Background: DNA hypomethylation has been suggested to cause genomic instability and increase cancer risk. We aimed to test the hypothesis that DNA hypomethylation is associated with increased risk of bladder cancer. Methods: We measured cytosine methylation (5-mC) content in genomic DNA from blood cells from patients with bladder cancer enrolled in a large case-control study in Spain between Jan 1, 1998, and Dec 31, 2001. Cases were men and women with newly diagnosed and histologically confirmed urothelial carcinoma of the bladder. Controls were selected from patients admitted to the same hospital for diseases or conditions unrelated to smoking or other known risk factors for bladder cancer. Controls were individually matched to cases on age (within 5 years), sex, race, and area of hospital referral. 5-mC content was measured in leucocyte DNA by use of a combination of high-performance capillary electrophoresis, Hpa II digestion, and densitometry. Data on demographics, 34 polymorphisms in nine folate metabolism genes, and nutritional intake of six B vitamins (including folate), alcohol, and smoking were assessed as potential confounders. Relative 5-mC content was expressed as a percentage (%5-mC) with respect to the total cytosine content (the sum of methylated and non-methylated cytosines). The primary endpoint was median %5-mC DNA content. Findings: %5-mC was measured in leucocyte DNA from 775 cases and 397 controls. Median %5-mC DNA was significantly lower in cases (3·03% [IQR 2·17-3·56]) than in controls (3·19% [2·46-3·68], p=0·0002). All participants were subsequently categorised into quartiles by %5-mC content in controls. When the highest quartile of %5-mC content was used as the reference category (Q4), the following adjusted odds ratios (OR) and 95% CI were recorded for decreasing methylation quartiles: OR(Q3) 2·05 (95% CI 1·37-3·06); OR(Q2) 1·62 (1·07-2·44); and OR(Q1) 2·67 (1·77-4·03), p for trend <0·0001. The lowest cancer risk was noted in never smokers in the highest methylation quartile (never smokers in Q4). By comparison with never smokers in the highest quartile, current smokers in the lowest methylation quartile had the highest risk of bladder cancer (Q1: OR 25·51 [9·61-67·76], p for interaction 0·06). In analyses stratified by smoking, hypomethylation was a strong risk factor in never smokers (OR 6·39 [2·37-17·22]). Amount of methylation in controls were not associated with baseline characteristics, micronutrients, or selected genotypes in folate metabolism pathways. Interpretation: For the first time, to our knowledge, we have shown in a large case-control study that leucocyte DNA hypomethylation is associated with increased risk of developing bladder cancer, and this association is independent of smoking and the other assessed risk factors. Amount of global methylation in genomic DNA could provide a useful biomarker of susceptibility to certain cancer types and further research is warranted. Funding: Intramural Research Program of the National Institutes of Health, National Cancer Institute, Division of Cancer Epidemiology and Genetics, Bethesda, MD, USA, and Fondo de Investigacion Sanitaria, Spain (G03/174).
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