This report provides compelling evidence for an increased bladder cancer risk associated with the GSTM1 null and NAT2 slow acetylation genotypes. The association of the latter was particularly important among cigarette smokers. Although the relative risks for polymorphisms in NAT2 and GSTM1 genes are modest, they could account for a large percentage of bladder cancer cases because of their high prevalence in the population. Based on our data, we estimated that these polymorphisms are responsible for 31% (95% CI 20%-46%) of bladder cancer cases. In addition, we provide strong evidence against a substantial overall association for polymorphisms in other NAT and GST genes, with the possible exception of small to moderate associations for NAT1 *10/*10 and GSTP1 114Val/Val genotypes.
A new meta-analysis of studies of NAT2
slow acetylation and bladder cancer risk shows that this association is robust (p=5×10−8
) and similar for Caucasian and Asian populations. The fact that the association for Asian populations was not stistically significant might be explained by a substantially lower statistical power to detect associations in Asian studies due to a lower prevalence of NAT2
slow acetylators (11% for Asians versus 56% for Caucasians) along with a smaller number of cases available for the meta-analysis. We also show that NAT2
slow acetylators are especially susceptible to the adverse effects of cigarette smoking on bladder cancer risk. This gene-environment interaction has strong biological plausibility since NAT2
slow acetylators have a decreased capacity to detoxify aromatic monoamines by N-acetylation (13
), tobacco smoking is a primary source of exposure to aromatic amines in the general population, and aromatic amines are suspected of being the primary bladder carcinogen in tobacco smoke (12
). Although our data suggest that NAT2
slow acetylation might not increase bladder cancer risk among never smokers, although it does not rule out a small increase in risk in this group of subjects.
Because the content of aromatic amines is higher in black than in blond tobacco (51
), it is conceivable the effect of NAT2
slow acetylation may be stronger for smokers of black tobacco. Out data are consistent with this hypothesis, although differences were not statistically significant. The magnitude of the association between NAT2
slow acetylation and bladder cancer risk is similar for different levels of smoking intensity in our study population. Our meta-analysis of the interaction between smoking status and NAT2
slow acetylation genotype suggested a stronger interaction with ever/never smoking in European than in US studies. This could be due to a smaller number of studies conducted in the US than in Europe, or the lower aromatic amine content in blond tobacco generally smoked in the US compared to black tobacco commonly smoked in parts of Europe. Interestingly, a report from a population in the US recently reported an interaction between NAT2
slow acetylation genotype and smoking only for heavy smokers (47
Distinction of subjects with one and two copies of the GSTM1 gene, an issue that has not been adequately addressed in prior studies of bladder cancer, suggests the presence of a gene-dosage effect with relative risks of 1.2 (95% CI 0.8-1.7) and 1.9 (1.4-2.7) for subjects with one or no copies of GSTM1, respectively, compared to subjects with two copies (ptrend=3×10−8). Meta-analyses of the association between the deletion of two copies of the GSTM1 gene (null genotype) compared to subjects with one or two copies (present genotype), as presented in previous studies that could not distinguish between these two groups of subjects, indicated that this association is robust (p=9×10−15), and similar in magnitude and significant across different population subgroups.
The relative risk for GSTM1
null genotype and bladder cancer is similar for smokers and never smokers in our study population and meta-analysis within population subgroups, suggesting that the GSTM1
activity protects equally against tobacco-related and non-tobacco related bladder cancers. This finding indicates that GSTM1
may reduce the risk of bladder cancer through mechanisms that are not specific to the detoxification of polycyclic aromatic hydrocarbons (PAHs) in tobacco smoke. Other mechanisms of action for GSTM1
could be protection from oxidative damage through metabolism of reactive oxygen species (52
). Our data did not confirm previously suggested differences in risk for NAT2
slow acetylation and GSTM1
null genotypes by tumor grade or stage at presentation (26
). Our findings are consistent with a potential interaction between NAT2
slow acetylation and GSTM1
null genotypes; however, additional evidence is needed to confirm this interaction (17
Associations between bladder cancer risk and polymorphisms in genes coding for the NAT1 enzyme involved in the activation of aromatic amines by O-acetylation (13
), and other GST enzymes that play an important role in the detoxification of PAHs and other carcinogens (57
), have been less explored. Previous studies have provided inconsistent evidence for an association between bladder cancer risk and NAT1
*10 alone or in combination with NAT2
slow acetylation (14
null alone or in combination with GSTM1
null genotype (17
), and GSTP1 105 Val/Val
). Data from our study does not support a substantial association between GSTT1
genotypes and bladder cancer risk. We find no significant increases in bladder cancer risk associated with polymorphisms in NAT1
genes; however, our estimates do not exclude a small to moderate association for the NAT1*10/*10
compared to the NAT1*4/*4
genotype, or for genotypes with the GSTP1 114Val
allele compared to the 114Ala/Ala
Analyses using conventional logistic regression suggested a modification of the association between bladder cancer with NAT2, GSTM1 and NAT1 genotypes by gender. However, the modifications by gender are explained by unexpected differences in the genotype distribution for male and female controls.
Our study has several strengths of note: high participation rates, large sample size, high quality exposure and genotype information and use of state-of-the art statistical methods. Specifically, we made an effort to improve the precision in genotype estimation by genotyping the seven SNPs in NAT2
that likely account for virtually all genetic variation in Caucasian populations,(58
) and developed assays that sucesfully distinguished individuals with one or two copies of the GSTM1
genes. We also used the SPMLE method (37
) to increase power and reduce bias in the estimation of interactions, because of the strong evidence from previous studies for independence of NAT2
genotypes from cigarette smoking status (8
) and gender (38
) in the general population. In order to minimize selection bias, we carefully selected controls from patients admitted for a variety of diagnoses that were thought to be unrelated to exposures of interest including tobacco use. Genotype frequencies among the control population were similar to those previously published. We found no significant overall differences in genotype frequencies across control diagnoses that could have biased our results.
Although this is the largest study on the role of genetic polymorphisms and bladder cancer risk published to date and had adequate statistical power to detect small genotype associations, the power to detect small to moderate interactions was limited. Meta-analyses including previous studies improved our ability to make inferences on interactions, when there were an adequate number of previous studies with homogeneous results. A consortium of bladder cancer studies is currently being formed to facilitate the pooling of comparable data on environmental and genetic risk factors across studies that will help overcome the limited power of individual studies to evaluate complex interrelationships.
Overall, these findings are among the most consistent for common genetic polymorphisms and risk of any tumor site in the literature, and provide compelling evidence for the role of common polymorphisms in cancer risk. This report also illustrates that large and rigorous investigations are required to establish or dismiss effects of common polymorphisms on complex diseases.