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Polymorphisms of the CYP1B1 gene and hepatocellular carcinoma risk in a Chinese population
Gene 564 (2015) 14–20
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Research paper
Polymorphisms of the CYP1B1 gene and hepatocellular carcinoma risk in a Chinese population Fei Liu a, Li-Mei Luo b, Yong-Gang Wei a,⁎, Bo Li a, Wen-Tao Wang a, Tian-Fu Wen a, Jia-Yin Yang a, Ming-Qing Xu a, Lv-Nan Yan a a b
Department of Liver Surgery & Liver Transplantation Center, West China Hospital of Sichuan University, 37 Guo Xue Road, Chengdu 610041, Sichuan Province, China Department of Clinical Immunological Laboratory, West China Hospital of Sichuan University, 37 Guo Xue Road, Chengdu 610041, Sichuan Province, China
a r t i c l e
i n f o
Article history: Received 25 December 2014 Received in revised form 10 March 2015 Accepted 15 March 2015 Available online 18 March 2015 Keywords: Hepatocellular carcinoma Susceptibility CYP1B1 Polymorphism Case–control Study
a b s t r a c t Background: CYP1B1 is a P450 enzyme which is involved in the activation of pro-carcinogens to carcinogens as well as estrogen metabolism. We hypothesized that genetic variants in CYP1B1 may modify individual susceptibility to hepatocellular carcinoma (HCC). Methods: To test this hypothesis, we evaluated the associations of three CYP1B1 single nucleotide polymorphisms (SNPs) and HCC risk in a case–control study of 468 HCC cases and 515 cancer-free controls in a Chinese population. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry method and direct DNA sequencing were performed to detect these polymorphisms. Results: In overall analysis, we found that only the variant G allele of rs1056836 was associated with a significantly increased risk of HCC among the three SNPs (rs10012, rs1056836 and rs1800440). Moreover, we found that the variant genotypes containing the G allele of rs1056836 were associated with a significantly increased risk of HCC among HbsAg-positive individuals (adjusted OR = 2.13, 95% CI = 1.18, 3.86), but not among HbsAgnegative individuals. When stratifying by smoking status, we found that the variant GG genotype increased a 13.97-fold (95% CI = 1.28, 152.94) risk of HCC among smokers. Furthermore, high risk for liver cirrhosispositive clinical status was exhibited in HCC patients with rs1056836 CG and GG genotypes as compared with CC homozygotes. For the other two SNPs, we did not find any significant evidence of association with HCC risk in any subgroup. Conclusion: This study suggests that CYP1B1 rs1056836 polymorphism may be an important factor contributing to increased susceptibility and pathological development of HCC in Chinese population. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Primary liver cancer is one of the most common solid cancers worldwide, the second most frequent cause of cancer death in men, and the sixth in women (Jemal et al., 2011). About 748,300 new liver cancer cases and 695,900 cancer deaths are estimated to have occurred in 2008 worldwide (Jemal et al., 2011). Hepatocellular carcinoma (HCC) is the major histological subtype among primary liver cancers, which accounted for 70% to 85% of the total liver cancer burden worldwide (Perz et al., 2006). It's noteworthy that more than 75% of these cases occur in the Asia-Pacific region (Yuen et al., 2009) and China alone accounts for 55% cases of HCC worldwide (Parkin, 2001). Etiologically, carcinogenesis of HCC is a complex, multi-step and multi-factor process, in
Abbreviations: Cytochrome P450 1B1, CYP1B1; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; OR, odds ratio; CI, confidence interval; SNP, single nucleotide polymorphisms; HWE, Hardy–Weinberg equilibrium; AFP, alpha fetoprotein. ⁎ Corresponding author. E-mail address: [email protected] (Y.-G. Wei).
http://dx.doi.org/10.1016/j.gene.2015.03.035 0378-1119/© 2015 Elsevier B.V. All rights reserved.
which many factors are implicated. As we know, chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) is the most well established environmental risk factor for HCC worldwide. However, only a fraction of HBsAg carriers eventually develop HCC and only 2.5% of HCV infected individuals develop HCC later in life (Bowen and Walker, 2005). Thus, host genetic factors may affect HCC development. Identification of genetic factors related to susceptibility to HCC would help elucidate the complex process of hepatocarcinogenesis and improve the scientific basis for preventive interventions. Cytochrome P450 1B1 (CYP1B1) is a key P450 enzyme implicated in the metabolism of exogenous and endogenous substrates (Paracchini et al., 2007). A variety of studies have demonstrated that the metabolism of polycyclic aromatic hydrocarbons and other procarcinogens through CYP1B1 may well lead to the activation of the carcinogenic compounds (Shimada et al., 2001; Shimada and Fujii-Kuriyama, 2004). Meanwhile, CYP1B1 also participated in the hydroxylation of 17β-estradiol at the 2-OH and 4-OH positions, which can then be oxidized to semiquinones and quinones (Spink et al., 1994). The two electrophilic metabolites, semiquinones and quinones, can form DNA
F. Liu et al. / Gene 564 (2015) 14–20
adducts which could potentially introduce mutations into the genome (Chakravarti et al., 2001; Trubicka et al., 2010). This undesirable outcome alters the risk of malignancy by increasing the likelihood of introducing mutations into the genome (Trubicka et al., 2010). CYP1B1 gene is located on chr2p22-p21 and there are at least 179 different polymorphism sites in the gene (http://ncbi.nlm.nih.gov/ dbSNP). A number of single-nucleotide polymorphisms (SNPs) in this gene have been shown to affect the activity of the encoded protein (Bailey et al., 1998; Shimada et al., 1999). Three polymorphisms, occurring at codons rs10012 (48 C N G), rs1056836 (432 C N G) and rs1800440 (453 A N G), all of which result in single amino acid substitutions that result in an altered enzyme activity are of particular interest (Shimada et al., 1999). To predict the risk and prognosis of cancer, genotyping of these SNPs might provide a simple and valuable method. Although contributions of the CYP1B1 to the formation of many types of cancer are well known, its possible association with prediction of risk of HCC remains poorly investigated. In this study, the relationship between the three SNPs of CYP1B1 gene and HCC risk was investigated, and the impact of these SNPs on susceptibility and clinicopathological characteristics of HCC was also evaluated. 2. Materials and methods 2.1. Study population The detailed methods of this case–control study have been described previously (Liu et al., 2013a,b). Briefly, 500 patients with newly diagnosed, untreated HCC were enrolled in this study at the West China Hospital of Sichuan University from October 2007 to December 2011. The diagnosis of HCC cases included a combination of pathological examination, ultrasound and clinical manifestations. The patients who reportedly had previous cancer, other metastasized cancers and previous radiotherapy or chemotherapy were excluded. All subjects were genetically unrelated ethnic Han Chinese from Chengdu City and surrounding regions. Meanwhile, 550 cancer-free control subjects were recruited during the same period from the health examination individuals in West China Hospital. The controls were not blood relatives of the patients or each other and were frequency-matched with the cases by age at enrollment, sex, and residential area. Informed consents were obtained according to the Declaration of Helsinki. A written informed consent was obtained from each subject involved in the study. After informed consent was obtained, each subject was personally interviewed by trained interviewers using a pretested questionnaire, to obtain information on demographic data, lifestyles (alcohol consumption and cigarette smoking), and family history of cancer in first degree relatives (parents, siblings or children). After interview, a 5-mL venous blood sample was collected from each subject. The laboratory examinations, such as, serum alpha fetoprotein (AFP) levels, and hepatitis B virus (HBV) serological markers, were also collected from each subject. The study was approved by the Ethics Committee of Sichuan University. 2.2. DNA extraction Blood samples (5 mL) were collected in EDTA tubes after interview, and genomic DNA was isolated from whole blood samples using the whole blood DNA kit (Biotake corporation). DNA was extracted from 200 μL of the whole blood according to the manufacturer's protocol. The concentration of DNA was diluted to 20 ng/μL for working solutions and the isolated DNA was stored at −20 °C.
15
base extension were designed by using Assay Designer software package (Sequenom). Briefly, the DNA sample to be queried was diluted to 5 ng/μL, and 1 μL of DNA was combined with 0.95 μL of water, 0.625 μL of PCR buffer containing 15 mM MgCl2, 1 μL of 2.5 mM dNTP, 0.325 μL of 25 mM MgCl2, 1 μL of PCR primers and 0.1 μL of 5 units/μL HotStar Taq (Qiagen). The reaction was incubated at 94 °C for 15 min followed by 45 cycles at 94 °C for 20 s, 56 °C for 30 s, and 72 °C for 1 min, and a final incubation at 72 °C for 3 min. After PCR amplification, remaining dNTPs were dephosphorylated by adding 1.53 μL of water, 0.17 μL of SAP buffer, and 0.3 units of shrimp alkalinephosphatase (Sequenom). The reaction was placed at 37 °C for 40 min, and the enzyme was deactivated by incubating at 85 °C for 5 min. After shrimp alkaline phosphatase treatment, the single primer extension over the SNP was combined with 0.755 μL of water, 0.2 μL of 10× iPLEX buffer, 0.2 μL of termination mix, 0.041 μL of iPLEX enzyme (Sequenom), 0.804 μL of 10 μM extension primer. The single base extension reaction was carried out at 94 °C for 30 s and then 94 °C for 5 s, followed by 5 cycles of 52 °C for 5 s and 80 °C for 5 s, total 40 cycles, then 72 °C for 3 min. The reaction mix was desalted by adding 6 mg of cation exchange resin (Sequenom), mixed and resuspended in 25 μL of water. The completed genotyping reactions were spotted onto a 384 well spectroCHIP (Sequenom) using MassARRAY Nanodispenser (Sequenom) and determined by the matrixassisted laser desorption–ionization time-of-flight mass spectrometer. Genotype calling was performed in real time with MassARRAY RT software version 3.0.0.4 and analyzed using the MassARRAY Typer software version 3.4 (Sequenom). For quality control, genotyping was performed without knowing the subjects' case and control status, and a 5% random sample of cases and controls was genotyped twice for each locus. If a consensus on the tested genotype was not reached, two research assistants independently performed the repeated assays to achieve 100% concordance. To further validate the genotyping assay of MALDI-TOF MS for the three loci, about 5% of samples genotyped with MALDI-TOF MS were further confirmed by direct sequencing method using an automated sequencer (ABI 3730, Applied Biosystems). Among the 500 cases and 550 controls with DNA samples, the genotyping was successful for all three SNPs in 468 HCC cases and 515 controls, resulting in the overall success rate of 93.6%. Therefore, a total of 468 cases and 515 controls were included in the final analyses. 2.4. Statistical analysis Differences in the distributions of demographic characteristics, selected variables and genotype frequencies in the cases and controls were evaluated using the chi-square test or Fisher's exact test, since sample size was small for some subgroup analysis. The associations between CYP1B1 genotypes and risk of HCC were estimated by computing the odds ratios (ORs) and their 95% confidence intervals (CIs) from both univariate and multivariate logistic regression analyses with adjustment for age, sex, HBV carrier state, alcohol intake, smoking status, and family history of cancer. Hardy–Weinberg equilibrium was tested by a goodness-of-fit χ2 test, to compare the observed genotype frequencies to the expected ones among the control subjects. All statistical analyses were two sided and performed using SPSS version 16.0 for Windows statistical software (SPSS Inc., Chicago, IL, USA). A P-value of b0.05 was considered as statistically significant. 3. Results 3.1. Characteristics of studies
2.3. Genotype analyses SNP genotyping was performed using MassARRAY system (Sequenom, San Diego, CA, USA) by means of matrix assisted laser desorption ionization-time of flight mass spectrometry method (MALDI-TOF MS) according to the manufacturer's instructions. Primers for PCR and single
The demographic characteristics and risk factors in patients with HCC and controls are presented in Table 1. No significant differences between the two groups in terms of age and gender distribution (P = 0.45 for age and P = 0.86 for sex) suggested that matching of subjects based on these variables was adequate. There was no significant difference
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F. Liu et al. / Gene 564 (2015) 14–20
Table 1 Distributions of demographical characteristics and selected variables among HCC patients and controls. Variable
Age [n (%)] b55 years ≥55 years Gender [n (%)] Female Male Smoking status [n (%)] Never Ever Drinking status [n (%)] Never Ever HBV carrier state [n (%)] HbsAg (−) HbsAg (+) Family history of cancer [n (%)] No Yes
HCC
Controls
(n = 468)
(n = 515)
OR (95%CI)
266 (56.8) 202 (43.2)
305(59.2) 210(40.8)
1.00 1.10 (0.86, 1.42)
123 (26.3) 345 (73.7)
138(26.8) 377 (73.2)
1.00 1.03 (0.77, 1.36)
214 (45.7) 254 (54.3)
339 (65.8) 176 (34.2)
1.00 2.29 (1.77, 2.96)
338 (72.2) 130 (27.8)
394 (76.5) 121 (23.5)
1.00 1.25 (0.94, 1.67)
151 (32.3) 317 (67.7)
431 (83.7) 84 (16.3)
1.00 10.77 (7.95, 14.59)
P-value
0.45
0.86
b0.001
0.12
b0.001
0.64 424 (90.6) 44 (9.4)
471 (91.5) 44 (8.5)
1.00 1.11 (0.72, 1.72)
Abbreviations: HCC—hepatocellular carcinoma; OR—odds ratio; CI—confidence interval; HBV—hepatitis B virus; HBsAg—hepatitis B surface antigen.
