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To generate kilobase-scale CpG methylation maps of promoter sequence from non-invasively-collected clinical samples, for direct human studies in carcinogenesis, we developed a tag-modified bisulfite genomic sequencing (tBGS) method for construction of DNA methylation maps. This technology permits detailed inter-tissue and inter-individual comparisons of DNA methylation across kilobase-scale sequence, without cloning. Bisulfite-modified genomic DNA is followed by a two-round amplification strategy employing GC-tagged primers, yielding a PCR product amenable to direct cycle sequencing. As an application, the inter-tissue and inter-individual CpG methylation differences in ∼1.5 kb of promoter sequence for two cancer-related genes, cytochrome P450 CYP1B1 and glutathione S-transferase GSTP1, were explored across two human lung cell types in culture [normal human bronchial epithelial cells (NHBE); lung adenocarcinoma (A549) cells] and across four tissue types [peripheral blood mononuclear cells and exfoliated buccal cells, 40 subjects; paired nontumor−tumor lung tissues, 10 subjects]. For the blood, buccal and lung tissues examined, highly conserved methylation maps for these two gene promoters were apparent among all subjects, irrespective of case-control or smoking exposure status. Methylation mapping of the p16 promoter in exhaled breath condensate from human subjects will be presented. For cell culture studies, methylation maps for the GSTP1, but not CYP1B1, promoter differed between NHBE (non-methylated) and A549 cells (methylated) in an upstream region (-1695 to -988) of that promoter; this methylation difference correlated with differences in quantitative mRNA expression (p<0.01). Therefore, methylation patterns far upstream of the transcription start site may functionally impact on gene expression in normal and malignant tissues. This method offers a feasible approach to genomic methylation scanning in non-invasively-collected biospecimens.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]