Association of Intergenic and Intragenic MGMT Enhancer Methylation with MGMT Promoter Methylation, MGMT Protein Expression and Clinical and Demographic Parameters in Glioblastoma
Abstract
1. Introduction
2. Results
2.1. Overall Intergenic and Intragenic Enhancer Methylation
2.2. Association of Intergenic and Intragenic Enhancer Methylation with the Relative Position of CpGs
2.3. Association Between MGMT Promoter and Enhancer Methylation
2.4. Association of MGMT Promoter and Enhancer Methylation with MGMT Protein Expression
2.5. Association of MGMT Enhancer Methylation with Clinical and Demographic Parameters
2.6. Association of MGMT Enhancer Methylation with Overall Survival, Progression-Free Survival and Long-Term Survival
2.7. Association of MGMT Enhancer Methylation with TERT SNP rs2853669 and TERT Promoter Mutations C228T and C250T
2.8. Association of MGMT Enhancer Methylation with Ki-67 Index
2.9. Association of MGMT Enhancer Methylation with Age and Sex
3. Discussion
4. Materials and Methods
4.1. Samples and Cell Culturing
4.2. Determination of MGMT Protein Expression, Ki-67 Index, Genetic Variants, and Clinical Parameters
4.3. DNA Extraction and Bisulfite Conversion
4.4. Primer Design and PCR Conditions for DNA Methylation Analysis
4.5. PSQ of PCR Products
4.6. Data Analysis and Statistics
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
CpG | CpG dinucleotide |
EGFR | epidermal growth factor receptor |
GB | Glioblastoma |
GS | Gliosarcoma |
HRM | High-resolution melting |
IDH | Isocitrate dehydrogenase |
KPS | Karnofsky Performance Score |
MGMT | O6-methylguanine-DNA methyltransferase |
NCBI | National Center for Biotechnology Information |
OS | Overall survival |
PCR | Polymerase chain reaction |
PDCs | Patient-derived cells |
PSQ | Pyrosequencing |
TERT | Telomerase reverse transcriptase |
TMZ | Temozolomide |
WHO | World Health Organization |
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Patient | Age [y] | Sex | KPS [%] | Ki-67 [%] | OS [m] | PFS [m] | MGMT Exp | TERT Prom | TERT rs2853669 |
---|---|---|---|---|---|---|---|---|---|
GB01 | 64 | f | 60 | n.s. | 7.43 | 4.00 | 0.00 | C250T | TT |
GB02 | 85 | m | 40 | ≤50 (30) | 3.00 | n.s. | 0.00 | wt | TT |
GB03 | 53 | f | 100 | n.s. | 52.50 | 4.50 | 0.00 | C228T | CT |
GB04 | 67 | f | 70 | >50 (90) | 46.63 | 0.53 | 0.00 | C228T | TT |
GB05 | 57 | f | 100 | ≤50 (30) | 10.50 | 6.00 | 0.00 | C228T | TT |
GB06 | 46 | m | 90 | n.s. | 30.50 | 4.00 | 0.00 | C228T | TT |
GB07 | 50 | f | 90 | n.s. | 27.40 | n.s. | 0.00 | C250T | n.s. |
GB08 | 74 | f | 70 | ≤50 (30) | 7.79 | n.s. | 0.00 | C228T | CT |
GB09 | 48 | m | 60 | n.s. | 1.55 | n.s. | 0.00 | wt | TT |
GB10 | 64 | m | 80 | >50 | 11.70 | n.s. | 0.20 | C228T | CT |
GB11 | 73 | f | 60 | >50 | 10.60 | n.s. | 0.16 | C228T | TT |
GB12 | 44 | m | 80 | >50 | 13.00 | 9.00 | 1.10 | C250T | CT |
GB13 | 65 | m | 90 | ≤50 (15) | 9.27 | n.s. | 1.20 | C228T | TT |
GB14 | 69 | m | 90 | ≤50 (40) | 8.00 | n.s. | 0.04 | C250T | TT |
GB15 | 73 | f | 100 | n.s. | 7.00 | 2.63 | 1.10 | C250T | CT |
