Positive Association of Polymorphisms in Estrogen Biosynthesis Gene,CYP19A1,and Metabolism,GST,in Breast Cancer Susceptibility

Abstract

Purpose: This case–control study was conducted in order to evaluate the potential role of polymorphic genes encoding enzymes involved in estrogen biosynthesis (CYP19A1) and metabolism (GSTM1, GSTT1, and GSTP1), and their action in modulating individual susceptibility to breast cancer. Methods: Genomic DNA was extracted from blood samples of 101 patients with histological diagnosis of breast cancer and 121 healthy women. Genotyping analyses of CYP19A1 codon 39 Trp/Arg (T/C), GSTM1 and GSTT1 homozygous deletions, and GSTP1 codon 105 Ile/Val (A/G) were performed by polymerase chain reaction–based methods. Results: Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated by unconditional logistic regression. Significant statistical association of the TC/CC genotypes combined with breast cancer risk was found, with reference to TT genotype (OR=1.770; 95% CI=1.036–3.024; p=0.036). Also, CYP19A1 arginine allele in homozygosity or heterozygosity (TC/CC) was associated with a significant increased risk for breast cancer when associated to GSTM1 null genotype (OR=6.158; 95% CI=2.676–14.171; p<0.001) and GSTT1 null genotype (OR=4.870; 95% CI=2.216–10.700; p<0.001). The three-way combination of CYP19A1 TC/CC, GSTM1 null, and GSTT1 null polymorphism was related with significant increased risk for breast cancer (OR=11.429; 95% CI=3.590–36.385; p<0.001). Valine alleles compared with isoleucine alleles in codon 105 in GSTP1, in combination with CYP19A1 genotypes, were not associated with an increase of breast cancer development. Conclusions: Our results suggest that the effect of CYP19A1 T/C polymorphism in susceptibility to breast cancer development can be modulated by the presence of GSTM1 and GSTT1, but not GSTP1.