between cases and controls in the distribution of drinking status and family history of cancer. As shown in Table 1, smoking is a risk factor of HCC (P b 0.001). HbsAg-positive was also associated with significantly increased HCC risk (P b 0.001). 3.2. Genotypes and HCC risk We could detect three genotypes for the CYP1B1 rs10012 and rs1056836 polymorphisms, including CC, CG and GG, among the
Chinese population. Fig. 1 shows the three genotypes for rs1056836 polymorphism (detecting by MALDI-TOF MS). However, for the CYP1B1 1800440 polymorphism, we could only detect two genotypes (AG and AA) among the Chinese population. The genotype/allele distributions of CYP1B1 rs10012, rs1056836 and rs1800440 in the cases and the controls are shown in Table 2. The distributions of these genotype frequencies in controls were all in Hardy–Weinberg equilibrium except for rs10012 (P = 0.01, 0.48 and 0.77 for CYP1B1 rs10012, rs1056836 and rs1800440, respectively). According to the crude ORs with their 95% CIs for HCC of CYP1B1 gene polymorphisms, rs1056836 GG and CG/GG exhibited a significant higher risk of 3.23-fold (95% CI, 1.25, 8.35) and 1.43-fold (95% CI, 1.08, 1.90), respectively, to have HCC compared with the wild-type homozygote. However, the association disappeared after adjusting for other confounding factors. Nevertheless, the variant G allele of rs1056836 was associated with a significantly increased risk of HCC in both crude and adjust OR. In addition, the other two SNPs rs10012 and rs1800440 did not provide any significant evidence of association with HCC risk for both crude OR and adjust OR. The distributions of smoking status and HBV carrier state in cases and control groups were obviously screwy, indicating that subgroup analysis by the two factors was needed. When stratifying by HBV carrier state, we found that the variant genotypes of CYP1B1 rs1056836 were associated with a significantly increased risk of HCC among HbsAg-positive individuals but not HbsAg-negative people [adjusted OR (95% CI) = 1.89 (1.03, 3.48) for CG vs CC and 2.13 (1.18, 3.86) for CG/GG vs CC] (Table 3). We further analyzed the effects of these polymorphisms on HCC risk stratified by smoking status. As shown in Table 4, we found that only the variant genotype GG of CYP1B1 rs1056836 was associated with a significantly increased risk of HCC among smokers [adjusted OR (95% CI) = 13.97 (1.28, 152.94)] and but not among nonsmokers. To explore the impact of polymorphic genotype of CYP1B1 gene on susceptibility and pathological development of HCC, HCC patients were further classified into two subgroups, one subgroup with at least one polymorphic allele and the other subgroup with homozygous
Fig. 1. Genotyping of CYP1B1 rs1056836 polymorphism by MALDI-TOF. The horizontal axis represents the mass and the vertical axis represents the signal intensity. Around the 6000 Da in the horizontal axis, two lines (the C allele and the G allele) can be observed. If only a wave crest appeared at the C line, the genotype of this patient was CC (the left one); if only a wave crest appeared at the G line, the genotype of this patient was GG (the right one); if two wave crest appeared at both the C line and the G line, the genotype of this patient was CG (the middle one).
F. Liu et al. / Gene 564 (2015) 14–20
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Table 2 CYP1B1 genotype and allele frequencies of the cases and controls and their association with risk of HCC. Polymorphism CYP1B1
Genotypes/allele
HCC (n = 468)
Controls (n = 515)
Crude OR 95%CI
Adjusted ORa 95%CI
Rs10012
CC CG GG CG and GG C G CC CG GG CG and GG C G AA AG GG AG and GG A G
316 (67.5) 133 (28.4) 19 (4.1) 152 (32.5) 765 (81.7) 171 (18.3) 326 (69.7) 126 (26.9) 16 (3.4) 142 (30.3) 778 (83.1) 158 (16.9) 462 (98.7) 6 (1.3) 0 (0.0) 6 (1.3) 930 (99.4) 6 (0.6)
338 (65.7) 147 (28.5) 30 (5.8) 177 (34.3) 823 (79.9) 207 (20.1) 395 (76.7) 114 (22.1) 6 (1.2) 120 (23.3) 904 (87.8) 126 (12.2) 502 (97.5) 13 (2.5) 0 (0.0) 13 (2.5) 1017 (98.7) 13 (1.3)
1.00 0.96 [0.73, 1.28] 0.68 [0.37, 1.23] 0.92 [0.70, 1.20] 1.00 0.89 [0.71, 1.11] 1.00 1.34 [1.00, 1.80] 3.23 [1.25, 8.35] 1.43 [1.08, 1.90] 1.00 1.46 [1.13, 1.88] 1.00 0.50 [0.19, 1.33] – 0.50 [0.19, 1.33] 1.00 0.51 [0.19, 1.33]
1.00 0.93 [0.67, 1.30] 0.59 [0.29, 1.19] 0.87 [0.63, 1.19] 1.00 0.83 [0.64, 1.08] 1.00 1.24 [0.87, 1.75] 2.59 [0.88, 7.60] 1.30 [0.93, 1.83] 1.00 1.36 [1.08, 1.80] 1.00 0.33 [0.11, 1.04] – 0.33 [0.11, 1.04] 1.00 0.34 [0.13, 1.10]
Rs1056836
Rs1800440
P bvalue
0.67 0.14 0.39 0.28 0.24 0.08 0.13 0.002 0.06 – 0.06 0.14
The figures given in bold indicate statistically significant values. a Adjusted for age, gender, HBV carrier state, family history of cancer, smoking, and drinking status. b P value for adjusted OR and 95% CI.
wild-type alleles, to estimate the adjusted ORs with their 95% CIs for HCC of each CYP1B1 SNP. Adjusted ORs and their 95% CIs for the pathological characteristics of each SNP of CYP1B1 gene in HCC patients suggest that no significant differences between rs10012 and rs1800440 genotypic frequencies and any clinical pathological variables (data not shown), but a high risk of 1.63-fold (95% CI = 1.01, 2.61) for having liver cirrhosis appeared in HCC patients with rs1056836 CG and GG genotypes as compared with patients with rs1056836 CC genotype (Table 5). 4. Discussion In the present study, we examined the association between three CYP1B1 SNPs and the risk of HCC in Chinese Han patients. In the overall analysis, we found that only the variant G allele of rs1056836 was associated with a significantly increased risk of HCC among the three SNPs. When stratifying by HBV carrier status, we found that the variant genotypes containing the G allele of rs1056836 was associated with a
significantly increased risk of HCC among HbsAg-positive individuals. When stratifying by smoking status, we found that the variant GG genotype increased a 13.97-fold risk of HCC among smokers. Furthermore, high risk for liver cirrhosis clinical status was exhibited in HCC patients with rs1056836 CG and GG genotypes as compared with CC homozygotes. Although the exact biological mechanism remains to be explored, these findings provide some evidence that CYP1B1 rs1056836 polymorphism may be an important factor contributing to increased susceptibility and pathological development of HCC. The exact mechanism for the correlation between the SNP of CYP1B1 and the risk of HCC needs further exploring. However, previous published studies on the functions of the CYP1B1 gene together with their genetic variants may help us understand the potential roles of these polymorphisms. CYP1B1 is a key P450 enzyme implicated in the metabolism of exogenous and endogenous substrates (Paracchini et al., 2007). Among exogenous substrates, CYP1B1 is implicated in the metabolic activation of a number of environmental carcinogens, such as arylamines, heterocyclic amines, benzo(a)pyrene, and polycyclic aromatic hydrocarbons
Table 3 Stratified analyses between CYP1B1 polymorphisms and HCC risk by HBV carrier status. Gene variable
HBV carrier status HbsAg (+)
HbsAg (−) a
HCC n (%)
Control n (%)
Adjusted ORa
P
0.54 0.13 0.28
102 (67.5) 43 (28.5) 6 (4.0) 49 (32.5)
285 (66.1) 123 (28.5) 23 (5.4) 146 (33.9)
1.00 0.94 (0.61, 1.43) 0.76 (0.29, 1.96) 0.91 (0.61, 1.36)
0.76 0.56 0.65
1.00 1.89 (1.03, 3.48) 7.83 (0.95, 64.91) 2.13 (1.18, 3.86)
0.04 0.06 0.01
119 (78.8) 30 (19.9) 2 (1.3) 32 (21.2)
328 (76.1) 98 (22.7) 5 (1.2) 103 (23.9)
1.00 0.92 (0.58, 1.47) 1.22 (0.23, 6.54) 0.94 (0.59, 1.48)
0.73 0.82 0.78
1.00 0.27 (0.07, 1.07) – 0.27 (0.07, 1.07)
0.06 – 0.06
150 (99.3) 1 (0.7) 0 (0.0) 1 (0.7)
422 (97.9) 9 (2.1) 0 (0.0) 9 (2.1)
1.00 0.38 (0.05, 3.04) – 0.38 (0.05, 3.04)
HCC n (%)
Control n (%)
Adjusted OR
Rs10012 CC CG GG CG and GG
214 (67.5) 90 (28.4) 13 (4.1) 103 (32.5)
53 (63.1) 24 (28.6) 7 (8.3) 31 (36.9)
1.00 0.84 (0.48, 1.48) 0.46 (0.17, 1.27) 0.75 (0.45, 1.26)
Rs1056836 CC CG GG CG and GG
207 (65.3) 96 (30.3) 14 (4.4) 110 (34.7)
67 (79.8) 16 (19.0) 1 (1.2) 17 (20.2)
Rs1800440 AA AG GG AG and GG
312 (98.4) 5 (1.6) 0 (0.0) 5 (1.6)
80 (95.2) 4 (4.8) 0 (0.0) 4 (4.8)
The figures given in bold indicate statistically significant values. a Adjusted for age, gender, family history of cancer, smoking, and drinking status.
