasm, in the endoplasmic reticulum. A novel GS domain mutation, when overexpressed, reached the cell surface but is predicted to disrupt conformational changes owing to loss of a critical hydrogen bond. Two novel missense mutations were identified in ENDOGLIN.
Conclusions: The association of pulmonary arterial hypertension and HHT identifies an important disease complication and appears most common among subjects with defects in ALK-1 receptor signalling. Future studies should focus on detailed molecular analysis of the common cellular pathways disrupted by mutations of ALK-1 and BMPR2 that cause inherited pulmonary vascular disease.
PMCID: PMC1735342  PMID: 14684682
5.  Visceral manifestations in hereditary haemorrhagic telangiectasia type 2 
Journal of Medical Genetics  2003;40(7):494-502.
Hereditary haemorrhagic telangiectasia (HHT) is a genetic vascular disorder characterised by epistaxis, telangiectases, and visceral manifestations. The two known disease types, HHT1 and HHT2, are caused by mutations in the endoglin (ENG) and ALK-1 genes, respectively. A higher frequency of pulmonary arteriovenous malformations (AVMs) has been reported for HHT1 while HHT2 is thought to be associated with a lower penetrance and milder disease manifestations. In this study, we present 10 families with an ALK-1 genotype. Visceral manifestations were detected in 24 (26%) of the 93 HHT2 patients from nine of the families and included gastrointestinal bleeding (14%), intrahepatic shunts (6%), and AVMs in the lung (4%) and brain (3%). Gastrointestinal bleeding, the most frequent visceral manifestation, was reported in six of the 10 families, mostly in patients over the age of 50. These patients also had frequent epistaxis and suffered from anaemia, often requiring blood transfusions. The identification of ALK-1 mutations in subjects with a suspected diagnosis and without clinical signs of HHT argue in favour of a molecular diagnosis. We also analysed the data published on 44 families with HHT2 and conclude that visceral manifestations occur in 26 of these families and affect 30% of HHT2 patients. This is considered an underestimate given incomplete and variable screening for lung, brain, and/or liver involvement in different clinical centres. These findings, however, stress the need for an early diagnosis of HHT that can be useful for the early control of associated visceral involvement.
PMCID: PMC1735537  PMID: 12843319
6.  Investigation of Endoglin Wild-Type and Missense Mutant Protein Heterodimerisation Using Fluorescence Microscopy Based IF, BiFC and FRET Analyses 
PLoS ONE  2014;9(7):e102998.
The homodimeric transmembrane receptor endoglin (CD105) plays an important role in angiogenesis. This is highlighted by mutations in its gene, causing the vascular disorder HHT1. The main role of endoglin function has been assigned to the modulation of transforming growth factor β and bone morphogenetic protein signalling in endothelial cells. Nevertheless, other functions of endoglin have been revealed to be involved in different cellular functions and in other cell types than endothelial cells. Compared to the exploration of its natural function, little experimental data have been gathered about the mode of action of endoglin HHT mutations at the cellular level, especially missense mutations, and to what degree these might interfere with normal endoglin function. In this paper, we have used fluorescence-based microscopic techniques, such as bimolecular fluorescence complementation (BiFC), immunofluorescence staining with the endoglin specific monoclonal antibody SN6, and protein interaction studies by Förster Resonance Energy Transfer (FRET) to investigate the formation and cellular localisation of possible homo- and heterodimers composed of endoglin wild-type and endoglin missense mutant proteins. The results show that all of the investigated missense mutants dimerise with themselves, as well as with wild-type endoglin, and localise, depending on the position of the affected amino acid, either in the rough endoplasmic reticulum (rER) or in the plasma membrane of the cells. We show that the rER retained mutants reduce the amount of endogenous wild-type endoglin on the plasma membrane through interception in the rER when transiently or stably expressed in HMEC-1 endothelial cells. As a result of this, endoglin modulated TGF-β1 signal transduction is also abrogated, which is not due to TGF-β receptor ER trafficking interference. Protein interaction analyses by FRET show that rER located endoglin missense mutants do not perturb protein processing of other membrane receptors, such as TβRII, ALK5 or ALK1.
PMCID: PMC4117486  PMID: 25080347
7.  Directional Next-Generation RNA Sequencing and Examination of Premature Termination Codon Mutations in Endoglin/Hereditary Haemorrhagic Telangiectasia 
Molecular Syndromology  2013;4(4):184-196.