Introduction

I n Europe, breast cancer is the most common cancer affecting women, representing the leading cause of cancer death between 35 and 55 years old (Jemal et al., 2004). Breast cancer is the leading cause of death among women in developing countries. In Portugal, it presents the highest incidence and mortality rates among women diseases (Pinheiro et al., 2003). Although many risk factors for the development of breast cancer have been identified, the molecular mechanisms related to breast carcinogenesis remain unclear. Estrogens have been clearly identified as carcinogens, by inducing aneuploidy and structural chromosomal changes, and stimulation of breast cell proliferation has been proposed as the main effect of estrogens in breast carcinogenesis—as more rapidly cells proliferate, greater the chance of acquiring a potentially cancer-causing mutation (Zhu and Conney, 1998). Also, metabolic by-products of estrogens are responsible for free radical–mediated DNA damage, single-strand breaks, estrogen–DNA adducts formation, protein oxidation, and lipid peroxidation, which triggers genetic instability and cellular damage (Mueck and Seeger, 2007). Germline mutations in the so-called high penetrance genes to breast cancer susceptibility, BRCA1 and BRCA2, appear to account for the majority of hereditary breast cancer, but they represent only 5% to 10% of all breast cancer cases (Yang and Lippman, 1999). Thus, low penetrance genes, acting together with endogenous or life-style risk factors, can be linked with a significant percentage of breast cancer cases (Mitrunen and Hirvonen, 2003). Low penetrance genes can be found in several pathways like detoxification of environmental carcinogens, steroid hormone metabolism, and DNA damage repair pathways. Interindividual variability has been observed in estrogen biosynthesis and metabolic pathways, namely in genes encoding for proteins involved in estrogen biosynthesis, like CYP19A1, and in genes encoding for xenobiotic metabolizing enzymes, like Glutathione S-transferases (GSTs) (Thompson and Ambrosone, 2000). CYP19A1 gene, located on chromosome 15q21.1, encodes CYP19A1 or aromatase (EC 1.14.14.1), the enzyme that catalyzes three consecutive hydroxylation reactions converting C19 androgens, such as testosterone, to aromatic C18 estrogenic steroids (Bulun et al., 2004). Genetic variations at this locus may alter aromatase activity and thereby affect hormone levels. The most focused is a tetranucleotide repeat [TTTA]_n in intron 4 that may have a functional effect in the translated enzyme due to an alteration on the splice site; it has been reported to be associated with breast cancer risk among Caucasian women (Sourdaine et al., 1994; Kristensen et al., 1998; Hairman et al., 2000), although negative results also exist (Healey et al., 2000; Miyoshi et al., 2000; Baxter et al., 2001). Recently, a case–control study on the association of CYP19 Val(80) and [TTTA]_n polymorphisms and the risk of breast cancer in BRCA carriers and noncarriers suggests that the CYP19 Val(80) polymorphism is associated with increased breast cancer risk in young women from a population of Ashkenazi Jews with BRCA1 mutations (Raskin et al., 2009). Also, a C826T variation in exon 7 that gives rise to the nonconservative amino acid substitution Arg264Cys has been used in breast cancer studies despite not having an apparent effect on aromatase activity (Watanabe et al., 1997; Siegelmann-Danieli and Buetow, 1999; Long et al., 2006). Miyoshi et al. (2000) identified codon 39 Trp/Arg polymorphism in CYP19 gene and showed that it was significantly associated with breast cancer risk among Japanese women but there are no results available for other populations. Glutathione S-transferases (EC 2.5.1.18) constitute a superfamily of ubiquitous, multifunctional enzymes that play a key role in cellular detoxification (Ketterer, 1998). GSTs catalyze the conjugation of glutathione (GSH) to a wide variety of exogenous and endogenous chemicals with electrophilic functional groups like environmental carcinogens, reactive oxygen species (ROS), and chemotherapeutic agents. Conjugation with GSH renders metabolic products with higher hydrophilicity, which are easier to excrete (Hayes and Pulford, 1995). Human cytosolic GST family is the most complex and relevant to disease investigation. Cytosolic GSTs are subdivided into seven distinct classes that are designated as α (A), μ (M), π (P), σ (S), θ (T), ω (O), and ζ (Z). This classification is based on similarities in amino acid sequence, gene structures, and immunological crossreactivity (Hayes et al., 2005; Di Pietro et al., 2010). Polymorphic variants of three members of the GST family, GSTM1, GSTT1, and GSTP1, have been studied due to their potential importance in carcinogenesis. The human μ (M) class is encoded by a 100-kb gene cluster located on chromosome 1p13.3 (Landi, 2000) and the θ (T) class of GSTs consists of two genes located at 22q11.2 (Meyer et al., 1991). Homozygous gene deletion (null genotype) has been identified at both GSTM1 and GSTT1 (Xu et al., 1998; Alexandrie et al., 2002), and these deletion variants lead to the total absence of the codified protein, thus total absence of enzyme activity (Bruhn et al., 1998). The π (P) class of human GSTs is encoded by a gene located on chromosome 11q13.2 (Ali-Osman et al., 1997). The polymorphism in exon 5 A313G (Ile105Val) of the GSTP1 gene is in close proximity to the substrate binding site of GSTP1, and the Valine variant has been demonstrated to have either lower or higher specific activity and affinity than that of Ile variant, depending on the substrate (Ali-Osman et al., 1997; Sundberg et al., 1998). Associations of GSTM1 and GSTT1 null genotypes, and GSTP1 Ile105Val polymorphism with breast cancer have been reported with inconsistent results (Helzlsouer et al., 1998; Millikan et al., 2000; Gudmundsdottir et al., 2001; Mitrunen et al., 2001). Our group has previously shown, in a Portuguese population, that GSTM1 and GSTT1 null genotypes alone, both combined or combined with GSTP1 Valine alleles, are associated with higher susceptibility to breast cancer development (Ramalhinho et al., 2011). In light of this background, our aim is to analyze the influence of polymorphic genes encoding for enzymes involved in estrogen biosynthesis (CYP19A1) and in the conversion of estrogen metabolites and their by-products (GSTM1, GSTT1, and GSTP1) in breast cancer susceptibility.