P
0.36 0.36
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F. Liu et al. / Gene 564 (2015) 14–20
Table 4 Stratified analyses between CYP1B1 polymorphisms and HCC risk by smoking status. Gene variable
Smoking Status Smokers
Nonsmokers
HCC n (%)
Control n (%)
Adjusted ORa
P
HCC n (%)
Control n (%)
Adjusted ORa
P
Rs10012 CC CG GG CG and GG
170 (66.9) 71 (28.0) 13 (5.1) 84 (33.1)
112 (63.6) 51 (29.0) 13 (7.4) 64 (36.4)
1.00 0.98 (0.58, 1.65) 0.66 (0.25, 1.77) 0.92 (0.57, 1.49)
0.94 0.41 0.73
146 (68.2) 62 (29.0) 6 (2.8) 68 (31.8)
226 (66.7) 96 (28.3) 17 (5.0) 113 (33.3)
1.00 0.88 (0.56, 1.39) 0.54 (0.19, 1.60) 0.84 (0.54, 1.29)
0.59 0.27 0.42
Rs1056836 CC CG GG CG and GG
175 (68.9) 70 (27.6) 9 (3.5) 79 (31.1)
142 (80.7) 33 (18.8) 1 (0.5) 34 (19.3)
1.00 1.39 (0.79, 2.42) 13.97 (1.28, 152.94) 1.59 (0.92, 2.74)
0.25 0.03 0.10
151 (70.6) 56 (26.2) 7 (3.2) 63 (29.4)
253 (74.6) 81 (23.9) 5 (1.5) 86 (25.4)
1.00 1.08 (0.68, 1.71) 1.64 (0.46, 5.87) 1.10 (0.70, 1.73)
0.76 0.45 0.67
Rs1800440 AA AG GG AG and GG
251 (98.8) 3 (1.2) 0 (0.0) 3 (1.2)
172 (97.7) 4 (2.3) 0 (0.0) 4 (2.3)
1.00 0.20 (0.04, 1.13) – 0.20 (0.04, 1.13)
0.07 – 0.07
211 (98.6) 3 (1.4) 0 (0.0) 3 (1.4)
330 (97.3) 9 (2.7) 0 (0.0) 9 (2.7)
1.00 0.43 (0.10, 1.92) – 0.43 (0.10, 1.92)
0.27 0.27
The figures given in bold indicate statistically significant values. a Adjusted for age, gender, HBV carrier state, family history of cancer, and drinking status.
(Shimada et al., 2001; Kim et al., 1998). The change in amino acid from valine to leucine (causing by CYP1B1 polymorphism) has been shown to increase the activity of the CYP1B1 enzyme on a variety of substrates, including procarcinogens and gonadal steroid hormones (Shimada et al., 1999). Moreover, for the polymorphism C432G at codon 432 of exon 3 (rs1056836), it was reported that the 432 G allele increased the mutation of p53 (Ko et al., 2001). Therefore, it is reasonable to conceive that CYP1B1 rs1056836 polymorphism may affect the metabolism of environmental carcinogens and increase the mutation of p53 to alter susceptibility to
HCC. Among endogenous substrates, CYP1B1 is thought to have an important role in estrogen metabolism, which is mainly involved in the hydroxylation of 17β-estradiol at the 2-OH and 4-OH positions (Spink et al., 1994). Meanwhile, previous studies (Shimada et al., 1996, 1999; Hanna et al., 2000) reveal that the generation of 2-OH catechol estrogens does not appear to result in deleterious affects whereas the 4-OH catechol derivative has been shown to be associated with an increase in DNA single strand breaks. The rs1056836 polymorphism (Leu-Val transition at codon 432) of CYP1B1, which is located in the heme-binding domain of
Table 5 Adjusted OR and 95% CI of clinicopathological variables and CYP1B1 genotypic frequencies in HCC patients. Variable
CYP1B1 432 C/G, Rs1056836 CC (N = 326) n (%)
CG/GG (N = 142) n (%)
OR (95% CI)
Adjusted OR (95% CI)a
Pb value
Classification of TNM Stage I/II Stage III/IV
167 (51.2) 159 (48.8)
80 (56.3) 62 (43.7)
1.00 0.81 (0.55, 1.21)
1.00 0.82 (0.55, 1.23)
0.34
Tumor size ≤5 cm N5 cm
166 (50.9) 160 (49.1)
70 (49.3) 72 (50.7)
1.00 1.07 (0.72, 1.58)
1.00 1.13 (0.75, 1.70)
0.57
Tumor number Single Multiple
196 (60.1) 130 (39.9)
92 (64.8) 50 (35.2)
1.00 0.82 (0.54, 1.23)
1.00 0.84 (0.55, 1.27)
0.40
Lymph node metastasis No Yes
257 (78.8) 69 (21.2)
122 (85.9) 20 (14.1)
1.00 0.61 (0.36, 1.05)
1.00 0.61 (0.35, 1.06)
0.08
Distant metastasis No Yes
289 (88.7) 37 (11.3)
134 (94.4) 8 (5.6)
1.00 0.47 (0.21, 1.03)
1.00 0.49 (0.22, 1.09)
0.08
Portal vein tumor thrombus No Yes
269 (82.5) 57 (17.5)
115 (81.0) 27 (19.0)
1.00 1.11 (0.67, 1.84)
1.00 1.12 (0.67, 1.89)
0.67
AFP Not increased Increased
167 (51.2) 159 (48.8)
59 (41.5) 83 (58.5)
1.00 1.48 (0.99, 2.20)
1.00 1.36 (0.89, 2.06)
0.16
Liver cirrhosis Negative Positive
116 (35.6) 210 (64.4)
34 (23.9) 108 (76.1)
1.00 1.76 (1.12, 2.74)
1.00 1.63 (1.01, 2.61)
0.04
The figures given in bold indicate statistically significant values. a Adjusted for age, gender, HBV carrier state, family history of cancer, smoking, and drinking status. b Pvalue for adjusted OR and 95% CI.
