Elsevier

Methods

Volume 27, Issue 2, June 2002, Pages 162-169
Methods

Use of CpG island microarrays to identify colorectal tumors with a high degree of concurrent methylation

https://doi.org/10.1016/S1046-2023(02)00070-1Get rights and content

Abstract

We provide a comprehensive description of our microarray-based technique for the simultaneous detection of multiple CpG islands in cancer. Amplicons from tumor and control samples were pools of differentially methylated CpG island fragments hybridized to a panel of ∼8000 CpG island tags. Data analysis identified 694 CpG island loci hypermethylated in a group of 14 colorectal tumors. The Stanford hierarchical cluster algorithm segregated the tumors into two subgroups, one of which exhibited a high level of concurrent hypermethylation while the other had little or no methylation. This is in agreement with previous observations of a CpG island methylation phenotype present in colorectal tumors. The present study demonstrates that this microarray-based technique is useful in classifying tumors according to their methylation profiles.

Introduction

In addition to coding region mutations and deletions, hypermethylation of CpG islands located at the promoter and first exon of genes is now accepted as a key pathway leading to transcriptional silencing of critical genes in cancer [1]. As of today, the expression of ∼100 genes is shown to be down-regulated through this epigenetic process (an updated list is maintained on our website: http://www.missouri.edu/∼hypermet). This aberrant process can also occur in CpG islands located elsewhere in the genome although it is less clear how hypermethylation of these nonpromoter loci plays a role in conferring tumor growth [2].

Recent studies have suggested a generalized deregulation of CpG island methylation predisposing some tumors to this epigenetic process [3], [4]. This type of tumor usually displays a high degree of concordant methylation in multiple gene loci [5], [6]. Occurrence of such widespread hypermethylation was observed in retinoblastoma [5], acute myeloid leukemia [7], pancreatic adenocarcinoma [8], and colorectal [6], gastric [9], ovarian [10], [11], breast [12], and prostate and bladder [13] cancers. As further defined by Issa and colleagues [6], these tumors, characterized by a high degree of concordant CpG island hypermethylation, exhibit a so-called CpG island methylator phenotype (CIMP).

To date, methylation analysis is often performed using candidate gene approaches. Although it can provide detailed methylation status of designated CpG islands, this gene-by-gene approach in uncovering novel tumor suppressors may not be systematic and its ability to detect the prevalence of methylation alterations among different tumors is limited in scope. Analysis of DNA methylation on a global scale, however, can provide the broad-based information needed to achieve these goals. Such an approach may one day lead to the compilation of a human epigenomic map as well. Several methods capable of providing methylation status on a global scale include restriction landmark genome scanning [14], representational difference analysis [15], methylation-sensitive arbitrarily primed PCR [16], and methylated CpG island amplification [17]. In this paper, we used a microarray-based method, called differential methylation hybridization [18], to survey methylation alterations of ∼8000 CpG island loci in colorectal tumors. Methylation profiling identified a subset of the studied tumors displaying concordant methylation in multiple CpG island loci.

Section snippets

CpG island clones

The microarray panel contains short CpG island tags (0.2–2 kb) uniquely flanked by TTAA, the recognition site of a 4-base frequent cutter MseI. This genomic library, CGI, was originally constructed by Cross et al. [19] and later managed and distributed by the Human Genome Mapping Program Centre, United Kingdom (http://www.hgmp.mrc.ac.uk/). The steps taken to generate this library were as follows. First, male genomic DNA was restricted to short stretches by MseI. As its recognition sites rarely

Concluding remarks

In this study, we have shown that DMH is useful in identifying hypermethylated CpG islands in tumor cells and has a potential application in other biological conditions. This microarray-based assay was used to query the methylation status of ∼8000 GC-rich fragments in a group of colorectal tumors. Methylation profiling identified at least 58 CpG loci that segregate colorectal tumors with a high degree of concurrent methylation from tumors with low or no methylation in these loci. This

Acknowledgements

We thank Diane Peckham for assistance in the preparation of this manuscript and Dr. Sally H. Cross and colleagues from the United Kingdom Genome Mapping Project Center for providing the CGI library. This work was supported by National Cancer Institute Grants CA-69065 and CA-84701.

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