Glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) variants and breast cancer risk in Burkina Faso

Introduction

According to GLOBOCAN 2018 (Cancer database), there are about 2.1 million newly diagnosed female breast cancer cases worldwide each year, making up 11.6% of all cancer diagnoses. In 2018, breast cancer was the cause of 626,679 (6.6%) deaths, accounting for almost 1 in 4 cancer cases among women [2]. Thus, breast cancer is the most common cause of cancer mortality in women, and it constitutes an important health problem [2]. Research has suggested that risk factors for breast cancer are environmental, genetic, nutritional (diet), and hormonal. Several well-known risk factors for breast cancer development are family history of breast cancer, advanced age, early puberty, late menopause, nulliparity, obesity and hereditary transmission of certain predisposition genes, including the BRCA1 and BRCA2 genes (involved in 5-10% of breast cancer cases) and the CHEK2, TP53, ATM and PTEN genes [1, 3, 4]. In Burkina Faso, previous studies have described the distribution of breast cancer risk factors in a population [5] and shown that multiparity is associated with a decreased risk of breast cancer [6]. Genetic analyses targeting relevant exons in BRCA1 genes have also been performed [7]. Furthermore, environmental factors have been well-reported in many breast cancers studies. These environmental factors include carcinogens, xenoestrogens and chemical mutagens [3]. In chemical carcinogenesis, three enzyme systems - namely cytochrome P450 (CYP), antioxidant enzymes (AOEs), and glutathione S-transferases (GSTs) -- play an important role [8]. GSTs play a key role in the detoxification of electrophiles and potentially carcinogenic compounds by glutathione conjugation [9, 10]. In mammalian tissues, seven common classes of cytosolic GST enzymes have been identified (GST classes alpha(α), mu(μ), pi(π), omega(ω), theta(θ), sigma(σ) and zeta(ζ) ), and each class is encoded by a separate gene or gene family (respectively, GSTA, GSTM, GSTP, GSTO, GSTS, GSTT and GSTZ genes) [9, 11, 12, 13].

Many GST genes are polymorphic; thus, particular allelic variants are associated with altered risk (or outcome) of a variety of diseases [10]. These polymorphic variants in GST genes have been reported in different populations [4].

It has been demonstrated that GSTP protein level and GST activity in tumor tissue are significantly higher than in normal breast tissue [14, 15]. GSTs are also cancer chemotherapeutic agents, and thus contribute to tumor resistance to these agents [16]. Therefore, GSTs may be of clinical value in the case of some malignant cancers [17]. The μ (GSTM1:chromosome1p13.3) and θ (GSTT1: chromosome 22q11.23) members of this multigene family are candidate cancer susceptibility genes because of their ability to regulate the conjugation of carcinogenic compounds to excretable hydrophilic metabolites [8, 18]. GSTM1-null or GSTT1-null might then increase risk for deleterious effects of exposure to a wide range of environmental carcinogens [19]. Both variants are homozygous deletions (null genotype) and are therefore associated with the loss of enzyme activity and increased vulnerability to cytogenetic damage [16]. Previous findings have provided evidence that variants with GSTP1 and GSTM1 polymorphisms could influence breast cancer risk, response to chemotherapy, and overall survival in breast cancer patients treated with chemotherapy [20].

Rebbeck et al., (1997) have demonstrated that GSTM1 and GSTT1 are associated with variability in age at first breast cancer diagnosis in BRCA1 mutation carriers, with 22% difference across the observed age range (25–40 years) explained by the GSTT1 genotype [4]. Studies on polymorphisms in GSTM1 and GSTT1 showed a prevalence of 27.8% of GSTM1-null and 46.8% of GSTT1-null in Cameroun , 48.8% of GSTM1-null and 37.3% of GSTT1-null in Ethiopia , 49.2% of GSTM1-null and 28.3% of GSTT1-null in Italy, and 55.3% of GSTM1-null and 27.7% of GSTT1-null in Spain [21]. Other research has found that GSTM1-null individuals may have an increased risk of recurrent pregnancy loss [22], and an Indian meta-analysis found that female carriers of GSTT1 and GSTM1-null genotypes have a higher frequency of pregnancy loss [23].