Materials and Methods

Study population

A study group consisting of 101 women with histologically confirmed breast cancer, diagnosed at Child and Women Health Department, Gynecologic Oncology Division, Hospital Centre of Cova da Beira, Covilhã, Portugal, was enrolled between May 2008 and March 2009. A nonmalignant control group, 121 healthy female blood donors with no previous history of cancer and without family histories of cancers, was also studied. All subjects were Caucasian and had the same geographic area of residence. Both cases and controls completed a questionnaire that assessed parameters such as age, family history of breast cancer, parity, ethnicity, reproductive and menstrual history, and history of breast diseases and procedures. Informed consent was obtained from both patients and controls before entering in the study. The study was approved by the Institutional Review Board of Hospital Centre of Cova da Beira, Covilhã, Portugal.

DNA extraction

Genomic DNA of all cases and controls was isolated from either frozen or fresh blood samples using Wizard^® Genomic DNA Purification Kit (Promega) according to the manufacturer's instruction, and stored at 4°C.

Genotyping

CYP19A1 codon 39 Trp/Arg (T/C) polymorphism, GSTM1 and GSTT1 homozygous deletions, and GSTP1 codon 105 Ile/Val (A/G) polymorphism analyses were performed by polymerase chain reaction (PCR)–based methods. CYP19A1 genotyping was performed using PCR with confronting two-pair primers, modified from the procedure described by Hirose et al. (2004). Briefly, each PCR mixture was carried out in a total volume of 25 μL that contained 10 pmol of each primer, 1.5 mM of MgCl₂, 100 nM of each deoxynucleotide triphosphate, 1 unit of Taq DNA polymerase (Promega), and 100 ng of genomic DNA, using MyCycler Thermal Cycler (Bio-Rad). Reaction mixtures were preincubated for 10 min at 95°C. PCR conditions were 1 min at 95°C, 1 min at 56°C, and 1 min at 72°C, for 30 cycles. The final extension was at 72°C for 5 min. The amplified DNA was electrophoresed through 2% agarose gels stained with ethidium bromide. Genotypes were distinguished by the presence of a 200-bp band for T allele, a 264-bp band for C allele, and a 427-bp common band. GSTM1, GSTT1, and GSTP1 genotyping was performed as previously published by our group (Ramalhinho et al., 2011). Results were confirmed by re-genotyping 10% of randomly selected samples, and all the results were in agreement with the ones obtained previously.

Statistical analysis

To examine the association between genotypes and the development of breast cancer, we calculated odds ratios (ORs) and 95% confidence intervals (95% CIs) as estimates of relative risk, using logistic regression analysis with computer software SPSS for Windows (version 16.0). Chi-square test was applied to compare the allelic frequencies between normal controls and breast cancer patients. p-Values <0.05 were considered statistically significant.

