title: Mutation affecting the proximal promoter of Endoglin as the origin of hereditary hemorrhagic telangiectasia type 1.
abstract: 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.
authors.affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Ramiro de Maeztu 9, Madrid, 28040, Spain.
authors.forename: Virginia
authors.initials: V
authors.lastname: Albiñana
authors.affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Ramiro de Maeztu 9, Madrid, 28040, Spain.
authors.forename: Ma Paz
authors.initials: MP
authors.lastname: Zafra
authors.affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Ramiro de Maeztu 9, Madrid, 28040, Spain.
authors.forename: Jorge
authors.initials: J
authors.lastname: Colau
authors.affiliation: HHT Spanish Unit, Hospital Sierrallana and Centro de InvestigacionBiomedica en Red de Enfermedades Raras (CIBERER), Torrelavega, Santander, Spain.
authors.forename: Roberto
authors.initials: R
authors.lastname: Zarrabeitia
authors.affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Ramiro de Maeztu 9, Madrid, 28040, Spain.
authors.forename: Lucia
authors.initials: L
authors.lastname: Recio-Poveda
authors.affiliation: Secugen SL, Madrid, Spain.
authors.forename: Leticia
authors.initials: L
authors.lastname: Olavarrieta
authors.affiliation: Secugen SL, Madrid, Spain.
authors.forename: Julián
authors.initials: J
authors.lastname: Pérez-Pérez
authors.affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Ramiro de Maeztu 9, Madrid, 28040, Spain. cibluisa@cib.csic.es.
authors.forename: Luisa M
authors.initials: LM
authors.lastname: Botella
mesh_terms: D030301:Activin Receptors, Type II / Q000235:genetics; D000483:Alleles; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000071063:Endoglin / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D017930:Genes, Reporter; D005838:Genotype; D006801:Humans; D009000:Monocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D009154:Mutation; D010375:Pedigree; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D013683:Telangiectasia, Hereditary Hemorrhagic / Q000235:genetics* / Q000473:pathology; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism
chemical_list: D000071063:Endoglin; D014157:Transcription Factors; D004247:DNA; C425364:ACVRL1 protein, human; D030301:Activin Receptors, Type II
keywords: Endoglin promoter; Hereditary hemorrhagic telangiectasia (HHT); Rare disease; Transcription regulation