Association between Polymorphisms of Glutathione S-Transferase and Progression to Cervical Cancer in Women from Burkina Faso and Mali

Although persistence of high-risk human papillomavirus infection is the main risk factor, Glutathione S-Transferase highly polymorphic enzyme involved in the metabolism of xenobiotics, is a good candidate gene. The objective of this study was to compare the polymorphisms of Glutathione S-Transferase M1-null in women with cancerous lesions and without lesions. This study consisted of 322 uterine cervix samples of women from Mali and Burkina Faso with Cervical Intra-epithelial Neoplasia 2 and 3, adenocarcinoma and squamous cell carcinoma and 100 women with no lesions. Human Papillomavirus genotyping was performed by Real-time multiplex Polymerase Chain Reaction. Glutathione S-Transferase gene polymorphisms were determined using conventional Polymerase Chain Reaction followed by migration on agarose gel. A statistically significant association with high relative risks of 10.77 for the development of High grade Superficial or Squamous Intra-epithelial Lesion (95% CI = 5.59 20.72; p < 0.001), and 13.20 for cancer development (95% CI = 6.79 25.63; p < 0.001) was found in women with the null genotype of Glutathione S-Transferase M1 in the study population. In Burkina Faso and Mali, Glutathione S-Transferase M1-null presented relative risks of 9 and 11.05 for high-grade lesions, 15 and 11.40 for cancer. Similarly, How to cite this paper: Ouedraogo, T.-W.C., Djigma, F.W., Zohoncon, T.M., Idani, B., Ouattara, A.K., Sorgho, P.A., Obiri-Yeboah, D., Bado, P., Traore, M.A.E., Diarra, B., Yonli, A.T., Ouedraogo, C. and Simpore, J. (2020) Association between Polymorphisms of Glutathione S-Transferase and Progression to Cervical Cancer in Women from Burkina Faso and Mali. Journal of Biosciences and Medicines, 8, 12-25. https://doi.org/10.4236/jbm.2020.84002 Received: February 25, 2020 Accepted: March 28, 2020 Published: March 31, 2020 Copyright © 2020 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access T.-W. C. Ouedraogo et al. DOI: 10.4236/jbm.2020.84002 13 Journal of Biosciences and Medicines significant results had been observed in women with human papillomavirus positive and human papillomavirus negative. The results of the present study support the idea that the deletion of Glutathione S-Transferase M1 plays a crucial role in the progression of high-grade lesions and cervical cancer.


Introduction
Human papillomavirus (HPV) infection, a sexually transmitted disease, is the leading risk factor for cervical cancer [1] [2]. In 2018, the global incidence of cervical cancer was 569,847 cases and 311,365 deaths [3]. It is the fourth most common cancer and the second in terms of mortality in women worldwide [3]. According to some previous studies, 85% of cervical cancer cases are recorded in low and middle income countries [4] [5], with 87% of world deaths due to the disease as estimated by WHO. In West Africa, 31,955 cases and 23,529 deaths per year have been reported [6]. In Burkina Faso and Mali, where the disease is the first most common diagnosed cancer, the incidence was estimated at 2517 and 2206 cases respectively with 2081 and 1704 deaths per year [6] [7]. A frequency of 72.31% and 48.8% of HR-HPV were found in cancerous and precancerous lesions tissues of the uterine cervix in women in Burkina Faso [8] [9]. Although high-risk human papillomaviruses are identified as the major causative agent of cervical cancer, viral clearance has been observed in some women. This suggests that other cofactors (environmental or genetic) play an important role in viral persistence leading to progression to carcinogenesis [10]. Metabolized xenobiotic enzymes have been shown to be important risk modifiers in human carcinogenesis [11]. Phase I enzymes (cytochrome P450) activate procarcinogens, while reactive intermediates are inactivated by phase II enzymes [12] [13]. A possible mutation of the latter enzyme could have consequences on the carcinogenic compounds' elimination. Therefore, phase II metabolite enzymes polymorphisms have been designated according to the literature as cofactors in the HPV infections progression to precancerous and cancerous lesions.
Glutathione S-transferase (GST, E.C 2.5.1.18) is a super family of enzymes metabolizing xenobiotic substances with a high degree of specificity of conjugation of glutathione (GSH). These enzymes are essential for the metabolism of harmful electrophilic compounds and are responsible for the response to oxidative stress. There are 8 classes of enzymes: alpha (GSTA), mu (GSTM), theta (GSTT), pi (GSTP), sigma (GSTS), kappa (GSTK), omega (GSTO) and zeta (GSTZ), which are involved in detoxification of drug compounds, carcinogens, and for the inhibition of oxidative damage in tissues [14]. The GSTM1 and GSTT1 subclasses belonging respectively to the mu and theta classes, are highly polymorphic genes [15] [16] and the most encountered in the literature. There are two types of glutathione S-transferase M1 and T1 genes polymorphisms: nucleotide variation (G2619C for GSTM1 and A310C for GSTT1) and complete gene deletion or homozygous deletion (null genotype), associated with loss of enzyme activity. The nucleotide variations lead to two non-suppressive genotypes (called active or present genotypes) with an active presence of the enzyme.
GSTM1 and GSTT1 are located on chromosomes 1p13.3 and 22q11.23 respectively and the enzymes participate in conjugation and detoxification. Any variation in the activity of these enzymes could potentially have significant repercussions on the fate of drug, carcinogenic compounds and on the quantities of metabolites produced. Individuals with zero genotype have somewhat reduced overall detoxification capacity [16]. They are associated with the susceptibility to develop certain diseases, such as infectious diseases, cancers and others, probably because of increased susceptibility to the harmful effects of oxidative stress, environmental toxins and carcinogens [17]- [22].
In recent years, much data have been generated about the prevalence and the distribution of HPV genotypes especially in general population in West Africa and some suggest the emergence of non-HPV16/18 genotypes in precancerous lesions and cancers [23]. However, there are few association studies about HPV infections, genetics factors and progression to cervical cancer. The present case-control study aimed to compare the polymorphisms of GSTM1-null in women with cancerous lesions and without lesions in Burkina Faso and Mali.