Results

The mean age was 55.5 years (SD, 20.3 years; range, 17–84 years) for the controls and 62.8 years (SD, 12.9 years; range, 34–85 years) for the cases. Demographic data from cases and controls are shown in Table 1. The majority of study participants were postmenopausal (62.0% controls and 69.3% cases). The risk of breast cancer was higher for women with first degree family history of breast cancer (OR=2.532; 95% CI=2.133–3.005) and for women with history of benign breast disease (OR=1.959; 95% CI=1.146–3.350). Also, women with body mass index (BMI) superior to 25 seemed to have increased risk for breast cancer (OR=1.770; 95% CI=1.036–3.024). CYP19A1 codon 39 genotype results in cases and controls are presented in Table 2. Significant statistical association of the TC/CC genotypes combined with breast cancer risk, with reference to TT genotype, was documented (OR=1.770; 95% CI=1.036–3.024; p=0.036). Additionally, we analyzed the allele frequency in cases and controls (Table 3) and a positive correlation between C allele carriers (homozygous and heterozygous) and breast cancer risk was found (OR=1.540; 95% CI=1.013–2.341; p=0.043). Results regarding the genotype distribution of GSTM1, GSTT1, and GSTP1 in cases and controls in this population were previously published by our group, as well as the results of the association in two- and three-way combinations to evaluate the impact of gene–gene interaction (Ramalhinho et al., 2011). To investigate whether profiles of GSTs and CYP19A1 genotypes might be associated with the risk of breast cancer, we examined combinations of genotypes. The reference group consisted of individuals with all putative low-risk genotypes, that is, the presence of GSTM1 and GSTT1, the homozygous Ile/Ile genotype for GSTP1, and the homozygous TT genotype for CYP19A1. Heterozygous and homozygous individuals for the Val allele and also heterozygous and homozygous individuals for C allele were grouped for this analysis, due to the low number of individuals with homozygous Val allele and homozygous C allele, and so to increase the statistical power. We analyzed the two-way combination of GSTM1 and CYP19A1 genotypes (Table 4) and found that when GSTM1 is present, there is no significant association with breast cancer risk (OR=1.784; 95% CI=0.786–4.050; p=0.164) but we found a significant increase of breast cancer risk for carriers of GSTM1 null genotype along with CYP19A1 TT wild-type genotype (OR=3.115; 95% CI=1.362–7.124; p=0.006), and this risk is higher for carriers of both variants, GSTM1 null and CYP19A1 C allele, simultaneously (OR=6.158; 95% CI=2.676–14.171; p<0.001). We could find the same relation in the two-way analysis of GSTT1 and CYP19A1 genotypes (Table 5). There is no significant increase in breast cancer risk for carriers of GSTT1 present along with CYP19A1 C allele (OR=1.841; 95% CI=0.939–3.609; p=0.074), but for carriers of GSTT1 null genotype the risk is significantly higher in individuals with simultaneous CYP19A1 TT genotype (OR=4.599; 95% CI=1.792–11.804; p=0.001) or CYP19A1 TC/CC genotype (OR=4.870; 95% CI=2.216–10.700; p<0.001). Regarding the two-way analysis of CYP19A1 and GSTP1 genotypes (Table 6), we found no association of any genotype combination and risk of breast cancer. By analyzing the three-way combination of CYP19A1, GSTM1, and GSTT1 polymorphisms (Table 7), we found a positive association with breast cancer risk for women who carry C allele in CYP19A1 and null genotypes in GSTM1 (OR=5.000; 95% CI=1.491–16.771; p=0.007) or GSTT1 (OR=6.333; 95% CI=2.195–18.271; p=0.001). We also found that when both GSTs are deleted, the risk of breast cancer is higher, independently of CYP19A1 genotype, and the combination of all putative high-risk genotypes, CYP19A1 TC/CC, GSTM1 null, and GSTT1 null, carries a strong association with breast cancer risk (OR=11.429; 95% CI=3.590–36.385; p<0.001).

Table 1.