Hereditary haemorrhagic telangiectasia (HHT) is a disease characterised by abnormal vascular structures, and most commonly caused by mutations in ENG, ACVRL1 or SMAD4 encoding endothelial cell-expressed proteins involved in TGF-β superfamily signalling. The majority of mutations reported on the HHT mutation database are predicted to lead to stop codons, either due to frameshifts or direct nonsense substitutions. The proportion is higher for ENG (67%) and SMAD4 (65%) than for ACVRL1 (42%), p < 0.0001. Here, by focussing on ENG, we report why conventional views of these mutations may need to be revised. Of the 111 stop codon-generating ENG mutations, on ExPASy translation, all except one were premature termination codons (PTCs), sited at least 50-55 bp upstream of the final exon-exon boundary of the main endoglin isoform, L-endoglin. This strongly suggests that the mutated RNA species will undergo nonsense-mediated decay. We provide new in vitro expression data to support dominant negative activity of stable truncated endoglin proteins but suggest these will not generate HHT: the single natural stop codon mutation in L-endoglin (sited within 50-55 nucleotides of the final exon-exon boundary) is unlikely to generate functional protein since it replaces the entire transmembrane domain, as would 8 further natural stop codon mutations, if the minor S-endoglin isoform were implicated in HHT pathogenesis. Finally, next-generation RNA sequencing data of 7 different RNA libraries from primary human endothelial cells demonstrate that multiple intronic regions of ENG are transcribed. The potential consequences of heterozygous deletions or duplications of such regions are discussed. These data support the haploinsufficiency model for HHT pathogenesis, explain why final exon mutations have not been detected to date in HHT, emphasise the potential need for functional examination of non-PTC-generating mutations, and lead to proposals for an alternate stratification system of mutational types for HHT genotype-phenotype correlations.
PMCID: PMC3666459  PMID: 23801935
Alternative splicing; Nonsense-mediated decay; Pervasive transcription; Premature termination codons

8.  Gastric angiodysplasia in a hereditary hemorrhagic telangiectasia type 2 patient 
Hereditary hemorrhagic telangiectasia (HHT) is a rare autosomal-dominantly inherited disease that occurs in approximately one in 5000 to 8000 people. Clinical diagnosis of HHT is made when a person presents three of the following four criteria: family history, recurrent nosebleeds, mucocutaneous telangiectasis, and arteriovenous malformations (AVM) in the brain, lung, liver and gastrointestinal (GI) tract. Although epistaxis is the most common presenting symptom, AVMs affecting the lungs, brain and GI tract provoke a more serious outcome. Heterozygous mutations in endoglin, activin receptor-like kinase 1 (ACVRL1; ALK1), and SMAD4, the genes involved in the transforming growth factor-β family signaling cascade, cause HHT. We report here the case of a 63 year-old male patient who presented melena and GI bleeding episodes, proven to be caused by bleeding from multiple gastric angiodysplasia. Esophagogastroduodenoscopy revealed multiple angiodysplasia throughout the stomach. Endoscopic argon plasma coagulation was performed to control bleeding from a gastric angiodysplasia. The patient has been admitted several times with episodes of hemoptysis and hematochezia. One year ago, the patient was hospitalized due to right-sided weakness, which was caused by left basal ganglia hemorrhage as the part of HHT presentation. In family history, the patient’s mother and elder sister had died, due to intracranial hemorrhage, and his eldest son has been suffered from recurrent epistaxis for 20 years. A genetic study revealed a mutation in exon 3 of ALK1 (c.199C > T; p.Arg67Trp) in the proband and his eldest son presenting epistaxis.
PMCID: PMC3332300  PMID: 22553411
Hereditary hemorrhagic telangiectasia; Angiodysplasia; Intracranial hemorrhage; Epistaxis; Activin receptor-like kinase 1
9.  Hereditary haemorrhagic telangiectasia: mutation detection, test sensitivity and novel mutations 
Journal of Medical Genetics  2006;43(9):722-728.
Hereditary haemorrhagic telangiectasia (HHT) is a genetic disorder present in 1 in 8000 people and associated with arteriovenous malformations. Genetic testing can identify individuals at risk of developing the disease and is a useful diagnostic tool.
