Polymorphisms in the Tumor Necrosis Factor Genes Are Associated with Breast Cancer in the Moroccan Population

Abstract

Background:

The multifunctional cytokines of the tumor necrosis factor (TNF) family have been found to be involved in the promotion of inflammatory responses, and to play critical roles in the pathogenesis of inflammatory, autoimmune, and malignant diseases. The aim of the present study was to assess the associations among the TNFα −238 G > A (rs361525), TNFα −308 G > A (rs1800629), and TNFβ +252 A>G (rs909253) polymorphisms, and the breast cancer (BC) susceptibility in the Moroccan population.

Materials and Methods:

We conducted a case−control study, including 492 participants made up of 264 pathologically confirmed BC subjects, and 228 healthy women as controls. The samples were genotyped by means of polymerase chain reaction-restriction fragment length polymorphism analyses.

Results:

The TNFα −238 G > A and TNFα −308 G > A polymorphisms were significantly associated with increased risk of BC for the AA genotype, while, the AG genotype of TNFβ +252 A>G may offer a protective effect in this population. Haplotypic analyses showed that the GAA and AAG haplotypes increased the risk significantly for BC. Moreover, a significant association was observed between polymorphisms at the TNFα −238 A>G locus and the clinical profiles of the patients with regard to their estrogen-and progesterone-positive receptor status.

Conclusion:

These findings indicate that TNF gene polymorphisms are linked with the risk of BC in the Moroccan population. Further studies implementing a larger sample size are needed to support our findings.

Introduction

Breast cancer (BC) is the most common type of cancer among women, and leading cause of death worldwide (Bray et al., 2018). Its etiology is multifactorial, however, the family history is one of the most important and consistent risk factors for this disease. BC clinical diagnosis is generally based on the implementation of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor2 (Her2). Its immunohistochemical prognosis uses different subtypes, such as triple-negative BC, Her2⁺, luminal A, and luminal B BCs (Carey et al., 2006).

Additionally, the multifunctional cytokine tumor necrosis factor (TNF) was found to be involved in the promotion of the inflammatory responses, and plays critical roles in the pathogenesis of inflammatory, autoimmune, and malignant diseases, including BC (Mackay et al., 1998; Esquivel-Velázquez et al., 2015). Initially proposed to have anticarcinogenic effects, the TNF genes were later shown to be tumorigenic in both in vitro and in vivo studies (Coley 1991; Balkwill 2002; Cruceriu et al., 2020) Their protein effects depend presumably on their relative concentration and, the presence of other modulating factors, which make them important candidate genes in BC disorder (Miles et al., 1994).

TNFα and TNFβ genes are located within the highly polymorphic major histocompatibility complex (MHC) class III region, specifically on the short arm of chromosome 6. Their protein function is complex and it interacts with two receptors, TNFR1 and TNFR2, which participate in signal transduction pathways, and the signaling cascade of cellular responses such as apoptosis, proliferation, differentiation, migration, and angiogenesis (Cereda et al., 2012).

Changes in single nucleotides in coding regions of the TNFα and TNFβ loci, have been suggested to modify the binding site of specific transcription factors, and consequently, to affect transcriptional regulations by modulating their expression, which might make the host predispose to certain cancers, including BC (Hagihara et al., 1995; Kaluza et al., 2000; Hollegaard and Bidwell 2006). Therefore, it becomes imperative to consider the functional polymorphisms and haplotypes in TNFα and TNFβ loci, as useful immunological biomarkers in BC malignancy, owing to their elevated levels among tumors' cell environment, and to their implication in metastatic behavior of inflammatory breast carcinomas (Leek et al., 1998; Gaudet et al., 2007).

The TNFα −308 G > A (rs1800629) polymorphism is a G → A substitution in the promoter region of the TNF, and reportedly affects gene expression, since it is situated within the binding site of the Activator Protein1 and 2 (AP1-and AP2) repressive transcription factor (Wilson et al., 1997). In addition, the less common allele A has been associated with higher constitutive and inducible TNF-α expression, in comparison to the more common TNFα −308G allele, which is associated with relatively lower TNF-α expression (Wilson et al., 1997; Abraham and Kroeger 1999).

As to TNFα −238 G > A (rs361525) a substitution of G to A, was reported to decrease TNF transcription in vitro experiments (Hajeer and Hutchinson, 2001), whereas, the TNFβ gene is adjacent and structurally related to TNFα and shares many of its functions (Bazzoni and Beutler, 1996). Indeed, the TNFβ +252 A>G (rs909253) polymorphism located on the first intron of the TNFβ gene, affects the expression and the concentration of TNFα and TNFβ proteins in plasma (Messer et al., 1991; Pociot et al., 1993). It was also reported that the rare allele G has been correlated with a higher TNFβ expression, and the common allele A is associated with increased TNFα gene expression (Messer et al., 1991). However, the literature data on the association of both TNFα (−238 G>A; −308 G>A) and TNFβ +252 A>G polymorphism with BC are still controversial, and yielded contradictory results (Qidwai and Khan 2011; Aznag et al., 2018). The main objective of this study was to investigate for the first time, the association between the TNFα (−238 G>A; −308 G>A) and TNFβ +252 A>G polymorphisms and the BC disorder in the Moroccan population.

