O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme, involved in cellular defense against the biological effects of the mutagenic O6-methylguanine and O4-methylthymine in DNA. The MGMT enzyme catalyses transfer of methyl groups from O6-methylguanine and other methylated moieties of the damaged DNA to a cysteine residue in its own molecule. This will repair the toxic lesions, but will leave MGMT in an irreversible inactivated state. The repair of natural occurring DNA lesions as the O6-methylguanine back to guanine takes place under normal circumstances in the cell during DNA replication and transcription. The enzyme MGMT will therefore react when alkylating chemotherapy, as temozolomide, induces alkylation of the Oposition of guanine as part of the treatment. If MGMT is unable to repair mutations induced by temozolomide the cancer cell will eventually die as an effect of this potent carcinogen.

The MGMT promoter CpG island

The gene encoding MGMT is located at chromosome 10q26.3, where a CpG island of > 700 bp spans the promoter region and into the first exon. The CpG island constitutes 97 CpG dinucleotides, and two differentially methylated regions (DMR1 and DMR2) are defined as part of these CpG sites. For both DMRs, the methylation status correlates inversely with MGMT gene expression. DMR1 is upstream from the transcriptional start site (nucleotides -250 to -101), and DMR2 is situated within exon 1 (nucleotides +97 to +196). If any of the CpG dinucleotides within the DMR2 are changed, an effect is seen on MGMT expression, and if DMR2 is hypermethylated, DMR1 is too. The importance of these two DMRs is further strengthen, as it is shown that their methylation status is associated with a prolonged overall survival for glioblastoma patients treated with alkylating chemotherapy (Bady P et al. 2012).

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MGMT and Glioblastoma

The chromosomal region 10q26.3 is commonly deleted from one allele in glioblastomas, which means that only one allele drives the activity of MGMT in these cancer cells. Successful treatment of glioblastoma patients with alkylating chemotherapy is therefore dependent on epigenetic silencing of the remaining copy of the gene. Establishing the DNA methylation status of the MGMT promoter is therefore crucial for the choice of treatment for newly diagnosed glioblastoma patients. Please see the section: Glioblastoma methylation for further information.

Despite the evidence of a strong correlation between MGMT methylation status resulting in epigenetic silencing of the MGMT enzyme and benefit from temozolomide treatment, consensus has not yet been reached concerning the optimal detection method for MGMT promoter methylation analysis in glioblastoma patients. One obstacle is that the MGMT promoter CpG island can be heterogeneously methylated, which makes it difficult to assess the methylation status of the glioblastoma. The choice of methodology is therefore important to secure optimal treatment of the patient.

MGMT and other cancers

MGMT promoter methylation has been found in several other cancer types including colorectal cancer, lung cancer, gastric cancer, cervical carcinomas, and lymphoma.  As an example, methylation status of the MGMT promoter is shown to be significantly associated with gastric cancer risk, distant metastasis, and lymph node metastasis. Therefore, MGMT promoter methylation may play an important role in carcinogenesis and prognosis of gastric cancer. Likewise, methylation status of five CpG sites in the MGMT promoter region has a diagnostic value as a screening target for cervical cancer.

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How MethylDetect can assist your research on MGMT

MethylDetect provides more than 800 gene-specific ready-to-use kits for DNA methylation analysis. On www.MethylDetect.com you will find the EpiMelt MGMT test kits, which come in three kit sizes: 200, 500 and 2500 reactions.

The EpiMelt kits provide a fast and clear answer to whether your sample is heterogeneously methylated (See Daugaard I. et al 2018).

All EpiMelt kits contain a primer mix and a control set of 100% methylated, unmethylated, and an assay calibration control, to ensure the high sensitivity of your EpiMelt assays. Standard qPCR platforms with a high-resolution melting module can be used, please consult the protocol at https://www.methyldetect.com/methyldetect-kit-for-dna-methylation-assessment/, for further information on setting up the EpiMelt analysis in you laboratory.

If your target of interest is missing from our portfolio, we design and produce EpiMelt kits in collaboration with you, to ensure that the design fits your requirements for test material and expected methylation status (hypo- or hypermethylation). We design EpiMelt kits for DNA derived from sources as: FFPE tissue, liquid biopsies or high-quality DNA.

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Further Reading

Bady P et al. MGMT methylation analysis of glioblastoma on the in nium methylation BeadChip identi es two dis- tinct CpG regions associated with gene silencing and outcome, yielding a prediction model for comparisons across datasets, tumor grades, and CIMP-status. Acta Neuropathol. 2012;124(4):547–560.

Daugaard I et al. Chronic lymphocytic leukemia patients with heterogeneously or fully methylated LPL promotor display longer time to treatment. Epigenomics. 2018 Sep;10(9):1155-1166.

Mansouri A et al. MGMT promoter methylation status testing to guide therapy for glioblastoma: refining the approach based on emerging evidence and current challenges. Neuro-Oncology 21(2), 167–178, 2019

Yu W et al. O6-Methylguanine-DNA Methyltransferase (MGMT): Challenges and New Opportunities in Glioma Chemotherapy.Front. Oncol., 17 January 2020