Carrying SNP rs17506395 (T > G) in TP63 gene and CCR5Δ32 mutation associated with the occurrence of breast cancer in Burkina Faso

Abstract Genetic alterations in the TP63 (GenBank: NC_000003.12, ID: 8626) and CCR5 (receptor 5 chemokine co-receptor) (GenBank: NC_000003.12, ID: 1234) genes may increase the risk of developing breast cancer. The aim of this study was to investigate the probable involvement of polymorphisms rs17506395 in the TP63 (tumour protein 63) gene and the CCR5Δ32 mutation in the occurrence of breast cancer in Burkina Faso. This case–control study included 72 patients and 72 controls. Genotyping of SNP rs17506395 (TP63) was performed by polymerase chain reaction–restriction fragment length polymorphism, and genotyping of the CCR5Δ32 mutation was performed by allele-specific oligonucleotide polymerase chain reaction. For SNP rs17506395 (TP63), the genotypic frequencies of wild-type homozygotes (TT) and heterozygotes (TG) were, respectively, 27.72 and 72.22% in cases and 36.11 and 63.89% in controls. No mutated homozygotes (GG) were observed. For the CCR5Δ32 mutation, the genotypic frequencies of wild-type homozygotes (WT/WT) and heterozygotes (WT/Δ32) were 87.5 and 13.5%, respectively, in the cases and 89.29 and 10.71%, respectively, in the controls. No mutated homozygotes (Δ32/Δ32) were observed. None of the polymorphisms rs17506395 of the TP63 gene (OR = 1.47, 95% CI = 0.69–3.17, P = 0.284) and the CCR5Δ32 mutation (OR = 1.32, 95% CI = 0.46–3.77; P = 0.79) were associated with the occurrence of breast cancer in this study.


Introduction
Breast cancer is a major public health problem.In 2020, according to GLOBOCAN, it was the most frequently diagnosed cancer in women, causing the greatest loss of life in both developed and developing countries.In 2020, there were around 2,261,419 new cases and 684,996 deaths worldwide [1].In Burkina Faso, over the same period, 1,927 new cases were diagnosed, compared with 1,142 deaths, making it the leading cause of death from cancer [1].In addition, a number of studies have shown the involvement of sociodemographic, clinical, and behavioural factors (age, place of residence, occupation, and lifestyle) in the development of breast cancer [2].Familial forms are hereditary and result from a genetic predisposition.However, sporadic forms are the result of an association between both genetic and environmental factors [2,3].Polymorphisms or mutations in certain genes such as BRCA1 and BRCA2 [4,5] and ATM, BRIP1, CHEK2, NBS1, PALB2, and RAD50 [6] are also incriminated in the development of breast cancer.In addition to these polymorphisms and mutations, the SNP rs17506395 (TP63 gene) and the CCR5Δ32 mutation are thought to be involved in the development of breast cancer [7,8].
The TP63 gene (OMIM:603273, HUGO HGNC:15979) belongs to the p53 family and is located on chromosome 3q28.It codes for the p63 protein, which plays a crucial role in the maintenance of stem cells in several epithelial tissues and is required for the normal development of epithelial organs, including the mammary glands [9].The SNP rs17506395 (T > G) (189803530T > G,189521319T > G,211785T > G) of the TP63 gene, which was initially associated with fertility, has also been implicated in the development of several cancers, including breast cancer [10][11][12].
The CCR5 gene (OMIM:601373, HUGO HGNC:1606) is located on chromosome 3 and comprises three exons encoding the CCR5 protein.CCR5 is a chemokine receptor of the β-chemokine receptor family of integral membrane proteins [7].A 32-base pair deletion of the CCR5 gene (CCR5Δ32) leads to the formation of a non-functional receptor that causes significant defects in ligand-mediated chemotaxis and has been implicated in a variety of immune-mediated diseases [13,14].CCR5 may have an indirect effect on cancer progression by controlling the antitumour immune response [7].
The CCR5delta32 mutation (GenBank: NM_001394783.1)has been studied in several cancers including skin cancer, bladder cancer and cancer of the liver [15], cervical cancer [16], osteosarcoma [17], breast cancer [18,19], and oral cancer [20].These broad studies on different kinds of cancer, however, often suggest contradictory results that bring up the importance of further studying these mutations in different populations.
Genetic predisposition to breast cancer in the African population is less well studied [8].In Burkina Faso, previous studies have focused on certain genetic factors and breast and prostate cancers, in particular HLA-DRB1*11 1*12, TP53 and CHEK2, BRCA1, and 943ins10 [21][22][23]; exons of BRCA1 and BRCA2 [24]; R462Q (rs 486907) and D541E (rs 627928) of RNASEL gene [25]; Ser217Leu and Ala541Thr of ELAC2 gene [26]; and ERCC1 (rs3212986) and ERCC2 (rs1799793, rs13181) [27].However, no study has yet looked at the involvement or otherwise of the TP63 and CCR5 genes in the occurrence of cancer in Burkina Faso.However, a study has been conducted in Cameroon to elucidate the involvement of SNP rs17506395 in the development of breast cancer [8], and it was found that the rs17506395 of the TP63 gene was not involved in the development of breast cancer (OR = 0.86, P = 0.1269).It is in this context that this study aimed to investigate the probable involvement of the SNP rs17506395 of the TP63 gene and the CCR5Δ32 mutation in the occurrence of breast cancer in Burkina Faso.
This could contribute to not only knowledge of the genetic risk factors for breast cancer but also provide knowledge that could be used in a strategy to prevent the disease.

