MethyQESD, a robust and fast method for quantitative methylation analyses in HNPCC diagnostics using formalin-fixed and paraffin-embedded tissue samples

Abstract

Promoter hypermethylation occurs in various tumors and leads to silencing of tumor-relevant genes. Thus, promoter methylation analysis (MA) has been established as an important tool in cancer research and diagnostics. Here we present MethyQESD (methylation-quantification of endonuclease-resistant DNA) as a fast, easy, precise and reliable method for quantitative MA without the need of bisulfite-treatment or fluorescent probes. Though MethyQESD principally works with any gene promoter we established MethyQESD for the mismatch repair gene MLH1 and tested its utility to differentiate between sporadic microsatellite unstable (MSI-H) colorectal cancer and hereditary nonpolyposis colorectal cancer (HNPCC) by quantitative MLH1 MA. We investigated formalin-fixed and paraffin-embedded tissue samples from a previously published, well-characterized tumor collective comprising 25 HNPCC, 14 sporadic MSI-H CRC and 16 sporadic microsatellite stable (MSS) CRC. We found a high accuracy of MethyQESD by spiking experiments with dilution series of methylated (SW48 cancer cell line) and unmethylated (blood) DNA (Pearson's r ¼ 0.9997 (proximal MLH1 promoter region), r ¼ 0.9976 (distal MLH1 promoter region)). MethyQESD and conventional quantitative MA using of 96 formalinfixed and paraffin-embedded CRC showed a high degree of concordance of both methods (Pearson's r ¼ 0.885). HNPCC tumors showed either null MLH1 methylation or a significantly lower degree of MLH1 methylation than sporadic MSI-H CRC (Po0.001). MLH1 methylation was negative in all MSS tumors. Receiver operating characteristic (ROC) curve analyses defined a cutoff value of 16.5% MLH1 methylation for specific and sensitive identification of sporadic MSI-H CRC (area under ROC curve: 1.000; asymptotic significance: Po0.001). Thus, quantitative MLH1 MA by MethyQESD provides a simple, fast and valuable tool to identify HNPCC candidates. Furthermore, MethyQESD works reliably with formalin-fixed paraffin-embedded tissue and simplifies DNA MA both for research and diagnostic purposes.

Expression of the MAP kinase phosphatase DUSP4 is associated with microsatellite instability in colorectal cancer (CRC) and causes increased cell proliferation

“…Promoter methylation of the DUSP4 gene was analyzed in six CRC cell lines (HCT116, SW480, HT29, SW48, CaCo2 and LoVo) and tumor as well as matched normal tissue of 11 CRC patients according to the Methy‐QESD method 27. Genomic DNA was isolated using the MagNAPure LC system (Roche, Mannheim, Germany).…”

A randomized, double-blind, placebo-controlled, clinical trial on probiotic soy milk and soy milk: effects on epigenetics and oxidative stress in patients with type II diabetes

“…The promoter methylation analysis (MA), using methylation-specific digestion enzyme and real-time polymerase chain reaction (PCR), was done according to the guidelines provided by Bettstetter et al (2008). Two restriction digestion batches were prepared for every sample: (1) methylation quantification digestion (MQD), (2) methylation-independent calibrator digestion (CalD).…”

Quantitative detection of TUSC3 promoter methylation -a potential biomarker for prognosis in lung cancer

“…In contrast to the MethyQESD technique (36), previous studies used methods such as bisulfite-conversion, which bears the risk of incomplete conversion of unmethylated cytosines (39), leading to an overestimation of methylation. Furthermore, the use of methylation specific primers without enough discrimination capacity between methylated and unmethylated DNA can also result in false positive methylation results (39).…”

“…Methylation analysis was performed using MethyQESD (36), a combination of methylation-sensitive digestion and qPCR. A methylation specific quantification digestion (MQD) containing 40 units (U) of the methylation-sensitive endonuclease, Hin6I (Fermentas; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and a calibrator digestion (CalD) containing the methylation-independent endonucleases, XBaI (20 U) and DraI (20 U) (Fermentas; Thermo Fisher Scientific, Inc.), were set up for each sample.…”