Application of Multiplex Bisulfite PCR-Ligase Detection Reaction-Real-Time Quantitative PCR Assay in Interrogating Bioinformatically Identified, Blood-Based Methylation Markers for Colorectal Cancer

Abstract

The analysis of CpG methylation in circulating tumor DNA fragments has emerged as a promising approach for the noninvasive early detection of solid tumors, including colorectal cancer (CRC). The most commonly employed assay involves bisulfite conversion of circulating tumor DNA, followed by targeted PCR, then real-time quantitative PCR (alias methylation-specific PCR). This report demonstrates the ability of a multiplex bisulfite PCR-ligase detection reaction-real-time quantitative PCR assay to detect seven methylated CpG markers (CRC or colon specific), in both simulated (approximately 30 copies of fragmented CRC cell line DNA mixed with approximately 3000 copies of fragmented peripheral blood DNA) and CRC patient-derived cell-free DNAs. This scalable assay is designed for multiplexing and incorporates steps for improved sensitivity and specificity, including the enrichment of methylated CpG fragments, ligase detection reaction, the incorporation of ribose bases in primers, and use of uracil DNA glycosylase. Six of the seven CpG markers (located in promoter regions of PPP1R16B, KCNA3, CLIP4, GDF6, SEPT9, and GSG1L) were identified through integrated analyses of genome-wide methylation data sets for 31 different types of cancer. These markers were mapped to CpG sites at the promoter region of VIM; VIM and SEPT9 are established epigenetic markers of CRC. Additional bioinformatics analyses show that the methylation at these CpG sites negatively correlates with the transcription of their corresponding genes.

Figure 1

The scheme for identification and selection of CpG methylation markers tested for bisulfite PCR–ligase detection reaction–real-time quantitative PCR assay development. CRC, colorectal cancer; GEO, Gene Expression Omnibus; TCGA, The Cancer Genome Atlas.

Figure 2

Comparative methylation of the seven CpG markers (which the multiplex assay was designed for) in normal colon tissues [solid normal (SN)] and each of the three microsatellite (MS) subtype colorectal cancer primary tumors: high MS instability (MSI-H), low MSI (MSI-L), and MS stable (MSS).

Figure 3

Schematic of bisulfite PCR–ligase detection reaction–real-time quantitative PCR assay for detection of CpG methylation. 1. Bisulfite conversion. 2. Linear amplification (reverse primer only). 3. Digestion by uracil-N-DNA glycosylase (UDG). 4. PCR (add forward primer). 5. Ligase detection reaction. 6. Digestion by UDG. 7. ViiA7 real-time PCR.

Figure 4

Single-molecule detection of methylation of m_VIM by pixel PCR–ligase detection reaction–real-time quantitative PCR. A–C: Detection of 12 (A), 6 (B), and 0 (C) methylated molecules in presence of 2500 nonmethylated molecules. D: No template control. Rn, normalized reporter value.

Figure 5

Multiplexed detection of seven colorectal cancer (CRC) methylation markers by bisulfite PCR–ligase detection reaction–real-time quantitative PCR in simulated plasma sample (ie, average of 30 molecules of each methylated CpG marker mixed in 3000 unmethylated CpG markers from normal DNA; A), as well as plasma samples from CRC patients (B, D, and F) and healthy individuals (C, E, and G). cfDNA, cell-free DNA; Rn, normalized reporter value.