Study Framework and Sampling
The Our study was a cross-sectional case-control study with a retrospective collection of samples and socio-demographic data of women in Mali and a prospective collection of samples and data in Burkina Faso. Data was collected from archived patient files from different laboratories. Samples with lesions were considered cases.
In terms of controls, we selected the women who consented to the study and to being screened for cervical lesions by visual inspection with acetic acid and Lugol's iodine (VIA/VILI). A woman was included in the control group if her VIA/VILI was negative. It should be noted, however, that a fresh sample of the endocervix was taken from each woman before the VIA/VILI. All non-pregnant women and young girls in sexual activity were approached to participate in the study. Any virgin, menstruating or pregnant woman or women who had a total hysterectomy was not included in the study.

DNA Extraction
The DNA of the fresh samples was extracted using the commercial kit called "DNA-Sorb-A" from sacace biotechnologies® according to the manufacturer's protocol. The archived samples fixed and embedded in paraffin blocks were cut and DNA was extracted using the commercial FFPE DNA Purification kit from (NORGEN BIOTECH CORPORATION, Canada). Xylene was used for dewaxing the samples before the actual extraction according to the manufacturer's protocol. The DNA extracted from the two types of sample was stored at −20˚C for PCR amplifications.

HR-HPV Detection
The detection of HR-HPV was carried out with the HPV Genotypes 14

GSTM1 Polymorphism Detection
The conventional multiplex PCR method described by Chen et al. was used for the GSTM1 genotyping [27]. Each PCR well contained 10 μL of Taq Gold 360 Master Mix Ampli, 1 μL of each primer, 7 μL of sterile water and 2 μL of DNA.
The PCR amplification program was as follow: a denaturation step at 94˚C for 5 minutes followed by 35 cycles at 94˚C for 1 minute, 57˚C for 1 minute and 72˚C for 1 minute, ending with an extension at 72˚C for 7 minutes. PCR products were separated by electrophoresis through a 3% agarose gel containing ethidium bromide ( Figure 1). The GSTM1 and β-globin amplicons size were expected at 215 bp and 268, respectively. The absence of GSTM1 band in a positive β-globin sample was considered as null genotype and PCR was invalid in the absence of the β-globin band.

Ethical Considerations
The women enrolled in the present study gave their free and informed consent    software. The confidence interval was set at 95%. Fisher test and the odds ratio were used for comparison. The difference was statistically significant for p < 0.05.

Socio-Demographic Characteristics of the Women in the Study
The 232 women were found to have cervical lesions and had age ranging from 18 to 82 years with a mean of 44.24 ± 14.10 years while that of the 100 controls ranged from 18 to 57 years of age with a mean of 34.90 ± 9.2 years ( Nulliparous: woman without a child; Primiparous: woman with one child; Paupiparous: woman with two or three children; Multiparous: woman with four or five children; Great multiparous: woman with more than 5 children.

