title: Functional and splicing defect analysis of 23 ACVRL1 mutations in a cohort of patients affected by Hereditary Hemorrhagic Telangiectasia.
abstract: Hereditary Hemorrhagic Telangiectasia syndrome (HHT) or Rendu-Osler-Weber (ROW) syndrome is an autosomal dominant vascular disorder. Two most common forms of HHT, HHT1 and HHT2, have been linked to mutations in the endoglin (ENG) and activin receptor-like kinase 1 (ACVRL1or ALK1) genes respectively. This work was designed to examine the pathogenicity of 23 nucleotide variations in ACVRL1 gene detected in more than 400 patients. Among them, 14 missense mutations and one intronic variant were novels, and 8 missense mutations were previously identified with questionable implication in HHT2. The functionality of missense mutations was analyzed in response to BMP9 (specific ligand of ALK1), the maturation of the protein products and their localization were analyzed by western blot and fluorescence microscopy. The splicing impairment of the intronic and of two missense mutations was examined by minigene assay. Functional analysis showed that 18 out of 22 missense mutations were defective. Splicing analysis revealed that one missense mutation (c.733A>G, p.Ile245Val) affects the splicing of the harboring exon 6. Similarly, the intronic mutation outside the consensus splicing sites (c.1048+5G>A in intron 7) was seen pathogenic by splicing study. Both mutations induce a frame shift creating a premature stop codon likely resulting in mRNA degradation by NMD surveillance mechanism. Our results confirm the haploinsufficiency model proposed for HHT2. The affected allele of ACVRL1 induces mRNA degradation or the synthesis of a protein lacking the receptor activity. Furthermore, our data demonstrate that functional and splicing analyses together, represent two robust diagnostic tools to be used by geneticists confronted with novel or conflicted ACVRL1 mutations.
authors.affiliation: Genetics of rare diseases, University of Poitiers, Poitiers, France; Lebanese University Campus Hariri, Faculty of Science / EDST, Hadath, Lebanon.
authors.forename: Ferdos
authors.initials: F
authors.lastname: Alaa El Din
authors.affiliation: Genetics of rare diseases, University of Poitiers, Poitiers, France; Department of Genetics, University Hospital of Poitiers, Poitiers, France.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Patri
authors.affiliation: Genetics of rare diseases, University of Poitiers, Poitiers, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Thoreau
authors.affiliation: Genetics of rare diseases, University of Poitiers, Poitiers, France; Department of Genetics, University Hospital of Poitiers, Poitiers, France.
authors.forename: Montserrat
authors.initials: M
authors.lastname: Rodriguez-Ballesteros
authors.affiliation: Lebanese University Campus Hariri, Faculty of Science / EDST, Hadath, Lebanon.
authors.forename: Eva
authors.initials: E
authors.lastname: Hamade
authors.affiliation: INSERM U1036, CEA of Grenoble, Grenoble, France.
authors.forename: Sabine
authors.initials: S
authors.lastname: Bailly
authors.affiliation: Genetics of rare diseases, University of Poitiers, Poitiers, France; Department of Genetics, University Hospital of Poitiers, Poitiers, France; Competence Centre of Rendu-Osler, University Hospital of Poitiers, Poitiers, France.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gilbert-Dussardier
authors.affiliation: Lebanese University Campus Hariri, Faculty of Science / EDST, Hadath, Lebanon.
authors.forename: Raghida
authors.initials: R
authors.lastname: Abou Merhi
authors.affiliation: Genetics of rare diseases, University of Poitiers, Poitiers, France; Department of Genetics, University Hospital of Poitiers, Poitiers, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Kitzis
mesh_terms: D030301:Activin Receptors, Type II / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D015331:Cohort Studies; D055427:Growth Differentiation Factor 2 / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D021381:Protein Transport / Q000187:drug effects; D012326:RNA Splicing / Q000235:genetics*; D013347:Subcellular Fractions / Q000378:metabolism; D013683:Telangiectasia, Hereditary Hemorrhagic / Q000235:genetics*
chemical_list: D055427:Growth Differentiation Factor 2; C425364:ACVRL1 protein, human; D030301:Activin Receptors, Type II