Materials and Methods

Study samples and ethical considerations

A total of 264 histologically confirmed BC patients (i.e., cases) were recruited from the Regional Center of Oncology and Radiotherapy at Hospital Hassan II of Agadir City (Morocco), and 228 female controls without cancer, agreed to participate in this study. A written informed consent was obtained from all the participants. The study received approval from the Ethics Committee of Mohamed VI University Hospital, at the Cadi Ayyad University in Marrakech City (Morocco). Detailed data on the demographical, clinical, and pathological profile of each patient in terms of age, menopausal status, histopathological type, Scarff-Bloom-Richardson (SBR) tumor grading system, hormonal receptors, HER2 status, immunohistochemical and subtypes (IHC), were retrieved from the patient medical records. The control group was made up of healthy donors with no oncological, autoimmune, or inflammatory diseases. All women, both in case and control groups were Moroccans.

Determination of cytokine genotype

Genomic DNA was isolated from the peripheral blood leucocytes using the conventional salting-out chloroform-based method (Miller et al., 1988). The single nucleotide polymorphisms (SNPs) were carried out using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as described elsewhere (Cabrera et al., 1995).

Briefly, for the TNFα −238 G > A polymorphism, the forward primer was 5′-AAA CAG ACC ACA GAC CTG GTC-3′, and the reverse primer was 5′-CTC ACA CTC CCC ATC CTC CCG GAT C-3′, whereas the annealing temperature was 58°C. A PCR fragment of 150 base pairs (bp) was digested with the BamHI restriction enzyme, and the GG genotype was identified as 130 + 20 bp bands using a 3% agarose gel (Stayoussef et al., 2010).

For the TNFα −308 G > A polymorphism, the used PCR primers were 5′-GAG GCA ATA GGT TTT GAG GGC CAT-3′ and 5′-GGG ACA CAC AAG CAT CAA G-3′, whereas the annealing temperature was 61°C. A PCR fragment of 107 bp was digested with the NcoI restriction enzyme, the GG genotype was identified as 87 + 20 pb bands, and was revealed using a 3% agarose gel (Cabrera et al., 1995).

For the TNFβ +252 A>G polymorphism, the PCR primers used were 5′-CCG TGC TTC GTG CTT TGG ACT A3′ and 5′-AGA GCT GGT GGG ACA TGT CTG-3′, and the annealing temperature was 60°C. The GG genotype was identified as 586 + 196 bp bands, after digestion with the NcoI restriction enzyme, and revelation in 2% agarose gel (Cabrera et al., 1995).

Statistical analysis

Genotype frequencies of patient and control groups in each loci were tested for Hardy-Weinberg Equilibrium (HWE) departures using a chi-squared (χ²) test, differences were considered significant at p < 0.05. The distribution of the genotype and allelic frequencies of TNFα and TNFβ polymorphisms for patients and control subjects were compared using the χ² test. Odds ratio (OR) with respective confidence interval (95% CI) for disease susceptibility was also calculated. Patient tumor characteristics, such as menopausal status, histopathological type, SBR grading, hormonal receptors, HER2 status, and IHC were correlated and calculated with different genotypes. For each polymorphism, risk estimation was given as OR adjusted by age and menopausal status. All the statistical analyses were done using SNPStats software (Solé et al., 2006). Pairwise linkage disequilibrium (LD) of alleles at the three loci, was assessed in terms of coefficient of linkage disequilibrium (D), Lewontin's coefficient (D′), and Pearson's (r) statistics, using the Haploview software (Barrett et al., 2005). Haplotype frequencies of TNFα (−308 G > A −238G>A) and TNFβ +252 A>G polymorphisms were reconstructed using the two-stage Expectation Maximization algorithm, and subjects who had missing data in at least one polymorphism, were excluded from analysis. Association between haplotype and BC risk was further calculated using SNPStats software (Solé et al., 2006).

Results

The mean age of case and control groups were 48.26 and 54.41 years old, respectively. As for the menopausal status, no significant differences were observed between cases and controls, and nearly 60% of the patients were at the premenopause stage. The SBR tests showed that tumors with grade II were the most frequent (62.56%), followed by grade III (36.02), whereas those with grade I represented only 1.42%. The invasive ductal tumor was the major histological type, representing a percentage of 82.63. For the hormonal receptors, 68.18% of tumors were positive for ER, and 61.43% were positive for PR, whereas 69.27% of tumors did not express the HER2 on their cells. Furthermore, the IHC profile of breast carcinomas were classified into four groups; The Luminal A was the most predominant in BC cases with a percentage of 49.50%, followed by the Luminal B, which represented 20%, the triple-negative subtype with 19.50%, and the HER2/neu with only 11% (Table 1).

