Subjects and Family Members
Screening for BRCA1 promoter methylation was undertaken in the lymphocyte-derived DNA of 49 unrelated individuals from families affected by breast and/or ovarian cancer and with a Manchester score > 34 without a germline BRCA1 or BRCA2 pathogenic variant. A Manchester score represents the likelihood of detecting a pathogenic variant in BRCA1 or BRCA2.7, 18, 19 In our local population, 158 of 220 (71.8%) families with a Manchester score > 34 have had pathogenic variants in BRCA1 or BRCA2 identified by conventional genetic testing of DNA sequencing and multiplex ligation-dependent probe analysis (MLPA).
Blood, buccal mucosa, tumor, and hair samples were collected (where possible) from affected and unaffected family members with breast or ovarian cancer when BRCA1 promoter methylation was detected. Cancer diagnoses were confirmed from hospital records or through the North West (England) Cancer Intelligence Service, which has data on all individuals with any malignancy from 1960 onward. DNA was extracted from blood by Chemagen (Perkin Elmer), from hair with the QIAamp DNA Investigator Kit (QIAGEN), from buccal mucosa with the QIAGEN EZ1 system, and from tumor cells with the Cobas DNA Sample Preparation Kit (Roche). The study was approved by the Central Manchester Research Ethics Committee (10/H1008/24 and 11/H1003/3), and written informed consent was obtained from each participant.