F. Liu et al. / Gene 564 (2015) 14–20
exon 3, causes the enzyme display a higher 4-hydroxylation of estradiol (Li et al., 2000). Therefore, it is also possible that the CYP1B1 rs1056836 polymorphism is involved in hepatocarcinogenesis by increasing the likelihood of introducing mutations into the genome. Given the different activities of the CYP1B1 enzyme which strongly depends on the polymorphic form, it is biologically plausible that the CYP1B1 rs1056836 polymorphism may modulate the risk of HCC in Chinese Han population. Several studies, including meta-analysis, have examined the relationship between individual CYP1B1 polymorphisms and cancer risk, however, the results are conflicting. Yuan et al. (2008) have reported that CYP1B1 Val432Leu polymorphisms may not contribute significantly to the risk for HCC. Cui et al. (2012) included ten case-controlled studies to quantitatively summarize the association between CYP1B1 Leu432Val polymorphism and prostate cancer and found that CYP1B1 Leu432Val polymorphism was not associated with prostate cancer risk overall. Similarly, Xie et al. (2012) revealed that no association was found between the CYP1B1 Leu432Val polymorphism and risk of colorectal cancer among Caucasians. In contrast, in agreement with our present results, some reports also showed association between certain CYP1B1 polymorphism and risk for cancer. For instance, in recent, Li et al. (2013) found that CYP1B1 C4326G polymorphism may increase risk of cervical cancer in Chinese women, especially among young individuals with high-risk HPV infection. Xu et al. (2012) have reported that the CYP1B1 432GG, 119TT and 48GG genotypes were low-penetrance risk factors for developing lung cancer. Paracchini et al. (2007) assessed thirteen articles (7514 cases and 6817 controls) on the association between the CYP1B1 Leu432Val polymorphism and breast cancer risk and found that the CYP1B1 Leu432Val polymorphism was associated with an increased breast cancer risk among Caucasians. The conflicting results could be attributable to the differences in cancer types, demography, ethnicity, lifestyles, type of viral infections, and clinical settings. In addition, other methodologic factors in the studies, such as small sample size, inadequate adjustment for confounding factors, or lack of correction for multiple testing, could also cause the inconsistent results. As we know, chronic infection with HBV or HCV is the most well established environmental risk factor for HCC worldwide. In China, HBV infection is a major risk factor for developing HCC (Wang et al., 2012). In our study, the HBV infection rate was 67.7% in the HCC group, which was significantly higher than that of control group (16.3%). Moreover, stratification analysis by HBV carrier status, we found that the variant genotypes (CG + GG) of the CYP1B1 rs1056836 were associated with a significantly increased risk of HCC among HbsAg-positive individuals (adjusted OR = 2.13, 95% CI = 1.18–3.86), but not among those with HbsAg-negative, which indicated that variant genotypes of CYP1B1 and HBV infection interaction may influence the susceptibility to HCC development in the Chinese population. Tobacco smoking is an established risk factor for many cancers, including HCC (Cohen et al., 2000; Wei et al., 2000; Sasco et al., 2004), and has a destructive effect on human immune responses (Zeidel et al., 2002). Moreover, cigarette smoke contains a variety of carcinogenic compounds, such as polycyclic aromatic hydrocarbons, which are mainly metabolized by phase I and phase II xenobiotic metabolizing enzymes (Xu et al., 2012). A highly studied phase I gene is the CYP1B1, which plays a significant role in the oxidation of variety of carcinogens, including PAHs and arylamines (Shimada et al., 1999). Thus, smoking may interact with CYP1B1 polymorphisms to initiate and promote hepatocarcinogenesis. In the present study, the smoking rate was 54.3% in the HCC group, which was significantly higher than that of control group (34.2%). Moreover, stratification analysis by smoking status, we found that the variant genotype GG of the CYP1B1 rs1056836 increased a 13.97-fold risk of HCC among smokers, but not among non-smokers, which indicated that variant genotypes of CYP1B1 and smoking interaction may influence the susceptibility to HCC development in the Chinese population. The genotype distribution of CYP1B1 rs10012 deviated from Hardy– Weinberg proportions in controls (P = 0.01); however, the concordance
19
rate for the quality control samples (5% of the total sample size), which were randomly selected from cases and controls, was 100% for all the three polymorphisms, including the CYP1B1 rs10012. Therefore, we do not believe that the deviation from Hardy–Weinberg equilibrium for this polymorphism is due to genotyping error. In this case–control study, several limitations need to be addressed. First, the findings from the present study were only from a Chinese southwest population, so it is uncertain whether these results are generalizable to the general population of other ethnics. Second, the determination of the exact functional influence of the CYP1B1 variant allele was not performed in our study. Further studies on potential mechanisms are needed to elucidate how this CYP1B1 rs1056836 C → G substitution modifies HCC development. Finally, although this is a hospital based case control study, the selection bias may not be avoidable and the subjects may not be representative of the general population. However, potential confounding bias might have been minimized by matching the controls to the cases on age, sex, residential area and by further adjustment for the confounding factors in data analyses. In conclusion, our study demonstrated that CYP1B1 rs1056836 polymorphism might be an important factor contributing to increased susceptibility and pathological development of HCC in Chinese population. Validation of these findings with functional evaluation and larger studies with more rigorous study designs and inclusion of different ethnic populations are needed. Conflict of interest statement None declared. Acknowledgments This work was supported by grant from the Application Foundation Project of Science and Technology Agency in Sichuan Province (No 2012JY0079). References Bailey, L.R., Roodi, N., Dupont, W.D., Parl, F.F., 1998. Association of cytochrome P450 1B1 (CYP1B1) polymorphism with steroid receptor status in breast cancer. Cancer Res. 58, 5038–5041. Bowen, D.G., Walker, C.M., 2005. Adaptive immune responses in acute and chronic hepatitis C virus infection. Nature 436, 946–952. Chakravarti, D., Mailander, P.C., Li, K.M., Higginbotham, S., Zhang, H.L., Gross, M.L., Meza, J.L., Cavalieri, E.L., Rogan, E.G., 2001. Evidence that a burst of DNA depurination in SENCAR mouse skin induces error-prone repair and forms mutations in the H-ras gene. Oncogene 20, 7945–7953. Cohen, S.M., Shirai, T., Steineck, G., 2000. Epidemiology and etiology of premalignant and malignant urothelial changes. Scand. J. Urol. Nephrol. Suppl. 205, 105–115. Cui, L., Dillehay, K., Chen, W., Shen, D., Dong, Z., Li, W., 2012. Association of the CYP1B1 Leu432Val polymorphism with the risk of prostate cancer: a meta-analysis. Mol. Biol. Rep. 39, 7465–7471. Hanna, I.H., Dawling, S., Roodi, N., Guengerich, F.P., Parl, F.F., 2000. Cytochrome P450 1B1 (CYP1B1) pharmacogenetics: association of polymorphisms with functional differences in estrogen hydroxylation activity. Cancer Res. 60, 3440–3444. Jemal, A., Bray, F., Center, M.M., Ferlay, J., Ward, E., Forman, D., 2011. Global cancer statistics. CA Cancer J. Clin. 61, 69–90. Kim, J.H., Stansbury, K.H., Walker, N.J., Trush, M.A., Strickland, P.T., Sutter, T.R., 1998. Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by human cytochrome P450 1B1. Carcinogenesis 19, 1847–1853. Ko, Y., Abel, J., Harth, V., Bröde, P., Antony, C., Donat, S., Fischer, H.P., Ortiz-Pallardo, M.E., Thier, R., Sachinidis, A., Vetter, H., Bolt, H.M., et al., 2001. Association of CYP1B1 codon 432 mutant allele in head and neck squamous cell cancer is reflected by somatic mutations of p53 in tumor tissue. Cancer Res. 61, 4398–4404. Li, D.N., Seidel, A., Pritchard, M.P., Wolf, C.R., Friedberg, T., 2000. Polymorphisms in P450 CYP1B1 affect the conversion of estradiol to the potentially carcinogenic metabolite 4-hydroxyestradiol. Pharmacogenetics 10, 343–353. Li, Y., Tan, S.Q., Ma, Q.H., Li, L., Huang, Z.Y., Wang, Y., Li, S.W., 2013. CYP1B1 C4326G polymorphism and susceptibility to cervical cancer in Chinese Han women. Tumour Biol. 34, 3561–3567. Liu, F., Wei, Y.G., Luo, L.M., Wang, W.T., Yan, L.N., Wen, T.F., Xu, M.Q., Yang, J.Y., Li, B., 2013a. Genetic variants of p21 and p27 and hepatocellular cancer risk in a Chinese Han population: a case-control study. Int. J. Cancer 132, 2056–2064. Liu, F., Luo, L., Wei, Y., Wang, W., Li, B., Yan, L., Wen, T., 2013b. A functional NQO1 609C N T polymorphism and risk of hepatocellular carcinoma in a Chinese population. Tumour Biol. 34, 47–53.
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F. Liu et al. / Gene 564 (2015) 14–20
Paracchini, V., Raimondi, S., Gram, I.T., Kang, D., Kocabas, N.A., Kristensen, V.N., Li, D., Parl, F.F., Rylander-Rudqvist, T., Soucek, P., Zheng, W., Wedren, S., et al., 2007. Meta- and pooled analyses of the cytochrome P-450 1B1 Val432Leu polymorphism and breast cancer: a HuGE-GSEC review. Am. J. Epidemiol. 165, 115–125. Parkin, D.M., 2001. Global cancer statistics in the year 2000. Lancet Oncol. 2, 533–543. Perz, J.F., Armstrong, G.L., Farrington, L.A., Hutin, Y.J., Bell, B.P., 2006. The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. J. Hepatol. 45, 529–538. Sasco, A.J., Secretan, M.B., Straif, K., 2004. Tobacco smoking and cancer: a brief review of recent epidemiological evidence. Lung Cancer 45, S3–S9. Shimada, T., Fujii-Kuriyama, Y., 2004. Metabolic activation of polycyclic aromatic hydrocarbons to carcinogens by cytochromes P450 1A1 and 1B1. Cancer Sci. 95, 1–6. Shimada, T., Hayes, C.L., Yamazaki, H., Amin, S., Hecht, S.S., Guengerich, F.P., Sutter, T.R., 1996. Activation of chemically diverse procarcinogens by human cytochrome P450 1B1. Cancer Res. 56, 2979–2984. Shimada, T., Watanabe, J., Kawajiri, K., Sutter, T.R., Guengerich, F.P., Gillam, E.M., Inoue, K., 1999. Catalytic properties of polymorphic human cytochrome P450 1B1 variants. Carcinogenesis 20, 1607–1613. Shimada, T., Oda, Y., Gillam, E.M., Guengerich, F.P., Inoue, K., 2001. Metabolic activation of polycyclic aromatic hydrocarbons and other procarcinogens by cytochromes P450 1A1 and P450 1B1 allelic variants and other human cytochromes P450 in Salmonella typhimurium NM2009. Drug Metab. Dispos. 29, 1176–1182. Spink, D.C., Hayes, C.L., Young, N.R., Christou, M., Sutter, T.R., Jefcoate, C.R., Gierthy, J.F., 1994. The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on estrogen metabolism in MCF-7 breast cancer cells: evidence for induction of a novel 17 beta-estradiol 4hydroxylase. J. Steroid Biochem. Mol. Biol. 51, 251–258. Trubicka, J., Grabowska-Kłujszo, E., Suchy, J., Masojć, B., Serrano-Fernandez, P., Kurzawski, G., Cybulski, C., Górski, B., Huzarski, T., Byrski, T., Gronwald, J., Złowocka, E., Kładny, J.,
Banaszkiewicz, Z., Wiśniowski, R., Kowalska, E., Lubinski, J., Scott, R.J., 2010. Variant alleles of the CYP1B1 gene are associated with colorectal cancer susceptibility. BMC Cancer 10, 420. Wang, X., Zhang, X., Qiu, B., Tang, Y., Sun, H., Ji, H., Liu, Y., Shi, L., Song, G., Yang, Y., 2012. MDM2 SNP309T N G polymorphism increases susceptibility to hepatitis B virusrelated hepatocellular carcinoma in a northeast Han Chinese population. Liver Int. 32, 1172–1178. Wei, Q., Cheng, L., Amos, C.I., Wang, L.E., Guo, Z., Hong, W.K., Spitz, M.R., 2000. Repair of tobacco carcinogen-induced DNA adducts and lung cancer risk: a molecular epidemiologic study. J. Natl. Cancer Inst. 92, 1764–1772. Xie, Y., Liu, G.Q., Miao, X.Y., Liu, Y., Zhou, W., Zhong, D.W., 2012. CYP1B1 Leu432Val polymorphism and colorectal cancer risk among Caucasians: a meta-analysis. Tumour Biol. 33, 809–816. Xu, W., Zhou, Y., Hang, X., Shen, D., 2012. Current evidence on the relationship between CYP1B1 polymorphisms and lung cancer risk: a meta-analysis. Mol. Biol. Rep. 39, 2821–2829. Yuan, X., Zhou, G., Zhai, Y., Xie, W., Cui, Y., Cao, J., Zhi, L., Zhang, H., Yang, H., Zhang, X., Qiu, W., Peng, Y., et al., 2008. Lack of association between the functional polymorphisms in the estrogen -metabolizing genes and risk for hepatocellular carcinoma. Cancer Epidemiol. Biomarkers Prev. 17, 3621–3627. Yuen, M.F., Hou, J.L., Chutaputti, A., 2009. Hepatocellular carcinoma in the Asia pacific region. J. Gastroenterol. Hepatol. 24, 346–353. Zeidel, A., Beilin, B., Yardeni, I., Mayburd, E., Smirnov, G., Bessler, H., 2002. Immune response in asymptomatic smokers. Acta Anaesthesiol. Scand. 46, 959–964.
