Pre-Diagnostic Genome-Wide DNA Methylation in Blood and Risk of Bladder Cancer

BACKGROUND

Differential DNA methylation as measured in blood is a promising marker of bladder cancer susceptibility. In fact, established bladder cancer risk factors such as smoking and various germline genetic variants may promote carcinogenesis in bladder tissue through processes that are detectable as differential DNA methylation in blood.

METHODS

Genome-wide methylation was measured in pre-diagnostic blood samples, using the Illumina Infinium HumanMethylation450 Bead Array, among 440 bladder cancer cases with the transitional cell carcinoma (TCC) subtype and 440 matched controls from the Women’s Health Initiative (WHI) cohort. After normalization and probe filtering, methylation measurements at 361,184 CpG sites remained for each study participant. We used conditional logistic regression models adjusted for potential confounders and for cell type composition to test for associations between the methylation level at each remaining CpG site and bladder cancer risk (Chapter 1). For each of three smoking-associated CpG sites, we conducted a regression-based mediation analysis to assess whether current smoking affects bladder cancer risk through differential methylation at the CpG site, while accounting for the smoking-CpG interaction using the four-way decomposition approach (Chapter 2). Since there are four single nucleotide polymorphisms (SNPs) that are known to be associated with bladder cancer and are also associated with proximal DNA methylation changes, we performed a regression-based, multiple-mediator mediation analysis for each SNP, which allowed us to determine whether the associated methylation changes at CpG sites are a path through which the SNP causes bladder cancer (Chapter 3).

RESULTS

Increased methylation at cg22748573, located in a CpG island within the 5′-UTR/first exon of the CITED4 gene, was associated with an 82% decreased risk of bladder cancer (OR = 0.18, q-value = 0.05) (Chapter 1). The result was robust to sensitivity analyses accounting for time between enrollment and diagnosis, race, tumor subtype, and secondhand smoke exposure. Most of the excess relative risk (ERR) associated with current smoking for a 30 pack-year smoking history as compared to never smoking was mediated through cg05575921 in an enhancer-like regulatory element within AHRR and cg19859270 in the first exon of GPR15 (Chapter 2). The largest components were the mediated interactions for both cg05575921 (ERR component = 2.29, p = 0.05; percent of ERR = 72%, p = 0.02) and cg19859270 (ERR component = 1.89, p-value = 0.05; percent of ERR = 72%, p-value = 0.04), where the mediated interaction capture the indirect effect of smoking through differential methylation as well as the smoking-CpG interaction. There was little evidence that the effect of smoking on bladder cancer risk is mediated through cg03636183. Though not statistically significant, our results suggest that large proportions of the modest effects of rs401681 (NIE = 1.05; NIE percent = 98.5%) and of rs2294008 (NIE = 1.10; NIE percent = 77.6%) on bladder cancer risk occur through their associated CpG sites (Chapter 3). Based on exploratory analyses, cg27028750, which is located in a long terminal repeat element, may drive the indirect effect for rs401681. The effect of rs2294008 may be primarily mediated by cg24023258 near LY6K and by cg17252645 in LY6D among non-smokers and primarily mediated by cg03405983 and cg17888033 in LYNX1 and by cg06565975 near SLURP1 among smokers. There was little evidence supporting mediation through changes in DNA methylation for the associations of rs8102137 and rs798766 with bladder cancer risk.

CONCLUSIONS

While results need to be validated in additional prospective studies, differential methylation in the promoter region of CITED4, as measured in blood, is a promising marker of bladder cancer susceptibility. If confirmed, smoking may have effects on bladder cancer related to changes in AHRR and GPR15 expression. Our results also suggest that for some SNPs associated with bladder cancer, nearby methylation changes may be part of the underlying mechanisms of effect. Supplemental File: As supplementary information, “KM Jordahl Supplementary Table 1-1.xlsx” contains Supplementary Table 1.1 and provides the full results for all 361,184 CpG sites from the genome-wide study of DNA methylation and bladder cancer risk described in Chapter 1.