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Indian Journal of Medical Sciences, Vol. 63, No. 1, January, 2009, pp. 13-20 ORIGINAL CONTRIBUTION Association of CYP1A1*2 Polymorphisms with breast cancer risk : A case control study Surekha D, Sailaja K, Rao DNageswara, Padma T, Raghunadharao D, Vishnupriya S Department of Genetics, Osmania University, Hyderabad Code Number: ms09003 Abstract Background :The Cytochrome P-4501A1 (CYP1A1) gene, located on chromosome 15q, is involved in the metabolism of carcinogens mainly polycyclic aromatic hydrocarbons as well as estrogen. It is considered as candidate gene for low-penetrance breast cancer susceptibility. Hence the present study aims to discuss the role of CYP1A1 polymorphisms in breast cancer. Keywords: Association, breast cancer, CYP1A1FNx012, polymorphisms, receptor status Introduction CYP1A1 is a candidate gene for low-penetrance breast cancer susceptibility because it plays an important role in the metabolism of carcinogens such as polycyclic aromatic hydrocarbons (PAHs), as well as in the oxidative metabolism of estrogens. PAHs are known human carcinogens and have been found to cause mammary tumors in rodents. The CYP1A1 gene, located at 15q22-q24, comprises of 7 exons and 6 introns and spans 5,810 base pairs. [1] CYP1A1 expression occurs predominantly in extra-hepatic tissue. [2] In humans, CYP1A1 is under the regulatory control of the aryl hydrocarbon receptor, a transcription factor that regulates gene expression. [3] CYP1A1 messenger RNA has been detected in normal and cancerous breast tissue [4],[5] and can be induced in human breast-derived cell lines. The most common alleles of CYP1A1 resulting in amino acid substitutions are CYP1A1FNx012 (Ile462Val) and CYP1A1FNx014 (Thr461Asn). Several studies have reported association of CYP1A1 genotypes with an increased risk of certain types of cancer. Besides lung cancer, [6] these included cancers possibly related to CYP1A1-mediated estrogen activation such as breast cancer, [7] prostate cancer [8] and ovarian cancer. [9] Interestingly, when the burden of polychlorinated biphenyls, known potent inducers of CYP1A1, was high in the body, an increased risk of breast cancer associated with the presence of the CYP1A1FNx012 allele was reported. [10] Since the role of CYP1A1 in the metabolism of endogenous compounds such as estrogen, besides xenobiotics, including PAH, has been widely reported, [11] the significance of CYP1A1 polymorphism in modifying the cancer risk through differential metabolic activation of these compounds can be established by population-based studies. Several mutations have been identified in CYP1A1 gene and mainly four polymorphisms were studied in relation to breast cancer. The 3′ noncoding region of CYP1A1 showed 2 polymorphisms, viz., T3801C (M1) and T3205C (M3). Other mutations, at A2455G (M2) and C2453A (M4) in the heme binding region of exon 7, resulted in missense mutations of Ile462Val and Thr461Asp respectively. A case-control study of breast cancer in north Indian women revealed the association of M2 polymorphism of CYP1A1 gene with breast cancer development. [12] Other studies suggest that probably CYP1A1 (M1, M2, M3 and M4) polymorphism alone does not play a significant role in increasing the risk of breast cancer in north Indian women. [13] No association was found between any CYP1A1 Ile462Val polymorphism and breast cancer in a Korean study. [14] Hence our study aims to show the association of CYP1A1FNx012 polymorphism with breast cancer. Materials and Methods A group of 250 breast cancer patients were selected for the present study and 250 healthy and age-matched women without family history of breast cancer or any other cancers were selected to serve as the control group. The patients were chosen from Nizam′s Institute of Medical Sciences, Hyderabad, after confirmed diagnosis, and controls included healthy volunteers recruited by visiting colleges, social welfare organizations and surrounding villages in the period (2003-2005). The diagnosis of breast cancer was established by pathological examination, mammography, fine needle aspiration (FNAC) and biopsy. Epidemiological history including age at onset of breast cancer, diet, socioeconomic status, occupation, reproductive history, family history and consanguinity was taken by a personal interview with breast cancer patients using a specific format. The patients were screened for receptor status of estrogen, progesterone and HER-2/neu by immunohistochemical assay. Clinical history including size of the tumor, presence of auxiliary nodes, extent of metastasis, stage and type of the breast cancer, chemotherapeutic drugs used and prognosis of the disease was collected with the help of an oncologist. Informed consent was obtained from all patients and controls that were included in the study. The approval of the ethical committee was taken before initiation of the study. Five milliliters of blood was collected in an ethylenediaminetetraacetic acid (EDTA) vaccutainer from patients and controls. DNA was isolated [15] and used for amplification of CYP1A1FNx012 by polymerase chain reaction-restriction fragment length polymorphism PCR-RFLP. [16] CYP1A1FNx012 0 Polymorphism The polymorphism in exon 7 at codon position 462 arises due to transition of adenine by guanine (A2455G), which results in replacement of isoleucine with valine, which can be identified using PCR followed by restriction digestion with NcoI (New England Biolabs). Ile/Ile homozygous geotype was identified by the presence of 323 bp band, Ile/Val by 323, 216, 107 bp bands and Val/Val by 216, 107 bp bands on 2% agarose gel. The allele isoleucine is a wild type of allele and is common in both diseased persons and controls. Homozygotes and heterozygotes for valine have been found to increase enzyme activity, leading to production of toxins. Statistical Analysis The results were