A large-scale, population-based study with extensive coverage of common allelic DNA repair gene variants revealed a robust association between the minor allele of MGMT SNP rs2296675 and increased overall cancer risk. Carriers of the rs2296675 minor allele had 38% increased risk of developing any type of cancer and comprised 37% of the study population (33% heterozygotes and 4% homozygous rare genotype). Consistent with a genetically driven cancer risk, the association of rs2296675 with cancer risk was significantly stronger in younger individuals. The strength of the associations varied by cancer site, but all of the cancer site-specific associations were in the direction of increased risk.
MGMT directly repairs alkyl adducts that arise in the O
position of guanine. The O
position is important because O
-methylguanine tends to be read as adenine during replication, resulting in G:C to A:T transition mutations (21
). Germ-line MGMT
polymorphisms could potentially affect carcinogenesis via at least three mechanisms: (i) directly diminishing MGMT-mediated DNA repair via a functional coding change; (ii) the sequence variation could increase MGMT
’s susceptibility to epigenetic silencing of its transcription and (iii) other potential biologic pathways, such as, if this region of MGMT
was a target for RNA interference (RNAi), which can silence gene expression by degrading mRNA (22
Concerning the first, there is some evidence that at least one functional MGMT
SNP may be associated with overall cancer risk. For example, several functional MGMT
polymorphisms (not including rs2296675) were assessed for associations with overall cancer risk by using meta-analytic techniques that combined the results of site-specific cancer studies. The results indicated Leu84Phe (rs12917; but not Ile143Val, rs2308321) was associated with overall cancer risk, particularly among populations of European ancestry (7
). Although not directly relevant to rs2296675, the results of the meta-analysis lend credence to the notion that MGMT
functional polymorphisms could potentially contribute to human carcinogenesis in different tissues via putative effects on protein activity. On a cautionary note, however, the results of this study were null for rs12917, emphasizing the need for replication of our study findings with respect to rs2296675. The association observed for rs2296675 could be a consequence of being in high LD with a functional SNP. MGMT
SNP rs2296675 is an intronic SNP located on the 5ʹ side of exon 5, within 66 base pairs of rs2308321, a functional SNP in exon 5.
Hypermethylation of CpG islands in the MGMT
promoter blocks gene transcription (24
promoter hypermethylation is found in >20% of colon, lung, testicular, head-neck, retinoblastoma, cervical, lymphoma and brain tumors and 10–20% of esophageal, stomach, pancreatic and melanoma tumors (25
). The epigenetic silencing of MGMT
transcription renders cells unable to remove O
alkylguanine adducts, a defect that leads to increased mutations in key tumor suppressor genes and oncogenes, such p53 and K-ras (27
variants could plausibly impact susceptibility to a broad spectrum of cancers by enhancing MGMT
promoter hypermethylation, thereby inducing gene silencing, and creating a more permissive, procarcinogenic environment across multiple tissues. In support of this line of reasoning, germ-line MGMT
polymorphisms have been linked to both MGMT
promoter hypermethylation and gene silencing in colorectal cancer (28
). In a study of 182 colorectal tumors, the results for rs2296675 were not statistically significant but compared with those with two common alleles, MGMT
promoter hypermethylation in colorectal tumor tissue was 1.7 and 4.0 times more prevalent in those with one and two minor alleles, respectively (28
). Further, the rs2296675 minor allele was associated with loss of MGMT
expression, which increased from 28% to 38% to 50% in those with zero, one, and two minor alleles, respectively (28
). These are the only previously published data we know of for rs2296675; they raise the possibility that the rs2296675 minor allele could be a marker of susceptibility to MGMT
promoter hypermethylation and loss of gene expression, at least in the colorectum.
The levels of methylation in tumor compared with normal tissue may provide evidence to assess the likelihood that rs2296675 variants may be associated with cancer risk via a pathway that includes MGMT
promoter methylation. In healthy individuals without colorectal neoplasia, similar patterns of MGMT
promoter methylation in normal mucosa were seen as in tumor tissue from patients with colorectal cancer (29
), suggesting that promoter methylation may be involved early in the carcinogenic pathway in the colon and rectum. MGMT activity is inversely correlated with promoter methylation (30
), and there is more evidence available for MGMT activity in tumor compared with normal tissue (31
). Comparisons of MGMT activity in malignant tissue to normal tissue showed that for cancers relevant to this study’s findings (breast, colon and rectum and lung), MGMT activity was uniformly greater in the tumor tissue than the normal tissue. To the extent that MGMT activity serves as a useful proxy for promoter methylation, this differential between tumor and normal tissue does not support the hypothesis that germ-line variants alone determine enhanced susceptibility to promoter methylation.