Table 1.

Characteristics of Cases with Breast Cancer and Control Participants

Variable	Cases	Controls	p
Age (mean ± standard deviation)	48.26 ± 8.31	54.41 ± 11.11	<0.001
Menopausal status
Premenopause	158 (59.84%)	119 (52.19%)	0.0877
Postmenopause	106 (40.16%)	109 (47.81%)
SBR grade
I	3 (1.42%)	—
II	132 (62.56%)	—
III	76 (36.02%)	—
Histopathological type
Ductal	195 (82.63%)	—
Lobular	25 (10.59%)	—
Medullary	3 (1.27%)	—
Other	13 (5.51%)	—
ER status
Positive	150 (68.18%)	—
Negative	70 (31.82%)	—
PR status
Positive	137 (61.43%)	—
Negative	86 (38.57%)	—
HER 2 receptor
Positive	63 (30.73%)	—
Negative	142 (69.27%)	—
IHC subtypes
Luminal A	99 (49.50%)	—
Luminal B	40 (20%)	—
Triple negative	39 (19.5%)	—
HER 2/neu	22 (11%)	—

Statistically significant results are shown in bold.

SBR Grade I = well differentiated; Grade II = moderately differentiated; Grade III = poorly differentiated.

Luminal A: ER⁺ and/or PR⁺ ER⁻/PR⁺ with HER2⁻; Luminal B: ER⁺ and/or PR⁺ ER⁻/PR⁺ with HER2⁺; Triple negative: ER⁻, PR⁻, HER2⁻; HER2/neu: ER⁻, PR⁻ with HER2⁺.

ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; IHC, immunohistochemical; PR, progesterone receptor; SBR, Scarff-Bloom-Richardson.

Genotype frequency for all polymorphisms were found to be in concordance with HWE in both cases and controls (p > 0.05), except for TNFα −308 and TNFβ +252 polymorphisms, in case and control groups, respectively (Table 2).

Table 2.

Hardy-Weinberg Equilibrium Test for Genotype Frequencies, Genotype/Allele Distribution of the TNF Polymorphisms, and Their Association with Breast Cancer Risk

Locus	Genotypes/alleles frequencies	Cases	Controls	p	OR (95% CI)	p
TNFα −238 G > A(rs361525) HWEp	GGAGAAGA	128 (54%)86 (37%)21 (9%)342 (73%)128 (28%) 0.25	129 (64%)68 (33%)6 (3%)326 (80%)80 (20%) 0.51	0.3260.6380.0140.3540.043	1.001.50 (0.99-2.27)3.98 (1.51-10.48)1.52 (1.10-2.09)	0.004 0.009
TNFα −308 G > A(rs1800629) HWEp	GGAGAAGA	103 (42.2%)89 (36.5%)52 (21.3%)295 (60%)193 (40%) 0.00027	106 (52.7%)84 (41.8%)11 (5.5%)296 (74%)106 (26%) 0.36	0.1840.448<0.00010.0700.003	1.001.07 (0.72-1.61)4.40 (2.13-9.07)1.82 (1.37-2.43)	<0.0001 <0.0001
TNFβ +252 A>G(rs909253) HWEp	AAAGGGAG	117 (47%)108 (43%)26 (10%)342 (68%)160 (32%) 0.88	81 (39%)113 (54%)14 (7%)275 (66%)141 (34%) 0.0031	0.2960.1580.2070.0180.251	1.000.65 (0.44-0.96)1.27 (0.62-2.60)0.091 (0.69-1.20)	0.0360.516

Statistically significant results are shown in bold.

CI, confidence interval; HWE, Hardy-Weinberg equilibrium; OR, odds ratio; TNF, tumor necrosis factor.

When comparing genotype distributions, significant differences between cases and controls were observed in TNFα −238 G > A and TNFα −308 G > A for the AA genotypes (p < 0.05). For the A allele, significant differences were also detected between cases and controls in the three studied SNPs (Table 2).

The allelic frequency of TNFα −238A, TNFα −308A, and TNFβ +252G were 20%, 26%, and, 34%, respectively, in controls, whereas 28%, 40%, and, 32% in cases. None of the mutant variants was significantly associated with BC pathology. Whereas, AA genotypes of both TNFα −238 G > A and TNFα −308 G > A were significantly associated with increased risk of BC, AG genotype of TNFβ +252 A > G significantly decreased its susceptibility (Table 2). The three TNF polymorphisms were in strong linkage disequilibrium as illustrated in Figure 1.

FIG. 1.

Haploview plot depicting pairwise linkage disequilibrium between the three studied SNPs, in terms of Lewontin's coefficient (D′) and Pearson's (r) statistics. SNP, single nucleotide polymorphism; TNF, tumor necrosis factor.

