Quantitative analysis of promoter methylation in exfoliated epithelial cells isolated from breast milk of healthy women.
Promoter methylation analysis of genes frequently silenced in breast cancer is a promising indicator of breast cancer risk, as these methylation events are thought to occur long before presentation of disease. The numerous exfoliated epithelial cells present in breast milk may provide the breast epithelial DNA needed for detailed methylation analysis and assessment of breast cancer risk. Fresh breast milk samples and health, lifestyle, and reproductive history questionnaires were collected from 111 women. Pyrosequencing analysis was conducted on DNA isolated from the exfoliated epithelial cells immunomagnetically separated from the total cell population in the breast milk of 102 women. A total of 65 CpG sites were examined in six tumor suppressor genes: PYCARD (also known as ASC or TMS1), CDH1, GSTP1, RBP1 (also known as CRBP1), SFRP1, and RASSF1. A sufficient quantity of DNA was obtained for meaningful analysis of promoter methylation; women donated an average of 86 ml of milk with a mean yield of 32,700 epithelial cells per ml. Methylation scores were in general low as expected of benign tissue, but analysis of outlier methylation scores revealed a significant relationship between breast cancer risk, as indicated by previous biopsy, and methylation score for several CpG sites in CDH1, GSTP1, SFRP1, and RBP1. Methylation of RASSF1 was positively correlated with women's age irrespective of her reproductive history. Promoter methylation patterns in DNA from breast milk epithelial cells can likely be used to assess breast cancer risk. Additional studies of women at high breast cancer risk are warranted.
Wong
Chung M
CM
Department of Veterinary & Animal Science, University of Massachusetts at Amherst, MA, USA.
Anderton
Douglas L
DL
Smith-Schneider
Sallie
S
Wing
Megan A
MA
Greven
Melissa C
MC
Arcaro
Kathleen F
KF
D000328:Adult; D000367:Age Factors; D015703:Antigens, CD; D001940:Breast / Q000166:cytology / Q000378:metabolism; D001943:Breast Neoplasms / Q000235:genetics; D053468:CARD Signaling Adaptor Proteins; D015820:Cadherins / Q000235:genetics; D018899:CpG Islands; D003598:Cytoskeletal Proteins / Q000235:genetics; D019175:DNA Methylation; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D016147:Genes, Tumor Suppressor; D051549:Glutathione S-Transferase pi / Q000235:genetics; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D007774:Lactation / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D008875:Middle Aged; D008895:Milk, Human / Q000166:cytology* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D054840:Retinol-Binding Proteins, Cellular / Q000235:genetics; D012307:Risk Factors; D025521:Tumor Suppressor Proteins / Q000235:genetics; D055815:Young Adult; D000094742:Secreted Frizzled-Related Proteins
D015703:Antigens, CD; D053468:CARD Signaling Adaptor Proteins; D015820:Cadherins; D003598:Cytoskeletal Proteins; D051549:Glutathione S-Transferase pi; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D054840:Retinol-Binding Proteins, Cellular; D025521:Tumor Suppressor Proteins; C513384:CDH1 protein, human; C402202:PYCARD protein, human; C411762:RASSF1 protein, human; C518456:RBP1 protein, human; C117636:SFRP1 protein, human; D000094742:Secreted Frizzled-Related Proteins; C496556:GSTP1 protein, human