analyzed by SPSS version 14. Odds ratio was estimated in order to calculate the relative risk for each genotype to develop disease. Differences in genotype frequency distribution between patients and controls were studied using 2FNx012 2 and χ2 tests for heterogeneity. Results In the present study, association of CYP1A1FNx012 (Ile462Val) polymorphism with breast cancer was studied. Only one breast cancer patient was observed to be homozygous for Val allele but none among controls. The frequency of heterozygous Ile/Val genotype was found to be increased significantly in disease (68.1%) as compared to controls (51.0%). (χ2 : 16.638; P = .0002FNx01). The genotype distribution of CYP1A1FNx012 deviated from Hardy-Weinberg equilibrium in both breast cancer patients (χ2=64.08FNx01) and controls (χ2 =29.18FNx01), indicating that it is plausible that selective forces are operating in the population. Higher frequency of heterozygotes for Val allele was observed among premenopausal breast cancer patients (70.0%) as compared to postmenopausal patients (66.4%). Patients without family history of cancer showed increased frequency of heterozygosity for the Val allele (70.9%) when compared to familial cases. The frequency of heterozygotes for Val allele was found to be elevated in breast cancer patients with high BMI [Table - 1]. When HER2/neu status was compared, heterozygous frequency was found to be increased in HER2/neu-positive patients (95.8%). The frequency of Val allele was found to be slightly increased in breast cancer patients with respect to stage of the disease. The frequency of heterozygous Val genotype was found to be increased significantly in patients with node-negative status (74.7%) [Table - 2]. No significant association of CYP1A1 polymorphism was observed with occupation, estrogen receptor and progesterone receptor status of breast cancer patients.Discussion The CYP1A1 gene codes for aryl hydrocarbon hydroxylase (AHH) catalyze aromatic de-alkylation and other substances. [17] It is one of the candidate genes implicated in breast cancer susceptibility as it plays a significant role in the metabolism of carcinogens such as polycyclic aromatic hydrocarbons (PAH), as well as in the oxidative metabolism of endogenous or exogenous estrogens. [18] Polychlorinated biphenyls (PCBs) which are potent inducers of CYP1A1 gene might increase the risk of breast cancer by estrogenic tumor-promoting, [19] immune-modulating [20] and enzyme-inducing properties. [21] Several epidemiological studies demonstrated association of CYP1A1 genotypes with an increased risk of certain types of cancer. The CYP1A1 genotype was implicated in the origin of lung cancer, as well as estrogen-activated breast cancer, [7] prostate cancer [22] and ovarian cancer. [9] In the present study, association of CYP1A1FNx012 (Ile 462Val) polymorphism with breast cancer revealed interesting information. The frequency of heterozygous Ile/Val genotype was found to be increased significantly in breast cancer patients (68.1%) as compared to controls (51.0%). The presence of Val allele at 462 position results in a 1.5-fold increase in activity of aryl hydrocarbon hydroxylase, which in turn enhances the bioactivition of carcinogens, leading to breast cancer. An increased breast cancer risk was found to be associated with the CYP1A1FNx012 allele and smoking in Caucasian women, [23],[24] whereas Chinese and Japanese women carrying CYP1A1FNx012 allele had nil or significantly reduced risk of breast cancer as compared to carriers of the wild-type allele. [25],[26] The frequency of Val allele was found to be elevated in Asians (18%-33%) as compared to Caucasians (2%-10%). [27] The presence of more dietary flavanoids in the Asian diet as compared to western diet may be a beneficial environmental modifying factor in combination with CYP1A1 enzyme activity. [28] Higher frequency of heterozygotes for Val allele was observed among premenopausal breast cancer patients, suggesting that the presence of Val allele might influence the early onset of breast cancer, which is in agreement with a study that reported that polymorphism of CYP1A1 might affect the early onset of breast cancer. [29] In contrast to our results, previous studies have reported that the postmenopausal women with Val allele were more susceptible to develop breast cancer because of prolonged exposure to environmental carcinogens. [10] No significant association was observed with respect to occupation, estrogen receptor and progesterone receptor status. When HER2/neu status was compared, heterozygous frequency was found to be increased in HER2/neu-positive cases, indicating a possible role of environmental carcinogens with increased expression of HER2/neu, which further affects cell signaling or cell division. The frequency of Val allele was found to be slightly increased in breast cancer patients with respect to stage of the disease. The frequency of heterozygous Val genotype was found to be increased significantly in patients with node-negative status, which suggests that the presence of CYP1A1FNx012 polymorphism might not be contributing significantly to disease progression. In conclusion, our results suggest that the presence of CYP1A1FNx012 Val allele with high enzymatic activity when combined with exposure to environmental carcinogens as well as estrogens might predispose individuals to develop breast cancer. Our study also indicates that individuals with CYP1A1FNx012 Val allele and higher BMI carry greater risk to develop breast cancer. Acknowledgments This work was supported by the University Grants Commission, New Delhi, India; and Nizam′s Institute of Medical Sciences, Hyderabad, India. References
Copyright 2009 - Indian Journal of Medical Sciences The following images related to this document are available:Photo images[ms09003t1.jpg] [ms09003t2.jpg] |
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