CYP1B1 C4326G polymorphism and susceptibility to cervical cancer in Chinese Han women
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- Li, Y., Tan, S., Ma, Q. et al. Tumor Biol. (2013) 34: 3561. doi:10.1007/s13277-013-0935-4
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Cytochrome P450 1B1 (CYP1B1) is a key P450 enzyme, which could catalyze the formation of 4-hydroxy estrogen metabolites and play a role in estrogen-dependent cancers. We hypothesized that genetic variant in CYP1B1 may modify individual susceptibility to cervical cancer. The aim of this study was to evaluate the association between CYP1B1 C4326G polymorphism and cervical cancer risk in Chinese women. We extracted the peripheral blood samples in 250 patients with cervical cancer and 250 female controls. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry method and direct DNA sequencing were performed to detect the polymorphism. The frequencies of CC, CG, and GG genotypes of CYP1B1 C4326G in cases and controls were 66.0, 26.8, 7.2 % and 75.2, 21.6, and 3.2 %, respectively, and there was a significant difference between the two groups (P = 0.034). Compared with the wild-type CC genotype, the variant GG genotype was associated with a significantly increased risk of cervical cancer (adjusted OR = 2.30; 95 % CI = 1.02, 5.50). Moreover, stratification analysis by age, smoking, drinking, human papillomaviruses (HPV) 16 or 18 carrier status, and family history of cervical cancer, we found that the variant genotypes containing the G allele were associated with a significantly increased risk of cervical cancer among HPV 16 or 18-positive individuals (adjusted OR = 2.85; 95 % CI = 1.45, 5.62) and among women younger than 45 years old (adjusted OR = 1.87; 95 % CI = 1.03, 3.37). These results suggest that CYP1B1 C4326G polymorphism may increase risk of cervical cancer in Chinese women, especially among young individuals with high-risk HPV infection.
KeywordsCervical cancerCYP1B1PolymorphismCase–control study
Cytochrome P450 1B1
Polymerase chain reaction
Single nucleotide polymorphisms
Cervical cancer is the third most commonly diagnosed cancer and the fourth leading cause of cancer death in females worldwide, accounting for 9 % (529,800) of the total new cancer cases and 8 % (275,100) of the total cancer deaths among females in 2008 . Etiologically, carcinogenesis of cervical cancer is a complex, multistep, and multifactor process, in which many factors are implicated. Although age of first intercourse, number of sexual partners, more parities, cigarette smoking, race, and low socioeconomic status consistently have been shown as risk factors for cervical cancer, none has been shown to be as a significant and independent risk factor. At present, it is widely accepted that infection of oncogenic human papillomaviruses (HPVs), predominantly HPV-16 and HPV-18, is the major risk factor for the development of cervical cancer [3, 4]. However, only a small proportion of such HPV-infected cases progresses to cervical cancer , which indicates that HPV infection is a necessary, but not sufficient, condition to develop cervical cancer, and that other factors associated with increased susceptibility, such as genetic background, might be needed. Recently, host genetic factors, especially the single nucleotide polymorphisms (SNPs), are considered to explain the individual differences of susceptibility to specific malignant neoplasms.
Cytochrome P450 1B1 (CYP1B1) is a key P450 enzyme implicated in the metabolism of exogenous and endogenous substrates . It plays an important role in activating a variety of carcinogens, such as PAHs or heterocyclic amines, to reactive metabolites that cause DNA damage. Also, CYP1B1 is implicated in the metabolism of estrogen . Such as, it encodes for an enzyme that catalyzes the formation of both 2- and 4-hydroxyestrone and plays a role in estrogen-dependent cancers [8, 9]. Moreover, Meyer et al.  found that inherited alterations in the activity of CYP1B1, which lead to differences in estrogen metabolism, may explain interindividual differences in endometrial cancer risk.
