Tumor Biology

, Volume 37, Issue 10, pp 13571–13579 | Cite as

Identification of MGMT promoter methylation sites correlating with gene expression and IDH1 mutation in gliomas

  • Jie Zhang
  • Jian-hui Yang
  • Jia Quan
  • Xing Kang
  • Hui-juan Wang
  • Peng-gao Dai
Original Article


O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation was reported to be an independent prognostic and predictive factor in glioma patients who received temozolomide treatment. However, the predictive value of MGMT methylation was recently questioned by several large clinical studies. The purpose of this study is to identify MGMT gene promoter CpG sites or region whose methylation were closely correlated with its gene expression to elucidate this contradictory clinical observations. The methylation status for all CpG dinucleotides in MGMT promoter and first exon region were determined in 42 Chinese glioma patients, which were then correlated with MGMT gene expression, IDH1 mutation, and tumor grade. In whole 87 CpG dinucleotides analyzed, three distinct CpG regions covering 28 CpG dinucleotides were significantly correlated with MGMT gene expression; 10 CpG dinucleotides were significantly correlated with glioma classification (p < 0.05). Isocitrate dehydrogenase 1 (IDH1) mutation and MGMT gene hypermethylation significantly co-existed, but not for MGMT gene expression. The validation cohort of gliomas treated with standard of care and comparison of the CpGs we identified with the current CpGs used in clinical setting will be very important for gliomas individual medicine in the future.


Glioma MGMT Methylation IDH1 Pyrosequencing Correlation analysis 



This work was supported by the National Natural Science Foundation of China (31271353) and the Program for New Century Excellent Talents in University (NCET121048).

Compliance with ethical standards

Conflicts of interest


Supplementary material

13277_2016_5153_MOESM1_ESM.docx (19 kb)
Table S1 Sequences of PCR primer and sequencing primers used for pyrosequencing reactions, annealing temperatures, and number of CpG islands covered by each primer for the respective PCR amplifications (DOCX 19 kb)


