Abstract
BackgroundCST6 promoter is highly methylated in cancer, and its detection can provide important prognostic information in breast cancer patients. The aim of our study was to develop a Methylation-Sensitive High Resolution Melting Analysis (MS-HRMA) assay for the investigation of CST6 promoter methylation.MethodsWe designed primers that amplify both methylated and unmethylated CST6 sequences after sodium bisulfate (SB) treatment and used spiked control samples of fully methylated to unmethylated SB converted genomic DNA to optimize the assay. We first evaluated the assay by analyzing 36 samples (pilot training group) and further analyzed 80 FFPES from operable breast cancer patients (independent group). MS-HRMA assay results for all 116 samples were compared with Methylation-Specific PCR (MSP) and the results were comparable.ResultsThe developed assay is highly specific and sensitive since it can detect the presence of 1% methylated CST6 sequence and provides additionally a semi-quantitative estimation of CST6 promoter methylation. CST6 promoter was methylated in 39/80 (48.75%) of FFPEs with methylation levels being very different among samples. MS-HRMA and MSP gave comparable results when all samples were analyzed by both assays.ConclusionsThe developed MS-HRMA assay for CST6 promoter methylation is closed tube, highly sensitive, cost-effective, rapid and easy-to-perform. It gives comparable results to MSP in less time, while it offers the advantage of additionally providing an estimation of the level of methylation.
Highlights
Cystatin M gene (CST6) promoter is highly methylated in cancer, and its detection can provide important prognostic information in breast cancer patients
We have previously demonstrated that CST6 is hypermethylated in breast cancer tissues and that CST6 promoter methylation provides important prognostic information in patients with operable breast cancer [26]
Analytical sensitivity and specificity The developed Methylation-Sensitive High Resolution Melting Analysis (MS-High-Resolution Melting Analysis (HRMA)) assay for CST6 methylation is highly specific for sodium bisulfate (SB) treated DNA since under these experimental conditions only SB treated DNA is amplified
Summary
CST6 promoter is highly methylated in cancer, and its detection can provide important prognostic information in breast cancer patients. Sodium bisulfite (SB) modification of DNA is necessary for DNA methylation assays that are based on PCR amplification, since DNA polymerase does not recognize methylated nucleotides, and as a result methylation information is lost during amplification. The second approach is based on primers that amplify a region of the desired template including CpG islands, no matter what its methylation status is. In this case, Methylation Independent PCR (MIP) is firstly performed and information on the methylation status of that region is obtained through postPCR analyses techniques like bisulfite sequencing, restriction digestion, single-strand conformation analysis, and high-resolution melting [6]
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