Developing DNA methylation-based diagnostic biomarkers

Abstract

An emerging paradigm shift for disease diagnosis is to rely on molecular characterization beyond traditional clinical and symptom-based examinations. Although genetic alterations and transcription signature were first introduced as potential biomarkers, clinical implementations of these markers are limited due to low reproducibility and accuracy. Instead, epigenetic changes are considered as an alternative approach to disease diagnosis. Complex epigenetic regulation is required for normal biological functions and it has been shown that distinctive epigenetic disruptions could contribute to disease pathogenesis. Disease-specific epigenetic changes, especially DNA methylation, have been observed, suggesting its potential as disease biomarkers for diagnosis. In addition to specificity, the feasibility of detecting disease-associated methylation marks in the biological specimens collected noninvasively, such as blood samples, has driven the clinical studies to validate disease-specific DNA methylation changes as a diagnostic biomarker. Here, we highlight the advantages of DNA methylation signature for diagnosis in different diseases and discuss the statistical and technical challenges to be overcome before clinical implementation.

Keywords: Biomarker; Brain disorders; Cancer; DNA methylation; Epigenetics; Liquid biopsy; Molecular diagnosis.

Fig. 1. Cytosine modifications in mammalian genome

The fifth position of cytosine can be methylated by DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) to generate 5-methylcytosine (5mC). The methyl group of 5mC can be oxidized by ten-eleven translocation (TET) family enzymes (TET1, TET2, and TET3), generating 5-hydroxymethylcytosine (5hmC), 5-formlycytosine (5fC), and 5-carboxycytosine (5caC). While 5mC is maintained by the interaction between the replication machinery and DNMT1, no maintenance mechanisms exist for the oxidative derivatives; therefore, the levels of 5hmC, 5fC and 5caC are diminished over the replication (passive dilution). In addition, 5fC and 5caC can be excised by thymine DNA glycosylase (TDG), and eventually replaced with cytosine (active demethylation by base excision repair (BER)). Both passive and active demethylation mechanisms contribute to dynamics of cytosine modification.

Fig. 2. Clinical application of DNA methylation marks specific to disease status

DNA methylation is specific to tissue of origin. Even cell-free DNA (cfDNA) found in the circulating system retains DNA methylation marks. However, these modifications are frequently altered during disease progression. Epigenetic alterations are one of cancer hallmarks and hypo- or hyper-methylation at particular promoter regions are biomarkers of neurodegenerative disorders. Differential DNA methylation regions (DMRs) associated with disease can be identified in an unbiased manner including whole-genome bisulfite sequencing (WGBS) and microarray-based techniques (e.g., Illumina 850K array), which can be clinically used as diagnostic markers.