In models controlling for smoking status, gender and age, we have found a significant 1.8-fold increased risk of bladder cancer among subjects with mean LINE1 methylation levels in the lowest decile of methylation, ranging from 57.89 to <74.25%, as compared to subjects with >74.25% LINE1 methylation. This suggests that LINE1 hypomethylation is an independent risk factor for bladder cancer and that it may be an excellent biomarker. Our results are consistent with a previous report that also noted a significantly increased risk of bladder cancer amongst patients with lower global DNA methylation levels, as assessed by the radioactive methyl-incorporation assay (27
). Interestingly, unlike that report we did not observe a dose-dependent association of LINE1 methylation extent with bladder cancer risk, but rather saw only an association with disease risk at the lowest level, a result consistent with our previous work examining LINE1 methylation in head and neck cancer (18
We also observed that the risk of non-invasive bladder cancer was increased almost two fold for subjects in the lowest LINE1 methylation decile, while there was no significant association in invasive cases. This is of interest, because it suggests that LINE1 methylation may be a specific biomarker of noninvasive disease. The mechanism responsible for this apparent distinction is unclear but it is consistent with the fact that there are well known differences in this disease associated with an invasive pathology (4
In examining the predictors of lowest tenth decile of LINE1 methylation we found that women were greater than two-fold significantly more likely to have reduced LINE1 methylation extent. This finding is consistent with a study which found that females had statistically lower methylation levels than males in LINE1 and Alu repeats measured from whole blood (34
) as well as with our previous work in head and neck cancer (18
). Two other studies did not find a significant association between LINE1 methylation and gender in leucocytes and in adenomas; however, these studies did not use a large number of subjects and may have been underpowered to observe this association (17
). Our gender-stratified analyses further suggest that the risk for bladder cancer conferred by low LINE1 methylation extent is significant only among women, and is consistent with Moore et al. (27
) who observed a similar increased risk of bladder cancer among women with reduced overall global DNA methylation levels.
Our finding of reduced LINE1 methylation in relation to arsenic exposure is consistent with experimental data showing that arsenic and its inorganic salt, arsenite, induce DNA hypomethylation in cultures of human lung and rat hepatocytes, as well as in rat livers through feeding studies (35
). A study in Bangladesh, with endemic, high level arsenic exposures, observed that reduced DNA methylation was a risk factor for arsenic-associated skin lesions (38
) but found a positive relationship between urinary arsenic concentrations and global DNA methylation in peripheral blood leucocytes (using the radioactive methyl-incorporation assay) (39
). This different finding may be related to the different measures of DNA methylation and the far greater exposures in Bangladesh compared with ours. Although the methylation extent of the LINE1 region has been correlated with overall cellular 5-methycytosine content (40
), and thus serve as a surrogate marker, changes in the extent of methylation of these elements may lead to specific functional consequences on their expression. Expression of these elements can lead to their retrotransposition to various regions of the genome, possibly leading to downstream effects on other genes in these cells (42
). It is unclear if the changes in methylation extent being measured lead to changes in expression of specific functional LINE1 elements. Going forward, it will be important to delineate the biological nature of this association, as LINE1 measurement may be, in some cases, in the causal pathway for disease.
We did not find an interaction between LINE1 methylation and smoking status, unlike previous reports examining methylation by smoking intensity (27
). The non-significant interaction in our model could be attributable to differential smoking patterns between the populations.
Strengths of this study included the population-based nature of the study, as well as the use of quantitative pyrosequencing for LINE1 methylation determination and detailed exposure assessment including the use of toenail analysis of arsenic concentrations. Limitations of this study included the exclusion of cases and controls without methylation data which could lead to a selection bias if included and excluded cases and controls differ from one another. However, we did not note any differences between included and excluded cases and controls in any of our demographic information and use of tobacco products. Another limitation of this study was the low number of invasive cases available; a larger study is necessary to further understand the impact of global hypomethylation on the risk of development of non-invasive as compared to invasive bladder cancer. Another limitation would be the poor age matching done in this study; however we do not think this impacted the results as age is not found to be significant in any of our analyses. Further research on hormonal influences on global methylation and risk of bladder cancer is also warranted related to our findings of differential risk amongst women.
In summary, this study adds to the growing literature on the role of epigenetics in human cancer etiology, and suggests LINE1 hypomethylation as a risk factor for bladder cancer, with potential utility as a disease biomarker. In addition, this study shows an increased risk of LINE1 hypomethylation with high levels of arsenic, raising important questions about the nature of the causal pathway for alterations in DNA repeat methylation to contribute to cancer risk. These results also suggest that women demonstrate lower levels of LINE1 methylation, and may suggest distinct etiologies of bladder cancer between men and women, which may also aid in explaining the disparate incidence of this disease in men and women. Further work is needed to better clarify how exposures important to the disease play a role in LINE1 region methylation, and to clarify the specific subgroups where this factor may be most predictive of disease risk.