Real-time methylation-specific PCR for the evaluation of methylation status of MGMT gene in glioblastoma
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Masaki Yoshioka1,2, Tomoo Matsutani1, Ayaka Hara1, Seiichiro Hirono1, Takaki Hiwasa2, Masaki Takiguchi2 and Yasuo Iwadate1
1Department of Neurological Surgery, Chiba University Graduate School of Medicine, Chiba, Japan
2Department of Biochemistry and Genetics, Chiba University Graduate School of Medicine, Chiba, Japan
Yasuo Iwadate, email: [email protected]
Keywords: glioma; MGMT; methylation; real-time PCR; MSP
Received: February 07, 2018 Accepted: May 09, 2018 Published: June 12, 2018
The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) gene is a strong predictor for the efficacy of temozolomide chemotherapy and survival periods. However, the correlation between the extent of methylation and the difference in survival times has not been fully clarified. Simple and quantitative evaluations of the methylation status in the promotor region of the MGMT gene are expected to be worldwide standardized diagnostics. We applied real-time semi-quantitative methylation-specific polymerase chain reaction (SQ-MSP) of the MGMT gene promoter region to 84 glioblastoma patients. The SQ-MSP result showed that the ΔCt value, which represents the difference between uCt and mCt (uCt value – mCt value), is inversely correlated with overall survival. With adequate cutoff setting, this assay showed that those patients suffering from a tumor with low ΔCt (methylated) survived significantly longer than those having tumors with high ΔCt (un-methylated). The most significant difference was observed when the cutoff was set at a ΔCt of 2. Using this cutoff point, the result of MGMT immunohistochemical analysis was also significantly correlated with the methylation status examined with real-time SQ-MSP. These results collectively show that MGMT promoter methylation status actually affects patients’ survival and protein expression depending on its methylation level, and the extent of methylated CpGs would be better assessed with real-time SQ-MSP than with the standard gel-based MSP. This method is cost- and labor-saving compared with pyrosequencing, and significantly contributes to the accurate and objective prediction of patient survival.
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