The CYP1B1 gene maps to chromosome 2p22–p21  and contains three exons and two introns . Meanwhile, CYP1B1 is a polymorphic gene in the human population, and there are at least 179 different polymorphism sites in the gene (http://ncbi.nlm.nih.gov/dbSNP). Of these, four are known to result in amino acid substitutions including codon Arg48Gly (rs10012), codon Ala119Ser (rs1056837), codon Leu432Val (4326 C > G, rs1056836), and codon Asn453Ser (rs1800440) [13, 14]. Importantly, these polymorphic variants have been associated with enhanced catalytic activity when compared to the wild-type allele [15, 16]; it has been postulated that this functional finding may confer susceptibility towards cancer at a certain extent . Especially, the Leu432Val polymorphism seems to have the largest impact on the catalytic properties of the enzyme; the Val432 allele displays threefold higher 4-hydroxylase activity than the Leu432 allele . To date, a number of case–control studies have been conducted to investigate the associations of the Leu432Val polymorphism with different types of cancer susceptibility, including colorectal cancer, breast cancer, endometrial cancer, and lung cancer [6, 18–20]. However, to the best of our knowledge, no studies have examined the effects of CYP1B1 Leu432Val variant on cervical cancer susceptibility. Given the important role that CYP1B1 plays in activating a variety of carcinogens, we hypothesized that polymorphism in CYP1B1 may modify individual susceptibility to cervical cancer. Therefore, we evaluated the association between the CYP1B1 Leu432Val polymorphism and cervical cancer risk in a hospital-based case–control study.
Materials and methods
This study includes 250 newly diagnosed patients with cervical cancer and 250 cancers-free controls. From January 2009 to February 2010, the patients with histologically confirmed primary cervical cancer were recruited in West China Second University Hospital of Sichuan University. Control subjects were randomly selected from healthy volunteer women, who underwent a routine cancer screening program for early detection of cervical cancer conducted in the same regions during the same period, when the case patients were recruited, and no evidence of cervical lesions was found by cytology test. The controls were frequency-matched with the cases by age at enrollment (within ±5 years) and residential area. All subjects were genetically unrelated ethnic Han Chinese from Chengdu City and surrounding regions. A written informed consent was obtained from each subject involved in the study according to the Declaration of Helsinki. 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 cervical cancer. After interview, a 5-ml venous blood sample was collected from each subject for DNA preparation. To detect high-risk HPV infection (types 16 and 18), DNA was obtained from cervical biopsies of the patients with cervical cancer and from exfoliated cervical cells of the control women. The research protocol was approved by the institutional review board of West China Second University Hospital.
White blood cells were separated from peripheral blood samples by washing three times in phosphate-buffered saline. Then, genomic DNA was extracted with phenol/chloroform and precipitated with 4 °C ethanol. The concentration of DNA was diluted to 15 ng/μL for working solutions, and the isolated DNA was stored at −20 °C.
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 according to the manufacturer's instructions. Primers for PCR and single-base extension were designed by using Assay Design 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 adding1.53 μl of water, 0.17 μl of SAP buffer, and 0.3 units of shrimp alkaline phosphatase (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 10X iPLEX buffer, 0.2 μl of termination mix, 0.041 μl of iPLEX enzyme (Sequenom), and 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 of 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 matrix-assisted laser desorption–ionization time-of-flight mass spectrometer. Genotype calling was performed in real time with MassARRAY RT software version 126.96.36.199 and analyzed using the MassARRAY typer software version 3.4 (Sequenom). For quality control, the genotyping analysis was done blind with regard to the subjects. The selected PCR-amplified DNA samples (5 % of the total samples) were also examined by DNA sequencing to confirm the genotyping results, and the results were 100 % concordant.
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. Differences in age, cigarette smoking, alcohol consumption, HPV carrier status, and family history between cervical cancer patients and controls were evaluated using the χ2 test. The association between the polymorphism and risk of development of cervical cancer was estimated by odds ratios (ORs) and their 95 % confidence intervals (CIs) calculated by unconditional logistic regression models. P < 0.05 was considered statistically significant. All analyses were done using SPSS version 16.0 for Windows statistical software (SPSS Inc., Chicago, IL, USA).
Characteristics of study subjects
Frequency distribution of selected variables among CC patients and controls
OR (95 %CI)
(n = 250)
(n = 250)
Age [n (%)]
1.03 (0.72, 1.47)
Cigarette smoking [n (%)]
1.59 (0.84, 3.01)
Alcohol consumption [n (%)]
0.81 (0.52, 1.27)
HPV-16 or -18 [n (%)]
6.89 (4.66, 10.21)
Family history of CC [n (%)]
1.11 (0.60, 2.05)
Distributions of CYP1B1 C4326G polymorphism
CYP1B1 C4326G genotype and allele frequencies of the cases and controls and their association with risk of CC
n = 250
n = 250
1.41 (0.93, 2.14)
1.18 (0.74, 1.88)
2.56 (1.09, 6.05)
2.30 (1.02, 5.50)
CG and GG
1.56 (1.06, 2.30)
1.29 (0.89, 2.00)
1.59 (1.14, 2.22)
1.32 (1.04, 2.08)
The association between CYP1B1 polymorphism and cervical cancer risk
Compared with the wild-type CC genotype, the variant GG genotype and genotypes containing the G allele (CG/GG) significantly increased the risk of developing cervical cancer; the crude OR was 2.56 (95 % CI = 1.09–6.05) and 1.56 (95 % CI = 1.06–2.30), respectively. Similarly, compared with the C allele, the variant G allele significantly increased the risk of developing cervical cancer (OR = 1.59; 95%CI = 1.14–2.22). However, multivariate logistic regression analyses revealed that only the variant GG genotype and the variant G allele significantly increased the risk of cervical cancer [adjusted OR = 2.30 (95 % CI = 1.02, 5.50) for GG vs. CC; 1.32 (95 % CI = 1.04, 2.08) for the allele G vs. C].