The most common haplotype of the TNFα −308 G > A, TNFβ +252 A>G, and TNFα −238 G>A in the studied population was GAG, followed by GAA, AGG, AAG, and GGG. The remaining haplotypes AAA, GGA, and AGA were considered rare because of their low frequencies. Association between the eight constructed haplotypes and BC risk revealed that the GAA and AAG haplotypes increased significantly the risk of BC (OR = 1.94; p = 0.014 and OR = 2.28; p = 0.0034, respectively) (Table 3).

Table 3.

Haplotype Frequencies of TNF Polymorphisms and Their Associations with the Breast Cancer Risk

Haplotype associations
	TNFα −308 G > A	TNFβ +252 A>G	TNFα −238 G > A	Frequencies	OR (95% CI)	p
1	G	A	G	0.3486	1.00	—-
2	G	A	A	0.1677	1.94 (1.15-3.29)	0.014
3	A	G	G	0.1632	1.54 (0.98-2.44)	0.062
4	A	A	G	0.1263	2.28 (1.32-3.94)	0.003
5	G	G	G	0.1227	0.88 (0.46-1.66)	0.69
6	A	A	A	0.0314	3.13 (0.92-10.59)	0.067
7	G	G	A	0.0255	2.27 (0.55-9.29)	0.26
8	A	G	A	0.0148	1.97 (0.27-14.28)	0.5

Statistically significant results are shown in bold.

Considering the dominant genetic model, associations between clinical profiles of participants and TNF polymorphisms were investigated as shown in Table 4. Significant relationships were detected only at the TNFα −238 A>G locus, particularly for the ER⁺ and PR⁺ receptors (OR = 1.61; p = 0.03 and OR = 1.68; p = 0.02, respectively). Furthermore, within the patient group (i.e., cases), a statistically significant link was found at the TNFα −308 G > A SNP, with regard to the wild and mutant genotypes (OR = 1.52; p = 0.02). In addition, significant association was observed at the TNFβ +252 A>G locus, specially for the luminal A subtype of BC (OR = 0.57; p = 0.026). For the other characteristics, no associations were observed between the wild and mutant genotypes in our population.

Table 4.

Genotype Distribution of the TNF Polymorphisms Under the Dominant Genetic Model in Cases and Controls

Characteristics	TNFα −238 G > A				TNFα −308 G > A				TNFβ +252 A>G
Category	GG	AG+AA	OR (95% CI)	p	GG	AG+AA	OR (95% CI)	p	AA	AG+GG	OR (95% CI)	p
Controls	129	74	1.00		106	95	1.00		81	127	1.00
Cases	128	107	1.4572 (0.9924-2.1399)	0.0547	103	141	1.5274 (1.0490-2.2242)	0.0272	117	134	0.7305 (0.5030-1.0608)	0.0989
Grade SBR
I	1	2	3.4865 (0.3108-39.108)	0.3113	1	2	0.5579 (0.0498-6.2516)	0.6360	2	1	0.3189 (0.0285-3.5742)	0.3540
II	64	52	1.4164 (0.8903-2.2534)	0.1417	65	52	0.8926 (0.5647-1.4110)	0.6268	54	70	0.8268 (0.5265-1.2984)	0.4087
III	39	30	1.3410 (0.7697-2.3363)	0.3003	24	26	1.2088 (0.6502-2.2472)	0.5490	32	41	0.8172 (0.47631.4020)	0.4635
IHC subtypes
Luminal A	48	44	1.5980 (0.9702-2.6321)	0.0656	45	50	1.2398 (0.7605-2.0212)	0.3888	50	45	0.5740 (0.3517-0.9368)	0.0263
Luminal B	16	18	1.9611 (0.9436-4.0760)	0.0712	18	21	1.3018 (0.6544-2.5896)	0.4523	17	23	0.8629 (0.4345-1.7136)	0.6736
Triple-neg	17	14	1.4356 (0.6694-3.0788)	0.3529	15	20	1.4877 (0.7209-3.0701)	0.2825	16	21	0.8371 (0.4125-1.6986)	0.6224
HER2/neu	11	9	1.4263 (0.5649-3.6011)	0.4524	9	13	1.6117 (0.6593-3.9400)	0.2953	12	9	0.4783 (0.1929-1.1861)	0.1115
ER and PR status
ER⁺	69	64	1.6169 (1.0369-2.5214)	0.0340	63	76	1.3460 (0.8723-2.0771)	0.1794	68	75	0.7035 (0.4573-1.0820)	0.1093
ER⁻	33	25	1.3206 (0.7298-2.3898)	0.3581	26	37	1.5879 (0.8953-2.8160)	0.1137	30	36	0.7654 (0.4376-1.3385)	0.3484
PR⁺	61	59	1.6861 (1.0665-2.6656)	0.0254	56	71	1.4147 (0.9052-2.2109)	0.1278	59	73	0.7891 (0.5072-1.2278)	0.2937
PR⁻	44	30	1.1886 (0.6892-2.0499)	0.5344	33	45	1.5215 (0.8976-2.5790)	0.1190	40	40	0.6378 (0.3794-1.0721)	0.0897
HER⁺	27	27	1.7432 (0.9518-3.1929)	0.0719	27	34	1.4051 (0.7897-2.4999)	0.2473	29	32	0.7038 (0.3962-1.2502)	0.2308
HER⁻	68	58	1.4869 (0.9461-2.3369)	0.0855	60	73	1.3575 (0.8746-2.1072)	0.1730	67	68	0.6473 (0.4179-1.0027)	0.0514

Statistically significant results are shown in bold.

