Detection of MSH 2 Gene Methylation in Extramammary Paget's Disease by Methylation-Sensitive High-Resolution Melting Analysis

Abstract

Background: Extramammary Paget's disease (EMPD) is a rare skin tumor. Hypermethylation in the MSH2 promoter resulting in the downregulation of its protein expression shows a high detection rate in EMPD tumor tissue, which indicates that the methylation of MSH2 may play an important role in the pathogenesis of EMPD.

Objective: This study aims to establish a rapid analysis strategy based on the methylation-sensitive high-resolution melting curve (MS-HRM) to detect the methylation level of the MSH2 promoter.

Methods: With the use of universal methylated human DNA products, we established the MS-HRM standard curve to quantitatively detect the methylation level of the MSH2 promoter. Then, all 57 EMPD tumor DNA samples were analyzed. Pyrosequencing assay was also carried out to test the accuracy and efficacy of MS-HRM. Besides, a total of 54 human normal and other cancerous tissues were included in this study to test the reliability and versatility of the MS-HRM standard curve.

Results: In this study, by using the established MS-HRM, we found that 96.5% (55/57) EMPD tumor samples had varying methylation levels in the MSH2 promoter ranging from 0% to 30%. Then, the methylation data were compared to the results obtained from pyrosequencing, which showed a high correlation between these two techniques by Pearson's correlation (r = 0.9425) and Bland-Altman plots (mean difference = -0.1069) indicating that the methylation levels analyzed by MS-HRM were consistent with DNA pyrosequencing. Furthermore, in 23 normal and 31 other cancerous tissue samples, there were two colorectal cancer (CRC) tissues that tested MSH2 methylation positive (1% and 5%) which confirmed that our established MS-HRM can be widely applied to various types of samples.

Conclusion: MS-HRM standard curve can be used for the detection of the methylation level of MSH2 in EMPD tumor samples and other cancerous tissues potentially, which presents a promising candidate as a quantitative assay to analyze MSH2 promoter methylation in routine pathological procedure.

Figure 1

Using the MS-HRM standard curve to detect the methylation level of the MSH2 gene promoter. Standard curves are shown, from top to bottom: 100%, 50%, 30%, 10%, 5%, 1%, and 0% of standard methylated DNA. The lines in black and grey are two example results of EMPD samples.

Figure 2

Profile of MSH2 methylation (%) in 57 EMPD samples with both MS-HRM and pyrosequencing techniques.

Figure 3

Bland–Altman plot analysis: each point represents a sample, the horizontal axis represents its average value, and the vertical axis indicates the difference between the two methods. The 95% limits of agreement were adopted and marked as dotted lines.