Based on studies investigating the association of GSTT1 and GSTM1 variants with breast cancer risk, as well as on the role of GSTs in inactivating endogenous metabolites during oxidative stress and its influence on the normal functions of mammalian tissues, we investigated the distribution of GSTM1 and GSTT1 variants in patients with histologically diagnosed breast cancer in comparison to controls to explore the possible association of GST genotypes and risk of breast cancer development. The present case-control study is based on data acquired from a population in Burkina Faso.

Materials and methods

Extraction of genomic DNA and Characterization of deletions of GSTM1 and GSTT1 genes by multiplex PCR

Genomic DNA was extracted from whole blood using the “DNA Rapid Salting-Out” method described by [24] and stored at - 80°C until use. Multiplex PCR was performed on the GeneAmp PCR system 9700 (Applied Biosystem, USA) according to the method described by [25] with a reaction volume of 25 µL, including 10 µL of Maxter Mix Ampli Taq Gold® (Applied Biosystems, USA), 1 µL of each primer pair (20 µM) (Applied Biosystems, USA) GSTM1 ( Forward5’-GAACTCCCTGAAAAGCTAAAGC-3’; Reverse 5’GTTGGGCTCAAATATACGGTGG-3’) GSTT1 (Forward 5’ TTCCTTACTGGTCCTCACATCTC-3’, Reverse 5’-TCACCGGATCATGGCCAGCA-3’), 7µL of nuclease-free water, and 2 µL of DNA. All reagents were purchased from Applied Biosystem (ABI, Appleura International Inc., Foster City, CA, USA). The amplification program was as follows: an activation phase at 94°C for 5 min; 40 cycles of a series of denaturation at 94°C for 1 min, hybridization at 57°C for 1 min, elongation at 72°C for 1 min; and extension at 72°C for 7 min. The PCR products were subjected to 2% agarose gel electrophoresis and visualized under UV light at 312 nm using the Gene Flash rRevelation (Syngenge Bio Imaging, USA) PCR amplification was considered successful if the sample had a band corresponding to b-globin gene fragment (5’-CAACTTCATCCACGTTCACC-3’, 5’-GAAGAGCCAAGGACAGGTAC-3’) (Figure 1).

Figure 1

PCR-multiplex electrophoresis gel. M = Molecular weight marker (100 bp); 1; 7 et 14 = Double present genotypes of GSTM1 and GSTT1; 2; 5; 10; 12 et 13 = Genotypes of GSTM1 present and GSTT1 null; 3; 4; 11= Double genotypes null of GSTM1 and GSTT1; 6; = invalid; 8GSTM1 null and GSTT1 present.

Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the Burkina Faso Health Research Ethics Committee.

Informed consent: Informed consent has been obtained from all individuals included in this study

Results

In this study, we examined the influence of two GST genes, GSTM1 and GSTT1, in breast cancer susceptibility. The study is cross-sectional, including breast cancer patients and healthy females without breast cancer as controls.

Discussion

The relationship between GSTM1- and GSTT1-null genotypes and breast cancer risk was examined in a population-based case-control study in Burkina Faso.

The results for our study showed a frequency of 14.89 % of GSTM1/GSTT1 double null, while GSTM1-null occurred in 28.75% and 18.00% of the cases and the controls respectively. The GSTT1-null genotype was more frequent in cases (30.00%) than in controls (15.00%). Finally, 14.89% of cases and 6.49% of controls had a GSTM1/GSTT1 double null genotype. Studies by Garte et al.; (2001) and Bu et al. (2004) found the frequency of the GSTM1-null genotype to be 42-60% among Caucasians, 42-54% among Asians, 16-36% among Africans, and 54.6% among Arabs. They also found that the frequency of the GSTT1-null genotype was 13-26% for Caucasians, 35-52% for Asians, 15-26% for Africans, and 25% for Arabs. The frequency of the GSTM1/GSTT1 double null genotype was 10.4% in Caucasians, 24.6% in Asians, and 12.6% in Africans) [13, 26]. Indeed, our proportions of the GSTM1-null, GSTT1-null and GSTM1/GSTT1 double null genotypes are relatively close to the reported prevalence in African populations. Furthermore, these two GST variants have been investigated for their association with susceptibility to human cancers such as breast cancer. Often, such studies have resulted in low penetrance or high prevalence associations between cancer risk and the GSTM1/GSTT1 double null variant [9]. In our study, GSTM1 and GSTT1-null genotypes were not associated with increased breast cancer risk. Indeed, on the strong association between GSTM1- and GSTT1-null genetic variants and breast cancer, study results have not yet converged [27, 28, 29]. Thus, in a meta-analysis of 10,067 cancer cases and 12,276 controls, GSTM1-null and GSTT1-null variants are associated with an increased risk of breast cancer in Asians [30]. Also, the GSTM1-null genotype was associated with increased breast cancer risk, but no associations between the GSTT1-null genotype and neoplasia risk were found in populations in Mexico [28]. Likewise, in a Portuguese population, researchers found an increased breast cancer risk associated with GSTM1-null and GSTT1-null genotypes both alone and in combination with GSTP1 valine alleles (rs1695/rs1138272) [31].

