Research article

Gene promoter hypermethylation in ductal lavage fluid from healthy BRCA gene mutation carriers and mutation-negative controls

Imogen Locke^1,2 , Zsofia Kote-Jarai^1,2 , Mary Jo Fackler³ , Elizabeth Bancroft^1,2 , Peter Osin⁴ , Ashutosh Nerurkar⁴ , Louise Izatt⁵ , Gabriella Pichert⁵ , Gerald PH Gui⁶ and Rosalind A Eeles^1,2

¹  Translational Cancer Genetics Team, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK

²  Department of Cancer Genetics, Royal Marsden NHS Foundation Trust, Fulham Road, London SW3 6JJ, UK

³  Department of Oncology, Johns Hopkins University School of Medicine, 401 North Broadway, Baltimore, MD 21231, USA

⁴  Department of Pathology, Royal Marsden NHS Foundation Trust, Fulham Road, London SW3 6JJ, UK

⁵  Department of Genetics, Guy's and St Thomas' NHS Foundation Trust, St Thomas Street, London SE1 9RT, UK

⁶  Department of Surgery, Royal Marsden NHS Foundation Trust, Fulham Road, London SW3 6JJ, UK

author email corresponding author email

Breast Cancer Research 2007, 9:R20doi:10.1186/bcr1657

Published:	26 February 2007

Abstract

Introduction

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-β, 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).

Conclusion

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.