Alkylating agents are potent carcinogens that originate from a variety of endogenous and exogenous sources (32
. Many endogenous alkylating agents (or precursors) have been identified, including S
-adenosyl-methionine, a common methyl donor in biochemical reactions, nitrosated amines or bile acids produced enzymatically by Escherichia coli
, and nitrosated alkaloids possibly produced by endogenous nitrosating agents such as Nox
). With respect to exogenous sources of exposure to alkylating agents, N
-nitroso compounds are common environmental alkylating agents found in tobacco products and prepared foods, or formed during natural and industrial processes (33
). Active cigarette smoking is a major environmental source of exposure to alkylating agents (33
). Cigarette smoke contains many N
-nitroso compounds that have been shown to produce O
-meG or other methylated bases after metabolic activation (34
). Other than smoking status, a limitation of this study was the lack of information on potential sources of alkylating agent exposure.
The results observed in this study, indicating that carrying the minor rs2296675 allele was robustly associated with increased risk of all cancers, as well as several specific cancer sites, raises the question as to why this SNP, or others in LD with it, have not previously been reported to be associated with cancer in prior genome-wide association studies (GWAS) of specific cancer sites. One factor contributing to this apparent discrepancy may be a loss of power in GWAS due to corrections for multiple comparisons. That is, SNPs in high LD with rs2296675 may have been associated with risk of specific cancer sites, but these associations were not statistically significant after correcting for multiple comparisons. For some cancer sites, the associations in this study were large enough to be detected in GWAS. Differences in defining the cancer phenotype may have contributed to important differences in the magnitude of the observed associations between this study and GWAS. Typically, individuals with a history of NMSC are not excluded from the control group. However, it is now becoming well established that NMSC is associated with increased risk of malignancy in virtually all other tissues (35
). In this study, rs2296675 was shown to be weakly associated with NMSC risk; this association would have been masked had NMSC patients been classified as controls. Including NMSC cases in the control group could therefore have introduced misclassification that biased associations toward the null. This bias could be substantial because the prevalence of NMSC among Caucasian populations is very high. Misclassification of NMSC patients is an issue that needs to be carefully addressed in future validation studies.
A consideration of these issues emphasizes the need for replication of our study finding before strong inferences can be made about the observed association between rs2296675 and overall cancer risk. In the absence of replication, an important limitation is the possibility that the results of this study could be false-positive findings. Concerns about the possibility of a false-positive finding could be heightened by the fact that the P
-value distribution was slightly enriched for lower P
-values, possibly due to genotyping error rates leading to type I error inflation. However, as is evident in , this deviation from the expected null distribution is not severe and cannot reasonably explain the P
-values of the most significant findings, a marker with an appreciable MAF of 0.21. As previously discussed, the lack of any published supportive data from GWAS nonetheless adds to the index of suspicion of a false-positive finding. False-positive findings from epidemiology studies have been a source of growing concern (36
. Replication studies could come in the form of taking a similar cohort study approach as in this study, or alternatively pooling existing data across different cancer sites from case–control or nested case–control studies. The approach of pooling across case–control studies has the advantage of more cancer site-specific matching for potential confounding variables, whereas the cohort study approach has the advantage of being inclusive of all malignancies occurring in a population, in keeping with the goal of this study. Even though replication is lacking, this study does contain the appropriate safeguards of accounting for multiple comparisons and appropriately cautious inferences, taking into account previous findings and biologic plausibility (37
In summary, the hypothesis that germ-line SNPs in DNA repair genes were associated with risk of all cancers combined was tested. Compared with those with two common alleles, carriers of the minor allele of MGMT SNP rs2296675 had 38% greater risk of any cancer, an association that was highly statistically significant even after accounting for multiple comparisons. This overall increased cancer risk was not due to associations with a few malignancies but rather associations in the direction of increased risk were observed for all of the specific cancer sites studied. This implies that the association, if it is in actual fact genuine, may be relevant to a broad spectrum of malignancies. Replication of this finding in other study populations is needed before more definitive inferences concerning the potential scientific and public health importance of this finding can be made.