Rare variants in BRCA2 and CHEK2 are associated with the risk of urinary tract cancers

Previous studies have shown that two rare variants, rs11571833 in BRCA2 and rs17879961 in CHEK2 were associated with lung cancer. However, the associations between these two variants and urinary tract cancers risk remain largely unexplored. We applied imputation of three genome-wide association studies published in the database of Genotypes and Phenotypes (dbGaP). Unconditional logistic regression analysis and meta-analysis were performed to assess the association between these two variants and the risk of urinary tract cancers. Our results showed that rs11571833[T] had an effect on urinary tract cancers predisposition (ORmeta = 1.45, Pmeta = 0.013), especially associated with increased the risk of bladder cancer (ORmeta = 1.60, Pmeta = 0.010). Moreover, rs17879961[C] had a protective effect on the urinary tract cancers (ORmeta = 0.67, Pmeta = 1.0 × 10−3) and was mostly associated with a lower incidence of renal cell carcinoma (ORmeta = 0.51, Pmeta = 2.0 × 10−3). Together, our study indicates that BRCA2 and CHEK2 play an important role in the genetic susceptibility to urinary tract cancers.

lung, prostate and bladder cancer [24][25][26][27][28][29] . Furthermore, mounting evidence shows chemical carcinogens and reactive oxygen can induce damage to DNA in urothelial cells and polymorphisms in DNA repair genes might modify urothelial carcinoma risk 30 . In addition, the exposure of carcinogens was thought to be similar throughout the urinary tract and the urinary tract cancers are often multifocal 31 . We performed imputation analysis from dbGaP database to explore the association between rs11571833 and rs17879961 polymorphism and the risk of urinary tract cancers in the populations of European descent.

Results
The characteristics of GWAS studies for urinary tract cancers. A total of three GWAS studies including 6,064 cases and 8,661 controls were enrolled in our study. The detailed characteristics of GWAS studies are shown in Table 1. In addition, the principal component analysis (PCA) showed there were no abnormal outlier samples in the study population ( Supplementary Fig. 1). Tables 2 and 3. The imputation info of the two rare variants was mostly more than 0.9, which indicated the accuracy of our imputation result was high. The rs11571833[T] showed significant association with bladder cancer in Illumina 610 array (OR = 1.87, 95%CI = 1.21-2.87, P = 4.5 × 10 −3 ). Simultaneously, rs17879961 [C] was related with decreased the risk of renal cell carcinoma in Illumina 610 array and bladder cancer in Illumina 610 array (OR = 0.50, 95%CI = 0.33-0.77, P = 1.5 × 10 −3 and OR = 0.71, 95%CI = 0.52-0.98, P = 0.036, separately).

Association analysis for individual GWAS data and joint (pooled) GWAS data. Our logistic regression results are applied in
In consideration of the relative small sample, we attempted to combine the original GWAS data to implement joint analyses. We merged genotype data across each cancer (three for bladder cancer and three for renal cell carcinoma) to increase the sample size. Totally, the sample size is 3591 cases and 4132 controls for bladder cancer, 1322 cases and 3428 controls for renal cell carcinoma, 1151 cases and 1101 controls for prostate cancer. Our results indicated that rs11571833[T] was significantly associated with increased the risk of bladder cancer (OR combined = 1.70, 95%CI = 1.19-2.42 and P combined = 3.6 × 10 −3 ), but showed non-significant association between renal cell carcinoma and prostate cancer ( Table 2). rs17879961[C] had a correlation with renal cell carcinoma (OR combined = 0.63, 95%CI = 0.44-0.89 and P combined = 1.0 × 10 −2 ). There were no remarkable associations between rs17879961 and the other two cancers (Table 3). For the total three cancers, the results revealed that rs11571833[T] could increase 47% risk of urinary tract cancers (OR combined = 1.47, 95%CI = 1.12-1.94 and P combined = 5.7 × 10 −3 ) and rs17879961[C] was associated with a significantly lower risk of urinary tract cancers (OR combined = 0.73, 95%CI = 0.59-0.90 and P combined = 3.7 × 10 −3 ). In conclusion, rs11571833 may play a risk factor in urinary tract cancers predisposition, especially be associated with the risk of bladder cancer. Meanwhile,  rs17879961 is associated with decreased risk of urinary tract cancers and mostly associated with the risk of renal cell carcinoma.

Meta-analysis across multiple GWAS databases.
Except for joint analysis, we also applied meta-analysis to assess the association between the two rare variants and urinary tract cancers. For rs11571833, we performed a meta-analysis from seven imputation association results and found that the association between rs11571833[T] and the risk of urinary tract cancers was significant (OR meta = 1.45, 95%CI = 1.08-1.94 and P meta = 0.013). In addition, a significant association between rs11571833[T] and bladder cancer was observed in the subgroup meta-analysis (OR meta = 1.60, 95%CI = 1.12-2.29 and P meta = 0.010; Table 4 and Fig. 1a). The result showed that rs17879961[C] was associated with decreased the risk of urinary tract cancers (OR meta = 0.67, 95%CI = 0.53-0.85 and P meta = 1.0 × 10 −3 ) and had a protective effect on renal cell carcinoma (OR meta = 0.51, 95%CI = 0.34-0.78 and P meta = 2.0 × 10 −3 ; Table 5 and Fig. 1b) by fixed effect model.