Setting and study population
This was a descriptive and analytical case-control study in Ouagadougou, Burkina-Faso.The study population consisted of 144 women, 72 of whom were patients with breast cancer (cases) and 72 healthy women without breast cancer (controls) who attended consultations at two University Hospitals: Yalgado OUE-DRAOGO (CHU-YO) and Bogodogo (CHU-B) and two medical centres with a surgical unit: Schiphra and Paul VI. Various biomolecular tests were carried out at the Laboratory of Molecular and Genetic Biology (LABIOGENE), Joseph KI-ZERBO University, and Pietro Annigoni Biomolecular Research Centre (CERBA).In this study, any participant who received anatomopathological confirmation was considered a "case."Eligible controls were women with no breast abnormality on ultrasound examination.These controls shared the same socio-demographic framework as the patients in this study.
Any participant without anatomopathological confirmation of breast cancer and those with breast cancer who had not given their consent to take part in the study were excluded.
Informed consent: Informed consent has been obtained from all individuals included in this study.

Ethical approval:
The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance with the tenets of the Helsinki Declaration, and has been approved by the Health Research Ethics Committee (CERS) of Burkina Faso (Deliberation No. 2019-5-067 of May 15, 2019).

Data collection and sampling
A questionnaire was administered to the patients to collect their socio-demographic, anthropometric, and clinical data.Five millilitres (5 mL) of venous blood was then collected in an EDTA (ethylene-diamine-tetra-acetic acid) tube and centrifuged at 3,500 rpm for 15 min.The plasma and pellet were stored separately at −20°C.

DNA extraction, quantification, and purity testing
The participant's genomic DNAs were extracted using the QIAamp®DSP DNA Blood Mini kit (QIAGEN, GmbH, QIAGEN Strabe1, D-40724 Hilden, Germany).The Nanodrop (Thermo Fisher Scientific) was used to quantify and check the purity of the extracted DNA, the concentration of which was adjusted to 10 ng/μL.

Genotyping of SNP rs17506395 (T > G) in TP63 and CCR5Δ32 mutation
For the rs17506395 polymorphism of the TP63 gene, polymerase chain reaction-restriction fragment length polymorphism was used, and for the Δ32 mutation of the CCR5 gene, allele-specific oligonucleotide polymerase chain reaction (ASO-PCR).

SNP rs17506395 (T > G) in TP63
A reaction mixture with a total volume of 25 μL (per sample) composed of 4 μL of 5X FIREPOL ® Master Mix (Solis BioDyne), 0.5 μL of each primer (0.5 μM), 14 μL of sterile water, and 5 μL of DNA (10 ng/μL).The sequences of the primer pairs used are recorded in Table 1 (Guleria et al., 2012;Tiofack et al., 2020).Amplification was carried out using the Gene Amp ® PCR System 9700 thermal cycler (Applied Biosystems) following the amplification programme: initial denaturation at 94°C for 5 min followed by 40 amplification cycles (denaturation at 94°C, 30 s, hybridisation at 57°C for 30 s, and elongation at 72°C, 30 s), and final elongation at 72°C for 7 min.The PCR products were then enzymatically digested with the MboII enzyme at 37°C for 3 h.Finally, the digestion products were subjected to electrophoretic migration for 45 min (100 V) on a 2% agarose gel containing 2 μL of ethidium bromide (10 mg/mL) and visualised using the "Vilber" apparatus (vilber Lourmat).

Statistical analysis
Data were entered using Excel 2016 and analysed using SPSS software version 21.0, R software version 4.2.1, and Epi Info software version 7. The chi-square test was used for frequency comparisons.Odds ratios (OR) and 95% confidence intervals (CI) were calculated to assess risk.Results were considered statistically significant for a P-value of less than 0.05.

