CYP2A13 Genetic Polymorphisms in Relation to the Risk of Bladder Cancer in Japanese Smokers

Abstract

Tobacco-specific nitrosamines including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), which can be activated by the metabolic enzyme CYP2A13, are potent procarcinogens. Smoking plays a role in carcinogenesis in the human bladder, which expresses CYP2A13 at a relatively high level. Numerous genetic polymorphisms of CYP2A13 causing amino acid substitution might reduce CYP2A13 metabolic activity toward NNK and NNN, resulting in decreased susceptibility to bladder cancer. The aim of this study was to reveal any association between bladder cancer development and CYP2A13 genetic polymorphisms in Japanese smokers. The CYP2A13 genotype of each subject (163 bladder cancer patients and 161 controls) was determined by next-generation sequencing (NGS) of the full CYP2A13 gene. All samples were genotyped for five CYP2A13 variant alleles (CYP2A13*2, *3, *4, *6, *7). Based on biological logistic regression, the odds ratio (95% confidence interval) for the CYP2A13*1/*2 genotype was 0.34 (0.17–0.69). Thus, CYP2A13 genetic polymorphisms might play important roles in the development of bladder cancer in Japanese smokers.

Human CYP2A6 and CYP2A13 catalyze the metabolism of tobacco-specific nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN).¹⁾ The carcinogenicity of each compound was previously evaluated by the International Agency for Research on Cancer.²⁾ NNK induces susceptibility to lung tumors in F-344 rats, and detection of tobacco-specific nitrosamines in the urine of smokers indicates that they accumulate in the urinary bladder.^3,4) Therefore, NNK and NNN are expected to be activated by metabolic enzymes, such as CYP2A6 and CYP2A13, expressed in the human bladder.⁵⁾

Although the association between CYP2A6 polymorphisms and the risk of developing cancer such as lung and bladder cancer was reported in numerous case-control studies, our recent study revealed no significant association between the development of bladder cancer and CYP2A6 genetic polymorphisms in Japanese smokers.^6–8) CYP2A13 is expressed at higher levels than CYP2A6 in the human bladder and plays a larger role than CYP2A6 in the metabolic activation of NNK in a reconstituted system (intrinsic clearance (CL_int) of CYP2A6 and CYP2A13 was 0.008 and 0.36 µL/min/nmol CYP, respectively).^5,9) Furthermore, NNK alpha-hydroxylation by CYP2A13 is higher than that by CYP2A6 in human lung cells.¹⁰⁾ To date, 10 CYP2A13 allelic variants have been reported (http://www.cypalleles.ki.se/cyp2a13.htm), and most of them affect the metabolism of NNK and NNN.¹¹⁾ Cancer development caused by local generation of short-lived and carcinogenic metabolites from procarcinogens such as NNK occurs in organs with high expression of CYP2A13.¹²⁾ Thus, investigation of the effect of individual differences in CYP2A13 enzymatic activity on the risk of developing cancer would be valuable. However, the role of CYP2A13 in cancer development in Japanese smokers has not been investigated to our knowledge. Given that the risk of small-cell lung carcinoma is significantly lower for patients with the nonsense mutation (CYP2A13*7; Arg101Stop), we hypothesized that CYP2A13 genetic polymorphisms might also be associated with the development of bladder cancer in smokers because it exhibits the highest expression levels in the bladder, followed by the lung.^5,13)

To evaluate this hypothesis, we conducted a case-control study in Japanese smokers to examine the contribution of CYP2A13 polymorphisms to the risk of bladder cancer using next-generation sequencing (NGS) and Sanger sequencing methods.

SUBJECTS AND METHODS

Subjects

This case-control study involved 163 patients with bladder cancer and 161 healthy controls matched by age, gender, and smoking behavior. All patients with bladder cancer and 116 of the control subjects were used in our previous study.⁸⁾ The 45 control subjects were additionally recruited from Tohoku University Hospital (Sendai, Japan) in this study. The participants provided written informed consent according to the protocols approved by the ethical review board of Tohoku University Hospital, the Ethics Committees of Tokyo Metropolitan Geriatric Hospital, and Hamamatsu University School of Medicine.