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Research paper
Polymorphisms of the CYP1B1 gene and hepatocellular carcinoma risk in a Chinese population Fei Liu a, Li-Mei Luo b, Yong-Gang Wei a,⁎, Bo Li a, Wen-Tao Wang a, Tian-Fu Wen a, Jia-Yin Yang a, Ming-Qing Xu a, Lv-Nan Yan a a b
Department of Liver Surgery & Liver Transplantation Center, West China Hospital of Sichuan University, 37 Guo Xue Road, Chengdu 610041, Sichuan Province, China Department of Clinical Immunological Laboratory, West China Hospital of Sichuan University, 37 Guo Xue Road, Chengdu 610041, Sichuan Province, China
a r t i c l e
i n f o
Article history: Received 25 December 2014 Received in revised form 10 March 2015 Accepted 15 March 2015 Available online 18 March 2015 Keywords: Hepatocellular carcinoma Susceptibility CYP1B1 Polymorphism Case–control Study
a b s t r a c t Background: CYP1B1 is a P450 enzyme which is involved in the activation of pro-carcinogens to carcinogens as well as estrogen metabolism. We hypothesized that genetic variants in CYP1B1 may modify individual susceptibility to hepatocellular carcinoma (HCC). Methods: To test this hypothesis, we evaluated the associations of three CYP1B1 single nucleotide polymorphisms (SNPs) and HCC risk in a case–control study of 468 HCC cases and 515 cancer-free controls in a Chinese population. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry method and direct DNA sequencing were performed to detect these polymorphisms. Results: In overall analysis, we found that only the variant G allele of rs1056836 was associated with a significantly increased risk of HCC among the three SNPs (rs10012, rs1056836 and rs1800440). Moreover, we found that the variant genotypes containing the G allele of rs1056836 were associated with a significantly increased risk of HCC among HbsAg-positive individuals (adjusted OR = 2.13, 95% CI = 1.18, 3.86), but not among HbsAgnegative individuals. When stratifying by smoking status, we found that the variant GG genotype increased a 13.97-fold (95% CI = 1.28, 152.94) risk of HCC among smokers. Furthermore, high risk for liver cirrhosispositive clinical status was exhibited in HCC patients with rs1056836 CG and GG genotypes as compared with CC homozygotes. For the other two SNPs, we did not find any significant evidence of association with HCC risk in any subgroup. Conclusion: This study suggests that CYP1B1 rs1056836 polymorphism may be an important factor contributing to increased susceptibility and pathological development of HCC in Chinese population. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Primary liver cancer is one of the most common solid cancers worldwide, the second most frequent cause of cancer death in men, and the sixth in women (Jemal et al., 2011). About 748,300 new liver cancer cases and 695,900 cancer deaths are estimated to have occurred in 2008 worldwide (Jemal et al., 2011). Hepatocellular carcinoma (HCC) is the major histological subtype among primary liver cancers, which accounted for 70% to 85% of the total liver cancer burden worldwide (Perz et al., 2006). It's noteworthy that more than 75% of these cases occur in the Asia-Pacific region (Yuen et al., 2009) and China alone accounts for 55% cases of HCC worldwide (Parkin, 2001). Etiologically, carcinogenesis of HCC is a complex, multi-step and multi-factor process, in
Abbreviations: Cytochrome P450 1B1, CYP1B1; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; OR, odds ratio; CI, confidence interval; SNP, single nucleotide polymorphisms; HWE, Hardy–Weinberg equilibrium; AFP, alpha fetoprotein. ⁎ Corresponding author. E-mail address: [email protected] (Y.-G. Wei).
http://dx.doi.org/10.1016/j.gene.2015.03.035 0378-1119/© 2015 Elsevier B.V. All rights reserved.
which many factors are implicated. As we know, chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) is the most well established environmental risk factor for HCC worldwide. However, only a fraction of HBsAg carriers eventually develop HCC and only 2.5% of HCV infected individuals develop HCC later in life (Bowen and Walker, 2005). Thus, host genetic factors may affect HCC development. Identification of genetic factors related to susceptibility to HCC would help elucidate the complex process of hepatocarcinogenesis and improve the scientific basis for preventive interventions. Cytochrome P450 1B1 (CYP1B1) is a key P450 enzyme implicated in the metabolism of exogenous and endogenous substrates (Paracchini et al., 2007). A variety of studies have demonstrated that the metabolism of polycyclic aromatic hydrocarbons and other procarcinogens through CYP1B1 may well lead to the activation of the carcinogenic compounds (Shimada et al., 2001; Shimada and Fujii-Kuriyama, 2004). Meanwhile, CYP1B1 also participated in the hydroxylation of 17β-estradiol at the 2-OH and 4-OH positions, which can then be oxidized to semiquinones and quinones (Spink et al., 1994). The two electrophilic metabolites, semiquinones and quinones, can form DNA
F. Liu et al. / Gene 564 (2015) 14–20
adducts which could potentially introduce mutations into the genome (Chakravarti et al., 2001; Trubicka et al., 2010). This undesirable outcome alters the risk of malignancy by increasing the likelihood of introducing mutations into the genome (Trubicka et al., 2010). CYP1B1 gene is located on chr2p22-p21 and there are at least 179 different polymorphism sites in the gene (http://ncbi.nlm.nih.gov/ dbSNP). A number of single-nucleotide polymorphisms (SNPs) in this gene have been shown to affect the activity of the encoded protein (Bailey et al., 1998; Shimada et al., 1999). Three polymorphisms, occurring at codons rs10012 (48 C N G), rs1056836 (432 C N G) and rs1800440 (453 A N G), all of which result in single amino acid substitutions that result in an altered enzyme activity are of particular interest (Shimada et al., 1999). To predict the risk and prognosis of cancer, genotyping of these SNPs might provide a simple and valuable method. Although contributions of the CYP1B1 to the formation of many types of cancer are well known, its possible association with prediction of risk of HCC remains poorly investigated. In this study, the relationship between the three SNPs of CYP1B1 gene and HCC risk was investigated, and the impact of these SNPs on susceptibility and clinicopathological characteristics of HCC was also evaluated. 2. Materials and methods 2.1. Study population The detailed methods of this case–control study have been described previously (Liu et al., 2013a,b). Briefly, 500 patients with newly diagnosed, untreated HCC were enrolled in this study at the West China Hospital of Sichuan University from October 2007 to December 2011. The diagnosis of HCC cases included a combination of pathological examination, ultrasound and clinical manifestations. The patients who reportedly had previous cancer, other metastasized cancers and previous radiotherapy or chemotherapy were excluded. All subjects were genetically unrelated ethnic Han Chinese from Chengdu City and surrounding regions. Meanwhile, 550 cancer-free control subjects were recruited during the same period from the health examination individuals in West China Hospital. The controls were not blood relatives of the patients or each other and were frequency-matched with the cases by age at enrollment, sex, and residential area. Informed consents were obtained according to the Declaration of Helsinki. A written informed consent was obtained from each subject involved in the study. After informed consent was obtained, each subject was personally interviewed by trained interviewers using a pretested questionnaire, to obtain information on demographic data, lifestyles (alcohol consumption and cigarette smoking), and family history of cancer in first degree relatives (parents, siblings or children). After interview, a 5-mL venous blood sample was collected from each subject. The laboratory examinations, such as, serum alpha fetoprotein (AFP) levels, and hepatitis B virus (HBV) serological markers, were also collected from each subject. The study was approved by the Ethics Committee of Sichuan University. 2.2. DNA extraction Blood samples (5 mL) were collected in EDTA tubes after interview, and genomic DNA was isolated from whole blood samples using the whole blood DNA kit (Biotake corporation). DNA was extracted from 200 μL of the whole blood according to the manufacturer's protocol. The concentration of DNA was diluted to 20 ng/μL for working solutions and the isolated DNA was stored at −20 °C.
15
base extension were designed by using Assay Designer software package (Sequenom). Briefly, the DNA sample to be queried was diluted to 5 ng/μL, and 1 μL of DNA was combined with 0.95 μL of water, 0.625 μL of PCR buffer containing 15 mM MgCl2, 1 μL of 2.5 mM dNTP, 0.325 μL of 25 mM MgCl2, 1 μL of PCR primers and 0.1 μL of 5 units/μL HotStar Taq (Qiagen). The reaction was incubated at 94 °C for 15 min followed by 45 cycles at 94 °C for 20 s, 56 °C for 30 s, and 72 °C for 1 min, and a final incubation at 72 °C for 3 min. After PCR amplification, remaining dNTPs were dephosphorylated by adding 1.53 μL of water, 0.17 μL of SAP buffer, and 0.3 units of shrimp alkalinephosphatase (Sequenom). The reaction was placed at 37 °C for 40 min, and the enzyme was deactivated by incubating at 85 °C for 5 min. After shrimp alkaline phosphatase treatment, the single primer extension over the SNP was combined with 0.755 μL of water, 0.2 μL of 10× iPLEX buffer, 0.2 μL of termination mix, 0.041 μL of iPLEX enzyme (Sequenom), 0.804 μL of 10 μM extension primer. The single base extension reaction was carried out at 94 °C for 30 s and then 94 °C for 5 s, followed by 5 cycles of 52 °C for 5 s and 80 °C for 5 s, total 40 cycles, then 72 °C for 3 min. The reaction mix was desalted by adding 6 mg of cation exchange resin (Sequenom), mixed and resuspended in 25 μL of water. The completed genotyping reactions were spotted onto a 384 well spectroCHIP (Sequenom) using MassARRAY Nanodispenser (Sequenom) and determined by the matrixassisted laser desorption–ionization time-of-flight mass spectrometer. Genotype calling was performed in real time with MassARRAY RT software version 3.0.0.4 and analyzed using the MassARRAY Typer software version 3.4 (Sequenom). For quality control, genotyping was performed without knowing the subjects' case and control status, and a 5% random sample of cases and controls was genotyped twice for each locus. If a consensus on the tested genotype was not reached, two research assistants independently performed the repeated assays to achieve 100% concordance. To further validate the genotyping assay of MALDI-TOF MS for the three loci, about 5% of samples genotyped with MALDI-TOF MS were further confirmed by direct sequencing method using an automated sequencer (ABI 3730, Applied Biosystems). Among the 500 cases and 550 controls with DNA samples, the genotyping was successful for all three SNPs in 468 HCC cases and 515 controls, resulting in the overall success rate of 93.6%. Therefore, a total of 468 cases and 515 controls were included in the final analyses. 2.4. Statistical analysis Differences in the distributions of demographic characteristics, selected variables and genotype frequencies in the cases and controls were evaluated using the chi-square test or Fisher's exact test, since sample size was small for some subgroup analysis. The associations between CYP1B1 genotypes and risk of HCC were estimated by computing the odds ratios (ORs) and their 95% confidence intervals (CIs) from both univariate and multivariate logistic regression analyses with adjustment for age, sex, HBV carrier state, alcohol intake, smoking status, and family history of cancer. Hardy–Weinberg equilibrium was tested by a goodness-of-fit χ2 test, to compare the observed genotype frequencies to the expected ones among the control subjects. All statistical analyses were two sided and performed using SPSS version 16.0 for Windows statistical software (SPSS Inc., Chicago, IL, USA). A P-value of b0.05 was considered as statistically significant. 3. Results 3.1. Characteristics of studies
2.3. Genotype analyses SNP genotyping was performed using MassARRAY system (Sequenom, San Diego, CA, USA) by means of matrix assisted laser desorption ionization-time of flight mass spectrometry method (MALDI-TOF MS) according to the manufacturer's instructions. Primers for PCR and single
The demographic characteristics and risk factors in patients with HCC and controls are presented in Table 1. No significant differences between the two groups in terms of age and gender distribution (P = 0.45 for age and P = 0.86 for sex) suggested that matching of subjects based on these variables was adequate. There was no significant difference
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F. Liu et al. / Gene 564 (2015) 14–20
Table 1 Distributions of demographical characteristics and selected variables among HCC patients and controls. Variable
Age [n (%)] b55 years ≥55 years Gender [n (%)] Female Male Smoking status [n (%)] Never Ever Drinking status [n (%)] Never Ever HBV carrier state [n (%)] HbsAg (−) HbsAg (+) Family history of cancer [n (%)] No Yes
HCC
Controls
(n = 468)
(n = 515)
OR (95%CI)
266 (56.8) 202 (43.2)
305(59.2) 210(40.8)
1.00 1.10 (0.86, 1.42)
123 (26.3) 345 (73.7)
138(26.8) 377 (73.2)
1.00 1.03 (0.77, 1.36)
214 (45.7) 254 (54.3)
339 (65.8) 176 (34.2)
1.00 2.29 (1.77, 2.96)
338 (72.2) 130 (27.8)
394 (76.5) 121 (23.5)
1.00 1.25 (0.94, 1.67)
151 (32.3) 317 (67.7)
431 (83.7) 84 (16.3)
1.00 10.77 (7.95, 14.59)
P-value
0.45
0.86
b0.001
0.12
b0.001
0.64 424 (90.6) 44 (9.4)
471 (91.5) 44 (8.5)
1.00 1.11 (0.72, 1.72)
Abbreviations: HCC—hepatocellular carcinoma; OR—odds ratio; CI—confidence interval; HBV—hepatitis B virus; HBsAg—hepatitis B surface antigen.
