Abstract 60: Constitutional MGMT promoter methylation and allelic expression imbalance are associated with the c.-56C>T (rs16906252) variant

Abstract

Abstract Loss of expression of the DNA repair protein MGMT in cancer is associated with a mutator phenotype and frequently occurs due to promoter methylation. The c.-56C>T SNP (rs16906252), located within an enhancer element in the 5’UTR of MGMT, is strongly associated with promoter methylation in colorectal cancer (CRC) and at lower levels in normal tissues, including normal colorectal mucosa and peripheral blood. We aimed to investigate the mechanistic link between the c.-56C>T SNP and MGMT methylation in CRC development with a focus on the differential methylation and expression of the two alleles in normal tissues from CRC patients and healthy controls. Genotyping revealed the T allele frequency to be similar in CRC cases (7%) and controls (8%). In the peripheral blood DNA of CRC cases, MGMT methylation was detected at a higher frequency in heterozygous cases (66.6%) compared to homozygous C/C cases (17.1%)(p<0.0001), as well as at higher levels (p<0.0001). Similarly in the blood of healthy controls, MGMT methylation was detected at a higher frequency in heterozygous individuals (43.5%) compared to C/C homozygotes (14.9%)(p<0.002), as well as at higher levels (p<0.0001). Furthermore, allelic sequencing of products from methylation-specific PCR demonstrated that MGMT methylation was linked exclusively to the T allele in 6/6 blood and 15/16 normal colorectal mucosa samples from CRC cases. The relative levels of allelic expression derived from the C and T alleles at the c.-56C>T variant were examined in the normal colorectal mucosa from 14 patients (8 C/T, 6 C/C) informative for a second downstream expressible SNP (rs1803965) by allele-quantification pyrosequencing. Allelic expression imbalance was observed in all eight heterozygotes, with allelic expression ratios (C/T) ranging from 1.79-4.51. In contrast, the six C/C cases showed balanced expression levels (range 1.08-1.54), indicating that reduced transcription was significantly associated with the T allele (p<0.001). Sequencing of cloned cDNA products confirmed the allele expressed at lower levels at the downstream SNP was linked exclusively to the T allele at the c.-56C>T SNP. Finally, we found that in six highly methylated (>30%) CRC specimens that retained MGMT immunoexpression, methylation was predominant on the T allele. Our findings indicate the c.-56T allele is associated with constitutively reduced transcriptional activity in the normal colorectal mucosa of CRC patients. This mechanism may predispose to secondary methylation of the T allele, most likely via altered interaction with cis-acting transcription factors. Monoallelic methylation of MGMT may explain the discordance between presence of MGMT methylation and retention of protein expression in tumors. If commonplace, these findings suggest that MGMT methylation status serves as a poor predictor of patient responsiveness to alkylating drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 60. doi:10.1158/1538-7445.AM2011-60