Pocket Oncology (Pocket Notebook Series), 1st Ed.

EPIGENETIC TESTING

Jacob L. Glass and Omar Abdel-Wahab

Background

Current epigenetic testing is generally performed in the research setting. A wide variety of techniques are used, ranging from whole genome surveys to high resolution locus-specific evaluation. These techniques rely on several basic molecular-biology building blocks:

Bisulfite conversion: A reaction converting unmethylated cytosine (C) in DNA to uracil (U). When amplified by PCR, uracil is replaced by thymidine (T) due to the symmetry of A-T/A-U pairing. The net result is (C->T). DNA breakage also occurs as a consequence of the reaction.

Restriction enzyme digestion: Restriction enzymes cut DNA at specific seq, some of w/c include recognition of a methylated cytosine. Eg, the HpaII enzyme cleaves DNA at CCGG seq where the 2nd ‘C’ is methylated whereas the MSPI enzyme cleaves at CCGG sites regardless of methylation status.

Immunoprecipitation: A means of selecting specific molecules using Ab recognition. Methylated DNA or a protein bound to DNA (such as a specific modified histone residue or transcription factor) are typical targets. Also abbreviated as ChIP.

Mass spectrometry: A method to identify the molecular composition of a sample by computational analysis of the mass/charge ratio signatures of its components

Microarray analysis: A means of identifying specific DNA seq w/in a sample by their homology to DNA fragments affixed to a glass slide or chip. The set of DNA fragments used is highly customizable, but the scope of identifiable seq is limited to those that are placed on the chip. Bioinformatic quality control & analysis is essential.

High-throughput seq: A technology in w/c individual DNA molecules w/in a sample are seq directly. This technology is also limited to short seq w/c are reassembled bioinformatically.

DNA Methylation Analysis

Bisulfite seq: The seq of bisulfite-converted DNA is compared to its unconverted source & the positions at w/c there is a T:C mismatch are identified as methylated sites. This procedure is repeated several times to derive the %methylation at each position. This can be extended using microarrays or high-throughput seq.

Bisulfite pyroseq: An extension of bisulfite seq using fluorophores in w/c the %methylation at a given position can be derived reliably from a single experiment by the relative T:C fluorescence. Highly reliable but also restricted to short seq.

MassARRAY: A proprietary extension of bisulfite seq from Sequenom that computes base-specific DNA methylation percentages by analysis of bisulfite-converted DNA mass spectrometry results (Bioinformatics 2009;2164–2170)

MeDIP-chip, MeDIP-seq: Methylated DNA is broken up into small fragments & immunoprecipitated using an Ab specific for methylated DNA. The resulting DNA fragments are then analyzed either using a microarray or “chip” (Nat Genet 2005;853:862) or by high-throughput seq (BMC Genomics 2010;137). Bioinformatic analysis is required.

Restriction digest analysis: Several variants of this approach exist. One example is the HELP assay, in w/c a subset of methylatable sites are analyzed by comparison of HpaII & MSPI restriction enzyme digests. The digest is either analyzed by microarray (Genome Res 2006;1046:1055) or high-throughput seq (Methods 2010;218:222). Sophisticated bioinformatic analysis is required.

Illumina methylation assay: A proprietary platform in w/c bisulfite-converted DNA is hybridized to a commercial predesigned microarray containing a subset of possible methylation sites in the human genome, w/paired analogs to the bisulfite-converted methylated & unmethylated seq. A single nucleotide seq step is performed followed by a series of stains & proprietary software analysis to identify the ratio of methylated to unmethylated DNA (Epigenomics2009;177:200).

Comparison of DNA methylation analysis techniques*

Histone Analysis

ChIP-chip: A means of evaluating the genomic positioning of proteins such as specifically modified histones. Proteins are cross-linked to DNA, & the histone-DNA complexes of interest are selected w/a specific Ab. The cross-linking is then reversed, & the resulting DNA is then hybridized to a microarray (chip) for analysis. Bioinformatic processing of the microarray results is essential.

ChIP-seq: Similar to ChIP-chip w/high-throughput seq replacing microarray analysis (chip). Bioinformatic processing is essential.

Western blot: A classic means to detect the presence of specific proteins such as specifically modified histones. Proteins are separated by gel electrophoresis, transferred to a membrane, & identified by homology to an Ab wash. Only one Ab can be used at a time, & sequential Ab stripping/rehybridization is limited by sample degradation.

ELISA: Another classic Ab-mediated method to identify specific proteins. Input protein samples are placed in test tubes & hybridized to a set of antibodies that produce a color change if a specific protein is present.

Mass spectrometry: The presence of multiple modified histones in a sample can be identified in parallel by mass spectrometry. Similar to massArray analysis of DNA methylation, this technique depends heavily on computational analysis.