title: Gene promoter hypermethylation in ductal lavage fluid from healthy BRCA gene mutation carriers and mutation-negative controls.
abstract: BACKGROUND
  Female germline BRCA gene mutation carriers are at increased risk for developing breast cancer. The purpose of our study was to establish whether healthy BRCA mutation carriers demonstrate an increased frequency of aberrant gene promoter hypermethylation in ductal lavage (DL) fluid, compared with predictive genetic test negative controls, that might serve as a surrogate marker of BRCA1/2 mutation status and/or breast cancer risk.
  
  
  METHODS
  The pattern of CpG island hypermethylation within the promoter region of a panel of four genes (RAR-beta, HIN-1, Twist and Cyclin D2) was assessed by methylation-specific polymerase chain reaction using free DNA extracted from DL fluid.
  
  
  RESULTS
  Fifty-one DL samples from 24 healthy women of known BRCA mutation status (7 BRCA1 mutation carriers, 12 BRCA2 mutation carriers and 5 controls) were available for methylation analysis. Eight of 19 (42.1%) BRCA mutation carriers were found to have at least one hypermethylated gene in the four-gene panel. Two BRCA mutation carriers, in whom aberrant methylation was found, also had duct epithelial cell atypia identified. No hypermethylation was found in DL samples from 5 negative controls (p = 0.13).
  
  
  CONCLUSIONS
  We found substantial levels of aberrant methylation, with the use of a four-gene panel, in the fluid from the breasts of healthy BRCA mutation carriers compared with controls. Methylation analysis of free DNA in DL fluid may offer a useful surrogate marker for BRCA1/2 mutation status and/or breast cancer risk. Further studies are required for the evaluation of the specificity and predictive value of aberrant methylation in DL fluid for future breast cancer development in BRCA1/2 mutation carriers.
authors.affiliation: Translational Cancer Genetics Team, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK. imogen.locke@icr.ac.uk
authors.forename: Imogen
authors.initials: I
authors.lastname: Locke
authors.forename: Zsofia
authors.initials: Z
authors.lastname: Kote-Jarai
authors.forename: Mary Jo
authors.initials: MJ
authors.lastname: Fackler
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Bancroft
authors.forename: Peter
authors.initials: P
authors.lastname: Osin
authors.forename: Ashutosh
authors.initials: A
authors.lastname: Nerurkar
authors.forename: Louise
authors.initials: L
authors.lastname: Izatt
authors.forename: Gabriella
authors.initials: G
authors.lastname: Pichert
authors.forename: Gerald P H
authors.initials: GP
authors.lastname: Gui
authors.forename: Rosalind A
authors.initials: RA
authors.lastname: Eeles
mesh_terms: D000328:Adult; D016022:Case-Control Studies; D018899:CpG Islands; D056742:Cyclin D2; D016213:Cyclins / Q000235:genetics; D016207:Cytokines / Q000235:genetics; D019175:DNA Methylation; D005260:Female; D019398:Genes, BRCA1; D024522:Genes, BRCA2; D018095:Germ-Line Mutation; D006801:Humans; D042361:Mammary Glands, Human / Q000737:chemistry*; D008875:Middle Aged; D009687:Nuclear Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D018168:Receptors, Retinoic Acid / Q000235:genetics; D007507:Therapeutic Irrigation; D025521:Tumor Suppressor Proteins / Q000235:genetics; D051879:Twist-Related Protein 1 / Q000235:genetics
chemical_list: C080336:CCND2 protein, human; D056742:Cyclin D2; D016213:Cyclins; D016207:Cytokines; D009687:Nuclear Proteins; D018168:Receptors, Retinoic Acid; C434732:SCGB3A1 protein, human; C103173:TWIST1 protein, human; D025521:Tumor Suppressor Proteins; D051879:Twist-Related Protein 1; C082925:retinoic acid receptor beta