Tumor Necrosis Factor Alpha rs1800629 Polymorphism and Risk of Cervical Lesions: A Meta-Analysis

Background Tumor necrosis factor- alpha (TNF-α) is an inflammatory cytokine which may play important role on the immune response may control the progression of cervical lesions. There is a possible association between TNF-α rs1800629 G/A polymorphism and cervical lesions, but previous studies report conflicting results. We performed a meta-analysis to comprehensively assess the association between TNF-α rs1800629 polymorphism and cervical lesions risk. Methods Literature searches of Pubmed, Embase, Web of Science, and Wanfang databases were performed for all publications on the association between TNF-α rs1800629 polymorphism and cervical lesions through December 15, 2012. The pooled odds ratios (ORs) with their 95% confidence interval (95%CIs) were calculated to assess the strength of the association. Results Twenty individual case-control studies from 19 publications with a total of 4,146 cases and 4,731 controls were finally included into the meta-analysis. Overall, TNF-α rs1800629 polymorphism was significantly associated with increased risk of cervical lesions under two main genetic comparison models (For A versus G: OR 1.22, 95%CI 1.04–1.44, P = 0.017; for AA versus GG: OR 1.32, 95%CI 1.02–1.71, P = 0.034). Subgroup analysis by ethnicity further showed that there was a significant association between TNF-α rs1800629 polymorphism and increased risk of cervical lesions in Caucasians but not in Asians. Subgroup analysis by the types of cervical lesions showed that there was a significant association between TNF-α rs1800629 polymorphism and increased risk of cervical cancer (For A versus G: OR 1.24, 95%CI 1.05–1.47, P = 0.011; for AA versus GG: OR 1.31, 95%CI 1.01–1.70, P = 0.043; for AA/GA versus GG: OR 1.25, 95%CI 1.01–1.54, P = 0.039). Conclusion The meta-analysis suggests that TNF-α rs1800629 polymorphism is associated with increased risk of cervical lesions, especially in Caucasians.


Introduction
Cervical cancer is the third most common cancer and the fourth leading cause of cancer death among females, accounting for nearly 10% of the total newly-diagnosed cancer cases and 8% of the total cancer deaths [1]. Global incidence of cervical cancer has increased from about 378,000 cases per year in 1980 to about 454,000 cases per year in 2010 [2]. Risk factors of cervical cancer include human papillomavirus (HPV) and smoking, and infection of carcinogenic HPV is a main cause of almost all cases with cervical lesions [3]. Preinvasive lesions of cervical cancer have also been classified in terms of squamous intraepithelial lesions (SILs). Cervical cancer is considered to be a preventable disease because of its relatively long period of precancerous lesions, such as cervical intraepithelial neoplasia (CIN) [4]. Several cytokines that modulate the immunologic response have been implicated in the development of cervical cancer [5][6][7]. It has been well accepted that cervical cancer is mainly initiated by HPV infection, and tumor necrosis factor-alpha (TNF-a) is an inflammatory cytokine which may play important roles in the immune response of cervical lesions [5,7]. TNF-a is located on chromosome 6 (region p21.3), and it is a potent proinflammatory cytokine playing an important role in the development of the immune response [5,6]. Currently, there are several common single nucleotide polymorphisms (SNP) in the TNF-a gene which can regulate the transcription and production of TNF-a, such as TNF-a rs1800629 and TNF-a rs361525 [8,9]. TNF-a rs1800629 is the most studied polymorphism, which is a G to A transition in the promoter at position 2308 and is associated with the level of TNFa expression [10,11]. Therefore, TNF-a rs1800629 polymorphism may be related with risk of cervical lesions [10,11]. However, results from previous studies examining the association between TNF-a rs1800629 polymorphism and risk of cervical lesions were contradictory [12][13][14][15][16][17][18][19][20][21][22][23][24][25]. These inconclusive results may due to the limited sample size of single study or the different characteristics among studies, such as ethnicity, pathological types, and sources of controls. Therefore, we performed a meta-analysis to comprehensively assess the association between TNF-a rs1800629 polymorphism and cervical lesions risk.

Search strategy
An electronic search of the Pubmed, Embase, Web of Science, and Wanfang databases was performed to identify the eligible studies assessing the association between TNF-a rs1800629 polymorphism and risk of cervical lesions. There were no language restrictions and the last search time was the November 16, 2012. We used the keywords and subject terms: (''cervical carcinoma'', ''cervical cancer'', ''cervix carcinoma'' or ''cervix cancer'' or cervical neoplasm, cervical lesions, cervical lesion, cervical intraepithelial lesion, cervical intraepithelial lesions, cervical dysplasia, cervical intraepithelial neoplasia) and (''polymorphism'' or ''variant'' or ''genotype'' or ''mutation'' or) and (''tumor necrosis factor'' or ''TNF'' or ''308 G/A'' or ''TNF-a'' or ''rs1800629''). All eligible studies were retrieved, and their bibliographies were checked for other relevant publications.

