Use of DNA from human stools to detect aberrant CpG island methylation of genes implicated in colorectal cancer.
Hypermethylation of cytosine residues in the CpG islands of tumor suppressor genes is a key mechanism of colorectal carcinogenesis. Detection and quantification of CpG island methylation in human DNA isolated from stools might provide a novel strategy for the detection and investigation of colorectal neoplasia. To explore the feasibility of this approach, colorectal biopsies and fecal samples were obtained from 32 patients attending for colonoscopy or surgery, who were found to have adenomatous polyps, colorectal cancer, or no evidence of neoplasia. A further 18 fecal samples were obtained from healthy volunteers, with no bowel symptoms. Isolated DNA was modified with sodium bisulfite and analyzed by methylation-specific PCR and combined bisulfite restriction analysis for CpG island methylation of ESR1, MGMT, HPP1, p16(INK4a), APC, and MLH1. CpG island methylation was readily detectable in both mucosal and fecal DNA with methylation-specific PCR. Using combined bisulfite restriction analysis, it was established that, in volunteers from whom biopsies were available, the levels of methylation at two CpG sites within ESR1 assayed using fecal DNA were significantly correlated with methylation in DNA from colorectal mucosa. Thus, noninvasive techniques can be used to obtain quantitative information about the level of CpG island methylation in human colorectal mucosa. The methods described here could be applied to a much expanded range of genes and may be valuable both for screening purposes and to provide greater insight into the functional consequences of epigenetic changes in the colorectal mucosa of free-living individuals.
Belshaw
Nigel J
NJ
Institute of Food Research, Norwich Research Park, Colney, Norwich, United Kingdom.
Elliott
Giles O
GO
Williams
Elizabeth A
EA
Bradburn
David M
DM
Mills
Sarah J
SJ
Mathers
John C
JC
Johnson
Ian T
IT
D018256:Adenomatous Polyps / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D000483:Alleles; D001706:Biopsy; D016022:Case-Control Studies; D003082:Colectomy; D003113:Colonoscopy; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation; D017931:DNA Primers / Q000235:genetics; D044127:Epigenesis, Genetic; D005240:Feasibility Studies; D005243:Feces / Q000737:chemistry; D005260:Female; D016147:Genes, Tumor Suppressor; D006801:Humans; D007413:Intestinal Mucosa / Q000473:pathology; D008297:Male; D008403:Mass Screening; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D018570:Risk Assessment
D017931:DNA Primers