Selected Characteristics of the Population Studied

	Controls, n (%)	Cases, n (%)	OR (95% CI)
Total	121 (100)	101 (100)
Age at menarche
≤12 years	57 (47.1)	63 (62.4)	1.0
13–14 years	41 (33.9)	25 (24.7)	0.552 (0.299–1.018)
≥15 years	23 (19.0)	13 (12.9)	0.511 (0.237–1.103)
Age at first full term pregnancy
≤25 years	51 (53.1)	40 (50.6)	1.0
26–30 years	19 (19.8)	21 (26.6)	0.710 (0.337–1.496)
≥31 years	26 (27.0)	18 (22.8)	1.133 (0.546–2.350)
Number of full term pregnancies
Nulliparous	25 (20.7)	22 (21.8)	1.0
1	19 (15.7)	26 (25.7)	1.555 (0.682–3.543)
2	45 (37.2)	29 (28.7)	0.732 (0.350–1.533)
3+	32 (26.4)	24 (23.8)	0.852 (0.391–1.859)
Menopausal status
Premenopausal	46 (38.0)	31 (30.6)	1.0
Postmenopausal	75 (62.0)	70 (69.3)	1.385 (0.791–2.424)
First degree family history of breast cancer
No	121 (100)	79 (78.2)	1.0
Yes	0 (0)	22 (21.8)	2.532 (2.133–3.005)
History of benign breast disease
No	74 (61.2)	45 (44.5)	1.0
Yes	47 (38.8)	56 (55.4)	1.959 (1.146–3.350)
BMI
≤25	65 (53.7)	40 (39.6)	1.0
>25	56 (46.3)	61 (60.4)	1.770 (1.036–3.024)

OR, odds ratio; CI, confidence interval; BMI, body mass index.

Table 2.

Distribution of CYP19 Codon 39 Polymorphisms in Patients and Controls

	Controls, n (%)	Cases, n (%)	OR (95% CI)	p-Value
TT	65 (53.7)	40 (39.6)	1.0
TC/CC	56 (46.3)	61 (60.4)	1.770 (1.036–3.024)	0.036

Table 3.

CYP19 Codon 39 Allele Frequency and Genotype, in Cases and Controls

	Controls, n (%)	Cases, n (%)	OR (95% CI)	p-Value
T, n (%)	185 (76.4)	137 (67.8)	1.0
C, n (%)	57 (23.5)	65 (32.2)	1.540 (1.013–2.341)	0.043

Table 4.

Association Between GSTM1 and CYP19 Genotype Combinations and Breast Cancer

GSTM1	CYP19	Controls, n (%)	Cases, n (%)	OR (95% CI)	p-Value
+	TT	39 (32.2)	13 (12.9)	1.0
+	TC/CC	37 (30.6)	22 (21.8)	1.784 (0.786–4.050)	0.164
−	TT	26 (21.5)	27 (26.7)	3.115 (1.362–7.124)	0.006
−	TC/CC	19 (15.7)	39 (38.6)	6.287 (2.676–14.171)	<0.001

Table 5.

Association Between GSTT1 and CYP19 Genotype Combinations and Breast Cancer

GSTT1	CYP19	Controls, n (%)	Cases, n (%)	OR (95% CI)	p-Value
+	TT	56 (46.3)	23 (22.8)	1.0
+	TC/CC	41 (33.9)	31 (30.7)	1.841 (0.939–3.609)	0.074
−	TT	9 (7.4)	17 (16.8)	4.599 (1.792–11.804)	0.001
−	TC/CC	15 (12.4)	30 (29.7)	4.870 (2.216–10.700)	<0.001

Table 6.

Association Between GSTP1 and CYP19 Genotype Combinations and Breast Cancer

GSTP1	CYP19	Controls, n (%)	Cases, n (%)	OR (95% CI)	p-Value
Ile/Ile	TT	34 (28.1)	26 (25.7)	1.0
Ile/Val + Val/Val	TT	31 (25.6)	14 (13.9)	0.591 (0.262–1.130)	0.202
Ile/Ile	TC/CC	23 (19.0)	28 (27.7)	1.592 (0.751–3.376)	0.224
Ile/Val + Val/Val	TC/CC	33 (27.3)	33 (32.7)	1.308 (0.648–2.640)	0.454

Table 7.

