Epigenetic Diagnostics: Locus-specific DNA Methylation Testing Poised for Clinical Impact
Precision medicine holds great potential to make a direct positive impact in the clinic. A growing number of diagnostic panels addressing a list of disease indications are now available for testing. Currently most tests are designed for genetic analysis of sample DNA to help inform treatment decisions. With a few notable exceptions, epigenetic analysis of sample DNA lags far behind in terms of tests and panels with the potential for clinical applications. However that all might be about to change, as an international consortium of researchers reported in a paper published in Nature Biotechnology last summer.
Epigenome-wide studies provide evidence that DNA methylation pattern changes can correctly identify clinically relevant information. However, those genome-wide discoveries need to be followed-up with validation studies of the most promising candidate regions. A collection of 18 research groups, from seven different countries, systematically tested the most common locus-specific validation assays in order to benchmark the relative strengths, weaknesses, and best practices for implementation of targeted DNA methylation analysis methods into validation studies. The study, led by Christoph Bock, a Principal Investigator at CeMM Research Center for Molecular Medicine in Austria, and Stephan Beck, Professor at University College London, gave participating labs 32 reference samples that were designed to mimic sample types often encountered in a clinical setting. Samples included DNA from matched tumor and normal colon origin, serial dilutions of in vitro methylated DNA spiked into an unmethylated background, DNA taken from cell lines treated with specific drugs targeting normal epigenetic function, and more.
The labs were then asked to design assays targeting several dozen pre-determined genomic loci using their preferred method of choice.
The investigation methods which the various labs used can be broken down into 3 broad categories:
- Absolute DNA methylation assays; including amplicon bisulfite sequencing (BS), enrichment bisulfite sequencing, mass spectrometric analysis, and bisulfite pyrosequencing.
- Relative DNA methylation assays; including MethyLight probe-based assessment, methylation-specific melting or melt-curve assays, and quantitative methylation-specific PCR.
- Global DNA methylation assays
Evaluation of participant data showed that most methods were accurate and reproducible with general agreement between different platform types and contributing labs. However, the absolute quantification methods led by amplicon BS and pyrosequencing performed better than relative methylation analysis assay methods in terms of accuracy & reproducibility, sensitivity & specificity, sample throughput, and overall cost. The authors noted that global assays suffered from noisy data and divergent results between technologies, while standard clonal bisulfite sequencing was labor intensive and subject to a high level of technical noise.
Overall the study demonstrates that locus-specific DNA methylation assays could be considered a mature technology ready for widespread use in biomarker development and clinical applications. Of the available methods, absolute DNA methylation assays are the recommended choice when validating DNA methylation differences in large cohorts or developing epigenetic biomarkers. The study concluded by emphasizing that DNA methylation assays will become widely useful for diverse applications including early diagnostics, companion diagnostics, precision medicine, forensic testing, and more.
BLUEPRINT consortium. (2016). Quantitative comparison of DNA methylation assays for biomarker development and clinical applications. Nature biotechnology, 34 (7), 726-37 PMID: 27347756