Using nanobiotechnology to increase the prevalence of epigenotyping assays in precision medicine

Abstract

Epigenetic silencing of genes that are important for DNA repair, cell cycle control, apoptosis, and cellular interactions with the extracellular matrix has been causally linked to several subtypes of cancer. Translating this knowledge of the implications of promoter methylation to wide and routine use in clinical pathology laboratories has been more challenging than the case of genetic analyses because epigenetic modifications do not change the underlying sequence of the affected nucleic acid, rendering polymerase chain reaction analysis alone uninformative. Two epigenotyping assays that detect promoter methylation are currently standard of care in treatment of two distinct tumor types in only a few top hospitals across the United States. Both rely on a harsh chemical step that degrades over 90% of tumor DNA samples, which are often available in limited quantities, and imparts the potential for false-negative or false-positive results if the reaction conditions are not exactly correct. Using nanotechnology and biotechnology to devise practical new analysis techniques that avoid the drawbacks of current techniques represents a powerful approach that is likely to significantly increase the clinical use of this class of biomarkers in the coming years. WIREs Nanomed Nanobiotechnol 2017, 9:e1407. doi: 10.1002/wnan.1407 For further resources related to this article, please visit the WIREs website.

An examination of critical parameters in hybridization‐based epigenotyping using magnetic microparticles

“…The titration curve in the presence of genomic DNA looks very similar to that without genomic DNA, and the limit of detection in the presence of genomic DNA was determined to be 5 pM target DNA based on a two‐sample Wilcoxon test at a significance level of 0.01. However, further improvement in sensitivity is still required in order for the assay to be useful for clinical samples, where only 10 6 copies of the genome may be available and sub‐pM sensitivity is required . Such improvements could potentially be made by either using a system with improved transport properties to concentrate the target DNA on a smaller number of beads, thus increasing the amount of target captured and MBD bound per bead, or by using more sensitive detection mechanisms.…”

“…Currently, all clinical methylation testing uses bisulfite conversion of DNA followed by PCR or sequencing . In bisulfite conversion, unmethylated cytosines are chemically converted to uracil while methylated cytosines remain unchanged.…”

“…Several alternatives to MS‐PCR have been proposed . Some of these methods rely on bisulfite conversion but offer alternatives to PCR and gel electrophoresis.…”

Engineering affinity agents for the detection of hemi-methylated CpG sites in DNA

“…2–4 The utility of promoter methylation as a biomarker for cancer diagnostics is demonstrated by the commercially available tests Cologuard ® and Epi ProColon ® for colorectal cancer screening. 5–7 While there are only a few gene promoters that are routinely evaluated for methylation at this time, 8,9 studies have shown tumor-associated promoter methylation of more than 45 genes in various types of cancers. 10 …”

“…9,11 Such methods have numerous drawbacks. For example, large quantities of DNA are required because 90% can be degraded during the conversion step, and without careful optimization and control of the reaction conditions, false positives and negatives are common.…”