title: Mutation scanning the GJB1 gene with high-resolution melting analysis: implications for mutation scanning of genes for Charcot-Marie-Tooth disease.
abstract: BACKGROUND
  X-linked Charcot-Marie-Tooth type 1 disease has been associated with 280 mutations in the GJB1 [gap junction protein, beta 1, 32 kDa (connexin 32, Charcot-Marie-Tooth neuropathy, X-linked)] gene. High-resolution melting analysis with an automated instrument can be used to scan DNA for alterations, but its use in X-linked disorders has not been described.
  
  
  METHODS
  A 96-well LightScanner for high resolution melting analysis was used to scan amplicons of the GJB1 gene. All mutations reported in this study had been confirmed previously by sequence analysis. DNA samples were amplified with the double-stranded DNA-binding dye LC Green Plus. Melting curves were analyzed as fluorescence difference plots. The shift and curve shapes of melting profiles were used to distinguish controls from patient samples.
  
  
  RESULTS
  The method detected each of the 23 mutations used in this study. Eighteen known mutations provided validation of the high-resolution melting method and a further 5 mutations were identified in a blind study. Altered fluorescence difference curves for all the mutations were easily distinguished from the wild-type melting profile.
  
  
  CONCLUSIONS
  High-resolution melting analysis is a simple, sensitive, and cost-efficient alternative method to scan for gene mutations in the GJB1 gene. The technology has the potential to reduce sequencing burden and would be suitable for mutation screening of exons of large multiexon genes that have been discovered to be associated with Charcot Marie Tooth neuropathy.
authors.affiliation: Northcott Neuroscience Laboratory, ANZAC Research Institute, Concord NSW, Australia. marinak@anzac.edu.au
authors.forename: Marina L
authors.initials: ML
authors.lastname: Kennerson
authors.forename: Trent
authors.initials: T
authors.lastname: Warburton
authors.forename: Eva
authors.initials: E
authors.lastname: Nelis
authors.forename: Megan
authors.initials: M
authors.lastname: Brewer
authors.forename: Patsie
authors.initials: P
authors.lastname: Polly
authors.forename: Peter
authors.initials: P
authors.lastname: De Jonghe
authors.forename: Vincent
authors.initials: V
authors.lastname: Timmerman
authors.forename: Garth A
authors.initials: GA
authors.lastname: Nicholson
mesh_terms: D002607:Charcot-Marie-Tooth Disease / Q000235:genetics*; D017630:Connexins / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D009154:Mutation; D000097002:Gap Junction beta-1 Protein
chemical_list: D017630:Connexins; D000097002:Gap Junction beta-1 Protein