Discussion
Genome stability is critical for preventing tumorigenesis. DNA damages can result in the activation of oncogenes and inactivation of tumor suppressor genes. DNA damage repaired systems involve in maintenance of genome stability and supply a crucial defense function against DNA-damaging agent, such as exposure of cigarette smoking and ultraviolet component of ionizing radiation, sunlight and genotoxic substance 22,32 . The main DNA damage repaired pathways include nucleotide-excision repair (NER), base-excision repair (BER), homologous recombination and end joining 33 . Previous studies have highlighted that inherited defects in the DNA repaired pathways may predispose to malignancy 22 .
Recently, an imputation study identified two rare variants rs11571833 in BRCA2 and rs17879961 in CHEK2 were associated with lung cancer. CHEK2 (checkpoint kinase 2 checkpoint homologue) plays an important role in encoding a pluripotent kinase which can induce cell cycle arrest or apoptosis in response to unrepaired DNA damage 34,35 . The missense variant rs17879961 (p.Ile157Thr) changes Isoleucine to Threonine at position 157 of the protein and it locates in a region coding for a functionally important FHA domain of CHEK2 and injures binding of principal substrates. The rs17879961 (CHEK2 p.Ile157Thr) substitution may alter its ability to bind p53, BRCA1 and Cdc25A proteins 4,36,37 . BRCA2 (breast cancer early onset 2) is a widely known anti-oncogene and associated with the risk of breast cancer and ovarian cancer 38 . BRCA2 also involves in the maintenance of genome stability through interacting with RAD51 recombinase, specifically in the homologous recombination pathway for DNA repair [39][40][41] . The variant rs11571833 (p.Lys3326X) leads to a stop codon, which results in loss of the final 93 amino acids of the BRCA2 protein. The interaction of RAD51 and BRCA2 plays a crucial role in BRCA2-mediated double strand-break repair. The C-terminus of BRCA2 contains a RAD51 binding domain and small protein sequence incorporating p.Lys3326X (amino acids 3265-3330) is capable of binding RAD51. Besides, an important serine involved in BRCA2-mediated repair process is close to this truncating mutation [41][42][43] . Above evidence invites that the SNP rs11571833 is functional in DNA damage repair pathway thus alter the genetic susceptibility of cancers. Previous studies have found that rs11571833 was associated with the risk of upper aerodigestive tract cancer (OR = 2.53) 21 . Besides, some studies suggest that rs11571833 have an association with risk of breast cancer 42 . Meanwhile, rs17879961 have been reported to be associated with a significantly lower incidence of lung or upper aerodigestive tract cancer, but increased the risk of thyroid cancer 34,44,45 . However, the effect of these two rare variants on urinary tract cancers remains largely unexplored.
In this study, we investigated the associations between rare genetic variants of rs11571833 and rs17879961 and the risk of urinary tract cancers (including bladder cancer, prostate cancer and renal cell carcinoma) and found that the rare variant rs11571833 (c.9976A > T) was associated with increased risk of urinary tract cancers, especially associated with bladder cancer. Concurrently, rs17879961 (c.470T > C) played a protective role in the urinary tract cancers carcinogenesis and mostly decreased the risk of renal cell carcinoma. Our meta-analyses result show highly consistency with joint analyses, which strengthen our conclusion that the rs11571833 is  It is noteworthy that the rare variants rs17879961 may impair function of CHEK2, but it was associated with decreased the risk of renal cell cancer. A speculation for the possible protective mechanism is that CHEK2 can have two opposite effects on damaged stem cells and it impedes stem cell division until DNA damage has been repaired or actives apoptosis if unrepaired DNA damage happened. Accumulating evidence has shown that in the circumstance of continued DNA damage by long-term exposure of tobacco, the normal stem cell defenses that involve CHEK2 can be attenuated by reducing the CHEK2 activity as a result of rs17879961 (p.Ile157Thr) 17,32,34 .
There are several limitations in our study. Firstly, the available GWAS data published in dbGaP database provides relatively little samples, which may influence the power to identify the association between the rare variants and diseases. Furthermore, our analysis based on public database is short of relevant demography information applied in stratification analysis. Additionally, our study is an imputation-based analysis from public database and the ORs of two rare variants are relatively modest. Besides, the sample sizes of controls are unevenly matched with cases in some cohorts, which may result in sample bias. Further large well-designed studies in other independent populations and functional studies are needed to validate our findings. Meanwhile, the interaction between environmental exposures and genetic susceptibility should be also considered in the future research 32 .
In conclusion, our imputation analysis results indicated that the rare variant rs11571833 (c.9976A > T) showed an effect on urinary tract cancers predisposition, especially associated with increased the risk of bladder cancer. However, rs17879961 (c.470T > C) may play a protective role in the urinary tract cancers carcinogenesis and notably decrease the risk of renal cell carcinoma. Association signals of these two variants in urinary tract cancers are in the same direction to lung cancer. These results suggest that BRCA2 and CHEK2 play an important role in the genetic susceptibility to urinary tract cancers.

Materials and Methods
Study population. GWAS data from three cancer studies were available, which were requested from dbGaP.
Two genotype datasets were excluded because of low-density genotype panel and low sample size (These two datasets are genotyped by Illumina 240 array and Illumina 317 array separately, and their genome build are both build 35). In the final analysis, we included three bladder cancer studies, three renal cell carcinoma studies and one prostate cancer study, totally including 6,064 cases and 8,661 controls.

Statistical analyses.
An unconditional logistic regression model was applied to calculate the odds ratio (OR) and 95% confidence interval (CI) for each SNP in an additive model using SNPTEST software. We adjusted for some covariates such as age, gender, study and significant principle components (PCs). The principal component analysis (PCA) was carried out to assess for population stratification of study cohorts. Meta-analysis was conducted by Stata v.10 (Stata College, Taxas, US). Heterogeneity was evaluated by Cochran'Q and I 2 statistics. I 2 values ≥ 75% were considered to be significant heterogeneity. All statistical analyses were two-sided, and a P value < 0.05 was considered statistically significant.