To present a strategy for mutation detection in families clinically diagnosed with HHT.
An optimised strategy for detecting mutations that predispose to HHT is presented. The strategy includes quantitative multiplex polymerase chain reaction, sequence analysis, RNA analysis, validation of missense mutations by amino acid conservation analysis for the ENG (endoglin) and ACVRL1 (ALK1) genes, and analysis of an ACVRL1 protein structural model. If no causative ENG or ACVRL1 mutation is found, proband samples are referred for sequence analysis of MADH4 (associated with a combined syndrome of juvenile polyposis and HHT).
Data obtained over the past eight years were summarised and 16 novel mutations described. Mutations were identified in 155 of 194 families with a confirmed clinical diagnosis (80% sensitivity). Of 155 mutations identified, 94 were in ENG (61%), 58 in ACVRL1 (37%), and three in MADH4 (2%).
For most missense variants of ENG and ACVRL1 reported to date, study of amino acid conservation showed good concordance between prediction of altered protein function and disease occurrence. The 39 families (20%) yet to be resolved may carry ENG, ACVRL1, or MADH4 mutations too complex or difficult to detect, or mutations in genes yet to be identified.
PMCID: PMC2564570  PMID: 16690726
vascular disease; endoglin; ACVRL1; MADH4
10.  5'UTR mutations of ENG cause hereditary hemorrhagic telangiectasia 
Hereditary hemorrhagic telangiectasia (HHT) is a vascular disorder characterized by epistaxis, arteriovenous malformations, and telangiectases. The majority of the patients have a mutation in the coding region of the activin A receptor type II-like 1 (ACVRL1) or Endoglin (ENG) gene. However, in approximately 15% of cases, sequencing analysis and deletion/duplication testing fail to identify mutations in the coding regions of these genes. Knowing its vital role in transcription and translation control, we were prompted to investigate the 5'untranslated region (UTR) of ENG.
Methods and Results
We sequenced the 5'UTR of ENG for 154 HHT patients without mutations in ENG or ACVRL1 coding regions. We found a mutation (c.-127C > T), which is predicted to affect translation initiation and alter the reading frame of endoglin. This mutation was found in a family with linkage to the ENG, as well as in three other patients, one of which had an affected sibling with the same mutation. In vitro expression studies showed that a construct with the c.-127C > T mutation alters the translation and decreases the level of the endoglin protein. In addition, a c.-9G > A mutation was found in three patients, one of whom was homozygous for this mutation. Expression studies showed decreased protein levels suggesting that the c.-9G > A is a hypomorphic mutation.
Our results emphasize the need for the inclusion of the 5'UTR region of ENG in clinical testing for HHT.
PMCID: PMC3277489  PMID: 22192717
5'UTR region; ENG; c.-127C > T; c.-9G > A; homozygous
11.  Bioinformatic Analysis of Pathogenic Missense Mutations of Activin Receptor Like Kinase 1 Ectodomain 
PLoS ONE  2011;6(10):e26431.
Activin A receptor, type II-like kinase 1 (also called ALK1), is a serine-threonine kinase predominantly expressed on endothelial cells surface. Mutations in its ACVRL1 encoding gene (12q11-14) cause type 2 Hereditary Haemorrhagic Telangiectasia (HHT2), an autosomal dominant multisystem vascular dysplasia. The study of the structural effects of mutations is crucial to understand their pathogenic mechanism. However, while an X-ray structure of ALK1 intracellular domain has recently become available (PDB ID: 3MY0), structure determination of ALK1 ectodomain (ALK1EC) has been elusive so far. We here describe the building of a homology model for ALK1EC, followed by an extensive bioinformatic analysis, based on a set of 38 methods, of the effect of missense mutations at the sequence and structural level. ALK1EC potential interaction mode with its ligand BMP9 was then predicted combining modelling and docking data. The calculated model of the ALK1EC allowed mapping and a preliminary characterization of HHT2 associated mutations. Major structural changes and loss of stability of the protein were predicted for several mutations, while others were found to interfere mainly with binding to BMP9 or other interactors, like Endoglin (CD105), whose encoding ENG gene (9q34) mutations are known to cause type 1 HHT. This study gives a preliminary insight into the potential structure of ALK1EC and into the structural effects of HHT2 associated mutations, which can be useful to predict the potential effect of each single mutation, to devise new biological experiments and to interpret the biological significance of new mutations, private mutations, or non-synonymous polymorphisms.
