We evaluated the association of a panel of 276 SNPs in 38 oxidative-stress genes with the recurrence of NMIBC in patients treated with or without BCG. We identified 25 SNPs that were significantly associated with cancer recurrence in all (TUR- and BCG-treated) patients, 34 significant SNPs in TUR only patients, and 28 significant SNPs in BCG-treated patients. In addition, we found significant gene-dosage effects from the cumulative analysis, and identified one SNP associated with NMIBC progression.
The most significant SNP associated with recurrence in the BCG-treated group was NEIL2:
rs804256, which is located in the intron region. Compared with the wild-type genotypes, the recurrence risk for the homozygous variant CC genotype was increased more than four-fold. However, this SNP was not significant in TUR only subgroup, suggesting a potential interaction of rs804256 with BCG treatment. Five out of six of the most significant SNPs are located in NEIL2
, which highlights the importance of this gene in the recurrence risk. NEIL2
belongs to a class of DNA glycosylases homologous to bacterial Fpg/Nei family, which is essential for removing cytosine-derived lesions, particularly 5-hydroxyuracil and 5-hydroxycytosine 
. It has been reported that two 5′-UTR polymorphisms decrease NEIL2
expression and increase mutagen-induced genetic DNA damage 
. Downregulation of NEIL2
expression may lead to increased oxidative stress and genomic instability in rapidly proliferating tissues 
may also play important roles in repairing DNA damage induced by carcinogenic metals 
. One of the most significant SNPs associated with recurrence, rs4639, is located in 3′-UTR of NEIL2
. Luciferase reporter assay results showed that hsa-mir-421 and hsa-mir-1200 miRNAs had similar inhibitory effects on both wildtype and variant rs4639 expression. Since either miRNAs showed similar inhibitory effects on both wildtype and variant genotypes, it is possible that rs4639 may not be the causative variant but functions as a tagging SNP for other polymorphisms that may contribute to bladder cancer recurrence by altering NEIL2
expression or function. Alternatively, the variant allele of this SNP may affect targeting by other miRNAs. Future fine mapping of the regions surrounding the most significant tagSNPs are needed to identify the causal genetic variations and their molecular mechanisms.
We also identified a significant gene-dosage effect for the six tagging SNPs that showed significant main effects. Patients with the largest number of unfavorable genotypes had the highest risk of NMIBC recurrence, suggesting that additional risk genotypes within this key pathway were detrimental. This highlights the importance of assessing multiple SNPs within a shared pathway for clinical outcome assessment.
One polymorphism, UNG
: rs3890995, was significantly associated with NMIBC progression. Rs3890995 is located 1.87 kb upstream of UNG
5′ UTR. One important function of UNG is to recognize and remove uracil from DNA by cleaving the N-glycosylic bond and initiating the base-excision repair pathway. DNA uracil comes from cytosine deamination or misincorporation of dUMP residues 
. It is possible that the variant allele of SNP rs3890995 may affect gene transcription thus altering protein level. Alternatively, it may be linked to other causal variants in UNG
In summary, we showed that genetic polymorphisms of the oxidative stress pathway genes may modulate the risk of NMIBC recurrence and progression in BCG treated patients. Further, we have conducted a relatively comprehensive query of the oxidative stress pathway polymorphisms with detailed clinical information and analyses, which provided substantial evidence for the involvement of this pathway in the clinical outcomes of bladder cancer patients, particularly with BCG treatment. There are some limitations in this study. For example, only Caucasians were included. It would be interesting to exam these SNPs in minority populations. Additionally, sample size is not particularly large, although power calculation showed that our analysis had sufficient power (>80%) to detect the main effects analyzed. Although our data are largely internally validated, future replication studies in independent populations are needed to validate some of the results and to translate the findings to clinical trials.