Denaturing HPLC-based approach for detection of COL7A1 gene mutations causing dystrophic epidermolysis bullosa

Patrizia Posteraro, Monica Pascucci, Marina Colombi, Sergio Barlati, Alberto Giannetti, Mauro Paradisi, Aki Mustonen, Giovanna Zambruno, Daniele Castiglia

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)1391-1401
Number of pages11
JournalBiochemical and Biophysical Research Communications
Volume338
Issue number3
DOIs
Publication statusPublished - Dec 23 2005

Fingerprint

Epidermolysis Bullosa Dystrophica
High performance liquid chromatography
Genes
High Pressure Liquid Chromatography
Mutation
Mutation Rate
Exons
Collagen Type VII
Genotype
Scanning
Defects
DNA

Keywords

  • COL7A1
  • Denaturing high performance liquid chromatography
  • DHPLC
  • Dystrophic epidermolysis bullosa
  • Mutation detection
  • Type VII collagen

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Denaturing HPLC-based approach for detection of COL7A1 gene mutations causing dystrophic epidermolysis bullosa. / Posteraro, Patrizia; Pascucci, Monica; Colombi, Marina; Barlati, Sergio; Giannetti, Alberto; Paradisi, Mauro; Mustonen, Aki; Zambruno, Giovanna; Castiglia, Daniele.

In: Biochemical and Biophysical Research Communications, Vol. 338, No. 3, 23.12.2005, p. 1391-1401.

Research output: Contribution to journalArticle

Posteraro, Patrizia ; Pascucci, Monica ; Colombi, Marina ; Barlati, Sergio ; Giannetti, Alberto ; Paradisi, Mauro ; Mustonen, Aki ; Zambruno, Giovanna ; Castiglia, Daniele. / Denaturing HPLC-based approach for detection of COL7A1 gene mutations causing dystrophic epidermolysis bullosa. In: Biochemical and Biophysical Research Communications. 2005 ; Vol. 338, No. 3. pp. 1391-1401.
@article{4e60f2244f4d4cd58b56d37077411fd9,
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.",
keywords = "COL7A1, Denaturing high performance liquid chromatography, DHPLC, Dystrophic epidermolysis bullosa, Mutation detection, Type VII collagen",
author = "Patrizia Posteraro and Monica Pascucci and Marina Colombi and Sergio Barlati and Alberto Giannetti and Mauro Paradisi and Aki Mustonen and Giovanna Zambruno and Daniele Castiglia",
year = "2005",
month = "12",
day = "23",
doi = "10.1016/j.bbrc.2005.10.097",
language = "English",
volume = "338",
pages = "1391--1401",
journal = "Biochemical and Biophysical Research Communications",
issn = "0006-291X",
publisher = "Academic Press Inc.",
number = "3",

}

TY - JOUR

T1 - Denaturing HPLC-based approach for detection of COL7A1 gene mutations causing dystrophic epidermolysis bullosa

AU - Posteraro, Patrizia

AU - Pascucci, Monica

AU - Colombi, Marina

AU - Barlati, Sergio

AU - Giannetti, Alberto

AU - Paradisi, Mauro

AU - Mustonen, Aki

AU - Zambruno, Giovanna

AU - Castiglia, Daniele

PY - 2005/12/23

Y1 - 2005/12/23

N2 - 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.

AB - 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.

KW - COL7A1

KW - Denaturing high performance liquid chromatography

KW - DHPLC

KW - Dystrophic epidermolysis bullosa

KW - Mutation detection

KW - Type VII collagen

UR - http://www.scopus.com/inward/record.url?scp=27744537861&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=27744537861&partnerID=8YFLogxK

U2 - 10.1016/j.bbrc.2005.10.097

DO - 10.1016/j.bbrc.2005.10.097

M3 - Article

C2 - 16271705

AN - SCOPUS:27744537861

VL - 338

SP - 1391

EP - 1401

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

IS - 3

ER -