PMCID: PMC3196573  PMID: 22028876
12.  SMAD4 mutations found in unselected HHT patients 
Journal of Medical Genetics  2006;43(10):793-797.
Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant disease exhibiting multifocal vascular telangiectases and arteriovenous malformations. The majority of cases are caused by mutations in either the endoglin (ENG) or activin receptor‐like kinase 1 (ALK1, ACVRL1) genes; both members of the transforming growth factor (TGF)‐β pathway. Mutations in SMAD4, another TGF‐β pathway member, are seen in patients with the combined syndrome of juvenile polyposis (JP) and HHT (JP‐HHT).
We sought to determine if HHT patients without any apparent history of JP, who were undergoing routine diagnostic testing, would have mutations in SMAD4. We tested 30 unrelated HHT patients, all of whom had been referred for DNA based testing for HHT and were found to be negative for mutations in ENG and ALK1.
Three of these people harboured mutations in SMAD4, a rate of 10% (3/30). The SMAD4 mutations were similar to those found in other patients with the JP‐HHT syndrome.
The identification of SMAD4 mutations in HHT patients without prior diagnosis of JP has significant and immediate clinical implications, as these people are likely to be at risk of having JP‐HHT with the associated increased risk of gastrointestinal cancer. We propose that routine DNA based testing for HHT should include SMAD4 for samples in which mutations in neither ENG nor ALK1 are identified. HHT patients with SMAD4 mutations should be screened for colonic and gastric polyps associated with JP.
PMCID: PMC2563178  PMID: 16613914
hereditary haemorrhagic telangiectasia (HHT); juvenile polyposis (JP); endoglin (ENG); activin receptor‐like kinase 1 (ALK1, ACVRL1); SMAD4
13.  Immunohistochemical analysis of a merkeloma observed in a patient affected by hereditary haemorrhagic telangiectasia 
BMJ Case Reports  2010;2010:bcr09.2009.2251.
Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterised by epistaxis, telangiectases, and multiorgan vascular dysplasia. Mutations of the ENG and ACVRL1 genes cause at least 80% of cases. We report the first case of merkeloma found in a patient with HHT carrying an ENG mutation. We analysed the tumour with immunohistochemical methods using primary antibodies against CD105 (endoglin), TGF-β, Smad4, CD31 and CD34. Tumour cells were positive for Smad4, weakly positive for TGF-β, and negative for CD105. Vasal endothelial cells were highly positive for CD105, CD31 and CD34. No remarkable differences between cancer and normal cells in our patient or between the patient’s merkeloma and two control merkelomas were observed. The presence of a merkeloma in an HHT patient could be an occasional association, but to certainly assume it further investigations are needed.
PMCID: PMC3028296  PMID: 22315638
14.  Copy number variations in endoglin locus: mapping of large deletions in Spanish families with hereditary hemorrhagic telangiectasia type 1 
BMC Medical Genetics  2013;14:121.
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).
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.
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.
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.
PMCID: PMC4222255  PMID: 24267784
Hereditary hemorrhagic telangiectasia (HHT); Endoglin deletions; Multiplex ligation PCR assisted assay (MLPA); Copy number variation (CNV) arrays; Alu repetitive sequences
15.  A hereditary haemorrhagic telangiectasia family with pulmonary involvement is unlinked to the known HHT genes, endoglin and ALK-1 
Thorax  2000;55(8):685-690.
BACKGROUND—Pulmonary arteriovenous malformations (PAVMs) occur in over 25% of patients with the autosomal dominant disorder hereditary haemorrhagic telangiectasia (HHT). Mutations in two genes, endoglin and ALK-1, are known to cause HHT. Each encodes a protein expressed on vascular endothelial cells and involved in signalling by members of the transforming growth factor (TGF)-β superfamily. To date, PAVMs have not been detected in ALK-1 families. There is evidence from a single HHT family without pulmonary involvement that a third HHT gene may exist. To establish the existence of a further HHT gene responsible for PAVMs, linkage analyses were performed on an expanded PAVM-HHT family in which HHT did not result from endoglin mutations.
