title: Denaturing HPLC-based approach for detection of COL7A1 gene mutations causing dystrophic epidermolysis bullosa.
abstract: Dystrophic epidermolysis bullosa (DEB) is a rare clinically heterogeneous genodermatosis due to genetic defects in type VII collagen gene (COL7A1). Identification of COL7A1 mutations is a challenge since this gene comprises 118 exons and more than 300 mutations scattered over the gene have been reported. Here, we describe for the first time the use of denaturing high performance liquid chromatography (DHPLC) for COL7A1 mutation detection. To validate the method, exon-specific DHPLC conditions were applied to screen DNA samples from patients carrying known COL7A1 mutations. Abnormal DHPLC profiles were obtained for all known mutations. Subsequent DHPLC analysis of 17 DEB families of unknown genotype allowed the identification of 21 distinct mutations, 9 of which were novel. The DHPLC mutation detection rate was significantly higher compared with our mutation scanning rate with conventional techniques (97% vs 86%), indicating DHPLC as the method of choice for COL7A1 molecular characterization in DEB patients.
authors.affiliation: Department of Clinical and Molecular Pathology, IDI-Ospedale S. Carlo, Rome, Italy.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Posteraro
authors.forename: Monica
authors.initials: M
authors.lastname: Pascucci
authors.forename: Marina
authors.initials: M
authors.lastname: Colombi
authors.forename: Sergio
authors.initials: S
authors.lastname: Barlati
authors.forename: Alberto
authors.initials: A
authors.lastname: Giannetti
authors.forename: Mauro
authors.initials: M
authors.lastname: Paradisi
authors.forename: Aki
authors.initials: A
authors.lastname: Mustonen
authors.forename: Giovanna
authors.initials: G
authors.lastname: Zambruno
authors.forename: Daniele
authors.initials: D
authors.lastname: Castiglia
mesh_terms: D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003094:Collagen / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D016108:Epidermolysis Bullosa Dystrophica / Q000235:genetics*; D006801:Humans; D009154:Mutation / Q000235:genetics*; D009691:Nucleic Acid Denaturation
chemical_list: D003094:Collagen