between cases and controls in the distribution of drinking status and family history of cancer. As shown in Table 1, smoking is a risk factor of HCC (P b 0.001). HbsAg-positive was also associated with significantly increased HCC risk (P b 0.001). 3.2. Genotypes and HCC risk We could detect three genotypes for the CYP1B1 rs10012 and rs1056836 polymorphisms, including CC, CG and GG, among the
Chinese population. Fig. 1 shows the three genotypes for rs1056836 polymorphism (detecting by MALDI-TOF MS). However, for the CYP1B1 1800440 polymorphism, we could only detect two genotypes (AG and AA) among the Chinese population. The genotype/allele distributions of CYP1B1 rs10012, rs1056836 and rs1800440 in the cases and the controls are shown in Table 2. The distributions of these genotype frequencies in controls were all in Hardy–Weinberg equilibrium except for rs10012 (P = 0.01, 0.48 and 0.77 for CYP1B1 rs10012, rs1056836 and rs1800440, respectively). According to the crude ORs with their 95% CIs for HCC of CYP1B1 gene polymorphisms, rs1056836 GG and CG/GG exhibited a significant higher risk of 3.23-fold (95% CI, 1.25, 8.35) and 1.43-fold (95% CI, 1.08, 1.90), respectively, to have HCC compared with the wild-type homozygote. However, the association disappeared after adjusting for other confounding factors. Nevertheless, the variant G allele of rs1056836 was associated with a significantly increased risk of HCC in both crude and adjust OR. In addition, the other two SNPs rs10012 and rs1800440 did not provide any significant evidence of association with HCC risk for both crude OR and adjust OR. The distributions of smoking status and HBV carrier state in cases and control groups were obviously screwy, indicating that subgroup analysis by the two factors was needed. When stratifying by HBV carrier state, we found that the variant genotypes of CYP1B1 rs1056836 were associated with a significantly increased risk of HCC among HbsAg-positive individuals but not HbsAg-negative people [adjusted OR (95% CI) = 1.89 (1.03, 3.48) for CG vs CC and 2.13 (1.18, 3.86) for CG/GG vs CC] (Table 3). We further analyzed the effects of these polymorphisms on HCC risk stratified by smoking status. As shown in Table 4, we found that only the variant genotype GG of CYP1B1 rs1056836 was associated with a significantly increased risk of HCC among smokers [adjusted OR (95% CI) = 13.97 (1.28, 152.94)] and but not among nonsmokers. To explore the impact of polymorphic genotype of CYP1B1 gene on susceptibility and pathological development of HCC, HCC patients were further classified into two subgroups, one subgroup with at least one polymorphic allele and the other subgroup with homozygous
Fig. 1. Genotyping of CYP1B1 rs1056836 polymorphism by MALDI-TOF. The horizontal axis represents the mass and the vertical axis represents the signal intensity. Around the 6000 Da in the horizontal axis, two lines (the C allele and the G allele) can be observed. If only a wave crest appeared at the C line, the genotype of this patient was CC (the left one); if only a wave crest appeared at the G line, the genotype of this patient was GG (the right one); if two wave crest appeared at both the C line and the G line, the genotype of this patient was CG (the middle one).
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17
Table 2 CYP1B1 genotype and allele frequencies of the cases and controls and their association with risk of HCC. Polymorphism CYP1B1
Genotypes/allele
HCC (n = 468)
Controls (n = 515)
Crude OR 95%CI
Adjusted ORa 95%CI
Rs10012
CC CG GG CG and GG C G CC CG GG CG and GG C G AA AG GG AG and GG A G
316 (67.5) 133 (28.4) 19 (4.1) 152 (32.5) 765 (81.7) 171 (18.3) 326 (69.7) 126 (26.9) 16 (3.4) 142 (30.3) 778 (83.1) 158 (16.9) 462 (98.7) 6 (1.3) 0 (0.0) 6 (1.3) 930 (99.4) 6 (0.6)
338 (65.7) 147 (28.5) 30 (5.8) 177 (34.3) 823 (79.9) 207 (20.1) 395 (76.7) 114 (22.1) 6 (1.2) 120 (23.3) 904 (87.8) 126 (12.2) 502 (97.5) 13 (2.5) 0 (0.0) 13 (2.5) 1017 (98.7) 13 (1.3)
1.00 0.96 [0.73, 1.28] 0.68 [0.37, 1.23] 0.92 [0.70, 1.20] 1.00 0.89 [0.71, 1.11] 1.00 1.34 [1.00, 1.80] 3.23 [1.25, 8.35] 1.43 [1.08, 1.90] 1.00 1.46 [1.13, 1.88] 1.00 0.50 [0.19, 1.33] – 0.50 [0.19, 1.33] 1.00 0.51 [0.19, 1.33]
1.00 0.93 [0.67, 1.30] 0.59 [0.29, 1.19] 0.87 [0.63, 1.19] 1.00 0.83 [0.64, 1.08] 1.00 1.24 [0.87, 1.75] 2.59 [0.88, 7.60] 1.30 [0.93, 1.83] 1.00 1.36 [1.08, 1.80] 1.00 0.33 [0.11, 1.04] – 0.33 [0.11, 1.04] 1.00 0.34 [0.13, 1.10]
Rs1056836
Rs1800440
P bvalue
0.67 0.14 0.39 0.28 0.24 0.08 0.13 0.002 0.06 – 0.06 0.14
The figures given in bold indicate statistically significant values. a Adjusted for age, gender, HBV carrier state, family history of cancer, smoking, and drinking status. b P value for adjusted OR and 95% CI.
wild-type alleles, to estimate the adjusted ORs with their 95% CIs for HCC of each CYP1B1 SNP. Adjusted ORs and their 95% CIs for the pathological characteristics of each SNP of CYP1B1 gene in HCC patients suggest that no significant differences between rs10012 and rs1800440 genotypic frequencies and any clinical pathological variables (data not shown), but a high risk of 1.63-fold (95% CI = 1.01, 2.61) for having liver cirrhosis appeared in HCC patients with rs1056836 CG and GG genotypes as compared with patients with rs1056836 CC genotype (Table 5). 4. Discussion In the present study, we examined the association between three CYP1B1 SNPs and the risk of HCC in Chinese Han patients. In the overall analysis, we found that only the variant G allele of rs1056836 was associated with a significantly increased risk of HCC among the three SNPs. When stratifying by HBV carrier status, we found that the variant genotypes containing the G allele of rs1056836 was associated with a
significantly increased risk of HCC among HbsAg-positive individuals. When stratifying by smoking status, we found that the variant GG genotype increased a 13.97-fold risk of HCC among smokers. Furthermore, high risk for liver cirrhosis clinical status was exhibited in HCC patients with rs1056836 CG and GG genotypes as compared with CC homozygotes. Although the exact biological mechanism remains to be explored, these findings provide some evidence that CYP1B1 rs1056836 polymorphism may be an important factor contributing to increased susceptibility and pathological development of HCC. The exact mechanism for the correlation between the SNP of CYP1B1 and the risk of HCC needs further exploring. However, previous published studies on the functions of the CYP1B1 gene together with their genetic variants may help us understand the potential roles of these polymorphisms. CYP1B1 is a key P450 enzyme implicated in the metabolism of exogenous and endogenous substrates (Paracchini et al., 2007). Among exogenous substrates, CYP1B1 is implicated in the metabolic activation of a number of environmental carcinogens, such as arylamines, heterocyclic amines, benzo(a)pyrene, and polycyclic aromatic hydrocarbons
Table 3 Stratified analyses between CYP1B1 polymorphisms and HCC risk by HBV carrier status. Gene variable
HBV carrier status HbsAg (+)
HbsAg (−) a
HCC n (%)
Control n (%)
Adjusted ORa
P
0.54 0.13 0.28
102 (67.5) 43 (28.5) 6 (4.0) 49 (32.5)
285 (66.1) 123 (28.5) 23 (5.4) 146 (33.9)
1.00 0.94 (0.61, 1.43) 0.76 (0.29, 1.96) 0.91 (0.61, 1.36)
0.76 0.56 0.65
1.00 1.89 (1.03, 3.48) 7.83 (0.95, 64.91) 2.13 (1.18, 3.86)
0.04 0.06 0.01
119 (78.8) 30 (19.9) 2 (1.3) 32 (21.2)
328 (76.1) 98 (22.7) 5 (1.2) 103 (23.9)
1.00 0.92 (0.58, 1.47) 1.22 (0.23, 6.54) 0.94 (0.59, 1.48)
0.73 0.82 0.78
1.00 0.27 (0.07, 1.07) – 0.27 (0.07, 1.07)
0.06 – 0.06
150 (99.3) 1 (0.7) 0 (0.0) 1 (0.7)
422 (97.9) 9 (2.1) 0 (0.0) 9 (2.1)
1.00 0.38 (0.05, 3.04) – 0.38 (0.05, 3.04)
HCC n (%)
Control n (%)
Adjusted OR
Rs10012 CC CG GG CG and GG
214 (67.5) 90 (28.4) 13 (4.1) 103 (32.5)
53 (63.1) 24 (28.6) 7 (8.3) 31 (36.9)
1.00 0.84 (0.48, 1.48) 0.46 (0.17, 1.27) 0.75 (0.45, 1.26)
Rs1056836 CC CG GG CG and GG
207 (65.3) 96 (30.3) 14 (4.4) 110 (34.7)
67 (79.8) 16 (19.0) 1 (1.2) 17 (20.2)
Rs1800440 AA AG GG AG and GG
312 (98.4) 5 (1.6) 0 (0.0) 5 (1.6)
80 (95.2) 4 (4.8) 0 (0.0) 4 (4.8)
The figures given in bold indicate statistically significant values. a Adjusted for age, gender, family history of cancer, smoking, and drinking status.
