DNA methylation might have a significant role in preventing normal differentiation in pediatric cancers. We used a genomewide method for detecting regions of CpG methylation on the basis of the increased melting temperature of methylated DNA, termed denaturation analysis of methylation differences (DAMD). Using the DAMD assay, we find common regions of cancer-specific methylation changes in primary medulloblastomas in critical developmental regulatory pathways, including Sonic hedgehog (Shh), Wingless (Wnt), retinoic acid receptor (RAR), and bone morphogenetic protein (BMP). One of the commonly methylated loci is the PTCH1-1C promoter, a negative regulator of the Shh pathway that is methylated in both primary patient samples and human medulloblastoma cell lines. Treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-dC) increases the expression of PTCH1 and other methylated loci. Whereas genetic mutations in PTCH1 have previously been shown to lead to medulloblastoma, our study indicates that epigenetic silencing of PTCH1, and other critical developmental loci, by DNA methylation is a fundamental process of pediatric medulloblastoma formation. This finding warrants strong consideration for DNA demethylating agents in future clinical trials for children with this disease.hereas cancer is widely viewed as a genetic disease, the role of epigenetic modifications, especially cytosine methylation in the promoter regions of genes, has been a major research focus in attempting to delineate the mechanisms leading to the formation of cancer, as well as for biomarker discovery (1). Genomic DNA of cancer cells is generally hypomethylated compared to DNA of normal cells, but displays a striking hypermethylation in the promoter regions of a subset of genes. This DNA hypermethylation has been correlated with transcriptional repression, indicating that epigenetic silencing of tumor suppressor genes may be an early step in the process of carcinogenesis (2).In this report, we describe a genomewide DNA methylation assay that identifies CpG methylation on the basis of the biophysical property that 5-methylcytosine increases the melting temperature (T m ) of DNA. We show that this denaturation analysis of methylation differences method detects differentially methylated loci with high CpG density. Assessment of differential methylation in pediatric medulloblastomas compared to normal cerebellum DNA identifies cancer-specific methylation of genes associated with developmental processes. Of particular interest is cancer-specific methylation of the PTCH1-1C promoter, a negative regulator of the Sonic hedgehog (Shh) pathway. Whereas genetic mutations in PTCH1 have been described in human medulloblastomas, this demonstrates that epigenetic silencing by DNA methylation of PTCH1 contributes to the formation of this childhood cancer and suggests the use of DNA demethylating agents as a potential strategy for therapy.
Results
An Assay to Detect Palindrome Formation Enriches for CpG Methylation.Earlier work from our l...