Inclusion criteria and exclusion criteria
To be eligible for the inclusion criteria in the meta-analysis, the following criteria were used: 1) case-control studies comparing cervical lesions cases with healthy or non-cancer controls; 2) studies evaluating the association between TNF-a rs1800629 polymorphism and risk of cervical lesions; 3) Sufficient genotype data of TNF-a rs1800629 polymorphism were reported. 4) Studies was excluded if they were the following: 1) Case-only studies; 2) Case reports, letters, or reviews; 3) Incomplete data or no usable data were reported; 4) Studies containing overlapping data; 5) Family-based design or related cases and controls were contained.

Data extraction and quality assessment
The data extraction was performed independently by two reviewers, and the conflicting data extracted were settled by discussion. A standardized form was used in the data extraction from the published studies, and the following data were extracted: first author, year of publication, country, ethnicity, study design,  Table 1. Characteristics of twenty studies on the association between TNF-a rs1800629 polymorphism and cervical lesions risk. diagnostic criteria, source of cases and controls, number of cases and controls, types of cervical lesions, genotyping methods, frequencies of TNF-a rs1800629 genotypes, and the confirmation of Hardy-Weinberg equilibrium (HWE) in controls. The quality of included studies was assessed by the confirmation of HWE in controls, and studies without the confirmation of HWE in controls were defined as low-quality studies, while studies with the confirmation of HWE in controls were defined as high-quality studies.

Statistical analysis
Before the effect estimation of associations between the TNF-a rs1800629 polymorphism and risk of cervical lesions, we firstly tested whether the genotype frequencies of TNF-a rs1800629 genotypes in the controls were confirmed with HWE using the x 2 test. The strength of the associations between the TNF-a rs1800629 polymorphism and risk of cervical lesions was estimated using odds ratios (ORs) and their 95% confidence interval (95%CIs). The following contrasts for the associations between the TNF-a rs1800629 polymorphism and risk of cervical lesions were evaluated: comparison of the variant allele with ancestral allele (A vs. G); comparison of the variant homozygote with the ancestral homozygote (AA vs. GG); comparison of the variant homozygote combined with the heterozygote vs. ancestral homozygote (AA/GA vs. GG); comparison of the variant homozygote vs. ancestral homozygote combined with the heterozygote (AA vs. GA/GG). The I 2 statistic to quantify the proportion of the total variation due to heterogeneity were calculated, and a I 2 value of more than 50% was interpreted as significant heterogeneity among studies [26]. When the effects were assumed to be homogenous, the fixed-effects model was used (Mantel-Haenszel method) [27]. If obvious heterogeneity was present, the randomeffects model was used (DerSimonian-Laird method) [28]. Subgroup analysis based on ethnicity was used to explore the possible race-specific effect in the association. Subgroup analyses based on the types of cervical lesions and the quality of included studies were also performed. Potential publication bias was assessed by visual inspection of the funnel plots, in which the standard error of logOR of each study was plotted against its logOR, and an asymmetric plot suggested possible publication bias. In addition, We also performed Egger linear regression test at the P,0.05 level of significance to assess the funnel-plot's asymmetry [29]. All analyses were conducted using STATA (Version 11, StataCorp, College Station, TX). All P-values in the meta-analysis were two-sided, and statistical significance was considered when the P-value was less than 0.05.
Subgroup analysis of studies with high quality showed that there was still a significant association between TNF-a rs1800629 polymorphism and increased risk of cervical lesions under two main genetic comparison models (for AA versus GG: OR 1.81, 95%CI 1.26-2.61, P = 0.001; for AA versus GA/GG: OR 1.86, 95%CI 1.29-2.68, P = 0.001) ( Table 2). However, in the subgroup analysis of studies with low quality, there was no association between TNF-a rs1800629 polymorphism and increased risk of cervical lesions (Table 2). Subgroup analysis of Caucasians showed that there was a significant association between TNF-a rs1800629 polymorphism and increased risk of cervical lesions under all four genetic models (For A versus G: OR 1.33, 95%CI 1.07-1.65, P = 0.009; for AA versus GG: OR 1.47, 95%CI 1.08-2.00, P = 0.015; for GA/AA versus GG: OR 1.26, 95%CI 1.01-1.58, P = 0.045; for AA versus GA/GG: OR 1.45, 95%CI 1.06-1.97, P = 0.018). However, subgroup analysis of Asians showed that there was no association between TNF-a rs1800629 polymorphism and increased risk of cervical lesions in Asians (Table 2).
Subgroup analysis by the types of cervical lesions showed that there was a significant association between TNF-a rs1800629 polymorphism and increased risk of cervical cancer (For A versus G: OR 1.24, 95%CI 1.05-1.47, P = 0.011; for AA versus GG: OR 1.31, 95%CI 1.01-1.70, P = 0.043; for AA/GA versus GG: OR 1.25, 95%CI 1.01-1.54, P = 0.039). But there was no association between TNF-a rs1800629 polymorphism and risk of squamous intraepithelial lesions (Table 2).

