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.

Fig. 1.

A 777-bp O⁶-methylguanine-DNA methyltransferase gene (MGMT) cysteine-phosphate-guanine (CpG) island with 97 CpG sites, including the minimal promoter, first noncoding exon, and minimal enhancer, shown as black, white, and hatched bars, respectively. Nucleotides are numbered with respect to the transcription start site (TSS). The 52 CpG sites analyzed by pyrosequencing are indicated; 35 were upstream of the TSS, and 17 were downstream of the TSS. The methylation-specific PCR assay (MSP) region (118–137 and 174–195 for methylated primers) currently investigated is also indicated.

Fig. 2.

Hierarchical clustering of the 54 glioblastomas for the 52 cysteine-phosphate-guanine (CpG) sites analyzed by pyrosequencing. The darkness of the gray represents the methylation level at each CpG. CpG numbering starts at the CpG just before the transcription start site (TSS). A vertical solid line separates the two distinct patient classes provided by the hierarchical clustering. The vertical dashed line separates the intermediate and unmethylated groups. Methylation range averages for the 52 analyzed CpG sites are indicated for each group. At the bottom, the expression level of the O⁶-methylguanine-DNA methyltransferase gene is also indicated for each patient as a qualitative variable.

Fig. 3.

Distribution of O⁶-methylguanine-DNA methyltransferase gene (MGMT) expression levels for methylated (group 1), intermediate (group 2), unmethylated (group 3), and control groups (based on the analysis of all 52 cysteine-phosphate-guanine [CpG] sites). MGMT expression average of controls, which represent the cutoff value for low and high expressing groups, is indicated by a horizontal bar.