In this study, we assessed the effects of a comprehensive panel of 207 SNPs in 26 genes in the NER pathway on the risk of bladder cancer. ING2 gene was the most noteworthy finding, with four of six evaluated SNPs exhibiting significant associations with bladder cancer risk. Furthermore, we found potential gene-smoking interaction and higher-order interactions among these NER SNPs in the modulation of bladder cancer susceptibility.
Our findings of ING2
are biologically plausible. ING2
is a member of the inhibitor of growth (ING) gene family and encodes a putative tumor suppressor protein involved in the regulation of DNA repair, cell cycle progression, apoptosis, and epigenetic functions in a p53-dependent manner. ING2
gene was first cloned in 1998 as a homologue of the first ING family member, ING1 26
gene is 6 kb in length, located in chromosome region 4q35.1. The implication of ING2
in NER was first established by Wang et al., who found that overexpression of ING2
gene significantly enhanced the repair of UV-induced DNA damage 27
. This function of ING2 is dependent on the normal functions of p53 protein since small interfering RNA (siRNA)-mediated degradation of either ING2 or p53 abolished the observed repair capacity 27
. Furthermore, Want et al. showed that ING2 protein is not a component of the NER core protein complex; instead, ING2 enhance NER through recruiting XPA to the core complex 27
. The function of ING2 in DNA repair is also mediated by the interaction with the trimethylated and dimethylated H3K4, which stabilizes the mSin3a-HDAC1 protein complex to enhance the transcriptional activity of many relevant genes 28
. In addition to its involvement in NER, ING2 has also been implicated in G1 phase cell cycle arrest through increasing the transcriptional activation ability of p53 29
. Furthermore, ING2 interacts with phosphoinositides to activate p53-dependent apoptosis pathway 30
. In our study, all 4 significant ING2
SNPs, which are not in strong linkage, were associated with reduced risks of bladder cancer. Among them, 3 SNPs are located in the 3′ region of gene and one located in the 5′ region of ING2
, 5.5 kb upstream of the translation start codon. Since the promoter and enhancer sequences of ING2
have not been well-characterized experimentally, it remains to be determined whether these SNPs have any functional significance. It is more likely that they are tagging SNPs but not the causal variants. Therefore, high-density mapping in combination with functional characterizations are warranted to further elucidate the molecular mechanisms underlying the association between ING2
SNPs and bladder cancer risk observed in this study.
Another noteworthy gene we found in this study is DDB2. DDB2
gene contains 8 exons and spans approximately 24 kb in chromosomal 11p12-p11 31
. As a response protein to DNA damage induced by genotoxic agents such as UV-irradiation, DDB2 interacts with CUL4A, RBX1, and COPS2 to form a protein complex that binds to chromatin and initiates the NER process 32
. DDB2 enhances the DNA binding activity of DDB1 and serves as an crucial component in p53-mediated DNA repair process 33, 34
. Two SNPs of DDB2
exhibited a significant association with bladder cancer risk. One SNP, rs11039130, located ~7 kb upstream of transcription start site, was associated with a 1.64-fold increased risk under a recessive model. The other SNP, rs1685404, is located in the 3′ of the gene and conferred a reduced bladder cancer risk under a dominant model. Several studies have reported that the transcription of DDB2
gene is tightly regulated by a wide array of transcription factors such as E2F family, BRCA1, SP1, Myc, and NF1 35–37
. However, whether rs1685404 is a direct causative locus or surrogate remains to be determined through further fine mapping and functional characterization.
We performed stratified analysis by smoking status and tested SNP-smoking (smoking status and packyears) interactions. Due to the limited power to detect SNP-smoking interaction, this analysis was exploratory and further validation with larger sample size is needed. Interestingly, the effects of CCNH
SNPs were stronger in never smokers, whereas the effort of DDB2 SNP was only evident in ever smokers and other SNPs showed similar effects in never and ever smokers. This observation is in line with a recent genome-wide association study (GWAS) of bladder cancer in which the NAT2
slow acetylator genotype was associated with an increased bladder cancer risk in ever smokers but not never smokers, but the effect of GSTM1 null genotype was the strongest in never smokers and grew progressively weaker in former and current smokers 38
. Eight other GWAS-confirmed SNPs showed similar effect among never and ever smokers 38
. It is intriguing that different genotypes in carcinogen metabolism and DNA repair exhibited differential effect on bladder cancer risk among never and ever smokers. Other exposures may explain such interactions. Occupational exposure is the second major environmental risk factor for bladder cancer. Our previous publication has shown that prolonged exposure to diesel fuel or fumes on a regular basis, exposure to tar/mineral oil, dry cleaning fluids, leather and tanning solutions, rubber products, glues, pesticides, insecticides, or herbicides, fertilizers, arsenic, zinc, radioactive materials, and aromatic amine, were all associated with an increased risk of bladder cancer 39
. It would be interesting to assess the NER SNPs and bladder cancer risk in the context of these different DNA damaging exposures. Since only a small percentage of our study populations were exposed to these different occupational exposures, the power to detect significant associations in exposed populations was limited. Future studies are warranted to address this question.
We also conducted exploratory CART analyses to assess potential higher-order gene-gene interactions within the NER pathway genes. The process of tumorigenesis in sporadic cancers is a multifactorial and multistep process that involves complicated interactions of various low-penetrance genetic and environmental components. The CART analysis identified subsets of individuals with different cancer risks based on different combinations of genotypes, with the ORs of individuals in each terminal node ranging from 0.42 to 2.58 (). A paired interaction analysis supports some of the SNP-SNP interactions. These data suggest that gene-gene interactions play an important role in bladder cancer etiology. Nevertheless, given that CART analysis is a post-hoc data mining tool that was applied to the same dataset, the results are preliminary and should be interpreted with caution.
The strengths of our study include a large and homogenous study population, a comprehensive panel of genes in the relatively well-characterized NER pathway, and the use of an htSNP-based genotyping approach. The limitation of this study is that although we applied BFDP approach, one of several available statistical methods, to control for multiple testing, it is possible that some of our reported SNPs are false-positive findings. The main reason for our choice of BFDP is that the noteworthy threshold defined in BFDP approach accounts for the costs of false discovery and non-discovery. The other alternative approaches to correct for multiple testing, such as the Bonferroni correction for independent tests and the P(ACT) method to compute P
values adjusted for correlated tests 40
, do not consider the cost of non-discovery and are more conservative. Regardless of the method used for correcting multiple testing, the ultimate way to eliminate false positive findings is through independent validation. We reported both the significant SNPs after multiple testing correction by the BFDP method and the nominally significant SNPs in main effect and SNP-smoking interaction analyses. External validations in independent epidemiology studies with adequate sample sizes are warranted to confirm the results of our studies.