Identification of regions correlating MGMT promoter methylation and gene expression in glioblastomas

Abstract

The O(6)-methylguanine-DNA methyltransferase gene (MGMT) is methylated in several cancers, including gliomas. However, the functional role of cysteine-phosphate-guanine (CpG) island (CGI) methylation in MGMT silencing is still controversial. The aim of this study was to investigate whether MGMT CGI methylation correlates inversely with RNA expression of MGMT in glioblastomas and to determine the CpG region whose methylation best reflects the level of expression. The methylation level of CpG sites that are potentially related to expression was investigated in 54 glioblastomas by pyrosequencing, a highly quantitative method, and analyzed with respect to their MGMT mRNA expression status. Three groups of patients were identified according to the methylation pattern of all 52 analyzed CpG sites. Overall, an 85% rate of concordance was observed between methylation and expression (p < 0.0001). When analyzing each CpG separately, six CpG sites were highly correlated with expression (p < 0.0001), and two CpG regions could be used as surrogate markers for RNA expression in 81.5% of the patients. This study indicates that there is good statistical agreement between MGMT methylation and expression, and that some CpG regions better reflect MGMT expression than do others. However, if transcriptional repression is the key mechanism in explaining the higher chemosensitivity of MGMT-methylated tumors, a substantial rate of discordance should lead clinicians to be cautious when deciding on a therapeutic strategy based on MGMT methylation status alone.

Effect of O6‑methylguanine‑DNA methyltransferase methylation in medulloblastoma

“…Everhard et al reported CpG sites at +95, +113, +135 and +137 bp from the transcriptional start site (TSS) (CpG 1, 3, 7 and 8 in our study, respectively) and high concordance between methylation and expression of MGMT in their analysis of 53 CpG sites in 54 glioblastoma samples (15). Malley et al reported that individual or multiple consecutive methylation of CpG sites at +153, +185, +195 and +213 bp from the TSS (CpG 11, 14, 15 and 17 in our study, respectively) attenuated the activity of the MGMT promoter in their study of 98 CpG sites in xenografted glioblastoma samples and cell lines (6).…”

“…Furthermore, as MGMT promoter methylation is inversely correlated with MGMT expression (5,6), methylation-specific PCR (MSP) with bisulfate-treated DNA has been widely used to analyze MGMT promoter methylation status (7)(8)(9). However, MSP is only able to detect a limited number of methylated CpG sites in the primer region, and recent studies report that the region commonly investigated by MSP does not cover CpG sites that are most highly associated with expression of MGMT, and that MSP may not be well-suited for predicting the prognosis of patients with glioblastoma (15,16). Direct sequencing and pyrosequencing are alternative methods for quantitatively analyzing the methylation status of MGMT.…”

MGMT promoter methylation in malignant gliomas: ready for personalized medicine?

“…16 Some studies have provided insight into the relationship between gene expression and the patterns and localization of dense CpG methylation in the MGMT promoter. 16,17 Two regions that are prone to high levels of methylation have been identified, of which the region comprising the enhancer element seems to be more critical for the loss of MGMT gene expression upon methylation, on the basis of luciferase reporter assays interrogating different regions of the methylated promoter. 16,17 Hence, most methylation-specific tests are designed to interrogate this region ( Figure 2b).…”

Comparative assessment of 5 methods (methylation‐specific polymerase chain reaction, methylight, pyrosequencing, methylation‐sensitive high‐resolution melting, and immunohistochemistry) to analyze O6‐methylguanine‐DNA‐methyltranferase in a series of 100 glioblastoma patients

“…3 MGMT expression is mainly regulated at the epigenetic level through CpG island promoter methylation. 4,5 Promoter methylation tests are thus used for MGMT assessment at the DNA level. Most studies reporting a link between MGMT promoter methylation and survival in GBM patients have used methylation-specific polymerase chain reaction (MS-PCR).…”