NGS of the CYP2A13 Gene

PCR amplification was performed with genomic DNA samples (>10 ng), 2× KAPA HiFi HotStart ReadyMix (KAPA Biosystems, MA, U.S.A.), and 0.5 µM each primer (CYP2A13-S, 5′-GAC AGC TCT GGG TCA AAG CAA AAT C-3′; CYP2A13-AS, 5′-TTC CTC TCA TCA CAG CTC TGA AGG ACA TC-3′). PCR conditions involved an initial denaturation step at 95°C for 3 min; 30 cycles of denaturation at 98°C for 15 s, annealing at 68°C for 20 s, and extension at 72°C for 4 min; and a final extension at 72°C for 5 min. The purified PCR products (8329 bp) were quantified by the Qubit double stranded (ds)DNA BR assay with a Qubit 2.0 fluorometer (Thermo Fisher Scientific, CA, U.S.A.). Illumina paired-end sequencing libraries were constructed from 1 ng dsDNA using the Nextera XT preparation Kit (Illumina, CA, U.S.A.) and Nextera XT v2 Index Kit set A (Illumina) following the standard Illumina sample-preparation protocol. Sequencing libraries were analyzed on an Illumina MiSeq sequencing platform (Illumina) using MiSeq Reagent Kits v2 (Illumina).

Sample demultiplexing was performed on the MiSeq Reporter 2.4.60 (Illumina). Sequence quality score recalibration and single nucleotide variant (SNV) detection were conducted using the CLC Genomics Workbench version 6.0.1 (CLC Bio, Qiagen, Hilden, Germany); sequencing data were imported in FASTQ format according to the standard manufacturer’s protocol. Briefly, data for each read were trimmed based on the quality score and mapped separately to the reference sequence of the complete human CYP2A13 gene (GenBank accession number NG_007928) using specific parameter settings (length fraction=0.5, similarity=0.8). After removing duplicate mapped reads, quality-based variant detection was performed to detect SNVs. All SNVs were called if their frequency was at least 30% in each gene position. Samples with detected exon SNVs were confirmed using a Sanger sequencing-based genotyping method, and the CYP2A13 haplotypes were identified.

Sanger sequencing of the CYP2A13 gene was performed as previously described with slight modification to confirm the CYP2A13 genotypes in subjects with SNVs predicted by NGS.¹⁴⁾ PCR amplicons (0.2 ng) were amplified in 20-µL reactions containing 2× AmpliTaq Gold 360 Master Mix (Thermo Fisher Scientific) and each primer set as previously reported. The thermal cycling comprised denaturation at 95°C for 10 min; 20 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s; and a final extension at 72°C for 7 min. Column-purified PCR products were analyzed by Sanger sequencing.

Statistical Analyses

All statistical analyses were performed with IBM SPSS Statistics 22.0 (IBM, NY, U.S.A.). An unpaired t-test was used to compare the age and smoking behavior (cigarettes per d). For comparisons with respect to the gender ratio between cases and controls, the distribution of characteristics was estimated using the chi-squared test. Hardy–Weinberg equilibrium (HWE) in CYP2A13 genotypes was tested using the chi-squared test in the cases and controls. The crude odds ratio (OR) and 95% confidence interval (95% CI) were used to evaluate the distribution of allele frequency between bladder cancer cases and controls. The frequency of each genotype in cases and controls was assessed using binomial logistic regression to calculate the OR adjusted by age, gender, and smoking behavior, as well as the 95% CI. A two-tailed p-value <0.05 indicated statistical significance. Power calculation was performed using R version 3.0.3 (R core team (2014); R foundation for Statistical Computing, Vienna, Austria, http://www.R-project.org/).

RESULTS

The baseline characteristics of bladder cancer cases and controls are summarized in Table 1. There were no statistically significant differences in the mean age (p=0.105), smoking behavior (p=0.583), or gender ratio (p=0.200). All of these parameters were normally distributed.

Table 1. Baseline Characteristic of Cases with Bladder Cancer and Controls

Variable	Cases	Controls	p-Value
Total, n	163	161
Gender, n (%)
Male	154 (94.5)	145 (90.1)	0.200a)
Female	9 (5.5)	16 (9.9)
Mean age±S.D.	68.4±10.5	70.3±10.7	0.105b)
Cigarettes per d±S.D.	22.8±12.6	23.6±14.8	0.583b)

a) Chi-squared test. b) Unpaired t-test.