Discussion
The present case-control study compared GSTM1 deletion polymorphisms in HPV-infected women with normal cytology and those with precancerous lesions or cancer in order to determine the implication of GSTM1 polymorphisms in progression to cancer.
Glutathione S-Transferase M1-null was associated with the development of cancer in women HPV positive [28]. It was therefore obvious that the genetic polymorphisms of metabolic enzymes, by causing oxidative stress, would play a role in carcinogenesis modifying the cervical carcinogenic process induced by HPV infection.
In our study, the GSTM1-null genotype compared to the GSTM1-active genotype, we observed statistically significant frequencies with high relative risks for development of HSIL and cancer, respectively in Burkina (OR = 9 and 15), Mali (OR = 11.05 and 11.40) and in general study people (OR = 10.77 and 13.20). Another author found a low relative risk than ours. In India, some authors had observed 2.62 and 4.01 relative risks for progression to cancer respectively [29] [30]. Women with GSTM1-null genotype compared to the controls in Serbia, had statistically significant frequencies and important risk (OR = 2.26) [31] and in India (OR = 2.39) [32]. These risks were also lower than ours (OR = 11.92). The high relative risks in our study could be due to environmental factors such as inhaling smoke from firewood used for cooking, food hygiene, high risk HPV-infection other genital infection in low-and middle-income countries and increased GSTM1-null risk factor, for examples early sexual debut pregnancy, high parity [33], polygamy [34] and the high prevalence of HIV infection [35]. In addition, insufficient awareness and poor knowledge of cervical cancer [36], false beliefs about cancer [37], low proportion of women attending cancer screening [38], the lack of financial resources and facilities for cervical cancer  [42] and HSIL [29] [42] [43]. In Brazil, after a 60-month follow-up, Tacca et al. [44] determined 80.0% survival in women with GSTM1-active and 73.3% in women with GSTM1-null. Their results were also statistically no significant (p = 0.368). The possible explanation could be the existence of geographic differences according to the population studied, of the association of the GSTM1-null factor with cancer of the cervix. These results should lead us to exploit other factors that can be associated with GSTM1-null factor to increase the risk for cervical cancer in our countries.
In women with HPV+ or HPV− we also observed significant results with increased risks 11 to 19 in the null genotype of GSTM1 compared to the active genotype for HSILs as for cancers. Therefore, GSTM1-null risk factor for cervical cancer was no dependent on HPV-infection. In Hungary after 7 years of follow up, Cseh et al. [45] found significant results for the same genotype GSTM1-null in women with persistent HR-HPV infection (OR = 1.78). In India, an increased risk higher than ours was observed precisely in women infected with HPV16 (OR = 31.39).
The socio-demographic characteristics and samples size of the studies cited differ from one study to another (smoking status, smoke from firewood, etc.).
Several studies have used colposcopy and/or histology diagnostic test for cervical dysplasia. In our study, the VIA/VILI test was only used for controls. The difference of ethnic origin in the transmission of functional polymorphisms of genes metabolizing xenobiotics [46] could be a key factor in the differences in risks observed in Burkina Faso and Mali.
Blood samples were used considered by the different studies on the genotyping of GSTM1 polymorphisms. Our samples involved cells or tissues from the organ-infected or susceptible to be infected; allowing to study the GSTM1 polymorphisms on cells of the target organ of HPV. Since the virus infects cervical cells, DNA damage could be caused by the virus involving a high relative risk.
This could also be an advantage for our study, used endocervical and tissue samples of women.
Our study was carried out on samples of treated tissue, coated with paraffin and archived; this could be a limitation. It would be judicious to carry out a study in which the patients will be followed up as well as a comparison between genotyping of GSTM1-null blood and tissues, being able to constitute a track for possible research on polymorphisms.

Conclusion
The results of the present study demonstrated an association between glutathione S-transferase polymorphisms and progression to high-grade precancer-ous lesions and cervical cancer among women in Burkina Faso and Mali. Deep knowledge about the specific role of host GST genes polymorphisms in the progression of HPV infection to cancer through a longitudinal study is required in low income countries such as Burkina Faso and Mali with high prevalence of cervical cancer. Altogether the understanding of these mechanisms is of interest to improve HPV prevention and management especially in West African countries in the context of emergence of non-HPV16/18 genotypes suggested by recent studies.