METHODS—Family members were assessed clinically to assign HHT disease status and were screened for PAVMs. DNA was extracted from blood obtained from 20 individuals of known disease status. Short tandem repeat polymorphic markers spanning the intervals containing the endoglin and ALK-1 genes were amplified by the polymerase chain reaction using 33P-labelled oligonucleotide primers, separated by denaturing polyacrylamide gel electrophoresis (PAGE), and the resultant autoradiographs were examined for allele sizes. Linkage analyses were performed using MLINK and GENEHUNTER.
RESULTS—Twelve members spanning four generations were affected with HHT. Two had proven PAVMs, one with a classical appearance, the other exhibiting microscopic PAVMs exacerbated by pregnancy. Two point lod and multipoint lod scores significantly excluded linkage to endoglin and ALK-1 in this pedigree.
CONCLUSIONS—This study confirms the existence of a third HHT locus that accounts for disease in some HHT patients with pulmonary involvement.

PMCID: PMC1745830  PMID: 10899246
16.  Mutant endoglin in hereditary hemorrhagic telangiectasia type 1 is transiently expressed intracellularly and is not a dominant negative. 
Journal of Clinical Investigation  1997;100(10):2568-2579.
Endoglin (CD105), a component of the TGF-beta 1 receptor complex, is the target gene for the dominantly inherited vascular disorder hereditary hemorrhagic telangiectasia type 1 (HHT1). We have identified a novel endoglin splice site mutation, leading to an in-frame deletion of exon 3, in a new-born from a family with HHT. Expression of normal and mutant endoglin proteins was analyzed in umbilical vein endothelial cells from this baby and in activated monocytes from the affected father. In both samples, only normal dimeric endoglin (160 kD) was observed at the cell surface, at 50% of control levels. Despite an intact transmembrane region, mutant protein was only detectable by metabolic labeling, as an intracellular homodimer of 130 kD. In monocytes from three clinically affected HHT1 patients, with known mutations creating premature stop codons in exons 8 and 10, surface endoglin was also reduced by half and no mutant was detected. Overexpression into COS-1 cells of endoglin cDNA truncated in exons 7 and 11, revealed their intracellular expression, inability to be secreted and to form heterodimers at the cell surface. These results indicate that mutated forms of endoglin are transiently expressed intracellularly and not likely to act as dominant negative proteins, as proposed previously. A reduction in the level of functional endoglin is thus involved in the generation of HHT1, and associated arteriovenous malformations.
PMCID: PMC508458  PMID: 9366572
17.  A new locus for hereditary haemorrhagic telangiectasia (HHT3) maps to chromosome 5 
Journal of Medical Genetics  2005;42(7):577-582.
Patients with hereditary haemorrhagic telangiectasia (HHT, or Osler-Weber-Rendu syndrome) have variable presentation patterns and a high risk of preventable complications. Diagnostic tests for mutations in endoglin (HHT type 1) and ALK-1 (HHT type 2) are available. Some HHT patients are now known to have HHT-juvenile polyposis overlap syndrome due to Smad4 mutations. Families were ascertained following the presentation of probands for embolization of pulmonary arteriovenous malformations. Genome-wide linkage studies using over 700 polymorphic markers, and sequencing of candidate genes, were performed. In a previously described HHT family unlinked to endoglin or ALK-1, linkage to Smad4 was excluded, and no mutations were identified in the endoglin, ALK-1, or Smad4 genes. Two point LOD scores and recombination mapping identified a 5.4 cM HHT3 disease gene interval on chromosome 5 in which a single haplotype was inherited by all affected members of the pedigree. The remainder of the genome was excluded to a 2–5 cM resolution. We are currently studying a further family potentially linked to HHT3. We conclude that classical HHT with pulmonary involvement can result from mutations in an unidentified gene on chromosome 5. Identification of HHT3 should further illuminate HHT pathogenic mechanisms in which aberrant transforming growth factor (TGF)-ß signalling is implicated.