Clinical and socio-demographic characteristics
Our study included 144 women: 72 patients and 72 controls.Age ranged from 19 to 70 years, with an average of 41.08 ± 12.19 years.The majority of patients (68.06%) were aged strictly over 40.The majority of participants lived in urban areas (93.06% for cases and 100% for controls).Body mass index (BMI) was calculated according to the US National Institute of Health/National Heart Lung and Blood Institute criteria.A statistically significant association was found between obesity and the occurrence of breast cancer (OR = 3.88, 95% CI = 1.13-14.38,P-value = 0.015).There was no significant association between late menopause and the occurrence of breast cancer (Table 2).Approximately 15.27% of patients had a family history of breast cancer.

rs17506395 (TP63) polymorphism and risk
of breast cancer

Combined genotypes of SNP rs17506395 (T > G) in TP63 and CCR5Δ32 mutation and breast cancer risk
The combined effect of polymorphisms on breast cancer risk has been demonstrated with several combined  genotypes.However, no significant association was found between the combined genotypes of the polymorphisms and breast cancer (Table 5).

Socio-demographic and clinical characteristics
Our  [29] in Ivory Coast and Egypt in 2012 [30].This difference could be justified by the fact that Burkina Faso's population is characterised by its youth: around 80% of the population is aged under 35, according to the latest census published in 2020 by the National Institute of Statistics and Demography [31].No significant association was observed between family history and the development of breast cancer in this study.These results differ from those of Antoniou et al. [31] and Economopoulou et al. [32], who were able to establish a link between family history and breast cancer.
Our results showed a significant association between BMI and the occurrence of breast cancer (OR = 3.8, 95% CI: 1.13-14.38,P-value = 0.015).These results are consistent with those found in other studies in France [2,33].The probable explanation could be an accumulation of intraabdominal fat by these patients.This accumulation of fat leads to obesity, the causes of which are essentially linked to bad eating habits such as fast food, unlicensed alcohol consumption, and physical inactivity (sedentary lifestyle).This obesity is more prevalent in urban areas due to the proximity of these food products.In fact, women living in urban areas had somewhat stable economic situations and could therefore indulge in an excessively high-fat diet.This excessive weight gain, combined with a sedentary lifestyle, could lead to the onset of breast cancer.
In reality, the link between obesity and breast cancer is complex [34].Obesity induces metabolic dysfunctions, notably altered cellular metabolism and signalling pathways.It corresponds to excessive weight gain, and this intra-abdominal fat gain can lead to the formation of circulating oestrogens through an enzyme called aromatase.The accumulation of these oestrogens over a period of time in the body is thought to transform breast cells into cancer cells [35].Obesity is also thought to be associated with hyperinsulinism, an increase in high glucose levels leading to resistance to breast cancer treatment, which in turn favours the appearance of tumours [36].
Late menopause was not associated with a risk of breast cancer.This difference in our results can be explained by the fact that there are fewer menopausal women in our study population.This result differs from those found by Moroccan [37] and Algerian [38] researchers, who showed that late menopause was linked to an increased risk of developing breast cancer.The main cause was found to be prolonged exposure to ovarian hormones in their study populations.The longer a woman waits for menopause, the more she is exposed to oestrogens, which are likely to promote the development of breast cancer.It is also important to note the use of steroid hormones and carcinogenic products by these same populations.They can acquire them through hormone replacement therapy [39].The use of growth promoters for more than 5 years [40] and the use of hormonal contraceptives over a long period of time [41].

SNP rs17506395 (TP63) and CCR5Δ32 polymorphisms and breast cancer risk factors
Analysis of the genotypic and allelic frequencies of the study population showed that the different genotypes and alleles in the sample were in HWE for the Δ32 mutation of the CCR5 gene.This was observed in both cases and controls (P-value > 0.05) for the CCR5 polymorphism (x 2 = 1.125, P-value = 0.28 and x 2 = 1.16,P-value = 0.288).However, for the TP63 gene, the population was not in equilibrium in either cases or controls (x 2 = 23.27,P-value = 0.000001 and x 2 = 16.74,P-value = 0.000043).In fact, Hardy-Weinberg's law stipulates that within a population, allelic and genotypic frequencies remain constant from one generation to the next [42].The allelic and genotypic frequencies of the Δ32 mutation in the CCR5 gene are therefore constant from one generation to the next in Burkina Faso.However, the rs17506395 polymorphism does not respect Hardy Weinberg's law.In 2020, in Cameroon, a study was able to demonstrate that their study population was indeed in equilibrium with P-value = 1 [8].This difference could be explained by the relatively small size of our study population and also by the heterogeneity of this polymorphism (rs17506395).No association was observed between the rs17506395 polymorphism of the TP63 gene and the development of breast cancer in the study population (P-value > 0.05).Our results corroborate those found in Cameroon that tried to elucidate the involvement of this polymorphism in the occurrence of breast cancer but who did not find a link between this polymorphism and breast cancer regardless of the genotype considered, while another Cameroonian study found a link between the TP63 polymorphism and people aged under 40 (OR = 0.5, 95% CI = 0.26-0.94,P-value = 0.03) [8].Our results differ from those found in a Pakistani and Asian population, respectively, which had established an association between the TP63 polymorphism and the risk of developing breast cancer.Indeed, according to Fatima et al. in 2019 [43], the WT/Δ32  [12,44].According to a study in the same Asia, the GG mutant genotype of the TP63 polymorphism conferred protection against breast cancer [45].This genotype was not encountered in our study.We found no association between TG genotype and the development of breast cancer (OR = 1.47, 95% CI = 0.69-3.17,P-value = 0.284).In China, on the other hand, we found an association between breast cancer and the TG genotype in younger people [12].The discrepancy between these results and those of the present study could be explained by the fact that the majority of our patients were over 40 years of age.In our study, the frequency of the T allele was higher than that of the G allele, although this difference was not statistically significant.These results are similar to those obtained in 2018 [46].

