甲基化敏感性高分辨率熔解分析检测乳腺外佩吉特病中的2种基因甲基化

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

作者信息

Dong Liu, Zhu Yingfeng, Wu Liting, Zhang Qiaoan, Xu Feng, Zhang Xinju, Xu Xiao, Tang Yiting, Ren Guoqiang, Kang Zhihua, Guan Ming

机构信息

Department of Laboratory Medicine, Huashan Hospital, Shanghai Medical School, Fudan University, Shanghai, China.

Department of Pathology, Huashan Hospital North, Fudan University, Shanghai, China.

出版信息

J Oncol. 2021 Nov 1;2021:5514426. doi: 10.1155/2021/5514426. eCollection 2021.

DOI:10.1155/2021/5514426

PMID:34759969

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8575627/

Abstract

BACKGROUND

Extramammary Paget's disease (EMPD) is a rare skin tumor. Hypermethylation in the 2 promoter resulting in the downregulation of its protein expression shows a high detection rate in EMPD tumor tissue, which indicates that the methylation of 2 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 2 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 2 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 2 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 ( = 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 2 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 2 in EMPD tumor samples and other cancerous tissues potentially, which presents a promising candidate as a quantitative assay to analyze 2 promoter methylation in routine pathological procedure.

摘要

背景

乳腺外佩吉特病（EMPD）是一种罕见的皮肤肿瘤。2号启动子的高甲基化导致其蛋白表达下调，在EMPD肿瘤组织中检测率较高，这表明2号的甲基化可能在EMPD的发病机制中起重要作用。

目的

本研究旨在建立一种基于甲基化敏感高分辨率熔解曲线（MS-HRM）的快速分析策略，以检测2号启动子的甲基化水平。

方法

使用通用甲基化人类DNA产物，建立MS-HRM标准曲线以定量检测2号启动子的甲基化水平。然后，对所有57份EMPD肿瘤DNA样本进行分析。还进行了焦磷酸测序分析以测试MS-HRM的准确性和有效性。此外，本研究共纳入54份人类正常组织和其他癌组织样本，以测试MS-HRM标准曲线的可靠性和通用性。

结果

在本研究中，通过使用建立的MS-HRM，我们发现96.5%（55/57）的EMPD肿瘤样本在2号启动子中具有从0%到30%不等的甲基化水平。然后，将甲基化数据与焦磷酸测序结果进行比较，Pearson相关性分析（r = 0.9425）和Bland-Altman图（平均差异 = -0.1069）显示这两种技术之间具有高度相关性，表明MS-HRM分析的甲基化水平与DNA焦磷酸测序一致。此外，在23份正常组织和31份其他癌组织样本中，有两份结直肠癌（CRC）组织检测到2号甲基化呈阳性（1%和5%），这证实了我们建立的MS-HRM可广泛应用于各种类型的样本。