P
0.36 0.36
18
F. Liu et al. / Gene 564 (2015) 14–20
Table 4 Stratified analyses between CYP1B1 polymorphisms and HCC risk by smoking status. Gene variable
Smoking Status Smokers
Nonsmokers
HCC n (%)
Control n (%)
Adjusted ORa
P
HCC n (%)
Control n (%)
Adjusted ORa
P
Rs10012 CC CG GG CG and GG
170 (66.9) 71 (28.0) 13 (5.1) 84 (33.1)
112 (63.6) 51 (29.0) 13 (7.4) 64 (36.4)
1.00 0.98 (0.58, 1.65) 0.66 (0.25, 1.77) 0.92 (0.57, 1.49)
0.94 0.41 0.73
146 (68.2) 62 (29.0) 6 (2.8) 68 (31.8)
226 (66.7) 96 (28.3) 17 (5.0) 113 (33.3)
1.00 0.88 (0.56, 1.39) 0.54 (0.19, 1.60) 0.84 (0.54, 1.29)
0.59 0.27 0.42
Rs1056836 CC CG GG CG and GG
175 (68.9) 70 (27.6) 9 (3.5) 79 (31.1)
142 (80.7) 33 (18.8) 1 (0.5) 34 (19.3)
1.00 1.39 (0.79, 2.42) 13.97 (1.28, 152.94) 1.59 (0.92, 2.74)
0.25 0.03 0.10
151 (70.6) 56 (26.2) 7 (3.2) 63 (29.4)
253 (74.6) 81 (23.9) 5 (1.5) 86 (25.4)
1.00 1.08 (0.68, 1.71) 1.64 (0.46, 5.87) 1.10 (0.70, 1.73)
0.76 0.45 0.67
Rs1800440 AA AG GG AG and GG
251 (98.8) 3 (1.2) 0 (0.0) 3 (1.2)
172 (97.7) 4 (2.3) 0 (0.0) 4 (2.3)
1.00 0.20 (0.04, 1.13) – 0.20 (0.04, 1.13)
0.07 – 0.07
211 (98.6) 3 (1.4) 0 (0.0) 3 (1.4)
330 (97.3) 9 (2.7) 0 (0.0) 9 (2.7)
1.00 0.43 (0.10, 1.92) – 0.43 (0.10, 1.92)
0.27 0.27
The figures given in bold indicate statistically significant values. a Adjusted for age, gender, HBV carrier state, family history of cancer, and drinking status.
(Shimada et al., 2001; Kim et al., 1998). The change in amino acid from valine to leucine (causing by CYP1B1 polymorphism) has been shown to increase the activity of the CYP1B1 enzyme on a variety of substrates, including procarcinogens and gonadal steroid hormones (Shimada et al., 1999). Moreover, for the polymorphism C432G at codon 432 of exon 3 (rs1056836), it was reported that the 432 G allele increased the mutation of p53 (Ko et al., 2001). Therefore, it is reasonable to conceive that CYP1B1 rs1056836 polymorphism may affect the metabolism of environmental carcinogens and increase the mutation of p53 to alter susceptibility to
HCC. Among endogenous substrates, CYP1B1 is thought to have an important role in estrogen metabolism, which is mainly involved in the hydroxylation of 17β-estradiol at the 2-OH and 4-OH positions (Spink et al., 1994). Meanwhile, previous studies (Shimada et al., 1996, 1999; Hanna et al., 2000) reveal that the generation of 2-OH catechol estrogens does not appear to result in deleterious affects whereas the 4-OH catechol derivative has been shown to be associated with an increase in DNA single strand breaks. The rs1056836 polymorphism (Leu-Val transition at codon 432) of CYP1B1, which is located in the heme-binding domain of
Table 5 Adjusted OR and 95% CI of clinicopathological variables and CYP1B1 genotypic frequencies in HCC patients. Variable
CYP1B1 432 C/G, Rs1056836 CC (N = 326) n (%)
CG/GG (N = 142) n (%)
OR (95% CI)
Adjusted OR (95% CI)a
Pb value
Classification of TNM Stage I/II Stage III/IV
167 (51.2) 159 (48.8)
80 (56.3) 62 (43.7)
1.00 0.81 (0.55, 1.21)
1.00 0.82 (0.55, 1.23)
0.34
Tumor size ≤5 cm N5 cm
166 (50.9) 160 (49.1)
70 (49.3) 72 (50.7)
1.00 1.07 (0.72, 1.58)
1.00 1.13 (0.75, 1.70)
0.57
Tumor number Single Multiple
196 (60.1) 130 (39.9)
92 (64.8) 50 (35.2)
1.00 0.82 (0.54, 1.23)
1.00 0.84 (0.55, 1.27)
0.40
Lymph node metastasis No Yes
257 (78.8) 69 (21.2)
122 (85.9) 20 (14.1)
1.00 0.61 (0.36, 1.05)
1.00 0.61 (0.35, 1.06)
0.08
Distant metastasis No Yes
289 (88.7) 37 (11.3)
134 (94.4) 8 (5.6)
1.00 0.47 (0.21, 1.03)
1.00 0.49 (0.22, 1.09)
0.08
Portal vein tumor thrombus No Yes
269 (82.5) 57 (17.5)
115 (81.0) 27 (19.0)
1.00 1.11 (0.67, 1.84)
1.00 1.12 (0.67, 1.89)
0.67
AFP Not increased Increased
167 (51.2) 159 (48.8)
59 (41.5) 83 (58.5)
1.00 1.48 (0.99, 2.20)
1.00 1.36 (0.89, 2.06)
0.16
Liver cirrhosis Negative Positive
116 (35.6) 210 (64.4)
34 (23.9) 108 (76.1)
1.00 1.76 (1.12, 2.74)
1.00 1.63 (1.01, 2.61)
0.04
The figures given in bold indicate statistically significant values. a Adjusted for age, gender, HBV carrier state, family history of cancer, smoking, and drinking status. b Pvalue for adjusted OR and 95% CI.