Associations and stratification analysis of CYP1B1 C4326G polymorphism and CC risk
n = 250
n = 250
CG + GG
CG + GG
CG + GG
2.50 (0.61, 9.46)
CG + GG
1.13 (0.71, 1.79)
CG + GG
2.54 (0.84, 7.69)
CG + GG
1.19 (0.73, 1.93)
HPV-16 or 18
CG + GG
2.85 (1.45, 5.62)
CG + GG
0.58 (0.28, 1.20)
Family history of CC
CG + GG
3.70 (0.59, 16.21)
CG + GG
1.25 (0.79, 1.97)
In the present study, we examined the association between the CYP1B1 C4326G polymorphism and the risk of cervical cancer. To the best of our knowledge, this is the first case–control study that has investigated the association of CYP1B1 C4326G polymorphism and cervical cancer risk. In this hospital-based case–control analysis among Chinese women, we found that CYP1B1 C4326G polymorphism was associated with an increased risk for development of cervical cancer. Furthermore, when subgroup analysis was performed, the significant association was intensified in groups with high-risk HPV infection (types 16 or 18) and among women younger than 45 years old. Although the exact biological mechanism remains to be explored, our findings suggest that the CYP1B1 C4326G polymorphism may play a role in the development of cervical cancer in Chinese Han women, especially among young individuals with high-risk HPV infection (types 16 or 18).
Although the exact reason for the correlation between SNP of CYP1B1 and the risk of cervical cancer needs further exploring, we could understand the potential roles of the polymorphism by previous published studies on the structure and functions of the CYP1B1 gene together with their genetic variants. The CYP1B1 gene maps to chromosome 2p22–p21  and contains three exons and two introns . The entire coding sequence is contained in exons 2 and 3; exon 3 has a domain for heme binding. In terms of functionality, CYP1B1 appears to be implicated in the metabolic activation of a number of environmental carcinogens, such as arylamines, heterocyclic amines, benzo(a)pyrene, and polycyclic aromatic hydrocarbons [21, 22]. The change in amino acid from valine to leucine has been shown to increase the activity of the CYP1B1 enzyme on a variety of substrates, including procarcinogens and gonadal steroid hormones . Moreover, for the polymorphism Leu432Val at codon 432 of exon 3, it was reported that the 432 G allele increased the mutation of p53 . Therefore, it is reasonable to conceive that CYP1B1 Leu432Val polymorphism may affect the metabolism of environmental carcinogens and increase the mutation of p53 to alter susceptibility to cervical cancer. In addition to activating procarcinogens in the metabolism of environmental carcinogens, CYP1B1 is also thought to have an important role in estrogen metabolism, catalyzing principally the formation of 4-hydroxyestradiol, a carcinogenic metabolite that retains estrogenic activity . Furthermore, the Leu-Val transition at codon 432 of CYP1B1, which is located in the heme-binding domain of exon 3, causes the enzyme to display a higher 4-hydroxylation of estradiol . This leads to the formation of carcinogenic estradiol-3, 4-semiquinones, and quinones. It is possible that the CYP1B1 is involved in cervix carcinogenesis through the estrogenic activity of the 4-OH-E2 [15–17]. Given the different activity of the CYP1B1 enzyme, which strongly depends on the polymorphic form, it is biologically plausible that the CYP1B1 Leu432Val polymorphism may modulate the risk of cervical cancer in Chinese Han women.
