Novel tools for the science of change – refining epigenetic research

The total complexity of genetic information is not represented solely in the arrangement of the four nucleotide bases of primary sequence, but also in epigenetic modifications. Much research over the last few years has focused on two molecular mechanisms that mediate epigenetic phenomena: DNA methylation and histone modifications. These modifications are somatically heritable and maintained in subsequent cell divisions. Epigenetic processes are known to be essential for normal development and differentiation of higher organisms, but it is now increasingly recognized that environmental and social influences are engraved into our genetic make-up by epigenetic phenomena on a daily basis during our entire life span. In general, genetic sequence information can be read but not deliberately overwritten, while epigenetic information can be altered to affect gene regulation. The term “epigenetics” (literally “above, beside, or outside genetics”) is used to describe the study of these stable alterations that affect gene expression and do not involve sequence changes [1].

DNA methylation and related chromatin changes play essential roles in regulation of gene expression. Hypermethylation of CpG dinucleotides within regions known as CpG islands in the 5’ regulatory regions of genes is a well-characterized means of transcriptional silencing. The relevance of DNA methylation has been demonstrated in mammalian development, imprinting and X-chromosome inactivation, suppression of foreign DNA, and has been implicated in the development and progression of various malignancies.

In recent years, methylation detection and quantification studies have been actively pursued for development of cancer biomarkers for potential diagnosis and prognosis. However, most existing methylation analysis methods are inadequate for high resolution, quantitative fine mapping, and detecting small differences in methylation that may reflect biologically-relevant changes. Also, most methylation analysis methods are too laborious to accommodate high throughput screening. Thus, an efficient method enabling quantitative analysis for a broad throughput range is needed to accelerate studies on DNA methylation in cancer.

In addition, an atlas capturing DNA methylation changes in regions of epigenetic variability in commonly-studied genes will be beneficial for basic research as well as translational medicine. Hypothesis-free approaches to methylation analysis such as genome wide screening methods have generated promising results. However, these methods are limited by their quantitative accuracy and the number of CpG sites that can be assessed individually.

To gain insight into the mechanisms underlying developmental and pathological changes in DNA methylation, an increasing need has arisen for a quantitative, cost effective high throughput evaluation method. The Human Epigenome Project has also highlighted the need for a high throughput, high resolution and quantitative method for DNA methylation profiling. Although many technologies have been developed, accurate and quantitative fine mapping of DNA methylation levels has remained a challenge.

To overcome these challenges inherent to epigenomic research, Sequenom has developed a comprehensive suite of products and tools for methylation analysis. This editorial will highlight how the EpiTYPER® technology for the MassARRAY® Compact Analyzer and the EpiDesigner software [2] for experimental assay design provide the best quantitative solution for your methylation and epigenomic research challenges. We will also introduce Sequenom’s latest research tools, the Standard and Cancer EpiPanels.

Sequenom’s EpiTYPER® technology allows precise and quantitative DNA methylation detection based on bisulfite conversion, MassCLEAVE® chemistry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS, shown in Figure 1). Bisulfite treatment of genomic DNA converts non-methylated cytosine into uracil while methylated cytosines remain unchanged. Next, a PCR amplification step is carried out to yield an amplicon with a T7 promoter tag. The advantage of this method is that the PCR primers are independent of the genomic DNA methylation state, meaning they bind to both methylated and non-methylated template, as opposed to methylation-specific primers. Only two primers are needed to screen for methylation changes within a region of several hundred bases in a single experiment whereby the length of the PCR amplicon is only limited by the degradative side effects of the bisulfite treatment to the template [3].

Next, in vitro RNA transcription is performed on the reverse strand, followed by RNase A cleavage at specific bases (U or C). Cleavage products are generated for the reverse transcription reactions for both U (T) and C in separate reactions, and these are spotted on a chip containing a matrix compound to assist in laser deionization. Within the cleavage products, methylation-dependent C to T variation appears as G/A generated from the reverse strand by base-specific cleavage. These G/A variations result in a mass difference of 16 Da per CpG site, which is easily detected by the MassARRAY® Compact Analyzer, resulting in a signal pair pattern from the methylated and non-methylated template DNA.

The relative amount of methylation is determined by comparing the signal intensity between the mass signals of methylated and non-methylated template. EpiTYPER® generates quantitative results for each cleavage product analyzed. Each cleavage product encloses either one CpG site or an aggregate of multiple CpG sites. An analyzed unit containing one or multiple CpG sites is called a “CpG unit”. For both T and C reactions, the resulting cleavage products have the same length and differ only in their nucleotide composition.

