Technical advances in global DNA methylation analysis in human cancers

Abstract

Prototypical abnormalities of genome-wide DNA methylation constitute the most widely investigated epigenetic mechanism in human cancers. Errors in the cellular machinery to faithfully replicate the global 5-methylcytosine (5mC) patterns, commonly observed during tumorigenesis, give rise to misregulated biological pathways beneficial to the rapidly propagating tumor mass but deleterious to the healthy tissues of the affected individual. A growing body of evidence suggests that the global DNA methylation levels could serve as utilitarian biomarkers in certain cancer types. Important breakthroughs in the recent years have uncovered further oxidized derivatives of 5mC - 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), thereby expanding our understanding of the DNA methylation dynamics. While the biological roles of these epigenetic derivatives are being extensively characterized, this review presents a perspective on the opportunity of innovation in the global methylation analysis platforms. While multiple methods for global analysis of 5mC in clinical samples exist and have been reviewed elsewhere, two of the established methods - Liquid Chromatography coupled with mass spectrometry (LC-MS/MS) and Immunoquantification have successfully evolved to include the quantitation of 5hmC, 5fC and 5caC. Although the analytical performance of LC-MS/MS is superior, the simplicity afforded by the experimental procedure of immunoquantitation ensures it's near ubiquity in clinical applications. Recent developments in spectroscopy, nanotechnology and sequencing also provide immense promise for future evaluations and are discussed briefly. Finally, we provide a perspective on the current scenario of global DNA methylation analysis tools and present suggestions to develop the next generation toolset.

Keywords: 5-carboxylcytosine (5caC); 5-formylcytosine (5fC); 5-hydroxymethylcytosine (5hmC); 5-methylcytosine (5mC); Immunoquantitation; LC-MS/MS; Next generation toolset.

Fig. 1

Summary of the status of global levels of the DNA methylation derivatives in normal and tumorous tissue. (Refer to Table 1 for details and references)

Fig. 2

Schematic representation of methods for global analysis of DNA methylation derivatives based on (a) LC-MS/MS, (b) Immunoquantification, (c) FRET, (d) SPR, (e) Electrochemistry, (f) Nanofluidics and (g) Nanopore Sequencing

Fig. 3

Nanoparticle-driven optical tools for the detection of DNA methylation derivatives (a) (i) Surface enhanced Raman scattering (SERS) and (ii) Localized surface plasmon resonance (LSPR). SERS usually occurs on plasmonic nanostructures and dramatically enhances Raman scattering of adsorbed molecules. SERS efficiency is directly related to not only proximal distance among the particles shown here but also size, shape etc. LSPR that describes maximal optical absorption at the plasmon resonant frequency of nanoparticles can be distinguishably changed in the form of cluster formation of nanoparticles. b An illustration of quantification of subcellular 5caC (in the context of intact nucleus and single chromosome) with the help of local surface plasmon resonance (LSPR) properties of nanoprobes (nanoparticles conjugated antibody). The figs. on the left represent Hyperspectral dark-field imaging (HSDFI) of 5caC distribution, while the corresponding figs. on the right demonstrate reconstructed spectral maps of 5caC (scale bar = 5 μm) Reprinted with permission from [65] Copyright (2015) American Chemical Society

Fig. 4

Opportunities for innovating global 5mC analysis methods. a Comparison of tools based on LC-MS/MS with immunoquantification to perform global methylation analysis. The red check mark indicates the method that displays superiority on the basis of the indicated criterion. b The authors’ illustration of how the current global DNA methylation derivatives’ analysis methods fare on the basis of analytical superiority (aggregate of detection limit, input genomic DNA and robustness wherever available) and feasibility of translation (aggregate of time, complexity and cost)