Association between certain sociocharacteristics and polymorphisms (rs17506395 (TP63) and CCR5Δ32)
Our results show that there was no association between the TG genotype of the TP63 polymorphism or the WT/Δ32 genotype of the CCR5 gene and the risk of breast cancer in pre-menopausal women or those with a family history of breast cancer.In terms of pre-menopausal status, these two genotypes had P-value = 0.127 and P-value = 0.392, while family history had P-value = 0.372 and P = 0.515.These data differ from those of a study carried out in the Cameroonian population [8] who found an association between these polymorphisms of the TP63 gene and the Δ32 mutation of the CCR5 gene and the occurrence of breast cancer in women with a family history.Also, in 2014, a study conducted in China [12] showed that TT and TG polymorphisms were significantly associated with an increased risk of breast cancer in women under 40.The likely hypothesis is that these patients have been exposed for a long time to steroid hormones and carcinogens that act on breast cells.This exposure can initiate tumourigenesis by causing DNA damage, such as mutations; in fact, prolonged exposure to hormones induces endocrine disruption, leading to the dysfunction of the hormonal system.This imbalance results in the proliferation of cancer cells.Exposure to carcinogens also causes mutations, notably in the BRCA 1 and 2 genes, which can lead to breast cancer.
With regard to the family history of breast cancer and age at diagnosis, no risk was found between these factors and the genotypes of the rs17506395 polymorphisms of the TP63 gene and the CCR5Δ32 mutation.These results are similar to those of the studies carried out in Cameroon [8] and Iran [44].The same authors reported that there was no association between the rs17506395 (TP63) and CCR5Δ32 (CCR5 gene) polymorphisms and age at diagnosis on the one hand, and family history of breast cancer on the other hand.In our study, most of the people included had no family antecedents, which may explain the absence of any link between family history and the occurrence of breast cancer in our context [8].

Combined effects of rs17506395 (TP63) and CCR5Δ32 polymorphism genotypes and breast cancer risk
We believe that the association between the two polymorphisms would have a greater influence on the occurrence of breast cancer than the effect of a single polymorphism.However, analysis of our data showed no significant association between the combined genotypes of these two polymorphisms and the occurrence of breast cancer (OR = 1.35, 95% CI = 0.55-3.29,P-value = 0.41).In the literature, to our knowledge, no other study in the past has shown a probable link between the genotypes of the rs17506395 polymorphisms of the TP63 gene and the Δ32 mutation of the CCR5 gene and breast cancer.

Limitations of our study
The limitations of our study can be summed up essentially not only by the small size of our sample but also by a lack of socio-demographic and clinical information.It would therefore be more interesting to continue the study on a larger population in order to assess more objectively the real impact of these two polymorphisms on the occurrence of breast cancer in Burkina Faso.

Conclusion
This first study in Burkina Faso reported the presence of the [G] mutant allele of the TP63 gene in both cases and controls.Like the CCR5 Δ32 mutation, the [Δ32] mutant allele of the CCR5 gene was present in both cases and controls.Neither the (TG) genotype of the TP63 gene nor the (WT/Δ32) genotype of the CCR5 gene was associated with the development of breast cancer in our study population.

Figure 1 :
Figure 1: Electrophoretic profiles of the TP63 gene: Before (a) and after digestion (b).(M) 100bp molecular weight marker; (a) amplification result after conventional PCR; (b) amplification result after enzymatic digestion.

Table 1 :
Primers for amplification of SNP rs17506395 (T > G) in TP63 and CCR5Δ32 mutation

Table 2 :
Clinical characteristics of the study population OR: odd ratio; 95% CI: 95% confidence interval; BMI: body mass index.

Table 3 :
Distribution of allelic and genotypic frequencies of TP63 and CCR5

Table 4 :
Association between socio-characteristic and genotypes of the two polymorphisms

Table 5 :
Analysis of combined genotypes Ref: reference; OR: odds ratio; CI: confidence interval; NA: not applicable.