Association of TNF-α-308G/A and IL-18 Polymorphisms with risk of HPV infection among sexually active women in Burkina Faso

Human papillomavirus (HPV) infection is the most common sexually transmitted infection worldwide. Persistence infection can lead to the development of cervical cancer potentially due to some genetic factors such as polymorphisms in regulatory and coding regions of cytokine genes. The purpose of this study was to determine whether there is a relationship between TNF-308 G/A or IL18 polymorphisms and high-risk HPV infection among sexually active women from Burkina Faso. Ninety-one HPV infected and two hundred and nine HPV negative women (the latter used as healthy controls) were screened. TNFA-308 G/A and IL18-607 C/A polymorphisms were detected using the TaqMan allelic discrimination. HPV 52 (21.19%), HPV 39 (11.86%) and HPV 33 (11.02%) were the most common HPV genotypes. The TNFA-308A and IL18-607 C alleles were predominant in all women in the study. None of the TNFA and IL18 alleles were associated with HPV infection. The results suggest that there is no relationship between TNF-308 G/A or IL18-607C/A polymorphisms and HPV infection among women in the study.


Introduction
Every year, many women are affected by cervical cancer, worldwide. In 2018, there were an estimated 569,847 new cases and approximately 311,365 deaths from cervical cancer which is the second most common cancer in less-developed countries [1]. This cancer usually occurs following a persistent, high-risk, HPV infection in a small proportion of women who have contracted the infection in their lifetime. Risk factors such as the age at first sexual intercourse or the number of pregnancies, as well as genetic factors, would appear to be involved in the occurrence of this disease. Some authors suggest that the host's genetic 'make-up' may facilitate the persistence of HR-HPV infection [2] or even promote the progression of infection. Cytokines such as Tumor Necrosis Factor alpha (TNFα) and interleukin 18 (IL18) have been implicated in the process but the results remain controversial [3].
TNFα (TNFA) is a pro-inflammatory cytokine involved in systemic inflammation and acute phase reaction providing protection against infections. In humans, there is only one gene known for TNFα located on chromosome 6p21 within the locus of the major histocompatibility complex. TNFα exists in two forms, including a transmembrane form linked to the membrane which is the precursor of the soluble form [4]. Both forms are active but have different affinities for TNF receptors (TNFR-1 and TNFR-2). TNFR1 is the major receptor in the mediation of cell toxicity [5] and cell death [6]. TNFR2 is found on the surface of circulating T cells [7] but is also the primary mediator known to be involved in the self-regulation of TCD8 cell apoptosis [8]. TNFα presents several polymorphisms, including one in position -308 of the promoter by substitution of G in A. This single-Nucleotide Polymorphism (SNP), in addition to being involved in transcription regulation, appears to affect gene expression [9,10].
IL18 is an important regulator of the immune response produced by dendritic cells, Langerhans cells, macrophages and some B lymphocyte lines [11]. In the absence of any other stimulus, IL18 increases the production of TNF α by TCD4+ lymphocytes and NK cells [12]. The IL 18 gene contains several polymorphisms in the promoter region and SNPs at -137 G/C and -607 C/A positions appear to be the only ones that affect gene activity [13]. IL 18 -607C/A polymorphism was associated with protection against HPV infection while the TNFA -308A allele was associated with susceptibility to HPV infection among Brazilian women according to the study by Tavares et al. in 2016 [14]. Their study suggested that, it is necessary to replicate this kind of study in other populations with different ethnic backgrounds to better understand the effects of TNFA and IL18 genetic variants on HPV infection that is why we conducted this study, to investigate the distribution of TNF-308 G/A and IL18-607 C/A SNPs and their relationship to HPV infection in a cohort of women from Burkina Faso.

Study subjects
We collected endocervical samples from women who came for gynecological consultation in hospital of two cities in the west of Burkina Faso: Bobo-Dioulasso and Orodara. The DNA extraction was made using DNA-Sorb-A kit (Sacace Biotechnologies, Como, Italy). The genotyping of high-risk HPV was performed by real-time PCR using the kit "HPV Genotypes 14 Real-TM Quant" code V67-100FRT (Sacace Biotechnologies, Como, Italy), and the Sacycler-96 Real-Time PCR (Sacace Biotechnologies, Como, Italy). [15,16]. Following HPV genotyping, samples were separated in two groups to investigate the distribution of TNF-308 G/A and IL18-607 C/A SNPs: one which HPV infection and the other without HPV infection. Thus, this case control study involved 300 women with 91 women infected by HPV and 209 women without HPV.

Genotyping
The polymorphic regions of TNFα-308 G/A (rs 1800629) and IL 18-607 C/A (rs 1946518) were amplified by PCR, with a 20 μl reaction volume containing 5 μL template DNA, 4.5 μL purified water, 10 μL TaqMan Universal PCR Master Mix (2X) and 0.5 μL of each SNP mix (40X). SNP genotyping was performed using standard TaqMan SNP assays (ABI, Foster City, CA) carried out by a 7500 Fast Real-Time PCR Systems (Life Technologies, California, USA). PCR amplification program was as follow: 1 cycle of 95°C for 10 minutes, 40 cycles of 92°C for 15 seconds and 60°C for 1 minute.