Discussion

Recently, inflammatory responses have gained considerable interests among researchers and physicians studying and treating chronic diseases like cancer. The inflammatory cytokines have been reported to play critical roles at different stages of tumor development and progression (Landskron et al., 2014).

Several studies revealed significant demographic differences in the distribution of TNFα −238 G > A, TNFα −308 G > A, and TNFβ +252 A>G alleles, underlying the importance of population-specific references when evaluating the clinical relevance of these polymorphisms (Pooja et al., 2011; Aznag et al., 2018, 2019). In the present case−control study, we determined for the first time, the associations between potentially functional SNPs in the TNFα and TNFβ genes and BC susceptibility within the Moroccan population.

For the TNFα −238 G > A, the AA genotype significantly increased risk of BC in the premenopausal women. This finding was in concordance with an American study revealing that the AA genotype of the TNFα −238, was associated with an increased risk for BC, compared with the GG genotype (Gaudet et al., 2007). However, contrary to our finding, a Chinese study reported that the GA and AA genotypes significantly reduced BC risk (Wang et al., 2014). No association was found between TNFα −238 polymorphism and BC susceptibility in a large North European study (Azmy et al., 2004).

On the other hand, we have investigated the association between this polymorphism and hormonal receptor status among BC patients, as the level of these molecules in tumor tissue, in addition to other factors, are used to define the approach to treatment. We then observed, using the dominant genetic model, a significant association among patient with ER⁺, and PR⁺ statuses. According to literature, the correlation between steroid hormone receptors expression, and TNFα expression in BC have been demonstrated (Zhou et al., 2014). In support of our finding, it has also been reported that TNF can increase estrogen-dependent proliferation of tumor cells in the receptor-positive breast carcinoma (Rubio et al., 2006). Indeed, the TNFα −238 polymorphism (AG+AA) was found to be significantly associated with the levels of steroid receptor in BC patients, whereas, the AA high production genotype of TNFα −238 was more frequent in patients with invasive BC, than in those with fibrocystic changes (Sirotkovic-Skerlev et al., 2007). Finally, another study revealed that the absence of the AA genotype in BC patients was not involved in the initiation of malignancies, but it was a substantial factor of BC prognosis (Malivanova et al., 2013).

Interestingly, TNFα −308 G > A polymorphism was reported to affect the expression of TNFα, and the A allele produces high TNFα. So this allele acts as a major susceptibility allele for cancer. In the present study, the AA genotype of TNFα −308 G > A polymorphism was found to be significantly associated with increased risk of BC (OR = 4.40, 95% CI = 2.13-9.07, p < 0.0001). In addition, a significant association with TNFα −308 G > A polymorphism was observed under the dominant genetic model (OR = 1.5274, 95% CI = 1.04-2.22, p = 0.0272). Our finding was in agreement with studies from Mexico and India, reporting increased risk of BC in the presence of the AA genotype (Kohaar et al., 2009; Gomez Flores-Ramos et al., 2013). Another study reported that the allele and genotype frequencies at TNFα −308 G > A site were significantly different between cases and controls, such that the mutant allele, and the mutant genotypes (GA/AA), increased cancer risk among the Dravidian population (Pooja et al., 2011). Moreover, in Tunisian population, the AA genotypes of TNFα −308 G > A locus, were found to be independent risk factors of recurrence and death (Chouchane et al., 1997; Mestiri et al., 2001). Similarly to our finding, nonsignificant association was detected between the GG and GA genotype, and the risk of BC among Mexican and British populations (Smith et al., 2004; Gomez Flores-Ramos et al., 2013). Nevertheless, several studies have reported that TNFα −308 G > A polymorphism had no effect on BC susceptibility (Karakus et al., 2011; Li et al., 2015). Moreover, a meta-analysis study (Shen et al., 2011) found that no significant association was observed in all genotypes in worldwide populations, but stratification by ethnicity indicated that the TNFα A allele may be an important protective factor for BC in European individuals. It was further suggested that in African individuals, the AA genotype of TNFα −308 could constitute a risk factor for BC, which supports our finding (Shen et al., 2011).

