Methylation-Specific Oligonucleotide Microarray: A New Potential for High-Throughput Methylation Analysis

It is now clear that abnormally methylated cytosine within a CpG dinucleotide is a widespread phenomenon in cancer (Jones and Laird 1999). This epigenetic event has been observed in GC-rich regions, called CpG islands, frequently located in the promoter and exon 1 regions of genes. As a result of CpG island hypermethylation, chromatin structure in the promoter can be altered, preventing normal interaction with the transcriptional machinery (Baylin et al. 1998). If this occurs in genes critical to growth inhibition, the resulting silencing of transcription could promote tumor progression. In addition to classic genetic mutations, promoter CpG island hypermethylation has been shown to be a common mechanism for transcriptional inactivation of classic tumor suppressor genes (Baylin et al. 1998; Jones and Laird 1999) and genes important for cell cycle regulation (Costello et al. 1996; Nguyen et al. 2000), and DNA mismatch repair (Kane et al. 1997). At present, several molecular biology methods are routinely used to determine the methylation status of a CpG island. Among these, bisulfite nucleotide sequencing is a standard technique for detailed mapping of methylated cytosine residues within a gene promoter. This meticulous method, developed by Frommer and colleagues (Frommer et al. 1992), relies on the ability of sodium bisulfite to deaminate cytosine residues into uracil in genomic DNA, whereas the methylated cytosine residues are resistant to this modification. The target DNA is then amplified by PCR with specific primers to yield fragments in which all uracil residues are converted to thymine, whereas methylated cytosine residues are amplified as cytosine. The PCR products are sequenced and the methylation status of individual CpG sites is then analyzed by comparing it with the unmodified sequences of a given promoter. Using this conventional method, many investigators have addressed the importance of promoter CpG hypermethylation in the regulation of specific gene transcription in cancer (Hiltunen et al. 1997; Stirzaker et al. 1997; Rice et al. 1998; Melki et al. 1999). The method, which requires cloning and sequencing of individual inserts, can be labor intensive and is restricted to the evaluation of DNA methylation on a gene-by-gene basis. Such an approach has given researchers a limited picture of complex epigenetic alterations in cancer. Clearly, a compelling need exists to develop novel technologies for the next phase of epigenome research. Recently, considerable advances have been made in hybridization-based microarray technology for genome-wide analysis of gene mutations and single nucleotide polymorphisms (Ahrendt et al. 1999; Favis and Barany 2000; Wen et al. 2000). The technology uses thousands of short oligonucleotides arrayed on glass slides for detection of all possible nucleotide changes in target DNA. In this new approach, oligonucleotides are arrayed on solid supports that are known as probes, and the labeled complex DNA mixtures to be interrogated are known as targets (Hacia 1999). In this study, we have built on this cutting-edge technology by developing a novel technique called methylation-specific oligonucleotide (MSO) microarray for DNA methylation analysis. The targets were derived from PCR products of bisulfite-modified DNA, whereas the probes used a series of arrayed oligonucleotides that can discriminate between converted and unconverted nucleotides, that is, unmethylated and methylated cytosine, at specific sites. Herein, we describe the MSO procedure and its application for mapping methylated CpG sites in the human estrogen receptor (ER) α gene CpG island in breast cancer cells, normal fibroblasts, breast tumors, and normal control samples.