Abstract
Human papillomavirus persistent infection is the main risk factor for developing cervical cancer. Tumor necrosis factor α (TNFA) is a cytokine important for the development of immune response and is one of the primary cytokines released after HPV infection. In this context, we investigated the possible association of TNFA promoter polymorphisms (-238 G/A and -308 G/A) with cervical lesions in HPV infected women. Using fluorogenic allele-specific probes, we analyzed TNFA single nucleotide polymorphims (SNPs) -238 A/G (rs361525) and -308 A/G (rs1800629) in 435 women (259 women with normal cytological results and 176 women with cervical abnormalities) from North East Brazil. No significant differences in the distribution of -308 SNP allele and genotype frequencies were observed between the HPV-infected women group with cervical lesions and controls. With regard to the allele frequencies of TNFA -238 SNP, no statistically significant differences were identified between cases and controls, except when the HPV/LSIL subgroup was compared with controls (p = 0.03). Similarly, when we considered the genotype frequencies, no statistically significant differences were identified between cases and controls, except when the HPV/LSIL subgroup was compared with controls (p = 0.04). In our study, we found that the TNFA -308 A/G polymorphism had no effect on development of cervical lesions. With respect to TNFA -238 G/A polymorphism, we found that in the Northeastern Brazil population the polymorphism is associated with progression of cervical lesions. Our study provides a Northeastern Brazilian contribution to the investigation of genetic host factors involved with susceptibility to HPV infection, focusing on TNFA.
Introduction
Cervical cancer is considered the second most common cancer among women (1). Every year, over 530,000 new cases are diagnosed and about 275,000 people die from cervical cancer; more than 85% of the cases occur in developing countries (1, 2).
Human Papillomavirus (HPV) infection is the most common sexually transmitted disease. According to World Health Organization (WHO), HPV persistent infection is the main risk factor for developing cervical cancer and it is estimated that HPV oncogenic types are responsible for about 98% of the cases (4). However, high-risk HPV is a necessary but not sufficient risk factor to initiate malignant transformation of cervical cells and cervical cancer development (5). Environmental, genetic and immunological factors are also important to predisposition to the disease (5). Moreover, some studies suggest that the use of oral contraceptives may increase the risk of cervical carcinogenesis (6,7).
Regarding immunological factors, cytokines act in the defense against HPV-induced infections, modulating viral replication and polarizing the immune response to a Th1 or Th2 pattern, cellular and humoral, respectively (8). The development of cancer has been associated to several cytokines that are responsible for the modulation of immunological control, and genes involved in the immune response to viral infection have been already associated with susceptibility to develop cervical cancer (9).
Tumor necrosis factor α (TNFA) is a potent pro-inflammatory (Th1) cytokine secreted mainly by activated macrophages and it is known to play an important role in the development of the immune response (10). The TNFA gene is located on human chromosome 6 (region p21.3) (11) where functional single nucleotide polymorphisms (SNPs) have been identified within the gene sequence, especially at regulatory regions, including the -238 G/A (rs361525) and -308 G/A (rs1800629) promoter polymorphisms. These SNPs have been reported as able to modulate gene expression, being associated with differential levels of transcription (12) in the context of several diseases, including endometrial carcinoma (13, 14).
The present work aimed investigate the possible association of TNFA promoter polymorphisms (-238 G/A and -308 G/A) with cervical lesions in HPV infected women from Northeast Brazil.
Material and methods
Study group
The samples evaluated in this study were obtained by cervical scraping from 435 patients who volunteered to take part in cervical cancer screening at the Gynecological Clinic at the “Hospital das Clínicas” (HC) and “Hospital Universitário Oswaldo Cruz” (HUOC) in the metropolitan area of Recife, Pernambuco State, Northeastern Brazil. The control group consisted of 259 women with normal cytological results, HPV-negative (assessed by PCR) [Baldez da Silva et al., 2009] and not being treated with immunosuppressive medication (healthy controls), whereas the case group consisted of 176 women with cervical abnormalities; the latter group was divided into LSIL (79 – low-grade squamous intraepithelial lesions) and HSIL (97 – high-grade squamous intraepithelial lesions), all HPV-positive. All women (patients and controls) were from the same geographical area (North Eastern, Brazil). The ages of patients with HPV-positive cervical abnormality ranged from 16 to 82 (average 35.2 ± 12.2), and the age of the controls ranged from 16 to 65 years (average 36.0 ± 10.3).
All clinical investigation developed in this study was conducted according to the principles expressed in the Declaration of Helsinki. The patients enrolled were informed about the objectives of the research. We obtained approval of the Ethical Committee (Research Ethics Committee – Health Sciences Center/Federal University of Pernambuco – CEP/CCS/UFPE N° 491/11) and all women signed the consent.
DNA isolation and HPV analysis
The cervical cells collected with cytobrush were put in pH 7.4 phosphate-buffered saline (PBS) and maintained at -80°C until the time of DNA extraction. Genomic DNA was extracted from cervical cells using the DNeasy Blood and Tissue Kit (Qiagen), in accordance with the manufacturer's manual. HPV genotyping was performed using Bioplex (BioRad, Hercules, CA) technology following protocols already reported in the literature (15).
TNFA genotyping
Within TNFA functional promoter polymorphisms we have chosen the -238 G/A (rs361525) and -308 G/A (rs1800629) SNPs, based on previous literature association findings, as already mentioned in the introduction).
