Association of Intergenic and Intragenic MGMT Enhancer Methylation with MGMT Promoter Methylation, MGMT Protein Expression and Clinical and Demographic Parameters in Glioblastoma

The methylation status of the MGMT gene promoter is recognized as a key predictive biomarker for glioblastoma patients, influencing treatment decisions and outcomes. Emerging evidence suggests that enhancer methylation may also play a role in gene regulation and is associated with various clinical parameters, genetic variants, and demographic factors. This study aimed to assess DNA methylation levels in intergenic and intragenic MGMT enhancers to investigate their relationship with MGMT promoter methylation, MGMT protein expression, and clinical and demographic characteristics in glioblastoma. We developed 18 pyrosequencing assays to analyze 54 CpGs, including 34 in intergenic and 20 in intragenic enhancers. The assays were applied to tumor cells derived from 38 glioma patients. Intragenic enhancer CpGs showed significantly higher methylation than intergenic enhancer CpGs. Intragenic enhancer methylation showed a strong negative correlation with MGMT promoter methylation. For several CpGs in intragenic enhancers, an inverse L-shaped relationship between methylation levels and MGMT expression was observed. We identified distinct associations between enhancer methylation and clinical and demographic parameters. Intergenic enhancer methylation was primarily linked to the TERT SNP rs2853669 genotype, Ki-67 expression, age, and sex, whereas intragenic enhancer methylation was associated with MGMT promoter methylation, MGMT expression, overall survival, and progression-free survival. Further studies with larger patient cohorts are needed to validate the clinical relevance of intergenic and intragenic MGMT enhancer methylation in glioblastoma.