title: Heterogeneous DNA Methylation Patterns in the GSTP1 Promoter Lead to Discordant Results between Assay Technologies and Impede Its Implementation as Epigenetic Biomarkers in Breast Cancer.
abstract: Altered DNA methylation patterns are found in many diseases, particularly in cancer, where the analysis of DNA methylation holds the promise to provide diagnostic, prognostic and predictive information of great clinical value. Methylation of the promoter-associated CpG island of GSTP1 occurs in many hormone-sensitive cancers, has been shown to be a biomarker for the early detection of cancerous lesions and has been associated with important clinical parameters, such as survival and response to treatment. In the current manuscript, we assessed the performance of several widely-used sodium bisulfite conversion-dependent methods (methylation-specific PCR, MethyLight, pyrosequencing and MALDI mass-spectrometry) for the analysis of DNA methylation patterns in the GSTP1 promoter. We observed large discordances between the results obtained by the different technologies. Cloning and sequencing of the investigated region resolved single-molecule DNA methylation patterns and identified heterogeneous DNA methylation patterns as the underlying cause of the differences. Heterogeneous DNA methylation patterns in the GSTP1 promoter constitute a major obstacle to the implementation of DNA methylation-based analysis of GSTP1 and might explain some of the contradictory findings in the analysis of the significance of GSTP1 promoter methylation in breast cancer.
authors.affiliation: Department of Cancer genetics, Institute for Cancer Research, Oslo University Hospital, Radiumhospitalet, Oslo 0310, Norway. Grethe.I.Grenaker.Alnas@rr-research.no.
authors.forename: Grethe I Grenaker
authors.initials: GI
authors.lastname: Alnaes
authors.affiliation: Department of Cancer genetics, Institute for Cancer Research, Oslo University Hospital, Radiumhospitalet, Oslo 0310, Norway. Jo.Anders.Ronneberg@gmail.com.
authors.forename: Jo Anders
authors.initials: JA
authors.lastname: Ronneberg
authors.affiliation: Department of Cancer genetics, Institute for Cancer Research, Oslo University Hospital, Radiumhospitalet, Oslo 0310, Norway. v.n.kristensen@medisin.uio.no.|Institute of Clinical Medicine, University of Oslo, Oslo 0318, Norway. v.n.kristensen@medisin.uio.no.|Department of Clinical Molecular Biology (EpiGen), University of Oslo, Ahus, 1478 Lørenskog, Norway. v.n.kristensen@medisin.uio.no.
authors.forename: Vessela N
authors.initials: VN
authors.lastname: Kristensen
authors.affiliation: Laboratory for Epigenetics and Environment, Centre National de Génotypage, CEA-Institut de Génomique, Evry 91000, France. tost@cng.fr.
authors.forename: Jörg
authors.initials: J
authors.lastname: Tost
keywords: DNA methylation; GSTP1; MSP; MethyLight; biomarker; breast cancer; heterogeneity; mass spectrometry; method; pyrosequencing