PMCID: PMC1736109  PMID: 15994879
18.  HHT diagnosis by Mid-infrared spectroscopy and artificial neural network analysis 
The vascular disorder Hereditary Hemorrhagic Telangiectasia (HHT) is in general an inherited disease caused by mutations in the TGF-β/BMP receptors endoglin or ALK1 or in rare cases by mutations of the TGF-β signal transducer protein Smad4 leading to the combined syndrome of juvenile polyposis and HHT. HHT is characterized by several clinical symptoms like spontaneous and recurrent epistaxis, multiple telangiectases at sites like lips, oral cavity, fingers, nose, and visceral lesions like gastrointestinal telangiectasia, pulmonary, hepatic, cerebral or spinal arteriovenous malformations. The disease shows an inter- and intra-family variability in penetrance as well as symptoms from mild to life threatening. Penetrance is also depending on age. Diagnosis of the disease is based on the presence of some of the listed symptoms or by genetic testing. HHT diagnosis is laborious, time consuming, costly and sometimes uncertain. Not all typical symptoms may be present, especially at a younger age, and genetic testing does not always identify the disease causing mutation.
Infrared (IR) spectroscopy was investigated as a potential alternative to the current diagnostic methods. IR-spectra were obtained by Fourier-transform Mid-IR spectroscopy from blood plasma from HHT patients and a healthy control group. Spectral data were mathematically processed and subsequently classified and analysed by artificial neural network (ANN) analyses and by visual analysis of scatter plots of the dominant principal components.
The analyses showed that for HHT a disease specific IR-spectrum exists that is significantly different from the control group. Furthermore, at the current stage with the here used methods, HHT can be diagnosed by Mid-IR-spectroscopy in combination with ANN analysis with a sensitivity and specificity of at least 95%. Visual analysis of PCA scatter plots revealed an inter class variation of the HHT group.
IR-spectroscopy in combination with ANN analysis can be considered to be a serious alternative diagnostic method compared to clinical and genetically based methods. Blood plasma is an ideal candidate for diagnostic purposes, it is inexpensive, easy to isolate and only minimal amounts are required. In addition, IR-spectroscopy measurement times are fast, less than one minute, and diagnosis is not based on interpretation of may be uncertain clinical data. And last but not least, the method is inexpensive.
PMCID: PMC3698163  PMID: 23805858
ALK1; Artificial Neural Network; Diagnostic; Disease; Endoglin; Hereditary Hemorrhagic Telangiectasia; Mid-infrared Spectroscopy; Mutation
19.  Long Non-Coding RNA Expression Profiles in Hereditary Haemorrhagic Telangiectasia 
PLoS ONE  2014;9(3):e90272.
Hereditary Haemorrhagic Telangiectasia (HHT) is an autosomal dominantly inherited vascular disease characterized by the presence of mucocutaneous telangiectasia and arteriovenous malformations in visceral organs. HHT is predominantly caused by mutations in ENG and ACVRL1, which both belong to the TGF-β signalling pathway. The exact mechanism of how haploinsufficiency of ENG and ACVRL1 leads to HHT manifestations remains to be identified. As long non-coding RNAs (lncRNAs) are increasingly recognized as key regulators of gene expression and constitute a sizable fraction of the human transcriptome, we wanted to assess whether lncRNAs play a role in the molecular pathogenesis of HHT manifestations. By microarray technology, we profiled lncRNA transcripts from HHT nasal telangiectasial and non-telangiectasial tissue using a paired design. The microarray probes were annotated using the GENCODE v.16 dataset, identifying 4,810 probes mapping to 2,811 lncRNAs. Comparing HHT telangiectasial tissue with HHT non-telangiectasial tissue, we identified 42 lncRNAs that are differentially expressed (q<0.001). Using GREAT, a tool that assumes cis-regulation, we showed that differently expressed lncRNAs are enriched for genomic loci involved in key pathways concerning HHT. Our study identified lncRNAs that are aberrantly expressed in HHT telangiectasia and indicates that lncRNAs may contribute to regulate protein-coding loci in HHT. These results suggest that the lncRNA component of the transcriptome deserves more attention in HHT. A deeper understanding of lncRNAs and their role in telangiectasia formation possesses potential for discovering therapeutic targets in HHT.
