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Tools of the Trade

Thu, 01/10/2008 - 4:13pm
Marina Bibikova, PhD and Jian-Bing Fan, PhD, Illumina, San Diego, Calif.
Enhanced appreciation of cancer epigenetics fuels the demand for DNA methylation assays.

In recent years, a paradigm shift has occurred in the field of cancer research. Scientists can no longer consider only genetic changes linked with cancer. Epigenetic changes, or heritable alterations in gene function that do not affect DNA sequence, are rapidly gaining acceptance as co-conspirators in carcinogenesis. With burgeoning research being conducted on cancer epigenetics, numerous assays for detecting epigenetic changes have emerged. Researchers hope to use these tools to gain a better understanding of epigenetic processes, to identify biomarkers to assist in cancer diagnosis and treatment, and to clarify the complex interplay between genetic and epigenetic mechanisms in cancer.

Illumina’s Infinium Assay

click to enlarge
Illumina’s Infinium Assay “genotypes” the two genotypes created at CpG sites after bisulfite treatment of DNA samples. (Source: Illumina Inc.) 

The cancer connection
Three major types of epigenetic mechanisms have been identified in humans: DNA methylation, histone modification, and non-coding RNAs. DNA methylation appears to be the major epigenetic mechanism in humans and is the best characterized. DNA methyl-transferase enzymes can add a methyl group to the C-5 position of cytosine residues in the dinucleotide sequence CpG. Cytosine methylation does not affect DNA base-pairing, but it can influence DNA-protein interactions.

Long (>200-bp) stretches of CpG-rich sequence, termed "CpG islands," reside near the promoter regions of many genes. Methylation of cytosines in these regions can inactivate genes, probably by interfering with transcriptional initiation. Methyl-binding domain (MBD) proteins bind to heavily methylated sequences and recruit protein complexes that establish a repressive chromatin structure.

Most CpG islands near gene promoters in normal tissues are unmethylated. In contrast, CpG islands in the promoters of tumor suppressor genes are often hypermethylated in cancer cells. Whether aberrant DNA methylation is a cause or effect of cancer is controversial; however, there are clear differences in methylation patterns between cancerous and normal cells of the same cell type. In addition, different tumor types show unique patterns of DNA methylation.

Promise and problems
Researchers in the field of cancer epigenetics are exploring the relevance of DNA methylation patterns in human cancers. A major goal is to identify methylation biomarkers for different tumor types, which could aid in cancer diagnosis, prognosis, and treatment. Furthermore, drugs that inhibit DNA methylation are being investigated as potential cancer therapeutics. From a basic science standpoint, researchers would like to decipher the precise role of methylation in cancer development and progression and to unravel the interactions among various epigenetic and genetic factors.

To address these issues, researchers need techniques that can sensitively, accurately, and quantitatively detect changes in methylation patterns among different cancer specimens. Until fairly recently, problems inherent in the epigenetic analysis of cancer cells hindered the development of such assays. Unlike genetic changes, epigenetic alterations are not recorded in the genome in a manner that can be directly amplified, cloned, and sequenced. Therefore, the DNA sample must be fixed in the methylated state before amplification and the application of other technologies. A widely used method for “typesetting” epigenetic changes is bisulfite conversion of unmethylated cytosines. Treatment with bisulfite transforms unmethylated cytosine into uracil, whereas methylated cytosine is left intact.

Because there are millions of potential sites of cytosine methylation in the human genome, wide genome coverage is a formidable challenge. In the past, researchers have been forced to choose between detailed characterization of methylation sites in specific target genes, or broad but cursory sampling of methylation sites throughout the genome. Sample throughput is a major consideration in the implementation of any methylation assay because numerous samples must be analyzed to draw meaningful conclusions. In addition, methylation assays that are sufficiently robust to analyze DNA from paraffin-embedded biopsy samples, blood, or other body fluids are the most valuable for cancer epigenetic research.

Finally, it's important for a methylation assay to be quantitative. Often, a "yes or no" answer with regard to methylation status does not provide useful information. Different CpG sites within a promoter region may be methylated at different levels, so an assay with single CpG resolution is desirable. Also, relatively small differences (e.g., 50% versus 70%) in the methylation status of a given CpG site in a tumor sample can have big consequences in terms of gene expression.

In recent years, a variety of methods for probing the methylation state of genomic DNA have been established. Existing methods vary widely in genome coverage.

