Abstract 3004: Low levels of methylation in normal DNA mark a large subset of genes primed for hypermethylation in cancer

Abstract

Abstract Cytosine methylation of DNA is a vital component of epigenetic memory. Complex changes of DNA methylation in cancer permanently disturb epigenetic regulation and participate in neoplastic development. To characterize changes in methylome in myeloid neoplasms, we analyzed DNA methylation in 8 patients with myelodysplastic syndrome, 2 patients with acute myeloid leukemia (MDS/AML) and 3 healthy controls. Using Digital Restriction Enzyme Analysis of Methylation (DREAM), we performed quantitative mapping of DNA methylation status at 40,000 autosomal CpG sites. To measure methylation, we first create distinct DNA signatures at unmethylated or methylated CCCGGG sites by sequential restriction digests of gDNA with the SmaI and XmaI endonucleases and then resolve these signatures by massively parallel sequencing. The sequences are mapped back to the genome and methylation levels for individual CpG sites are calculated based on the numbers of sequencing reads with unmethylated or methylated signatures. We found that DNA methylation status had a binomial distribution with the majority of CpG sites showing either very low or very high methylation. CpG islands (CGI) were generally unmethylated, while non-CGI were predominantly methylated with the exception of gene transcription start sites (TSS). When we compared average methylation in MDS/AML with average methylation in controls, the picture was different in CGI and in non-CGI. DNA methylation was increased in MDS/AML in CGI at all distances from gene transcription start sites (TSS) by 2-3%. In contrast, methylation in non-CGI was decreased in MDS/AML by approximately 3% with the exception of regions within 1 kb from TSS. Interestingly, low levels of CGI methylation detectable in blood DNA from healthy controls were associated with a propensity for hypermethylation in leukemia. Methylation levels 0-1% in controls were observed in 5,680 genes. Of those, 51 genes (1%) showed average methylation >20% in MDS/AML. Methylation 1-5% in controls was seen in 1,912 genes. Of those, 192 genes (10%) showed average methylation >20% in MDS/AML. Methylation 5-10% affected 364 genes in controls, while 131 (36%) of these genes had >20% methylation in MDS/AML (P=8.3E-240). The same pattern was observed when we restricted our analysis to methylation in CGI close to TSS (−1 kb to +1 kb). Methylation 0-1% in controls was found in 4,743 genes. Of those, only 24 genes (0.5%) showed average methylation >20% in MDS/AML. Methylation 1-5% in controls was detected in 1,204 genes. Of those, 94 genes (8%) showed average methylation >20% in MDS/AML. Methylation 5-10% was observed in 158 genes in controls, while 55 (35%) of these had >20% methylation in MDS/AML (P=7.7E-172). We conclude that low levels of methylation in CGI present in DNA of healthy individuals mark a large subset of genes destined for hypermethylation in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3004. doi:10.1158/1538-7445.AM2011-3004