Our results were consistent with some previous published meta-analyses based on other cancer. For example, Xu et al.  assessed 10 case–control studies included 7,067 cases and 9,374 controls of the association between CYP1B1 SNPs of Leu432Val, Asn453Ser, Ala119Ser, Arg48Gly, and the risk of lung cancer. They found that the CYP1B1 432GG, 119TT, and 48GG genotypes are low-penetrance risk factors for developing lung cancer. Wang et al.  evaluated twelve studies (3,605 cases and 5,692 controls) on the association of CYP1B1 gene polymorphisms with endometrial cancer risk, and found that CYP1B1 gene R48G, L432V, and N453S polymorphisms were associated with endometrial cancer risk. Paracchini et al.  assessed thirteen articles (7,514 cases and 6,817 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. However, Cui et al.  included 10 case-controlled studies (3,221 cases and 3,447 controls) to quantitatively summarize the association between CYP1B1 Leu432Val polymorphism and prostate cancer, and found CYP1B1 Leu432Val polymorphism was not associated with prostate cancer risk overall. Similarly, Xie et al.  revealed that no association was found between the CYP1B1 Leu432Val polymorphism and risk of colorectal cancer among Caucasians. These inconsistent results may be due to differences between studies of cancer types, patient population, and different risk factors for various types of cancers. Other factors in the studies such as inclusion of different ethnic groups in a single study, gene–gene or gene–environment interactions, or inadequate adjustment for confounding factors could also cause the inconsistent results.
According to several epidemiological and experimental studies, infection with certain oncogenic types of HPV, predominantly HPV-16 and HPV-18, is the most important risk factor in carcinogenesis of the uterine cervix [3, 4], and HPV DNA is found in most of the cervical cancers . Therefore, we studied the role of high-risk HPV (16 or 18) infection in the development of cervical cancer in the present study. In our study, the high-risk HPV (16 or 18) infection rate was 71.2 % in the cervical cancer group, which was significantly higher than that of control group (26.4 %). Moreover, stratification analysis by HPV carrier status, we found that the variant genotypes (CG + GG) of the CYP1B1 C4326G were associated with a significantly increased risk of cervical cancer among high-risk HPV 16 or 18-positive individuals (adjusted OR = 2.85; 95 % CI = 1.45, 5.62), but not among those with HPV-negative, which indicated that variant genotypes of CYP1B1 and high-risk HPV infection interaction may influence the susceptibility to cervical cancer development in Chinese women.
HPV is thought to be an essential but not a sufficient cause of cervical cancer ; therefore, the other factors associated with increased susceptibility, such as genetic background and environmental factors, might be needed. Tobacco smoking has been reported as an epidemiological risk factor for uterine cervical cancer in many studies [28–30], and it may be one of the previously mentioned “environmental factors,” along with HPV in cervical carcinogenesis. However, the cervical cancer group and control group failed to show any significant difference in the distribution of cigarette smoking in this study. Moreover, when stratifying by cigarette smoking, we found that CYP1B1 C4326G polymorphism could not alter cervical cancer risk among both smokers and nonsmokers. The discrepancies between our results and the previous published results may stem from an inadequate sample size of this study.
Variation in allele frequency between different ethnic groups has been observed for CYP1B1 C4326G polymorphism. For example, the frequency of 4,326 G allele was varied from 0.378 to 0.558 in Caucasians [31, 32], and it was only 0.087–0.212 in Asians [8, 33]. The frequency of the 4,326 G allele was 0.140 in our controls, which is similar to 0.133 that was reported for Chinese Han population in the National Center for Biotechnology Information SNP database. This indicates that the samples in our hospital-based study are unlikely to produce significant bias in estimating the genotype-specific ORs. However, our results should be interpreted with caution because the frequency of 4,326 G allele was varied among different ethnic groups.
In this case–control study, several limitations need to be addressed. Firstly, the cervical cancer cases were enrolled from the hospitals, and the controls were selected from the cancer screening individuals, inherent selection bias could not be completely excluded. However, by matching on age and residential area, potential confounding factors might have been minimized. Secondly, determination of the exact functional influence of the CYP1B1 C4326G variant allele was not performed in our study. Further studies on potential mechanisms are needed to elucidate how this CYP1B1 C → G substitution modifies cervical cancer development. Finally, the findings from the present study were only from Chinese population, so it is uncertain whether these results are generalizable to the general population of other ethnics.
In conclusion, our study demonstrated that the CYP1B1 C4326G polymorphism might play a role in the development of cervical cancer in Chinese Han women, especially among young individuals with high-risk HPV infection (types 16 or 18). 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
We state that we have not received any funding.