PMCID: PMC3946172  PMID: 24603890
20.  Oxidative Stress Contributes to Endothelial Dysfunction in Mouse Models of Hereditary Hemorrhagic Telangiectasia 
Hereditary hemorrhagic telangiectasia (HHT) is a vascular dysplasia caused by mutations in endoglin (ENG; HHT1) or activin receptor-like kinase (ALK1; HHT2) genes, coding for transforming growth factor-β (TGF-β) superfamily receptors. We demonstrated previously that endoglin and ALK1 interact with endothelial NO synthase (eNOS) and affect its activation. Endothelial cells deficient in endoglin or ALK1 proteins show eNOS uncoupling, reduced NO, and increased reactive oxygen species (ROS) production. In this study, we measured NO and H2O2 levels in several organs of adult Eng and Alk1 heterozygous mice, to ascertain whether decreased NO and increased ROS production is a generalized manifestation of HHT. A significant reduction in NO and increase in ROS production were found in several organs, known to be affected in patients. ROS overproduction in mutant mice was attributed to eNOS, as it was L-NAME inhibitable. Mitochondrial ROS contribution, blocked by antimycin, was highest in liver while NADPH oxidase, inhibited by apocynin, was a major source of ROS in the other tissues. However, there was no difference in antimycin- and apocynin-inhibitable ROS production between mutant and control mice. Our results indicate that eNOS-derived ROS contributes to endothelial dysfunction and likely predisposes to disease manifestations in several organs of HHT patients.
PMCID: PMC3540964  PMID: 23320130
21.  Clinical Features and Mutations in the ENG, ACVRL1, and SMAD4 genes in Korean Patients with Hereditary Hemorrhagic Telangiectasia 
Hereditary hemorrhagic telangiectasia (HHT) is an inherited disorder that is characterized by abnormal communication between the arteries and veins in the skin, mucosa, and various organs. HHT has been reported to show significant phenotypic variability and genetic heterogeneity with wide ethnic and geographic variations. Although mutations in the endoglin (ENG) and activin A receptor type II-like 1 (ACVRL1) genes have been known to cause HHT for more than 10 yr, little is known about the clinical features or genetic background of Korean patients with HHT. In addition, mutations in mothers against decapentaplegic homolog 4 (SMAD4) are also seen in patients with the combined syndrome of juvenile polyposis and HHT. This study examined five Korean patients with the typical manifestations of HHT such as frequent epistaxis and pulmonary arteriovenous malformations. Direct sequencing of the ENG and ACVRL1 genes revealed one known mutation, ENG c.277C>T, in one patient and two novel mutations, ENG c.992-1G>C and ACVRL1 c.81dupT in two patients, respectively. The remaining two patients with negative results were screened for SMAD4 mutations as well as gross deletions of ENG and ACVRL1 using multiple ligation-dependent probe amplification, but none was detected. Despite the small number of patients investigated, we firstly report Korean patients with genetically confirmed HHT, and show the genetic and allelic heterogeneity underlying HHT.
PMCID: PMC2650970  PMID: 19270816
Telangiectasia, Hereditary Hemorrhagic; ENG; ACVRL1; SMAD4; Mutation; Korean
22.  Mutation analysis of "Endoglin" and "Activin receptor-like kinase" genes in German patients with hereditary hemorrhagic telangiectasia and the value of rapid genotyping using an allele-specific PCR-technique 
BMC Medical Genetics  2009;10:53.
Hereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber syndrome, is an autosomal dominant disorder which is clinically characterised by recurrent epistaxis, mucocutaneous telangiectasia and visceral arteriovenous malformations. Genetic linkage studies identified two genes primarily related to HHT: endoglin (ENG) on chromosome 9q33-34 and activin receptor-like kinase1 (ACVRL1) on chromosome 12q13. We have screened a total of 41 unselected German patients with the suspected diagnosis of HHT. Mutation analysis for the ENG and ACVRL1 genes in all patients was performed by PCR amplification. Sequences were then compared to the HHT database sequences of the ENG mRNA (accession no. BC014271.2) and the ACVRL1 mRNA (accession no. NM000020.1).
We identified 15 different mutations in 18 cases by direct sequencing. Among these mutations, one novel ENG mutation could be detected which has not yet been described in the literature before. The genotype-phenotype correlation was consistent with a higher frequency of pulmonary arteriovenous malformations in patients with ENG mutations than in patients with ACVRL1 mutations in our collective.
For rapid genotyping of mutations and SNPs (single nucleotide polymorphisms) in ENG and ACVRL1, allele-specific PCR methods with sequence-specific primers (PCR-SSP) were established and their value analysed.
PMCID: PMC2701415  PMID: 19508727
23.  Genotype‐phenotype relationship in hereditary haemorrhagic telangiectasia 
Journal of Medical Genetics  2005;43(4):371-377.
Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterised by vascular malformations in multiple organ systems, resulting in mucocutaneous telangiectases and arteriovenous malformations predominantly in the lungs (pulmonary arteriovenous malformation; PAVM), brain (cerebral arteriovenous malformation; CAVM), and liver (hepatic arteriovenous malformation; HAVM). Mutations in the ENG and ALK‐1 genes lead to HHT1 and HHT2 respectively. In this study, a genotype‐phenotype analysis was performed. A uniform and well classified large group of HHT patients and their family members were screened for HHT manifestations. Groups of patients with a clinically confirmed diagnosis and/or genetically established diagnosis (HHT1 or HHT2) were compared. The frequency of PAVM, CAVM, HAVM, and gastrointestinal telangiectases were determined to establish the genotype‐phenotype relationship. The analysis revealed differences between HHT1 and HHT2 and within HHT1 and HHT2 between men and women. PAVMs and CAVMs occur more often in HHT1, whereas HAVMs are more frequent in HHT2. Furthermore, there is a higher prevalence of PAVM in women compared with men in HHT1. In HHT1 and HHT2, there is a higher frequency of HAVM in women. HHT1 has a distinct, more severe phenotype than HHT2. There is a difference in the presence of symptoms between men and women. With these data, genetic counselling can be given more accurately when the family mutation is known.
PMCID: PMC2563220  PMID: 16155196
hereditary haemorrhagic telangiectasia; Rendu‐Osler‐Weber disease;  ENG ;  ALK‐1 ; phenotype‐genotype relation; arteriovenous malformation
24.  Mutation study of Spanish patients with Hereditary Hemorrhagic Telangiectasia 
BMC Medical Genetics  2008;9:75.
Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant and age-dependent vascular disorder characterised mainly by mutations in the Endoglin (ENG) or activin receptor-like kinase-1 (ALK1, ACVRL1) genes.
Here, we have identified 22 ALK1 mutations and 15 ENG mutations, many of which had not previously been reported, in independent Spanish families afflicted with HHT.
We identified mutations in thirty-seven unrelated families. A detailed analysis of clinical symptoms was recorded for each patient analyzed, with a higher significant presence of pulmonary arteriovenous malformations (PAVM) in HHT1 patients over HHT2. Twenty-two mutations in ALK1 and fifteen in ENG genes were identified. Many of them, almost half, represented new mutations in ALK1 and in ENG. Missense mutations in ENG and ALK1 were localized in a tridimensional protein structure model.
Overall, ALK1 mutations (HHT2) were predominant over ENG mutations (HHT1) in our Spanish population, in agreement with previous data from our country and other Mediterranean countries (France, Italy), but different to Northern Europe or North America. There was a significant increase of PAVM associated with HHT1 over HHT2 in these families.
PMCID: PMC2518546  PMID: 18673552
25.  Novel Biochemical Pathways of Endoglin in Vascular Cell Physiology 
Journal of cellular biochemistry  2007;102(6):1375-1388.
The broad role of the transforming growth factor beta (TGFβ) signaling pathway in vascular development, homeostasis, and repair is well appreciated. Endoglin is emerging as a novel, complex, and poorly understood regulatory component of the TGFβ receptor complex, whose importance is underscored by its recognition as the site of mutations causing hereditary hemorrhagic telangiectasia (HHT) [McAllister et al., 1994]. Extensive analyses of endoglin function in normal developmental mouse models [Bourdeau et al., 1999; Li et al., 1999; Arthur et al., 2000] and in HHT animal models [Bourdeau et al., 2000; Torsney et al., 2003] exemplify the importance of understanding endoglin’s biochemical functions. However, novel mechanisms underlying the regulation of these pathways continue to emerge. These mechanisms include modification of TGFβ receptor signaling at the ligand and receptor activation level, direct effects of endoglin on cell adhesion and migration, and emerging roles for endoglin in the determination of stem cell fate and tissue patterning. The purpose of this review is to highlight the cellular and molecular studies that underscore the central role of endoglin in vascular development and disease.
PMCID: PMC2199238  PMID: 17975795
endoglin; transforming growth factor-beta receptors; hereditary hemorrhagic telangiectasia; vascular pathology; vascular endothelium; vascular smooth muscle

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