One gene at a time
One of the earliest and still widely used techniques for analyzing genomic DNA methylation is methylation-specific PCR (MSP). One such method is the MethyLight assay, developed by Peter Laird and coworkers at the University of Southern California School of Medicine, Los Angeles. This method uses bisulfite conversion of unmethylated cytosines followed by PCR amplification with primers specific for methylated (unconverted) CpG sequences. Because methylated sequences are amplified by real-time PCR with a methylation-specific fluorescent probe, the MethyLight assay is semi-quantitative. This assay is best for examining one or a few genes at a time because separate primers and fluorescent probes must be designed for every CpG island of interest.

Biotage AB (Uppsala, Sweden) uses a pyrosequencing method for examining methylation patterns in target genes. After bisulfite treatment and PCR amplification, target DNA is sequenced by a DNA polymerase. A fluorescent burst signals the incorporation of each nucleotide, such that the amount of methylated (unconverted) versus unmethylated (converted to uracil) cytosines can be determined. Pyrosequencing can be applied to fixed, paraffin-embedded samples. The MassARRAY system by Sequenom, Inc. (San Diego, Calif.) uses bisulfite conversion of DNA followed by PCR, cleavage with restriction enzymes, and MALDI-TOF mass spectrometry to determine the methylation status of CpGs in ~600-bp stretches at a time.

CpG island hopping
Several methods for genome-wide interrogation of methylation patterns are based upon the fragmentation of genomic DNA by methylation-sensitive restriction enzymes, usually followed by selective PCR amplification of methylated sequences. Although these methods provide an overview of genome-wide methylation patterns, they tend to be labor-intensive and relatively low-throughput, and they cannot be used for partially degraded DNA samples extracted from archived tumor tissues. Other disadvantages of restriction-enzyme-based methods include incomplete digestion and the inability to analyze CpG sites lacking a nearby restriction enzyme recognition sequence.

Array-based approaches represent the "second generation" of genome-wide methylation mapping techniques. Although the existing commercial array platforms share common targeted regions such as CpG islands, they vary slightly in sample preparation approaches and probe design. In the DNA methylation assay by NimbleGen Systems, Inc., (Madison, Wis.), DNA is fragmented, and methylated DNA is immunoprecipitated with an antibody against 5-methyl cytidine. The immunoprecipitated DNA is then hybridized to a NimbleChip array. NimbleGen offers several microarrays suitable for methylation analyses, including a whole genome tiling array consisting of 385,000 probes, a promoter sequence array, and custom arrays. Cancer and normal DNA samples are differentially labeled and hybridized to a single array to identify sequences that are aberrantly methylated in cancer cells. The CpG Island Microarray by Agilent Technologies (Santa Clara, Calif.) also requires immunoprecipitation of methylated DNA, and this array contains ~240,000 probe sequences from CpG islands.

Another approach to array-based methylation analysis, the GoldenGate Assay for Methylation from Illumina, Inc. (San Diego, Calif.), monitors differences in methylation at specific CpG sites with single-nucleotide resolution. Bisulfite-treated genomic DNA is hybridized to primers that target specific CpG sites, either methylated or unmethylated. After primer extension and ligation, each CpG sequence is PCR amplified with fluorescently-labeled, common primers. Methylated and umethylated DNA is labeled with Cy5 and Cy3, respectively, to allow quantitative measurement of methylation at each CpG site. Labeled DNA is then hybridized to an Illumina universal bead array. A Methylation Cancer Panel I primer pool was designed to quantitatively analyze the methylation status of 1505 different CpG sites from 807 cancer-related genes in a high-throughput (96 samples per array) format. In addition, the GoldenGate Assay can be used to analyze paraffin-embedded samples.

The future?
Armed with an impressive toolkit of assays to analyze the methylation status of cancer cells, researchers are poised as never before to attain new insights into how changes in DNA methylation contribute to cancer development. In addition to discovering new cancer biomarkers and treatments, researchers hope to clarify the relationship between cancer genetics and epigenetics. Clearly, a multifaceted approach will be required to probe the importance of the myriad genetic and epigenetic changes that transpire in cancer cells. Quite possibly, epigenetic mechanisms will comprise a valuable, and until now missing, piece of the complex puzzle of carcinogenesis.

About the Authors
Marina Bibikova, PhD, has over 15 years of experience in molecular biology, biochemistry, and genetics, has authored over 30 peer-reviewed publications, and is currently a scientist at Illumina, leading development of array-based DNA Methylation analysis technologies for the company.

Jian-Bing Fan, PhD, Director of Scientific Research at Illumina, has over 15 years of experience in human genome research and array technology development with over 60 peer-reviewed publications.  His research focuses on the genotyping of single nucleotide polymorphisms (SNPs), gene expression profiling/splicing monitoring, and genome-wide DNA Methylation detection.

This article was published in Drug Discovery & Development magazine: Vol. 11, No. 1, January, 2008, pp. 50-52.

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