Statistical analysis
The data were processed and analyzed using SPSS software version 20.0. Version 3.25 of the Power-Marker software was used for the determination of the Hardy-Weinberg equilibrium and the calculation of allele and genotype frequencies. Changes were considered statistically significant at p < 0.05, using the Fisher Exact test. Odds ratio (OR) and confidence intervals (CI) at 95% were calculated to estimate the associations of HPV infection with the rs 1800629 and rs 1946518 polymorphisms using Epi Info 7.
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 Ethics Committee for Health Research of Burkina Faso (CERS) (Deliberation No. 2014-9-110).
Informed consent: Informed consent has been obtained from all individuals included in this study

Participant demographics
The characteristics of the two groups of women are given in Table 1. No statistically significant differences were found between cases and controls regarding age, age at first intercourse or number of pregnancies (p values were respectively 0.631; 0.103 and 0.513)

Prevalence of TNFα (rs 1800629) and IL 18 (rs 1946518) polymorphisms
The allele and genotype frequencies of TNFA -308 (rs1800629) and IL18 -607 C/A (rs1946518) polymorphisms for cases and controls are listed in Table 2. Results demonstrated that the two SNPs rs 1800629 and rs 1946518 genotype are all in Hardy-Weinberg equilibrium (HWE).
The rs 1800629 A allele for TNFα was present in all women in the 2 study groups with a frequency of 84.61% and 84.69% respectively in cases and controls (Table  II). None of the TNFA-308A or TNFA-308G alleles were associated with carrying HPV infection (p=0.92).
The C allele of interleukin 18 was the most predominant in both cases (67.6%) and controls (61.5%). However, we have not found an association between the IL-607 C or IL-607 A allele and HPV infection in women.

Discussion
The 3 most common HPV genotypes found in cases in this study was HPV 52, HPV 39 and HPV 33. In contrast, HPV 16 has been found as the most common genotype in other studies [17,18], yet HPV16 was not detected in our study. This result agrees with previous findings reported in Burkina Faso [19,20] where HPV 16 and HPV 18 were not among the most frequent genotypes.
We investigated the distribution of allele and genotype of TNFα -308 (rs 1800629) and IL 18-607 (rs 1946518). The results showed that TNFα-308A was the predominant allele among all the women in the study. The frequency of this allele was 84.6% and 84.7% respectively in cases (women with positive HPV results) and controls (no HPV). There was no statistically significant difference for the A and G alleles of TNF α (p=0.92). It has been suggested that TNF α may be able to directly fight against HPV infection by inducing apoptosis of virus-infected cells [21]. The frequencies of the TNFα -308 G/G and TNFα -308 A/A genotypes were not statistically different between cases and controls (p=0.6 for G/G and p=0.8 for AA). There was therefore no association between these genetic variants and susceptibility to HPV infection. In 2019, Chagas et al. [22] indicated in their study that the association of TNFα -308 represents a risk to the susceptibility in the development of cervical cancer in women infected by HPV  58. Therefore, our results could be explained by the fact that this genotype was among the least frequent in the women of our study. In Brazil, Tavares et al. had found that the TNFα -308 G/G genotype and the G allele were associated with increased susceptibility to HPV infection [14]. Meanwhile, it has also been reported that , the polymorphism of TNFα -308 G/A is associated with cancerous lesions. For example, in Zimbabwe, the TNFα -308 G/G genotype was more common in HPV positive women with cancer than in controls [23], while a study on Portuguese women with the A allele showed that those women were twice as likely to develop invasive cervical cancer [24]. Also, the authors of the Zimbabwe study found that the A allele as well as the A/A genotype were less common in their study than in populations in Europe. This is not the case for the women in our study. The A allele of TNF α is known as TNF2, which would be associated with an increase in the production of this cytokine [9] and the level of TNF α production in an individual would depend on his genetic predispositions [23].
Concerning the IL18 gene, the allele C was most common in women in both cases (67.6%) and controls (65.1%). Similarly, the predominant genotype was -607 CC in both cases (49.4%) and controls (45.0%). However, there were no significant differences in the frequencies obtained between the genetic variants -607 CC (p=0.56) and -607 AA (p=0.96) for cases and controls. None of these genetic variants therefore had an influence on HPV infection in our study. Tavares et al. [14] reported in their 2016 study that -607 C/A polymorphism in the IL18 promoter was associated with protection against HPV infection. According to these authors, the -607 A/A genotype provided protection against HPV infection but did not affect progression to cancerous lesions. However, the results of a meta-analysis showed that -607 C/A polymorphism was associated with the risk of developing all types of cancer [25]. This polymorphism leads to a decrease in IL18 production due to a deficiency in the immune response against HPV, which would increase the risk of infection.
It is possible that the expression levels of the TNFα and IL18 genes may partly affect the persistence of HPV infection [21] while it is linked to progression to cervical cancer. The effect of these genes on HPV infection appears to be influenced by the cytokine gene products. As the production level of these is genetically controlled, the results of one study will be true for a given population whereas they would be different in another.
To our knowledge this is the first study investigating the effects of these two genes TNFA and IL18 on HPV infection among women in Burkina Faso The study demonstrated that TNF-308 G/A and IL-18 C/A SNPs were not associated with HPV infection in the studied population. The results can be useful to identify the host genetic factors of HPVrelated diseases. But it would therefore be important to complete the genetic association studies with the characterization of the effects of these SNPs by measuring serum cytokine levels bigger population sample size.