As shown in Table 2, all samples were genotyped for five CYP2A13 variant alleles (CYP2A13*2, *3, *4, *6, *7) on the Human Cytochrome P450 Allele Nomenclature Database. The average quality score was 33.8, and more than 80% of total sequencing reads were obtained with Q30, which is logarithmically related to the base-calling error probabilities (Supplementary Fig. 1). Sanger sequencing was conducted for 68 samples with detected exon SNVs from cases and control subjects to confirm the concordance rate. For exon SNVs, the NGS results were the same as those from Sanger sequencing (100% concordance rate).

Table 2. CYP2A13 Allelic Variants Detected in This Study

Alleles	Nucleotide changes	Amino acid changes	Allele frequency (%)
			Present study	Previous reports
CYP2A13*1			89.04
CYP2A13*2b)	74G>A, 3375C>T	Arg25Gln, Arg257Cys	7.25	4.8,a) 5.6,15) 7.316)
CYP2A13*3	1634_1635insACC, 1706C>G	133_134insThr, Asp158Glu	2.47	1.4,a) 1.8,15) 4.916)
CYP2A13*4	579G>A	Arg101Gln	0.16	0.1,15) 0.316)
CYP2A13*6	7465C>T	Arg494Cys	0.62	0.5,a) 0.3,15) 1.016)
CYP2A13*7	578C>T	Arg101Stop	0.46	0.5,a) 0.415)

a) Allele frequency in Japanese population of 1000 Genomes Project phase 1 data. b) CYP2A13*2A and CYP2A13*2B, both of which code the same CYP2A13 variant protein, were defined as CYP2A13*2 because some intron regions in CYP2A13 gene were not mapped correctly by NGS platform.

The CYP2A13 genotype distribution in both bladder cancer patients and healthy controls were not different from HWE (χ²=1.94, 5.21, p=0.997, 0.990, respectively). The allele distributions of CYP2A13 in bladder cancer cases and controls are summarized in Table 3. The frequency of CYP2A13*2 in bladder cancer cases was lower than control subjects (Crude OR=0.39, 95% CI=0.21–0.75). Table 4 shows the relationship between bladder cancer susceptibility and CYP2A13 genotypes. Individuals who possessed the CYP2A13*1/*2 genotypes exhibited decreased risk of bladder cancer (adjusted OR=0.34, 95% CI=0.17–0.69). The genotype distribution did not depart from HWE for any of the genotypes.

Table 3. Allele Frequency of CYP2A13 Polymorphisms among Cases and Controls and Their Association with Bladder Cancer

Allele	Cases, n (%) n=326	Controls, n (%) n=322	Crude ORa) (95% CI)	p-Value	Power
CYP2A13*1	299 (91.7)	278 (86.3)	1 (Reference)
CYP2A13*2	14 (4.3)	33 (10.3)	0.39 (0.21–0.75)	0.006	92.1
CYP2A13*3	9 (2.8)	7 (2.2)	1.20 (0.44–3.25)	0.912	8.5
CYP2A13*4	0 (0)	1 (0.3)
CYP2A13*6	2 (0.6)	2 (0.6)	0.93 (0.13–6.65)	1.000	5.6
CYP2A13*7	2 (0.6)	1 (0.3)	1.86 (0.17–20.62)	1.000	39.5

OR, odds ratio. CI, confidence interval. a) Chi-squared test.

Table 4. Genotype Frequency of CYP2A13 Polymorphisms among Cases and Controls and Their Association with Bladder Cancer

Genotype	Cases, n (%) n=163	Controls, n (%) n=161	Adjusted ORa) (95% CI)	p-Value	Power
CYP2A13*1/*1	137 (84.0)	119 (74.0)	1 (Reference)
CYP2A13*1/*2	13 (8.0)	31 (19.3)	0.34 (0.17–0.69)	0.003	99.7
CYP2A13*1/*3	8 (4.9)	5 (3.1)	1.20 (0.38–3.83)	0.754	10.8
CYP2A13*1/*4	0 (0)	1 (0.6)
CYP2A13*1/*6	2 (1.2)	2 (1.2)	1.10 (0.14–8.44)	0.928	6.6
CYP2A13*1/*7	2 (1.2)	1 (0.6)	1.72 (0.15–19.35)	0.661	50.9
CYP2A13*2/*3	1 (0.7)	2 (1.2)	0.38 (0.03–4.23)	0.428	98.6

OR, odds ratio. CI, confidence interval. a) Logistic regression analysis adjusted by age, gender, and smoking behavior.