Association Between GSTM1, GSTT1, and CYP19 Genotype Combinations and Breast Cancer

GSTM1	GSTT1	CYP19	Controls, n (%)	Cases, n (%)	OR (95% CI)	p-Value
+	+	TT	32 (26.4)	8 (7.9)	1.0
		TC/CC	29 (24.0)	12 (11.9)	1.655 (0.593–4.618)	0.333
+	−	TT	7 (5.8)	5 (4.9)	2.857 (0.715–11.410)	0.128
		TC/CC	8 (6.6)	10 (9.9)	5.000 (1.491–16.771)	0.007
−	+	TT	24 (19.8)	15 (14.8)	2.500 (0.912–6.851)	0.071
		TC/CC	12 (9.9)	19 (18.8)	6.333 (2.195–18.271)	0.001
−	−	TT	2 (1.6)	12 (11.9)	24.000 (4.448–129.490)	0.001
		TC/CC	7 (5.8)	20 (19.8)	11.429 (3.590–36.385)	<0.001

Discussion

High estradiol levels have been consistently associated with an increased breast cancer risk (Dorgan et al., 1996; Key et al., 2002; Missmer et al., 2004; Zeleniuch-Jacquotte et al., 2004), as estrogen promotes cellular growth and contributes to tumor growth by promoting the proliferation of cells with existing mutations or perhaps by increasing the opportunity for mutations (Zhu and Conney, 1998). Also, ROS have been related to the etiology of cancer, as they are known to be mitogenic to a variety of cells, and therefore capable of tumor promotion (Mitrunen and Hirvonen, 2003). Interindividual differences in estrogen biosynthetic and metabolic pathways may define subpopulations of women with higher lifetime exposure to hormone-dependent growth promotion or to cellular damage from estrogens or their metabolites. Such variation could explain a portion of the cancer susceptibility associated with reproductive events and hormone exposure. In most studies, polymorphisms in estrogen biosynthesis and metabolism genes are considered separately (Helzlsouer et al., 1998; Kristensen et al., 1998; Haiman et al., 2000; Healey et al., 2000; Millikan et al., 2000; Miyoshi et al., 2000; Mitrunen et al., 2001; Baxter et al., 2001; Gudmundsdottir et al., 2001; Hirose et al., 2004; Raskin et al., 2009; Ramalhinho et al., 2011) but here we hypothesize that SNPs in these low penetrance genes can exhibit synergistic effects on modulating individual susceptibility to breast cancer. Furthermore, specific associations of polymorphisms in estrogen biosynthesis and metabolism genes could result in a high-risk profile, by influencing lifetime levels of estrogen that could influence breast cancer risk. The four genes analyzed in this study are considered as low penetrating genes. These genes, alone or in association, may identify subjects as poor or fast synthesizers, like subjects as poor or fast estrogen metabolizers. While fast synthesizers are prolonging their exposure to estrogen, with potential increased carcinogenic activity, poor metabolizers are more exposed to the formation of carcinogen-DNA adducts and/or mutations, which confers them higher susceptibility to complex genetic disorders such as cancer. Thus, women having the “fast synthesizer/poor metabolizer” profile may possess higher risk for developing breast cancer than women with other profiles. In this study we investigated, in a population-based, case–control study of Caucasian women from a Portuguese region, breast cancer risk in association with polymorphism in a gene involved in estrogen biosynthesis (CYP19A1) along with polymorphisms in other three genes implicated in estrogen metabolism (GSTM1, GSTT1, and GSTP1). To avoid potential biases, we selected cases and controls from a single institution that only accepts patients from the same geographic area, Central Eastern Portugal. Patients and controls were matched by ages and belonged to the same ethnic group (Caucasian). Regarding CYP19A1 codon 39 genotype distribution, our results were in agreement with the few previously published reports. Miyoshi et al. (2000) identified this polymorphism and performed a case–control study in order to evaluate its association with breast cancer risk in a Japanese population. They concluded that homozygous and heterozygous carriers of the variant Arg (C) allele at codon 39 had a decreased risk of breast cancer compared with the noncarriers. In our study, breast cancer patients had higher relative frequency of C allele and lower prevalence of Trp (T) allele than controls. Thus, for developing breast cancer, C allele carriers apparently have higher risk, while T allele carriers