Several factors have also been associated with increased BC risks, such as menopausal status (Trentham-Dietz et al., 2014). The analysis of BC risk association with menopausal status, revealed significant associations with BC risk at the premenopausal AA TNF-α −238 G > A genotype, and in both pre- and postmenopausal TNFα −308 G > A AA genotypes (data not shown). Similar result among premenopausal and obese patients was found in the Mexican population (Gomez Flores-Ramos et al., 2013). However, no statistically significant associations were observed between TNF α −308 A>G and the other clinical tumor characteristics within our population, which is in agreement with a Chinese study using a bigger population sample size (Li et al., 2015). In a large north European population also, the TNFα −308 G > A polymorphism was found to be associated with the presence of vascular invasion in breast tumors (Azmy et al., 2004). Furthermore, in a recent meta-analysis study, it was revealed that TNFα −308 A>G polymorphism might play a distinct role in the progression of triple-negative BC, especially in distant tumor metastasis (Li et al., 2015).

Concerning the TNFβ +252 A>G, allele frequencies of this polymorphism in control group was not in concordance with HWE. This deviation was also observed in a previous study among the general Moroccan population (Aznag et al., 2019), and was further believed to be a consequence of selection pressure, nonrandom mating, and/or inbreeding events (Parreira and Chikhi, 2015). Like the TNFα −238 G > A and TNFα −308 G > A, the TNFβ +252G allele has been reported to increase TNF-β production by phytohemagglutinin-activated mononuclear cells in vitro, and has higher secretary capacity and circulatory concentrations of TNFα (Messer et al., 1991; Pociot et al., 1993; Kohaar et al., 2009).

In the present study, the AG genotype had significantly decreased the risk of BC among patients, while several studies have previously reported that TNFβ +252 A>G polymorphism increased BC risk (Park et al., 2002) in such a way that the GG genotype was more frequent in patients than in controls (Park et al., 2002; Kohaar et al., 2009; Karakus et al., 2011). Moreover, in our study, no significant association was observed for this polymorphism with regard to menopausal status. Although in another study, it was reported that carrying the G allele increased postmenopausal BC risk (Lee et al., 2005). Besides, no association was found between TNFβ +252 A>G polymorphism, and BC risk in Iranian and American Polish population, even if they used bigger sample sizes (Kamali-Sarvestani et al., 2005; Gaudet et al., 2007).

Owing to the close proximity of the TNF genes in the MHC molecules, alleles are usually inherited in blocks (or haplotypes), as a consequence of restricted recombinations in this region (Anaya et al., 2013). This might explain the significant value of LD assessed in the present study. In fact, several studies demonstrated that the combinations of functionally relevant SNPs, could additively or synergistically contribute to the identification of predisposing factors of complex diseases (Park et al., 1998; Onay et al., 2006). Haplotypic associations among the TNFα −308 G > A, TNFβ +252 A>G, TNFα −238 G>A polymorphisms, and BC risk were then examined. The GAA and AAG haplotypes were found to increase significantly the risk of BC in the Moroccan population. This finding led to conclude that the presence of one mutant A allele in both TNFα −308 G > A and TNFα −238 G>A, as well as the A allele in TNFβ +252 A>G, were most probably responsible for this condition risk. Accumulating evidence confirmed that the presence of the mutant allele of both TNFα −308 G > A or TNFα −238 G > A increased significantly BC risk in European American and Indian population (Gaudet et al., 2007; Kohaar et al., 2009). Indeed, the common allele A of TNFβ +252 A>G was reported to be associated with increased TNF-α gene expression (Messer et al., 1991), and consequently with increased risk of BC.

Conclusion

The findings of the present study demonstrate for the first time, an eventual association between TNF gene polymorphisms and the risk of BC in the Moroccan population. Clearly, individual polymorphisms, together with haplotype analysis, revealed statistically significant associations with BC susceptibility in our population. Although, the limitation of the study was the small sample size obtained by stratification according to BC tumors characteristics. Consequently, further studies implementing a larger sample size are needed to support our findings.

Footnotes

Acknowledgments

The authors would like to express their gratitude to Dr. Mohammed Aghrouch from the Medical Analysis Laboratory at the Regional Hospital Hassan II, Agadir City, to the Dr. El Allali's Laboratory of Medical Analysis, and to all healthcare professionals at the Oncology and Radiotherapy Center of the Hospital Hassan II in Agadir, for supplying us with the blood samples.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

References

Abraham

, Kroeger

(1999) Impact of the −308 TNF promoter polymorphism on the transcriptional regulation of the TNF gene: relevance to disease. J Leukoc Biol, 66:562-566.

Anaya

, Shoenfeld

, Rojas-Villarraga

, et al. (2013) Major histocompatibility complex: antigen processing and presentation. In: Cruz-Tapias

, Castiblanco

, Anaya JM (eds) Autoimmunity: From Bench to Bedside. Bogota, Colombia: El Rosario University Press, pp 169-184.

Azmy

, Balasubramanian

, Wilson

, et al. (2004) Role of tumour necrosis factor gene polymorphisms (−308 and −238) in breast cancer susceptibility and severity. Breast Cancer Res, 6:R395-R400.