DISCUSSION

CYP2A13 plays an important role in metabolic activation of NNK and NNN, which is recognized as the cause of cancer development.¹⁾ The decline in NNK and NNN metabolic activation by CYP2A13 polymorphisms, leading to decreased catalytic efficacy, could account for the reduced bladder cancer risk observed herein. Thus, we performed a case-control study to reveal whether CYP2A13 genetic polymorphisms could have an effect on bladder cancer development in Japanese smokers.

CYP2A13 genotyping is generally performed by Sanger sequencing of the PCR product of each exon.^13–16) Because different PCR conditions or two-step PCR amplification is needed to amplify each of the nine exons, the Sanger sequencing-based genotyping approach to detect SNVs from exons and a few introns is complex and time-consuming. NGS might enable calling of global CYP2A13 SNVs, including exons and introns, using only one-time PCR amplification and library preparation. However, mapping in some intron regions has a high mismatch rate because of the presence of repetitive or similar sequences. Despite the mapping errors in some intron regions, we obtained a 100% concordance rate for SNVs in exon regions. Thus, the NGS platform is sufficient for genotyping at least the exon SNVs, which greatly simplifies the genotyping method.

The presence of CYP2A13*2 was associated with reducing the risk of bladder cancer in our study. This result is reasonable in the hypothesis of cancer development described previously. In contrast, Song et al. reported that a CYP2A13 variant allele (3375C>T) showed no significant association with the risk of bladder cancer in central China.⁷⁾ This conflicting result could arise from insufficient sample size (the number of bladder cancer cases and controls in smokers were 129 and 87, respectively).⁷⁾ Moreover, insufficient statistical power (10.8%) may mislead the results that there were no significant association between CYP2A13*2 and bladder cancer risk. Although a previous report suggested that several CYP2A13 allelic variants (CYP2A13*2–*8) reduced the metabolic activity of NNK and NNN, the association with the development of bladder cancer was observed for only CYP2A13*2.¹¹⁾ Herein, there were few cases with CYP2A13 variants CYP2A13*3, *4, *6, or *7. Consequently, no significant association with bladder cancer risk was recognized for minor alleles because of insufficient sample size.

Cancer development related to NNK might be associated with the CYP2A6 and CYP2A13 expression levels in the tissue. Our present study suggested that CYP2A13 genetic polymorphisms are significantly associated with bladder cancer risk in Japanese smokers. Cauffiez et al. reported that lack of CYP2A13 enzymatic function reduced the risk of small-cell lung cancer, which indicated higher CYP2A13 expression levels.^5,13) In contrast, CYP2A6 genetic polymorphisms were not significantly associated with the risk of bladder cancer in Japanese smokers because of the relatively low expression of CYP2A6 in the human bladder compared with that of CYP2A13.⁵⁾ Therefore, the expression levels of CYP2A13 in various human tissues including the bladder are expected to be related to cancer development due to NNK and NNN metabolic activation.

In conclusion, this case-control study suggested that the CYP2A13*1/*2 genotype decreased the risk of susceptibility to bladder cancer in Japanese smokers. To our knowledge, our study provides the first evidence of an association between the risk of bladder cancer and genetic polymorphisms of CYP2A13 in Japanese smokers. However, the statistical power was relatively small in some genotype groups with minor alleles even though the statistical power for the CYP2A13*1/*2 genotype groups reached 99.7%. Moreover, the lack of homozygous subjects for the CYP2A13*2 variant might lead to unclear association between the risk of bladder cancer and the presence of CYP2A13*2. Further studies examining thousands of cases and control subjects will be required to evaluate the association between bladder cancer risk and CYP2A13 genetic polymorphisms including minor alleles in more detail.

Acknowledgments

This study was supported by Grants from the Smoking Research Foundation, Ministry of Health, Labour and Welfare (MHLW) of Japan (“Initiative to facilitate the development of innovative drugs, medical devices, and cellular & tissue-based products”), the Japan Research Foundation for Clinical Pharmacology, the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan S001, and Japan Agency for Medical Research and Development (AMED).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

Supplementary Fig. 1. Distribution of Quality Scores for Illumina Paired-End Sequencing Libraries Including All Cases and Control Samples

Quality check was performed according to standard procedures using CLC Genomics Workbench version 6.0.1 (CLCbio).

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