TNF -308 G/A Polymorphism and Risk of Acne Vulgaris: A Meta-Analysis

Background The -308 G/A polymorphism in the tumor necrosis factor (TNF) gene has been implicated in the risk of acne vulgaris, but the results are inconclusive. The present meta-analysis aimed to investigate the overall association between the -308 G/A polymorphism and acne vulgaris risk. Methods We searched in Pubmed, Embase, Web of Science and CNKI for studies evaluating the association between the -308 G/A gene polymorphism and acne vulgaris risk. Data were extracted and statistical analysis was performed using STATA 12.0 software. Results A total of five publications involving 1553 subjects (728 acne vulgaris cases and 825 controls) were included in this meta-analysis. Combined analysis revealed a significant association between this polymorphism and acne vulgaris risk under recessive model (OR = 2.73, 95% CI: 1.37–5.44, p = 0.004 for AA vs. AG + GG). Subgroup analysis by ethnicity showed that the acne vulgaris risk associated with the -308 G/A gene polymorphism was significantly elevated among Caucasians under recessive model (OR = 2.34, 95% CI: 1.13–4.86, p = 0.023). Conclusion This meta-analysis suggests that the -308 G/A polymorphism in the TNF gene contributes to acne vulgaris risk, especially in Caucasian populations. Further studies among different ethnicity populations are needed to validate these findings.


Introduction
Acne vulgaris is a chronic inflammatory skin disease widely affecting adolescents and young adults [1]. The pathogenesis of acne vulgaris is a complex process in which several factors have been implicated, including hormonal effects, abnormal keratinocyte function, microbial components (Propionibacterium acnes), inflammation, environmental factors and genetics [2][3][4].
Inflammation plays one of the main roles in the development of acne vulgaris [5,6]. A research in vivo reported that a marked increase for tumor necrosis factor (TNF) gene transcripts was observed in acne lesions [7]. TNF is one of the main proinflammatory cytokines that play a central role in initiating and regulating the cytokine cascade during an inflammatory response [8].
Several single nucleotide polymorphisms (SNPs) in the TNF gene promoter have been identified [9,10], some of which may regulate TNF expression. One of these polymorphisms at position -308 (TNF -308 G/A) had been reported associated with regulation of TNF expression by, e.g., interfering with transcription factor binding sites or other regulatory elements [11].
A number of case-control studies were conducted to investigate the association of -308 G/A polymorphism in the TNF gene and the risk of acne vulgaris [12][13][14][15][16][17]. However, these studies reported conflicting results which may be due to the limitations in sample size and different ethnic populations in the corresponding investigation. Therefore, we perform present meta-analysis to systematically clarify the association between the TNF -308 G/A polymorphism and acne vulgaris risk based on all eligible casecontrol studies.

Literature Search
Two authors independently performed systematic searches in Pubmed, Embase, Web of Science and CNKI (China National Knowledge Infrastructure) databases to identify studies examining the association between the -308 G/A polymorphism in the TNF gene and acne vulgaris risk. A date limit of August 1, 2013 was applied. The search was performed without any restrictions on language. The search terms were as follows: ''Acne vulgaris'' or ''Acne'' in combination with ''TNF'' or ''tumor necrosis factor''. The reference lists of identified studies and review articles were manually searched to find additional relevant publications.

Study Selection
Studies were included in the meta-analysis if they satisfied the following inclusion criteria: (1) case-control studies focused on associations between TNF gene -308 G/A polymorphisms and acne vulgaris risk; (2) genotype frequencies were available for cases and controls; (3) the distribution of genotypes in the control group was consistent with Hardy-Weinberg equilibrium (HWE). (4) when publications involved the overlapping data sets, only the study with the largest number of participants was included. The supporting PRISMA checklist is available as Checklist S1.

Data Extraction
Two authors independently extracted data from the included studies. The following data were extracted: the name of the first author, year of publication, country of origin, ethnicity, sample size, genotyping method and genotype frequencies in acne vulgaris cases and controls. In case of conflicting evaluations, disagreements were resolved through discussion between the authors.

Statistical Analysis
The association between the TNF -308 G/A polymorphism and risk of acne vulgaris was assessed using odds ratios (ORs) and 95% confidence intervals (CIs). The significance of the pooled OR was determined using the Z-test and p,0.05 was considered statistically significant. We estimated the association based on four genetic models: allele model (A vs. G), dominant model (AA + AG vs. GG), recessive model (AA vs. AG + GG) and additive model (AA vs. GG).
To evaluate whether the association showed any ethnicityspecific effects, we analyzed the data for separate subgroups defined by ethnicity. Heterogeneity was evaluated using a x2based Q statistic and I 2 test, with p,0.10 or I 2 .50% considered statistically significant [18,19]. When p$0.10 or I 2 #50%, the pooled OR was calculated using a fixed effect model (Mantel-Haenszel method); otherwise, a random effect model (DerSimonian Laird method) was used.
Publication bias was assessed using Begg's funnel plots and Egger's test [20]. Sensitivity analysis was performed by excluding individual studies and recalculating the results in order to assess the stability of the results. Pearson's x2 test was used to determine whether the observed frequencies of genotypes in control group conformed to the HWE [21]. All statistical tests were performed using STATA 12.0 software.