  1. 1.
    Louis DN et al. The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol. 2007;114:97–109.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Wen PY et al. Malignant gliomas in adults. N Engl J Med. 2008;359:492–507.CrossRefPubMedGoogle Scholar
  3. 3.
    Cankovic M et al. The role of MGMT testing in clinical practice: a report of the association for molecular pathology. J Mol Diagn. 2013;15:539–55.CrossRefPubMedGoogle Scholar
  4. 4.
    Xu-Welliver M et al. Degradation of the alkylated form of the DNA repair protein, O(6)-alkylguanine-DNA alkyltransferase. Carcinogenesis. 2002;23:823–30.CrossRefPubMedGoogle Scholar
  5. 5.
    Malley DS et al. A distinct region of the MGMT CpG island critical for transcriptional regulation is preferentially methylated in glioblastoma cells and xenografts. Acta Neuropathol. 2011;121:651–61.CrossRefPubMedGoogle Scholar
  6. 6.
    Hegi ME et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med. 2005;352:997–1003.CrossRefPubMedGoogle Scholar
  7. 7.
    Skorpen F et al. The methylation status of the gene for O6-methylguanine-DNA methyltransferase in human Mer+ and Mer− cells. Carcinogenesis. 1995;16:1857–63.CrossRefPubMedGoogle Scholar
  8. 8.
    Hegi ME et al. Correlation of O6-methylguanine methyltransferase (MGMT) promoter methylation with clinical outcomes in glioblastoma and clinical strategies to modulate MGMT activity. J Clin Oncol Off J Am Soc Clin Oncol. 2008;26:4189–99.CrossRefGoogle Scholar
  9. 9.
    Everhard S et al. MGMT methylation: a marker of response to temozolomide in low-grade gliomas. Ann Neurol. 2006;60:740–3.CrossRefPubMedGoogle Scholar
  10. 10.
    Stupp R et al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol. 2009;10:459–66.CrossRefPubMedGoogle Scholar
  11. 11.
    Felsberg J et al. Promoter methylation and expression of MGMT and the DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2 in paired primary and recurrent glioblastomas. Int J Cancer. 2011;129:659–70.CrossRefPubMedGoogle Scholar
  12. 12.
    Maxwell JA et al. Quantitative analysis of O6-alkylguanine-DNA alkyltransferase in malignant glioma. Mol Cancer Ther. 2006;5:2531–9.CrossRefPubMedGoogle Scholar
  13. 13.
    Nakagawachi T et al. Silencing effect of CpG island hypermethylation and histone modifications on O6-methylguanine-DNA methyltransferase (MGMT) gene expression in human cancer. Oncogene. 2003;22:8835–44.PubMedGoogle Scholar
  14. 14.
    Mikeska T et al. Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis. J Mol Diagn. 2007;9:368–81.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Livak KJ et al. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 2001;25:402–8.CrossRefPubMedGoogle Scholar
  16. 16.
    Setty P et al. A pyrosequencing-based assay for the rapid detection of IDH1 mutations in clinical samples. J Mol Diagn. 2010;12:750–6.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Dong X et al. Correlation of promoter methylation in the MGMT Gene with glioma risk and prognosis: a meta-analysis. Mol Neurobiol. 2014;52:1–9.Google Scholar
  18. 18.
    Tang K et al. Clinical correlation of MGMT protein expression and promoter methylation in Chinese glioblastoma patients. Med Oncol. 2012;29:1292–6.CrossRefPubMedGoogle Scholar
  19. 19.
    Everhard S et al. Identification of regions correlating MGMT promoter methylation and gene expression in glioblastomas. Neuro-Oncology. 2009;11:348–56.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Shah N et al. Comprehensive analysis of MGMT promoter methylation: correlation with MGMT expression and clinical response in GBM. PLoS One. 2011;6:e16146.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Watanabe T et al. IDH1 mutations are early events in the development of astrocytomas and oligodendrogliomas. Am J Pathol. 2009;174:1149–53.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Jin G et al. Mutant IDH1 is required for IDH1 mutated tumor cell growth. Oncotarget. 2012;3:774–82.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Leu S et al. IDH/MGMT-driven molecular classification of low-grade glioma is a strong predictor for long-term survival. Neuro-Oncology. 2013;15:469–79.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Deimling A et al. The next generation of glioma biomarkers: MGMT methylation, BRAF fusions and IDH1 mutations. Brain Pathol. 2011;21:74–87.CrossRefGoogle Scholar
  25. 25.
    Turcan S et al. IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature. 2012;483:479–83.Google Scholar
  26. 26.
    Mulholland S et al. MGMT CpG island is invariably methylated in adult astrocytic and oligodendroglial tumors with IDH1 or IDH2 mutations. Int J Cancer. 2012;131:1104–13.CrossRefPubMedGoogle Scholar
  27. 27.
    Weller M et al. MGMT promoter methylation in malignant gliomas: ready for personalized medicine? Nat Rev Neurol. 2012;6:39–51.CrossRefGoogle Scholar
  28. 28.
    Weller M et al. Isocitrate dehydrogenase mutations: a challenge to traditional views on the genesis and malignant progression of gliomas. Glia. 2011;59:1200–4.CrossRefPubMedGoogle Scholar
  29. 29.
    Weller M et al. Molecular predictors of progression-free and overall survival in patients with newly diagnosed glioblastoma: a prospective translational study of the German Glioma Network. J Clin Oncol Off J Am Soc Clin Oncol. 2009;27:5743–50.CrossRefGoogle Scholar
  30. 30.
    Boots-Sprenger SH et al. Significance of complete 1p/19q co-deletion, IDH1 mutation and MGMT promoter methylation in gliomas: use with caution. Modn Pathol: Off J U S Can Acad Pathol. 2013;26:922–9.CrossRefGoogle Scholar
  31. 31.
    Minniti G et al. IDH1 mutation and MGMT methylation status predict survival in patients with anaplastic astrocytoma treated with temozolomide-based chemoradiotherapy. J Neuro-Oncol. 2014;118:377–83.CrossRefGoogle Scholar
  32. 32.
    Phillips HS et al. Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis. Cancer Cell. 2006;9:157–73.CrossRefPubMedGoogle Scholar
  33. 33.
    Verhaak RG et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell. 2010;17:98–110.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    McDonald KL et al. The T genotype of the MGMT C>T (rs16906252) enhancer single-nucleotide polymorphism (SNP) is associated with promoter methylation and longer survival in glioblastoma patients. Eur J Cancer. 2013;49:360–8.CrossRefPubMedGoogle Scholar
  35. 35.
    Lof-Ohlin ZM et al. Pyrosequencing assays to study promoter CpG site methylation of the O6-MGMT, hMLH1, p14ARF, p16INK4a, RASSF1A, and APC1A genes. Oncol Rep. 2009;21:721–9.PubMedGoogle Scholar
  36. 36.
    Stupp R et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005;352:987–96.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Jie Zhang
    • 1
  • Jian-hui Yang
    • 1
  • Jia Quan
    • 1
  • Xing Kang
    • 1
  • Hui-juan Wang
    • 1
  • Peng-gao Dai
    • 1
  1. 1.National Engineering Research Center for Miniaturized Detection Systems, School of Life SciencesNorthwest UniversityXi’anChina

Personalised recommendations