Another study has shown an increased breast cancer risk associated with the GSTM1-null genotype, while no association was found between the GSTT1-null genotype and overall breast cancer risk. These results suggest that variants in low penetrance genes ,such as GSTM1, GSTT1 and GSTP1, are associated with an increased breast cancer risk [32]. Indeed in a Turkish study, neither the GSTT1-null nor GSTM1-null genotype were associated with a significantly increased risk of developing breast cancer, but a combined genetic variability in members of the GST gene family may be associated with an increased susceptibility to breast cancer [33]. A meta-analysis from China provides strong support for earlier studies, showing no overall association for the GSTM1 and GSTT1 deletion variants [34]. In contrast, in an Indian population, the GSTT1/GSTM1 double null genotype was found to be protective against the development of carcinoma breast and did not show any association with response to chemotherapy. However, tumors more than 5 cm in size were associated with increased GSTM1 gene expression [35]. Thus our study, which does not find an association between breast cancer risk and these variants, is consistent with some of the results of previous studies.

No association was found between GSTM1- (OR =1.12; 95%CI 0.08-15.50) or GSTT1- (OR = 2.28; 95%CI 0.18-28.18) null genotypes and the disease stage of familial breast cancer patients or sporadic breast cancer patients (GSTM1, OR = 0.40; 95%CI 0.12-1.32) and (GSTT1, OR = 1.41; 95%CI 0.39-5.12). High penetrance genes and certain low penetrance genes may also play a role in the familial inheritance of breast cancer [29]. In previously conducted studies, no significant associations between GSTM1 or GSTT1 variants and disease stage were observed [19]. GSTM1 deletion was found to be significantly associated only with familial breast cancer; GSTT1 was associated only with sporadic breast cancer. However, familial breast cancer patients with a GSTM1-null genotype had a relatively higher risk of advanced disease stage [29].

In a previous study, the association with breast cancer were also null for GSTM1-null and GSTT1-null in pre- and postmenopausal women, or for early versus advanced stage breast cancer [34]. In the Carolina Breast Cancer Study for women with a history of breast cancer in one or more first-degree relatives, odds ratios were 2.1 (95% confidence interval, 1.0-4.2) for GSTM1-null and 1.9 (0.8-4.6) for GSTT1-null genotypes. Among these women with family histories, age at diagnosis was significantly earlier for those with the GSTM1-null genotype [19]. Furthermore, it has been noted that none of the allelic variants associated with sporadic breast cancer were associated with familial breast cancer [29]. Finally, there were no associations found between GSTM1-null (OR=0.60; 95%CI 0.21-1.68) and GSTT1-null (OR=0.60; 95%CI 0.21-1.68) genotypes in overweight/obese or normal/lean individuals with susceptibility to breast cancer. Few studies have directly addressed the relationship between GSTM1-null and GSTT1-null variants with breast cancer. Nevertheless, previous research has observed relationships between GSTM1 and BMI.

Postmenopausal women null for GSTM1 and having a BMI above 24.47 kg/m² have a sevenfold increase in breast cancer risk, while women null for GSTM1 and having a BMI less than 24.47 kg/m² were not at increased risk of breast cancer [36]. Our results contradict this data, but corroborate the results of Vogl et al., (2004) who found no association between those GST allelic variants, BMI, and the occurrence of breast cancer [19].

Conclusion

Our results suggest that no strong association exists between GSTM1-null, GSTT1-null, or GSTM1/GSTT1 double null genotypes and susceptibility to breast cancer development. The association studies between breast cancer risk and GSTM1 or GSTT1 variants could be investigated further, in agreement with most previous studies. The absence of positive associations for GSTM1-null and GSTT-null genotypes in women with either a family history of breast cancer or sporadic breast cancer and BMI indicate that further investigation is required to confirm a potential role for GST genotypes in both breast cancer prognosis and response to treatment.