title: DHPLC-based mutation analysis of ENG and ALK-1 genes in HHT Italian population.
abstract: Hereditary haemorrhagic telangiectasia (HHT or Rendu-Osler-Weber syndrome) is an autosomal dominant disorder characterized by localized angiodysplasia due to mutations in endoglin, ALK-1 gene, and a still unidentified locus. The lack of highly recurrent mutations, locus heterogeneity, and the presence of mutations in almost all coding exons of the two genes makes the screening for mutations time-consuming and costly. In the present study, we developed a DHPLC-based protocol for mutation detection in ALK1 and ENG genes through retrospective analysis of known sequence variants, 20 causative mutations and 11 polymorphisms, and a prospective analysis on 47 probands with unknown mutation. Overall DHPLC analysis identified the causative mutation in 61 out 66 DNA samples (92.4%). We found 31 different mutations in the ALK1 gene, of which 15 are novel, and 20, of which 12 are novel, in the ENG gene, thus providing for the first time the mutational spectrum in a cohort of Italian HHT patients. In addition, we characterized the splicing pattern of ALK1 gene in lymphoblastoid cells, both in normal controls and in two individuals carrying a mutation in the non-invariant -3 position of the acceptor splice site upstream exon 6 (c.626-3C>G). Functional essay demonstrated the existence, also in normal individuals, of a small proportion of ALK1 alternative splicing, due to exon 5 skipping, and the presence of further aberrant splicing isoforms in the individuals carrying the c.626-3C>G mutation.
authors.affiliation: Dipartimento di Biomedicina dell'età Evolutiva, Policlinico, Piazza Giulio Cesare, 70124, Bari, Italy.
authors.forename: Gennaro M
authors.initials: GM
authors.lastname: Lenato
authors.forename: Patrizia
authors.initials: P
authors.lastname: Lastella
authors.forename: Marilena C
authors.initials: MC
authors.lastname: Di Giacomo
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Resta
authors.forename: Patrizia
authors.initials: P
authors.lastname: Suppressa
authors.forename: Giovanna
authors.initials: G
authors.lastname: Pasculli
authors.forename: Carlo
authors.initials: C
authors.lastname: Sabbà
authors.forename: Ginevra
authors.initials: G
authors.lastname: Guanti
mesh_terms: D030301:Activin Receptors, Type II / Q000235:genetics*; D017398:Alternative Splicing; D016888:Angiodysplasia / Q000235:genetics*; D015703:Antigens, CD / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015331:Cohort Studies; D004252:DNA Mutational Analysis; D000071063:Endoglin; D006801:Humans; D007558:Italy; D009154:Mutation; D011110:Polymorphism, Genetic; D011446:Prospective Studies; D011956:Receptors, Cell Surface / Q000235:genetics*; D012189:Retrospective Studies; D017422:Sequence Analysis, DNA / Q000379:methods*; D013683:Telangiectasia, Hereditary Hemorrhagic / Q000175:diagnosis* / Q000235:genetics*
chemical_list: D015703:Antigens, CD; C064206:ENG protein, human; D000071063:Endoglin; D011956:Receptors, Cell Surface; C425364:ACVRL1 protein, human; D030301:Activin Receptors, Type II