title: Rapid screening of 4000 individuals for germ-line variations in the BRAF gene.
abstract: BACKGROUND
  The BRAF gene is frequently somatically altered in malignant melanoma. A majority of variations are at the valine 600 residue leading to a V600E substitution that constitutively activates the kinase. We screened 4000 patient and control DNAs for germ-line variations at the valine 600 residue.
  
  
  METHODS
  We developed a novel assay by adapting single-base variation assays and software for MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry to screen for all 5 reported variants at codon 600 of the BRAF gene. We screened a case-control collection comprising samples from 1082 melanoma patients and 154 of their unaffected relatives from 1278 families and from 2744 individuals from 659 unselected twin families with no history of melanoma. A panel of 66 melanoma cell lines was used for variation-positive controls.
  
  
  RESULTS
  All melanoma cell lines that we had found previously to carry a codon 600 variation were verified in this study. Three of the 4 possible variants (V600E n = 47, V600K n = 2, V600R n = 1) were detected, but no case of V600D was available. No germ-line variants were found in the samples from the 3980 melanoma patients or from the control individuals.
  
  
  CONCLUSIONS
  This new assay is a high-throughput, automated alternative to standard sequencing and can be used as a rapid initial screen for somatic variants associated with melanoma. Germ-line variants at valine 600 are unlikely to exist and do not contribute to the reported role of the BRAF gene in melanoma predisposition.
authors.affiliation: Queensland Institute of Medical Research, Brisbane, Australia. michaelJ@qimr.edu.au
authors.forename: Michael R
authors.initials: MR
authors.lastname: James
authors.forename: Troy
authors.initials: T
authors.lastname: Dumeni
authors.forename: Mitchell S
authors.initials: MS
authors.lastname: Stark
authors.forename: David L
authors.initials: DL
authors.lastname: Duffy
authors.forename: Grant W
authors.initials: GW
authors.lastname: Montgomery
authors.forename: Nicholas G
authors.initials: NG
authors.lastname: Martin
authors.forename: Nicholas K
authors.initials: NK
authors.lastname: Hayward
mesh_terms: D016022:Case-Control Studies; D045744:Cell Line, Tumor; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing; D005838:Genotype; D018095:Germ-Line Mutation; D006801:Humans; D008545:Melanoma / Q000235:genetics*; D016133:Polymerase Chain Reaction; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf