Abstract 3186: Methylation MGMT gene promoter analysis based on a high throughput method combines bisulfite conversion with amplicon sequencing
Authors: Zhang Yukun , Shi Min , Zheng Qiaosong , Shi Xiao , Chen Min , Li Le , Zhu Huiming , Jiang Liping , Ma Tonghui , Geng Sumin 
Published: September 2020

Abstract
Background: Methylation of the MGMT gene promoter was observed in approximately 50% of glioblastoma multiforme (GBM). Epigenetic silencing of the MGMT gene by promoter methylation results in decreased MGMT protein expression, reduced DNA repair activity, and potential increased sensitivity to alkylating agent-based chemotherapy. Although MGMT promoter methylation status has been widely evaluated by methylation-specific PCR and bisulfite pyrosequencing. The limitations to these methods include low quantitative accuracy and low sample throughput. Here, we developed a high throughput method (in-house One-Step Seq Method, Genetron) which combines bisulfite conversion with amplicon sequencing of MGMT gene promoter.
Methods: DNA was extracted from 144 FFPE or fresh frozen glioma tissues. For bisulfite pyrosequencing, MGMT promoter methylation status was analyzed using the MGMT Pyro Kit (Qiagen 970061) following the manufacturer's protocol. At the same time,the MGMT promoter methylation status was also analyzed by in-house One-Step Seq Methodfollowing bisulfite conversion. In briefly, the DNA was pre-treated by sodium bisulfite. Then Exon 1 of human MGMT gene was amplified from bisulfite conversed DNA. And library was constructed at the same time. High-throughput sequencing was performed on Ion GeneStudio S5. Reads mapping and methylation calling was handled by Bismark. The DNA sample was identified as MGMT methylation, if average methylation level of all CpG sites in exon 1 of MGMT was more than 21 %.
Results: Currently, bisulfite pyrosequencing was considered as the gold standard for DNA methylation analysis. Comparing the result of bisulfite pyrosequencing analysis with the result of in-house methylation status analysis, 94.4% of DNA samples from 144 glioma tissues showed consistent methylation status of MGMT gene promotor. 5 DNA samples were identified as non-methylation in bisulfite pyrosequencing, but with methylation in in-house methylation status analysis. While 3 DNA samples were identified as methylation in bisulfite pyrosequencing, but not in in-house methylation status analysis. Meanwhile, by in-house One-Step Seq Method following bisulfite conversion, both the bisulfite conversion and library construction could be completed injust 4 hours using 10 ng DNA, and MGMT gene promoter methylation status could be estimated in two days.
Conclusions: We developed a High-throughput sequencing based method to analyze MGMT status accurately combines bisulfite conversion with amplicon sequencing. This method shows high accuracy, high throughput, and easy manipulation for molecular classification of brain cancer.
Citation Format: Yukun Zhang, Min Shi, Qiaosong Zheng, Xiao Shi, Min Chen, Le Li, Huiming Zhu, Liping Jiang, Tonghui Ma, Sumin Geng. Methylation MGMT gene promoter analysis based on a high throughput method combines bisulfite conversion with amplicon sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3186.