Custom-designed methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) test for profiling of promoter methylation in ovarian and endometrial cancers

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http://urn.fi/URN:NBN:fi:hulib-202001141056
Title: Custom-designed methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) test for profiling of promoter methylation in ovarian and endometrial cancers
Author: Akhondzadeh, Soheila
Contributor: University of Helsinki, Faculty of Biological and Environmental Sciences, Faculty of Biological and Environmental Sciences
Publisher: Helsingin yliopisto
Date: 2016
Language: eng
URI: http://urn.fi/URN:NBN:fi:hulib-202001141056
http://hdl.handle.net/10138/309472
Thesis level: master's thesis
Discipline: perinnöllisyystiede
Genetics
genetik
Abstract: Background: Epithelial ovarian cancer is the most common type of ovarian cancer and is the most lethal gynecologic cancer due to its late diagnosis. Compared to ovarian cancer, endometrial carcinoma, as the most common gynecologic malignancy, is referred to as the “curable cancer”, as it can be detected early. As aberrant promoter methylation patterns are a common change in human cancer, detection of promoter methylation status may help in early diagnosis. In this study, we used a custom-designed methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay as a rapid and easy method, to simultaneously detect the methylation status of multiple genes in ovarian and endometrial cancer samples. Aims: To design and test an MS-MLPA assay for analyzing promoter methylation of four genes associated with ovarian and endometrial cancers. The selected genes were HNF1 homeobox B (HNF1β), Ten-eleven translocation 1(TET1), L1 cell adhesion molecule (L1CAM), and AT-rich interactive domain 1A (ARID1A). These genes are known to have expression changes by DNA methylation. Methods: The promoter DNA methylation patterns of these four genes were analyzed in 15 cancer cell lines and 5 normal cell lines and DNAs using bisulfite sequencing. Six synthetic probe pairs were designed and optimized by applying them to cancer and normal cell lines and normal DNAs and comparing the results with those of bisulfite sequencing. Finally, the MS-MLPA assay was performed on patient specimens according to the MRC-HOLLAND MS-MLPA general protocol and methylation frequencies were calculated from MS-MLPA data. Results and conclusion: The MS-MLPA assay gave accurate methylation results with the 170 samples assayed. The HNF1B, L1CAM, and TET1 Genes were observed methylated in tumor samples whereas they were not methylated in the normal samples or showed very little methylation, suggested to be favorable diagnostic markers. MS-MLPA robustly and sensitively detects the promoter DNA methylation status.
Subject: Ovarian cancer
Endometrial cancer
DNA methylation
MS-MLPA
Bisulfite sequencing


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