Genotyping of TNFA promoter polymorphisms (-238 G/A, rs361525 and -308 G/A, rs1800629) was performed with commercially available fluorogenic allele-specific probes (TaqMan® Probes, Applied Biosystems) using 50 ng of DNA and the ABI7500 Real-Time PCR platform (Applied Biosystems). SDS software 2.3 (Applied Biosystems) allowed allelic discrimination.
Statistical analysis
The statistical analysis was performed using open-source R software [R Core Team, 2014], the SNPassoc package and the genetics package (16). Conformity to Hardy-Weinberg equilibrium was assessed using chi-square test with Yates' continuity correction. Fisher’s exact test was used for pair-wise comparison of alleles and genotypes contingency tables. For all tests, the level of significance was set at p<0.05.
Results
All HPV positive individuals were tested for different viral subtypes. Of those, 16 different subtypes were found in this group (52.9% were infected by only one of any HPV type). We also detected co-infection of HPV subtypes that ranged from two to four different viral subtypes present in the same individual, making up to almost half of total cases (47.1%). The most prevalent HPV subtype was HPV-16 (21.0%) followed by HPV31 (11.9%), HPV58 (2.8%) and HPV33 (2.3%) (Table 1).
The distribution of TNFA polymorphisms in both cases and controls groups was in conformity to Hardy-Weinberg equilibrium (-238 G/A – p=0.2822; -308 G/A – p=0.3738). The distribution of genotypes and allele frequencies of TNFA -238 and -308 polymorphisms in patients HPV infected and healthy controls are displayed in Table 2.
When the TNFA -238 SNP was analyzed, the GG genotype was considered the reference genotype and the relative association of the GA and AA genotype with disease was expressed by calculating the odds ratios (OR) and their 95% CI (Table 2). In relation to the allele frequencies, no statistically significant differences were identified between cases and controls, except when the HPV/LSIL subgroup was compared with controls (G vs. A allele: OR = 3.51, CI: 1.06–11.62 and p = 0.03) (Table 2). Similarly, when we considered the genotype frequencies no statistically significant differences were identified, except when the HPV/LSIL subgroup was compared with controls (GG vs. GA/AA genotype: OR = 3.44, CI: 1.02–11.58 and p = 0.04) (Table 2).
With regard to the TNFA -308 SNP, the GG was selected as reference genotype and the relative disease association of GA and AA genotypes was expressed by calculating the odds ratios (OR) and their 95% CI (Table 2). Concerning the allele frequencies, no statistically significant difference was found between patients with HPV/cervical lesion and the healthy controls (Table 2). In the case of the genotypic frequencies, as specified earlier, no differences were identified between the control and case groups (Table 2).
Discussion
Cytokines are known to have an important role in tumor progression and therefore have importance as genetic host markers for the susceptibility to development of several cancers including cervical cancer (17).
TNFA is considered as one of the primary cytokines released in the HPV infection and its expression is regulated at transcriptional level (9, 12). The polymorphisms in the promoter region at positions -308 A/G and -238 A/G are the most commonly studied due to the association with different levels of expression, besides revealing a close association with inflammatory, infectious and autoimmune diseases (17, 18). The G to A substitution at position -308 in the TNFA gene has an effect on the expression of cytokine production, where the allele A produces higher level of TNFA than the allele G (19). The G to A substitution at position -238 in the TNFA gene might also affect TNFA expression, once a putative repressor site is located in a 25 bp stretch including the position -238 (20).
In our case-control study, we investigated the association among genetic variants of TNFA promoter and susceptibility to HPV and development of cervical lesions in a population from North East Brazil (Pernambuco). We compared two types of case subjects (LSIL and HSIL – women who were positive for HPV) with control subjects (women without HPV infection).
Several studies have shown that HPV16 is the most prevalent in Brazil (21, 22, 23). The current study showed that in Northeastern Brazil the HPV 16 was the most prevalent genotype followed by HPVs 31, 58 and 33. The results of HPV subtypes frequency found in this study were similar to other studies conducted in Northeastern Brazil (21, 22, 24, 25).
With respect to TNFA -308 SNP, our data did not show significant difference in the distribution of allele and genotype frequencies and the risk of development of cervical lesion among the Northeastern Brazil population. Our findings are similar to those related by Jang et al. (14), Stanczuk et al. (26) and Govan et al. (27).
Regarding to TNFA -238 SNP, our data showed a significant difference in the allele and genotype frequencies, when HPV/LSIL group was compared with healthy controls. This finding indicates that the -238A allele may be hypothesized as a risk factor of cervical lesions, furthermore, this allele has been shown to be associated with development of endometrial cancer (14). Our findings are similar to those related by Kohaar et al. (28) and Kirkpatrick et al. (29). In contrast, the overall results of a meta-analysis have suggested that TNFA -238 G/A polymorphism is associated with a decreased risk of cervical cancer (12).
The results from previous studies that were conducted to assess the association of TNFA -238 G/A and -308 G/A polymorphisms with cervical cancer risk are contradictory (14, 26, 30, 18, 27, 28, 31, 32, 33, 34, 35). Conflicting findings might be explained based upon the relative small sample size, which cannot assure sufficient statistical power or on ethnic differences among the studied populations.
Conclusions
In summary, in this case-control study, we found that the TNFA -308 polymorphism had no effect on development of cervical lesions. Being aware of the study limitations, represented by the small number of subjects analyzed for an association study, we hypothesized that in the Northeastern Brazil population the TNFA -238 A polymorphism is associated with progression of cervical lesions. Our study provides a Northeastern Brazilian contribution to the investigation of genetic host factors involved with susceptibility to HPV infection, focusing on TNFA, an important actor involved in the immune response.