Methylation-sensitive enrichment of minor DNA alleles using a double-strand DNA-specific nuclease

Abstract

Aberrant methylation changes, often present in a minor allelic fraction in clinical samples such as plasma-circulating DNA (cfDNA), are potentially powerful prognostic and predictive biomarkers in human disease including cancer. We report on a novel, highly-multiplexed approach to facilitate analysis of clinically useful methylation changes in minor DNA populations. Methylation Specific Nuclease-assisted Minor-allele Enrichment (MS-NaME) employs a double-strand-specific DNA nuclease (DSN) to remove excess DNA with normal methylation patterns. The technique utilizes oligonucleotide-probes that direct DSN activity to multiple targets in bisulfite-treated DNA, simultaneously. Oligonucleotide probes targeting unmethylated sequences generate local double stranded regions resulting to digestion of unmethylated targets, and leaving methylated targets intact; and vice versa. Subsequent amplification of the targeted regions results in enrichment of the targeted methylated or unmethylated minority-epigenetic-alleles. We validate MS-NaME by demonstrating enrichment of RARb2, ATM, MGMT and GSTP1 promoters in multiplexed MS-NaME reactions (177-plex) using dilutions of methylated/unmethylated DNA and in DNA from clinical lung cancer samples and matched normal tissue. MS-NaME is a highly scalable single-step approach performed at the genomic DNA level in solution that combines with most downstream detection technologies including Sanger sequencing, methylation-sensitive-high-resolution melting (MS-HRM) and methylation-specific-Taqman-based-digital-PCR (digital Methylight) to boost detection of low-level aberrant methylation-changes.

Figure 1.

Schematic workflow of Methylation-Sensitive Nuclease-assisted Minor-allele Enrichment, MS-NaME.

Figure 2.

Application of four-plex MS-NaME (ATM, RARb2, MGMT and GSTP1 gene promoters) on bisulfite-treated DNA to enrich methylated or unmethylated DNA. A 50:50 methylated: unmethylated DNA mix was used and results were assessed via MS-HRM (A) or sequencing (B). The MS-HRM curves are as follows: No-DSN control-green, U-antisense probes-blue, and M-antisense probes-red.

Figure 3.

Application of four-plex MS-NaME (ATM, RARb2, MGMT and GSTP1 gene promoters) on bisulfite-treated DNA to enrich serially diluted levels of methylated or unmethylated DNA. 0.1–10% methylated DNA or 0.1–10% unmethylated DNA were treated with U probes and M probes, respectively. (A and B) ATM and RARb2 were assessed via digital Methylight to determine changes in methylation and unmethylation levels as compared to no treatment. Error bars represent the standard error of the mean from three independent experiments. (C and D): Bisulfite Sanger sequencing for the four genes was conducted with 0.1% methylated DNA or 0.1% unmethylated DNA following MS-NaME treatment with U probes and M probes, respectively.

Figure 4.

Application of MS-NaME in clinical tumor and plasma samples. Lung tumor samples were treated with (A) four-plex and (B) 177-plex MS-NaME treatment and measured with RARb2 digital Methylight to quantify the methylation ratio change. Plasma circulating-DNA samples from two healthy donors (#21 and #25) which were unmethylated in ATM promtoter were spiked with 1% or 10% methylated DNA (final ratio), and then treated by 177-plex MS-NaME. Error bars represent the standard error of the mean from two independent experiments. (C) ATM MS-HRM was applied to sample 21a. The melting curves are as follows: 1% spike-in-No DSN-green, 1% spike-in-MS-NaME-green with square symbol, 10% spike-in-No DSN-red, 10% spike-in-MS-NaME-red with square symbol. (D) ATM digital Methylight was applied to plasma DNA samples 21a, 21b, 25a and 25b. a and b denote samples taken from the same healthy donor but spiked with different amounts of methylated DNA. Error bars represent the standard error of the mean from two independent experiments.