Publication bias
Publication bias was investigated by Begg's funnel plot, and funnel plot asymetry was further assessed by Egger's linear regeression test. As shown in the Figure 3, there was low possibility of asymmetry in the allele comparison model (A versus G) of this meta-analysis ( Figure 3). Besides, the Egger linear regression test also suggested there was no significant risk of publication bias (P = 0.532). Therefore, there was no risk of publication bias in the meta-analysis.

Discussion
Cervical cancer is mainly initiated by HPV infection, and TNFa is an inflammatory cytokine which may play important roles in the progression of cervical lesions [3,5]. There is a possible association between TNF-a rs1800629 G/A polymorphism and risk of cervical lesions, but previous studies report conflicting results [12][13][14][15][16][17][18][19][20][21][22][23][24][25]. We performed a meta-analysis to comprehensively assess the association between TNF-a rs1800629 polymorphism and cervical lesions risk. Twenty case-control studies with a total of 4,146 cases and 4,731 controls were finally included into the metaanalysis [13][14][15][16][17][18][19][20][21][22][23][24][25][30][31][32][33][34]. Overall, TNF-a rs1800629 polymorphism was significantly associated with increased risk of cervical lesions under two main genetic comparison models (Table 2). Subgroup analysis by ethnicity further showed that there was a significant association between TNF-a rs1800629 polymorphism and increased risk of cervical lesions in Caucasians but not in Asians (Table 2). Subgroup analysis by the types of cervical lesions showed that there was a significant association between TNF-a rs1800629 polymorphism and increased risk of cervical cancer (Table 2). Therefore, the meta-analysis suggests that TNF-a rs1800629 polymorphism is associated with increased risk of cervical lesions, especially in Caucasians.
Cervical cancer is the third most common cancer and the fourth leading cause of cancer death among female in the world. In recent years, the incidence of cervical cancer has increased gradually [2]. Since cervical cancer is considered to be a preventable disease because of its relatively long period of precancerous lesions, identifications of risk factors of cervical lesions can help us find some effective and preventive interventions. It has been well accepted that HPV and smoking are main causes of almost all cases with cervical lesions, and the genetic factors also play important roles in the development of cervical lesions [3]. Currently, several genetic polymorphisms have been suggested to be associated with risk of cervical cancer, such as p53 Arg72Pro mutation and MTHFR gene polymorphisms [40][41][42].
TNF-a is secreted mainly by activated macrophages, which is an extraordinarily pleiotropic cytokine with a central role in immune homeostasis, inflammation, and host defense [5]. The development of cervical cancer is induced by persistent HPV infection, and TNF-a may be involved in the susceptibility to HPV infection and development of cervical cancer by modulating viral replication [5]. TNF-a rs1800629 is the most studied polymorphism, which is a G to A transition in the promoter at position 2308 and is associated with the level of TNF-a expression [10,11]. Although a possible association of the TNF-a rs1800629 polymorphism with risk of cervical lesions was reported, and many case-control studies were further performed to identify the association, it was still unknown whether there was a significant association between TNF-a rs1800629 polymorphism and risk of cervical lesions [13][14][15][16][17][18][19][20][21][22][23][24][25][30][31][32][33][34]. In current meta-analysis, we conducted a systematic review and meta-analysis of previous published case-control studies to comprehensively evaluate the association between TNF-a rs1800629 polymorphism and cervical lesions risk. The findings from the meta-analysis suggest that TNFa rs1800629 polymorphism is associated with increased risk of cervical lesions and TNF-a plays an important role in the development of cervical cancer.
There were some limitations in our meta-analysis. Firstly, there was limited number of eligible studies in the meta-analysis of the association between TNF-a rs1800629 polymorphism and risk of squamous intraepithelial lesions. The limited sample size in the meta-analysis may fail to provide enough statistical power to detect a possible or weak effect of TNF-a rs1800629 polymorphism on squamous intraepithelial lesions. Therefore, more studies with large sample are needed to give a more precise estimation of the association between TNF-a rs1800629 polymorphism and risk of squamous intraepithelial lesions. Secondly, the meta-analysis was based on unadjusted data, as the ORs adjusted for the main confounding variables were not available from those studies. To provide a more reliable estimation of the association, more studies with well-design and large sample size are needed to further identify the association. Finally, gene-gene interactions were not fully addressed in the meta-analysis for the lack of relevant data. There are several polymorphisms associated with risk of cervical cancer, such as p53 Arg72Pro mutation and MTHFR gene polymorphisms [40][41][42]. But no studies investigated the gene-gene interactions in the association between TNF-a rs1800629 polymorphism and risk of cervical lesions. Future studies may further assess the possible gene-gene interactions in the association between TNF-a rs1800629 polymorphism and risk of cervical lesions.
In summary, our meta-analysis suggests that TNF-a rs1800629 polymorphism is associated with increased risk of cervical lesions, especially in Caucasians. However, more studies with large sample and well-design are needed to give a more reliable estimation of the association between TNF-a rs1800629 polymorphism is associated and risk of cervical lesions.

Author Contributions
Conceived and designed the experiments: ML HBW. Performed the experiments: ML YH YF HBW. Analyzed the data: ML TTW HBW. Contributed reagents/materials/analysis tools: ML YH TTW YF HBW. Wrote the paper: ML HBW.