A Single Nucleotide Polymorphism Chip-Based Method for Combined Genetic and Epigenetic Profiling: Validation in Decitabine Therapy and Tumor/Normal Comparisons

Abstract

Progress on several unresolved issues in cancer epigenetics will benefit from rapid and standardized methods for profiling DNA methylation genome-wide. In the area of epigenetic therapy, the demethylating drug decitabine (5-aza-2V-deoxycytidine) is increasingly used to treat acute myelogenous leukemia and myelodysplastic syndrome, but the mechanisms of its anticancer activity have remained unclear. Given the clinical efficacy of decitabine and the uncertainties about its mode of action, it will be useful to optimize methods for following DNA methylation as a biochemical response in individual patients. Here, we describe a single nucleotide polymorphism (SNP) chip-based method (MSNP) for profiling DNA methylation. Using this procedure, the extent of demethylation in bone marrow aspirates from patients with leukemia receiving decitabine can be assessed genome-wide using commercially available (Affymetrix) SNP chips. We validated the accuracy of MSNP by comparing the results with combined bisulfite restriction analysis and by sequencing cloned PCR products from bisulfite-converted DNA. We further validated MSNP in a Wilms' tumor/normal kidney comparison, comparing the results with methylation-sensitive Southern blotting. MSNP simultaneously detects aberrations in DNA copy number and loss of heterozygosity, making it a generally useful approach for combined genetic and epigenetic profiling in tissue samples from cancer patients. (Cancer Res 2006; 66(7): 3443-51)

Genome-wide analysis of DNA methylation patterns

“…Most of these are inhibited by methylation of their recognition site, whereas some, most notably McrBC, specifically digest methylated DNA. Many variations of restriction enzyme-based methods have been used in conjunction with genomic analysis (Khulan et al, 2006;Lippman et al, 2005;Rollins et al, 2006;Schumacher et al, 2006;Tompa et al, 2002;Yuan et al, 2006). Here we briefly describe the general logic behind all such approaches.…”

“…Using SNP arrays for DNA methylation analysis allows the genotyping of methylated DNA that has been isolated from polymorphic individuals (Hellman and Chess, 2007;Yuan et al, 2006). A recent study used Affymetrix SNP arrays to distinguish methylation of the active and inactive X chromosomes, and found that transcribed regions of genes were preferentially methylated on the active X (Hellman and Chess, 2007).…”

“…A study published in early 2006 estimated that DNA methylation of less than 0.1% of the human genome has been analyzed in detail (Schumacher et al, 2006). A number of recent reports have considerably expanded our knowledge of eukaryotic DNA methylation (Bibikova et al, 2006a;Eckhardt et al, 2006;Hellman and Chess, 2007;Keshet et al, 2006;Khulan et al, 2006;Rollins et al, 2006;Weber et al, 2007;Yuan et al, 2006;Zilberman et al, 2007). Nonetheless, we are just beginning to unravel genomic methylation patterns, particularly in the complex genomes of vertebrates.…”

A decade of genome-wide gene expression profiling in acute myeloid leukemia: flashback and prospects

“…97,98 The first results of genome-wide CpG methylation profiling of AML cell lines and primary cells have now been reported, and it is likely that more extensive investigations will follow. 99,100 Given the direct association between epigenetics and expression, much is to be expected from this field.…”

Single nucleotide polymorphism array analysis to predict clinical outcome in neuroblastoma patients

“…Array experiments were done according to the standard protocols for Affymetrix GeneChip Mapping 100K arrays [12]. Briefly, total genomic DNA was digested with a restriction enzyme (XbaI or HindIII), ligated to an appropriate adapter for each enzyme, and subjected to polymerase chain reaction amplification.…”