F. Liu et al. / Gene 564 (2015) 14–20
exon 3, causes the enzyme display a higher 4-hydroxylation of estradiol (Li et al., 2000). Therefore, it is also possible that the CYP1B1 rs1056836 polymorphism is involved in hepatocarcinogenesis by increasing the likelihood of introducing mutations into the genome. Given the different activities of the CYP1B1 enzyme which strongly depends on the polymorphic form, it is biologically plausible that the CYP1B1 rs1056836 polymorphism may modulate the risk of HCC in Chinese Han population. Several studies, including meta-analysis, have examined the relationship between individual CYP1B1 polymorphisms and cancer risk, however, the results are conflicting. Yuan et al. (2008) have reported that CYP1B1 Val432Leu polymorphisms may not contribute significantly to the risk for HCC. Cui et al. (2012) included ten case-controlled studies to quantitatively summarize the association between CYP1B1 Leu432Val polymorphism and prostate cancer and found that CYP1B1 Leu432Val polymorphism was not associated with prostate cancer risk overall. Similarly, Xie et al. (2012) revealed that no association was found between the CYP1B1 Leu432Val polymorphism and risk of colorectal cancer among Caucasians. In contrast, in agreement with our present results, some reports also showed association between certain CYP1B1 polymorphism and risk for cancer. For instance, in recent, Li et al. (2013) found that CYP1B1 C4326G polymorphism may increase risk of cervical cancer in Chinese women, especially among young individuals with high-risk HPV infection. Xu et al. (2012) have reported that the CYP1B1 432GG, 119TT and 48GG genotypes were low-penetrance risk factors for developing lung cancer. Paracchini et al. (2007) assessed thirteen articles (7514 cases and 6817 controls) on the association between the CYP1B1 Leu432Val polymorphism and breast cancer risk and found that the CYP1B1 Leu432Val polymorphism was associated with an increased breast cancer risk among Caucasians. The conflicting results could be attributable to the differences in cancer types, demography, ethnicity, lifestyles, type of viral infections, and clinical settings. In addition, other methodologic factors in the studies, such as small sample size, inadequate adjustment for confounding factors, or lack of correction for multiple testing, could also cause the inconsistent results. As we know, chronic infection with HBV or HCV is the most well established environmental risk factor for HCC worldwide. In China, HBV infection is a major risk factor for developing HCC (Wang et al., 2012). In our study, the HBV infection rate was 67.7% in the HCC group, which was significantly higher than that of control group (16.3%). Moreover, stratification analysis by HBV carrier status, we found that the variant genotypes (CG + GG) of the CYP1B1 rs1056836 were associated with a significantly increased risk of HCC among HbsAg-positive individuals (adjusted OR = 2.13, 95% CI = 1.18–3.86), but not among those with HbsAg-negative, which indicated that variant genotypes of CYP1B1 and HBV infection interaction may influence the susceptibility to HCC development in the Chinese population. Tobacco smoking is an established risk factor for many cancers, including HCC (Cohen et al., 2000; Wei et al., 2000; Sasco et al., 2004), and has a destructive effect on human immune responses (Zeidel et al., 2002). Moreover, cigarette smoke contains a variety of carcinogenic compounds, such as polycyclic aromatic hydrocarbons, which are mainly metabolized by phase I and phase II xenobiotic metabolizing enzymes (Xu et al., 2012). A highly studied phase I gene is the CYP1B1, which plays a significant role in the oxidation of variety of carcinogens, including PAHs and arylamines (Shimada et al., 1999). Thus, smoking may interact with CYP1B1 polymorphisms to initiate and promote hepatocarcinogenesis. In the present study, the smoking rate was 54.3% in the HCC group, which was significantly higher than that of control group (34.2%). Moreover, stratification analysis by smoking status, we found that the variant genotype GG of the CYP1B1 rs1056836 increased a 13.97-fold risk of HCC among smokers, but not among non-smokers, which indicated that variant genotypes of CYP1B1 and smoking interaction may influence the susceptibility to HCC development in the Chinese population. The genotype distribution of CYP1B1 rs10012 deviated from Hardy– Weinberg proportions in controls (P = 0.01); however, the concordance
19
rate for the quality control samples (5% of the total sample size), which were randomly selected from cases and controls, was 100% for all the three polymorphisms, including the CYP1B1 rs10012. Therefore, we do not believe that the deviation from Hardy–Weinberg equilibrium for this polymorphism is due to genotyping error. In this case–control study, several limitations need to be addressed. First, the findings from the present study were only from a Chinese southwest population, so it is uncertain whether these results are generalizable to the general population of other ethnics. Second, the determination of the exact functional influence of the CYP1B1 variant allele was not performed in our study. Further studies on potential mechanisms are needed to elucidate how this CYP1B1 rs1056836 C → G substitution modifies HCC development. Finally, although this is a hospital based case control study, the selection bias may not be avoidable and the subjects may not be representative of the general population. However, potential confounding bias might have been minimized by matching the controls to the cases on age, sex, residential area and by further adjustment for the confounding factors in data analyses. In conclusion, our study demonstrated that CYP1B1 rs1056836 polymorphism might be an important factor contributing to increased susceptibility and pathological development of HCC in Chinese population. Validation of these findings with functional evaluation and larger studies with more rigorous study designs and inclusion of different ethnic populations are needed. Conflict of interest statement None declared. Acknowledgments This work was supported by grant from the Application Foundation Project of Science and Technology Agency in Sichuan Province (No 2012JY0079). References Bailey, L.R., Roodi, N., Dupont, W.D., Parl, F.F., 1998. Association of cytochrome P450 1B1 (CYP1B1) polymorphism with steroid receptor status in breast cancer. Cancer Res. 58, 5038–5041. Bowen, D.G., Walker, C.M., 2005. Adaptive immune responses in acute and chronic hepatitis C virus infection. Nature 436, 946–952. Chakravarti, D., Mailander, P.C., Li, K.M., Higginbotham, S., Zhang, H.L., Gross, M.L., Meza, J.L., Cavalieri, E.L., Rogan, E.G., 2001. Evidence that a burst of DNA depurination in SENCAR mouse skin induces error-prone repair and forms mutations in the H-ras gene. Oncogene 20, 7945–7953. Cohen, S.M., Shirai, T., Steineck, G., 2000. Epidemiology and etiology of premalignant and malignant urothelial changes. Scand. J. Urol. Nephrol. Suppl. 205, 105–115. Cui, L., Dillehay, K., Chen, W., Shen, D., Dong, Z., Li, W., 2012. Association of the CYP1B1 Leu432Val polymorphism with the risk of prostate cancer: a meta-analysis. Mol. Biol. Rep. 39, 7465–7471. Hanna, I.H., Dawling, S., Roodi, N., Guengerich, F.P., Parl, F.F., 2000. Cytochrome P450 1B1 (CYP1B1) pharmacogenetics: association of polymorphisms with functional differences in estrogen hydroxylation activity. Cancer Res. 60, 3440–3444. Jemal, A., Bray, F., Center, M.M., Ferlay, J., Ward, E., Forman, D., 2011. Global cancer statistics. CA Cancer J. Clin. 61, 69–90. Kim, J.H., Stansbury, K.H., Walker, N.J., Trush, M.A., Strickland, P.T., Sutter, T.R., 1998. Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by human cytochrome P450 1B1. Carcinogenesis 19, 1847–1853. Ko, Y., Abel, J., Harth, V., Bröde, P., Antony, C., Donat, S., Fischer, H.P., Ortiz-Pallardo, M.E., Thier, R., Sachinidis, A., Vetter, H., Bolt, H.M., et al., 2001. Association of CYP1B1 codon 432 mutant allele in head and neck squamous cell cancer is reflected by somatic mutations of p53 in tumor tissue. Cancer Res. 61, 4398–4404. Li, D.N., Seidel, A., Pritchard, M.P., Wolf, C.R., Friedberg, T., 2000. Polymorphisms in P450 CYP1B1 affect the conversion of estradiol to the potentially carcinogenic metabolite 4-hydroxyestradiol. Pharmacogenetics 10, 343–353. Li, Y., Tan, S.Q., Ma, Q.H., Li, L., Huang, Z.Y., Wang, Y., Li, S.W., 2013. CYP1B1 C4326G polymorphism and susceptibility to cervical cancer in Chinese Han women. Tumour Biol. 34, 3561–3567. Liu, F., Wei, Y.G., Luo, L.M., Wang, W.T., Yan, L.N., Wen, T.F., Xu, M.Q., Yang, J.Y., Li, B., 2013a. Genetic variants of p21 and p27 and hepatocellular cancer risk in a Chinese Han population: a case-control study. Int. J. Cancer 132, 2056–2064. Liu, F., Luo, L., Wei, Y., Wang, W., Li, B., Yan, L., Wen, T., 2013b. A functional NQO1 609C N T polymorphism and risk of hepatocellular carcinoma in a Chinese population. Tumour Biol. 34, 47–53.
20
F. Liu et al. / Gene 564 (2015) 14–20
Paracchini, V., Raimondi, S., Gram, I.T., Kang, D., Kocabas, N.A., Kristensen, V.N., Li, D., Parl, F.F., Rylander-Rudqvist, T., Soucek, P., Zheng, W., Wedren, S., et al., 2007. Meta- and pooled analyses of the cytochrome P-450 1B1 Val432Leu polymorphism and breast cancer: a HuGE-GSEC review. Am. J. Epidemiol. 165, 115–125. Parkin, D.M., 2001. Global cancer statistics in the year 2000. Lancet Oncol. 2, 533–543. Perz, J.F., Armstrong, G.L., Farrington, L.A., Hutin, Y.J., Bell, B.P., 2006. The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. J. Hepatol. 45, 529–538. Sasco, A.J., Secretan, M.B., Straif, K., 2004. Tobacco smoking and cancer: a brief review of recent epidemiological evidence. Lung Cancer 45, S3–S9. Shimada, T., Fujii-Kuriyama, Y., 2004. Metabolic activation of polycyclic aromatic hydrocarbons to carcinogens by cytochromes P450 1A1 and 1B1. Cancer Sci. 95, 1–6. Shimada, T., Hayes, C.L., Yamazaki, H., Amin, S., Hecht, S.S., Guengerich, F.P., Sutter, T.R., 1996. Activation of chemically diverse procarcinogens by human cytochrome P450 1B1. Cancer Res. 56, 2979–2984. Shimada, T., Watanabe, J., Kawajiri, K., Sutter, T.R., Guengerich, F.P., Gillam, E.M., Inoue, K., 1999. Catalytic properties of polymorphic human cytochrome P450 1B1 variants. Carcinogenesis 20, 1607–1613. Shimada, T., Oda, Y., Gillam, E.M., Guengerich, F.P., Inoue, K., 2001. Metabolic activation of polycyclic aromatic hydrocarbons and other procarcinogens by cytochromes P450 1A1 and P450 1B1 allelic variants and other human cytochromes P450 in Salmonella typhimurium NM2009. Drug Metab. Dispos. 29, 1176–1182. Spink, D.C., Hayes, C.L., Young, N.R., Christou, M., Sutter, T.R., Jefcoate, C.R., Gierthy, J.F., 1994. The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on estrogen metabolism in MCF-7 breast cancer cells: evidence for induction of a novel 17 beta-estradiol 4hydroxylase. J. Steroid Biochem. Mol. Biol. 51, 251–258. Trubicka, J., Grabowska-Kłujszo, E., Suchy, J., Masojć, B., Serrano-Fernandez, P., Kurzawski, G., Cybulski, C., Górski, B., Huzarski, T., Byrski, T., Gronwald, J., Złowocka, E., Kładny, J.,
Banaszkiewicz, Z., Wiśniowski, R., Kowalska, E., Lubinski, J., Scott, R.J., 2010. Variant alleles of the CYP1B1 gene are associated with colorectal cancer susceptibility. BMC Cancer 10, 420. Wang, X., Zhang, X., Qiu, B., Tang, Y., Sun, H., Ji, H., Liu, Y., Shi, L., Song, G., Yang, Y., 2012. MDM2 SNP309T N G polymorphism increases susceptibility to hepatitis B virusrelated hepatocellular carcinoma in a northeast Han Chinese population. Liver Int. 32, 1172–1178. Wei, Q., Cheng, L., Amos, C.I., Wang, L.E., Guo, Z., Hong, W.K., Spitz, M.R., 2000. Repair of tobacco carcinogen-induced DNA adducts and lung cancer risk: a molecular epidemiologic study. J. Natl. Cancer Inst. 92, 1764–1772. Xie, Y., Liu, G.Q., Miao, X.Y., Liu, Y., Zhou, W., Zhong, D.W., 2012. CYP1B1 Leu432Val polymorphism and colorectal cancer risk among Caucasians: a meta-analysis. Tumour Biol. 33, 809–816. Xu, W., Zhou, Y., Hang, X., Shen, D., 2012. Current evidence on the relationship between CYP1B1 polymorphisms and lung cancer risk: a meta-analysis. Mol. Biol. Rep. 39, 2821–2829. Yuan, X., Zhou, G., Zhai, Y., Xie, W., Cui, Y., Cao, J., Zhi, L., Zhang, H., Yang, H., Zhang, X., Qiu, W., Peng, Y., et al., 2008. Lack of association between the functional polymorphisms in the estrogen -metabolizing genes and risk for hepatocellular carcinoma. Cancer Epidemiol. Biomarkers Prev. 17, 3621–3627. Yuen, M.F., Hou, J.L., Chutaputti, A., 2009. Hepatocellular carcinoma in the Asia pacific region. J. Gastroenterol. Hepatol. 24, 346–353. Zeidel, A., Beilin, B., Yardeni, I., Mayburd, E., Smirnov, G., Bessler, H., 2002. Immune response in asymptomatic smokers. Acta Anaesthesiol. Scand. 46, 959–964.