have lower risk, consequently T allele seems to be protective for carriers. A similar study, also in a Japanese population, was conducted by Hirose et al. (2004). They found, in homozygous and heterozygous carriers of the C allele, a significantly increased risk of breast cancer among premenopausal women with a late age at first full term pregnancy or a high BMI. In our study, we found positive statistical association of the TC/CC genotypes (C allele carriers) with breast cancer risk with no association with any particular demographic data. More recently, Tuzuneri et al. (2010) also found an association between C allele-bearing and breast cancer risk, and they propose TT haplotype as a protective haplotype. No published reports, regarding functional effects of CYP19 codon 39 Trp/Arg amino acid change, are of our knowledge. To explain our data, we can speculate about deleterious effect of Trp to Arg substitution in codon 39 of CYP19A1 gene; it seems that the encoded protein P450 aromatase variant tends to have higher activity, thereby raising estrogens levels, what leads to the association of C allele with breast cancer risk. Considering GSTs polymorphisms, our group has already demonstrated the significant increase of breast cancer risk associated with GSTM1 and GSTT1 null polymorphisms, both alone or in combination, in the same population, while GSTP1 genotypes seem to have no influence in breast cancer susceptibility (Ramalhinho et al., 2011). In that article we reported that GSTM1 null genotype was significantly more common among breast cancer cases compared with controls, so as GSTT1, and no significant increase of breast cancer risk associated with GSTP1 genotypes was found. We also observed an eight-fold increased breast cancer risk in women who carry null genotype both in GSTM1 and GSTT1, and we found that Ile/Ile genotype, so as presence of Val allele, seemed to be associated with risk of breast cancer when combined with both GSTM1 and GSTT1 null genotypes; thus, it appears that the increase of breast cancer risk was mainly linked to both GSTM1 and GSTT1 deletion, rather than to any genotype of GSTP1. Grouping CYP19A1 and GSTM1 genotypes, breast cancer susceptibility was not altered when GSTM1 was present. However, breast cancer risk for carriers of GSTM1 null genotype along with CYP19A1 TT wild-type genotype is increased, so as for simultaneous carriers of both variants, GSTM1 null and CYP19A1 C allele. Similar association was observed in the analysis of CYP19A1 and GSTT1 genotypes; independently of CYP19A1 polymorphism, breast cancer risk is lower for carriers of GSTT1. Again, no association between both CYP19A1 and GSTP1 genotypes taken together and risk of breast cancer was found. To our knowledge, this is the first study to analyze the potential role of CYP19A1 codon 39 polymorphism in Portuguese women, and its possible combined effect with GSTM1, GSTT1, and GSTP1 polymorphisms in the development of breast cancer. We determined that C allele, in homozygosity or heterozygosity, is significantly associated with an increased risk of breast cancer in the population studied. Furthermore, it seems that the effects of CYP19A1 T/C polymorphism in estradiol biosynthesis appear to be modulated by the presence of GSTM1 and GSTT1 in estrogen metabolic pathway, because breast cancer susceptibility is lower in carriers of GSTM1 and GSTT1, independently of CYP19A1 genotype. The results support the accepted hypothesis that estrogen formation and metabolism, which can be dependent of genetic and environmental variation, are determinant in defining breast cancer risk. Further studies are needed to evaluate the influence of the expression of these genes, especially in association with the expression of other known low penetrance genes in defining breast cancer risk.

Footnotes

Disclosure Statement

The authors declare that we have no conflict of interest.

Acknowledgments

The authors would like to thank all the technical staff from Centro Hospitalar Cova da Beira, Covilhã, Portugal, for their kindly cooperation in the collection of the blood samples and all the volunteers who agreed to participate in this study.

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