Aznag

, El kadmiri

, Izaabel

(2018) Tumor necrosis factor-alpha and tumor necrosis factor beta polymorphisms and risk of breast cancer: review. Genrep, 12:317-323.

Aznag

, Moutaoufik

, Korrida

, et al. (2019) Genetic distribution of the LTA +252 A>G and TNFA −308 G>A polymorphisms. Genet Test Mol Biomarkers, 23:871-876.

Balkwill

(2002) Tumor necrosis factor or tumor promoting factor?. Cytokine Growth Factor Rev, 13:135-141.

Barrett

, Fry

, Maller

, et al. (2005) Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics, 21:263-265.

Bazzoni

, Beutler

(1996) The tumor necrosis factor ligand and receptor families. N Engl J Med, 334:1717-1725.

Bray

, Ferlay

, Soerjomataram

, et al. (2018) Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 68:394-424.

10.

Cabrera

, Shaw

, Sharples

, et al. (1995) Polymorphism in tumor necrosis factor genes associated with mucocutaneous leishmaniasis. J Exp Med, 182:1259-1264.

11.

Carey

, Perou

, Livasy

, et al. (2006) Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA, 295:2492-3502.

12.

Cereda

, Gagliardi

, Cova

, et al. (2012) The role of TNF-alpha in ALS: new hypotheses for future therapeutic approaches. In: Maurer

(ed) Amyotrophic Lateral Sclerosi. Germany: In Tech, pp 414-429.

13.

Chouchane

, Ahmed

, Baccouche

, et al. (1997) Polymorphism in the tumor necrosis factor-alpha promotor region and in the heat shock protein 70 genes associated with malignant tumors. Cancer, 80:1489-1496.

14.

Coley

(1991) The treatment of malignant tumors by repeated inoculations of erysipelas. With a report of ten original cases. 1893. Clin Orthop Relat Res. 3-11.

15.

Cruceriu

, Baldasici

, Balacescu

, et al. (2020) The dual role of tumor necrosis factor-alpha (TNF-α) in breast cancer: molecular insights and therapeutic approaches. Cell Oncol Dordr, 43:1-18.

16.

Esquivel-Velázquez

, ostoa saloma

, Palacios-Arreola

, et al. (2015) The role of cytokines in breast cancer development and progression. J Interferon Cytokine Res, 35:1-16.

17.

Gaudet

MM.

, Egan

, Lissowska

, et al. (2007) Genetic variation in tumor necrosis factor and lymphotoxin-alpha (TNF-LTA) and breast cancer risk. Hum Genet, 121:483-490.

18.

Gomez Flores-Ramos

, Escoto-De Dios

, Puebla-Perez

, et al. (2013) Association of the tumor necrosis factor-alpha −308G>A polymorphism with breast cancer in Mexican women. Genet Mol Res, 12:5680-5693.

19.

Hagihara

, Shimura

, Sato

, et al. (1995) HLA and tumor necrosis factor β gene polymorphisms in Okinawa lung cancer patients: comparative study with Mainland Japan lung cancer patients. Hum Immunol, 43:95-100.

20.

Hajeer

, Hutchinson

(2001) Influence of TNFα gene polymorphisms on TNFα production and disease. Hum Immunol, 62:1191-1199.

21.

Hollegaard

, Bidwell

(2006) Cytokine gene polymorphism in human disease: on-line databases, Supplement 3. Genes Immun, 7:269-276.

22.

Kaluza

, Reuss

, Hug

, et al. (2000) Different transcriptional activity and in vitro TNF-α production in psoriasis patients carrying the TNF-α 238A promoter polymorphism. J Invest Dermatol, 114:1180-1183.

23.

Kamali-Sarvestani

, Gharesi-Fard

, Sarvari

, et al. (2005) Association of TNF-alpha and TNF-beta gene polymorphism with steroid receptor expression in breast cancer patients. Pathol Oncol Res, 11:99-102.

24.

Karakus

, Kara

, Ulusoy

, et al. (2011) Tumor necrosis factor alpha and beta and interferon gamma gene polymorphisms in Turkish breast cancer patients. DNA Cell Biol, 30: 371-377.

25.

KohaarI , Tiwari

, Kumar

et al. (2009) Association of single nucleotide polymorphisms (SNPs) in TNF-LTA locus with breast cancer risk in Indian population. Breast Cancer Res Treat, 114:347-355.

26.

Landskron

, De La Fuente

, Thuwajit

, et al. (2014) Chronic inflammation and cytokines in the tumor microenvironment. J Immunol Res, 2014:19.

27.

Lee

, Park

, Hamajima

, et al. (2005) Genetic polymorphisms of TGF-b1 & TNF-b and breast cancer risk. Breast Cancer Res Treat, 90:149-155.

28.

Leek

, Landers

, Fox

, et al. (1998) Association of tumour necrosis factor alpha and its receptors with thymidine phosphorylase expression in invasive breast carcinoma. Br J Cancer, 77:2246-2251.