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Other high throughput methods for quantitative DNA methylation analysis have been described, including several pyrosequencing-based approaches and a bead–based analysis method. Pyrosequencing is a real-time DNA sequencing by synthesis method that can be used after bisulfite treatment for direct quantitative methylation analysis. Methylation analysis using pyrosequencing results in sensitivity similar to that achieved using MassARRAY®, but the pyrosequencing method is limited by the read length of the sequence, resulting in a reduced number of CpG sites analyzed in a single reaction. Bead-based array methylation analysis is performed by incorporating multiplex analysis and is useful for analyzing multiple genes simultaneously, but is also limited in assessment of contiguous CpG sites. MassARRAY® EpiTYPER® technology represents a paradigm shift in quantitative DNA methylation analysis. The speed and accuracy of the MassARRAY® system enable cost-effective and quantitative analysis of the methylation status of multiple CpGs in one PCR amplicon. The data quality and reporting functions of EpiTYPER® make it the superior solution for methylation analysis, post-array validation or targeted gene promoter analysis.

Since the launch of EpiTYPER® in 2006, Sequenom has designed and validated assays for thousands of genomic regions. During the course of research and methylation service efforts over the past two years, we observed that a subset of genes is frequently analyzed. The methylation status of the genes in this subset is commonly subject to change, and is relevant to many areas of cancer research. In 2007, the Sequenom® Standard EpiPanel was compiled, representing the first high resolution, fine mapping panel for a subset of putative epigenetic targets. The Sequenom Standard EpiPanel offers the speed and convenience of using “off the shelf” validated amplicon designs. Using EpiTYPER® together with the amplicons found in the EpiPanel enables a first-of-its-kind combination of fast, inexpensive, and quantitative analysis that is not available elsewhere.

For each gene, PCR amplicons were designed to cover the majority of CpG-dense areas in close proximity to, or overlapping with the annotated transcription start. A total of four whole blood samples and four fully methylated (SssI treated) control samples were used to evaluate the assay performance. The results are shown for each gene locus separately. As shown in the example, (Figure 2A, below) our diagrams provide information about individual CpG methylation for the two control groups (upper panel), location of the amplification targets (middle panel) and gene structure (lower panel) within the context of the genomic annotation. For better visualization of methylation trends within each sample group across the genomic region, a principle curve analysis (blue and red dotted lines) is shown. In a subset of regions, elevated methylation levels occur in the whole blood samples. These findings indicate a physiological state, and are likely to be reproduced in other DNA samples derived from whole blood specimens.

In each Standard EpiPanel figure, a subset of amplicons to cover the entire CpG island is shown. All of these amplicon designs provide good quality results in our model system, but the preferred amplicon designs are indicated in dark red, while the others are indicated in pink. A detailed table lists the amplicon name, position, length and CpG coverage of each set (Figure 2B). A primer index lists the sequences to order for each amplicon (Figure 2C).

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Sequenom’s latest development in application-specific methylation panels is the Cancer EpiPanel. The Cancer EpiPanel contains quantitative DNA methylation profiles and assay information for more than 400 cancer related genes run on 62 cell lines (NCI60) that are derived from six different tissue types. This dataset can easily be expanded to develop a more comprehensive and ultimately more complete map of quantitative methylation changes. Our methylation data also provide an ideal starting point for further translational research. Results can be combined with existing large-scale datasets on mutational, transcriptional, and proteomic profiles to obtain a more comprehensive understanding of neoplastic transformations. The panel includes pre-validated assays covering over 12,000 CpG sites in promoter regions of genes known to be involved in neoplastic transformation and imprinting. The Cancer EpiPanel assays are designed for use with EpiTYPER® technology, and offer the first high throughput, quantitative, methylation profiling of a large set of cancer-related genes.

Assays for any genomic region of interest that are not currently included in either the Sequenom Standard EpiPanel or the Cancer EpiPanel, can be easily designed using Sequenom’s EpiDesigner software [2]. This program allows simple design of primers for bisulfite treated genomic DNA and recommends primer pairs for individual assays.

Advanced techniques for epigenetic analysis will provide the basis for translational research leading to novel medications which can target epimutations and likely provide an entirely new layer of therapeutic intervention for complex diseases (cancer, psychiatric disorders and beyond) [4] over the next decades. Already today epigenetics has proven itself as a very valuable source of biomarkers for diagnosis, prognosis and drug response prediction for existing therapies.

To whom correspondence should be addressed: Marijo Gallina, mgallina@sequenom.com

References:

1.   Lamarckism Revisited – Epigenetics and its Implications for Modern Health Care. Next Generation Pharmaceutical. May 2007

2.   Sequenom EpiDesigner

3.   Ehrich M, et al. A new method for accurate assessment of DNA quality after bisulfite treatment. Nucleic Acids Res. 2007; 35(5):e29.

4. Ptak C, Petronis A. Epigenetics and complex disease: from etiology to new therapeutics. Annu Rev Pharmacol Toxicol. 2008;48:257-76.