GB16 | 83 | m | 90 | ≤50 | 9.57 | 9.00 | 1.00 | C228T | CT |
GB17 | 74 | m | 90 | n.s. | 5.19 | n.s. | 1.32 | C250T | CT |
GB18 | 44 | m | 100 | n.s. | 23.15 | n.s. | 0.25 | wt | TT |
GB19 | 48 | m | 100 | >50 (60) | 18.67 | 4.54 | 0.09 | C228T | CT |
GB21 | 60 | m | 90 | ≤50 (40) | 13.00 | 3.00 | 0.40 | C228T | TT |
GB22 | 53 | m | 70 | >50 (60) | 0.89 | n.s. | 0.54 | C250T | TT |
GB23 | 47 | f | 70 | >50 (70) | 37.25 | 8.00 | 0.00 | C250T | CT |
GB24 | 64 | m | 90 | ≤50 (40) | 7.73 | n.s. | 0.49 | C228T | CT |
GB25 | 67 | f | 70 | >50 | 16.80 | 10.00 | 0.00 | C228T | TT |
GB26 | 75 | f | 70 | ≤50 (40) | 8.22 | n.s. | 0.00 | C228T | CT |
GB27 | 52 | f | 90 | >50 (70) | n.s. | n.s. | 0.00 | C250T | TT |
GB28 | 63 | m | n.s. | >50 | 21.80 | 8.00 | 0.00 | C250T | CT |
GB29 | 79 | f | 40 | >50 | 1.31 | n.s. | 0.00 | C228T | CT |
GB30 | 58 | m | 90 | >50 | 16.00 | 4.31 | 0.00 | C228T | TT |
GB31 | 58 | f | n.s. | >50 | 13.30 | 4.00 | 0.00 | C228T | TT |
GB32 | 71 | m | n.s. | ≤50 (20) | 1.25 | n.s. | 0.00 | C228T | TT |
GB33 | 57 | m | n.s. | n.s. | 12.70 | 6.00 | 0.20 | C250T | CT |
GB34 | 53 | m | n.s. | n.s. | 32.30 | 22.00 | 0.00 | C228T | TT |
GB35 | 64 | m | 40 | n.s. | 10.60 | n.s. | 0.00 | C228T | TT |
GB36 | 62 | m | 50 | ≤50 (25) | 1.48 | n.s. | 0.90 | wt | TT |
GB37 | 63 | m | 80 | >50 (60) | 19.50 | 9.01 | 0.11 | wt | TT |
GB38 | 76 | m | 30 | ≤50 (20) | 4.44 | n.s. | 0.29 | wt | CT |
GS01 | 43 | f | 80 | ≤50 (30) | 9 | n.s. | 0.00 | C250T | TT |
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Pühringer, K.; Czarda, P.; Iluca, S.; Zappe, K.; Weis, S.; Spiegl-Kreinecker, S.; Cichna-Markl, M. Association of Intergenic and Intragenic MGMT Enhancer Methylation with MGMT Promoter Methylation, MGMT Protein Expression and Clinical and Demographic Parameters in Glioblastoma. Int. J. Mol. Sci. 2025, 26, 3390. https://doi.org/10.3390/ijms26073390
Pühringer K, Czarda P, Iluca S, Zappe K, Weis S, Spiegl-Kreinecker S, Cichna-Markl M. Association of Intergenic and Intragenic MGMT Enhancer Methylation with MGMT Promoter Methylation, MGMT Protein Expression and Clinical and Demographic Parameters in Glioblastoma. International Journal of Molecular Sciences. 2025; 26(7):3390. https://doi.org/10.3390/ijms26073390
Chicago/Turabian StylePühringer, Katharina, Philipp Czarda, Sebastian Iluca, Katja Zappe, Serge Weis, Sabine Spiegl-Kreinecker, and Margit Cichna-Markl. 2025. "Association of Intergenic and Intragenic MGMT Enhancer Methylation with MGMT Promoter Methylation, MGMT Protein Expression and Clinical and Demographic Parameters in Glioblastoma" International Journal of Molecular Sciences 26, no. 7: 3390. https://doi.org/10.3390/ijms26073390
APA StylePühringer, K., Czarda, P., Iluca, S., Zappe, K., Weis, S., Spiegl-Kreinecker, S., & Cichna-Markl, M. (2025). Association of Intergenic and Intragenic MGMT Enhancer Methylation with MGMT Promoter Methylation, MGMT Protein Expression and Clinical and Demographic Parameters in Glioblastoma. International Journal of Molecular Sciences, 26(7), 3390. https://doi.org/10.3390/ijms26073390