29.

, Zhu

, Liu

, et al. (2015) Tumour necrosis factor-α gene polymorphism is associated with metastasis in patients with triple negative breast cancer. Sci Rep, 5:1244.

30.

Mackay

, Browning

, Lawton

, et al. (1998) Both the lymphotoxin and tumor necrosis factor pathways are involved in experimental murine models of colitis. Gastroenterology, 115:1464-1475.

31.

Malivanova

, Skoromyslova

, Yurchenko

, et al. (2013) Analysis of the −238(G/A)TNF polymorphism in breast-cancer patients. Mol Gen Mikrobiol Virusol, 28:52-55.

32.

Messer

, Spengler

, Jung

, et al. (1991) Polymorphic structure of the tumor necrosis factor (TNF) locus: an NcoI polymorphism in the first intron of the human TNF-beta gene correlates with a variant amino acid in position 26 and a reduced level of TNF-beta production. J Exp Med, 173:209-219.

33.

Mestiri

, Bouaouina

, Ahmed

, et al. (2001) Genetic variation in the tumor necrosis factor-alpha promoter region and in the stress protein Hsp70-2: susceptibility and prognostic implications in breast carcinoma. Cancer, 91:672-678.

34.

Miles

, Happerfield

, Naylor

, et al. (1994) Expression of tumour necrosis factor (TNFα) and its receptors in benign and malignant breast tissue. Int JCancer, 56:777-782.

35.

Miller

, Dykes

, Polesky

(1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res, 16:1215.

36.

Onay

, Briollais

, Knight

, et al. (2006) SNP-SNP interactions in breast cancer susceptibility. BMC Cancer, 6:114.

37.

Park

, Mok

, Ko

, et al. (2002) Polymorphisms of tumour necrosis factors A and B in breast cancer. Eur J Immunogenet, 29:7-10.

38.

Park

, Mok

, Rho

, et al. (1998) Analysis of TNFB and TNFA NcoI RFLP in colorectal cancer. Mol Cells, 8:246-249.

39.

Parreira

, Chikhi

(2015) On some genetic consequences of social structure, mating systems, dispersal, and sampling. Proc Natl Acad Sci U S A, 112:E3318-E3326.

40.

Pociot

, Briant

, Jongeneel

, et al. (1993) Association of tumor necrosis factor (TNF) and class II major histocompatibility complex alleles with the secretion of TNF-alpha and TNF-beta by human mononuclear cells: a possible link to insulin-dependent diabetes mellitus. Eur J Immunol, 23:224-231.

41.

Pooja

, Francis

, Bid

, et al. (2011) Role of ethnic variations in TNF-a and TNF-b polymorphisms and risk of breast cancer in India. Breast Cancer Res Treat, 126:739-747.

42.

Qidwai

, Khan

(2011) Tumour necrosis factor gene polymorphism and disease prevalence. Scand J Immunol, 74:522-547.

43.

Rubio

, Werbajh

, Cafferata

, et al. (2006) TNF-α enhances estrogen-induced cell proliferation of estrogen-dependent breast tumor cells through a complex containing nuclear factor-kappa B. Oncogene, 25:1367-1377.

44.

Shen

, Sun

, et al. (2011) Polymorphisms of tumor necrosis factor-alpha and breast cancer risk: a meta-analysis. Breast Cancer Res Treat, 126:763-770.

45.

Sirotkovic-Skerlev

, Cacev

, Krizanac

, et al. (2007) TNF alpha promoter polymorphisms analysis in benign and malignant breast lesions. Exp Mol Pathol, 83:54-58.

46.

Smith

, Bateman

, Fussell

, et al. (2004) Cytokine gene polymorphisms and breast cancer susceptibility and prognosis. Eur J Immunogenet, 31:167-173.

47.

Solé

, Guinó

, Valls

, et al. (2006) SNPStats: a web tool for the analysis of association studies. Bioinformatics, 22:1928-1929.

48.

Stayoussef

, Benmansour

, Al-Jenaidi

, et al. (2010) Identification of specific tumor necrosis factor-α-susceptible and -protective haplotypes associated with the risk of type 1 diabetes. Eur Cytokine Net, 21:285-291.

49.

Trentham-Dietz

, Sprague

, Hampton

, et al. (2014) Modification of breast cancer risk according to age and menopausal status: a combined analysis of five population-based case-control studies. Breast Cancer Res Treat, 145:165-175.

50.

Wang

, Liu

, Sun

, et al. (2014) Genetic polymorphisms in inflammatory response genes and their associations with breast cancer risk. Croat Med J, 55:638-646.

51.

Wilson

, Symons

, McDowell

, et al. (1997) Effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation. Proc Natl Acad Sci U S A, 94:3195-3199.

52.

Zhou

, Fan

, Yang

, et al. (2014) The clinical significance of PR, ER, NF-αB, and TNF-α in breast cancer. Dis Markers, 2014:7.