Characteristics of included studies
The flow chart that displays the study selection process was shown in Figure 1. A total of five publications evaluating the association between the TNF -308 G/A polymorphism and acne vulgaris risk were included in the meta-analysis, involving 1553 subjects (728 acne vulgaris cases and 825 controls) [12][13][14][15][16]. One study was excluded because the distribution of genotypes in the control group was inconsistent with HWE [17]. Among the eligible five studies, four studies were performed in Caucasian populations and one study was in Asian population. The diagnosis of acne was based on a thorough physical examination by dermatologists. The clinical grade of acne was assessed mostly based on the Global Acne Grading System [22]. Acne patients were classified based on severity of the disease into three subgroups: mild, moderate, and severe acne. The control group was chosen from healthy individuals without any systemic and dermatologic diseases. In general, studies were relatively small, and the mean number of cases was 146 (range, 84-229) and the mean number of controls was 165 (range, 75-390). The characteristics of the included studies were summarized in Table 1 and Table 2.

Meta-analysis results
With significant between-study heterogeneity by Q test and I 2 test under the majority of genetic models (I 2 .50%), the analysis was conducted using random effect model. The meta-analysis results showed that TNF -308 G/A polymorphism was linked to the risk of acne vulgaris under recessive and additive models ( AA vs. AG + GG: OR = 2.73, 95%CI: 1.37-5.44, p = 0.004; AA vs. GG: OR = 2.67, 95%CI: 1.28-5.57, p = 0.009; respectively) ( Figure 2). Results for these and other genetic models were summarized in Table 3.

Subgroup Analysis
In the subgroup analysis based on ethnicity, the results indicated that TNF -308 G/A polymorphism might significantly increase the risk of acne vulgaris among Caucasian populations (recessive model: AA vs. AG + GG, OR = 2.34, 95%CI: 1.13-4.86, p = 0.023; additive model: AA vs. GG, OR = 2.14, 95% CI: 1.02-4.47, p = 0.044; respectively). TNF -308 G/A polymorphism might show significant association with the increased risk of acne vulgaris in all genetic models among Asian populations (all p,0.05), but not enough reliability was established due to the result from a single study [15] (Table 3).

Sensitivity Analysis and Publication Bias
Sensitivity analysis were performed by sequential omission of individual studies for all subjects and subgroups. The corresponding pooled ORs were not significantly altered in all subjects and subgroups (data not shown). The results of sensitivity analysis indicated the stability of our results. Begg's funnel plot and Egger's test were used to assess publication bias. The shape of the funnel plots in all the genetic models seemed symmetrical, indicating that there were no evidences for obvious publication bias. Further, Egger's test provided similar results that there were no statistically significant publication bias in all genetic models (all p.0.05).

Discussion
The TNF gene is located on chromosome 6 (6p21.3) between HLA-B and DR within the class III region of the major histocompatibility complex [23]. There are several polymorphisms in the promoter region of the TNF gene (2863, 2857, 2850, 2575, 2375, 2308, 2274, 2238, 2237, 2162) [9,10]. The most common polymorphisms is in the promoter at position 2308. The polymorphism may affect cytokine production [11,24,25]. To date, several studies have been carried out to identify whether TNF -308 G/A polymorphism was associated with acne vulgaris risk. However, the conclusion was controversial. To the best of our knowledge, our article is the first meta-analysis evaluating the association between TNF -308 G/A polymorphism and acne vulgaris risk. We performed the present meta-analysis of eligible five independent case-control studies, including 728 cases and 825 controls. We were able to provide a more complete picture of the role of TNF -308 G/A polymorphisms in acne vulgaris risk, as comparing with that published in individual studies.
>When all the eligible studies were pooled into the metaanalysis, the results showed that -308 G/A polymorphism was associated with the risk of acne vulgaris under the recessive model, with AA homozygote at higher risk of acne vulgaris than AG heterozygote and GG homozygote. In addition, we performed a stratified analysis based on ethnicity. The results showed that -308 G/A polymorphism might increase the risk of acne vulgaris among Caucasian population under recessive model, and that the homozygotic AA genotype may be a risk factor for acne vulgaris. We also found the -308 G/A polymorphism to be a risk factor for acne vulgaris among Asians, but the result in Asian population was not reliable enough due to the estimation from a single study [15]. More studies are needed to perform in Asian population. Our finding that the -308 G/A polymorphism was a genetic risk factor for acne vulgaris both in Caucasians and Asians, suggested no population-specific genetic difference in acne vulgaris pathogenesis.  TNF -308 G/A polymorphism have been examined in several autoimmune and inflammatory diseases, such as psoriasis, lepromatous leprosy and systemic lupus erythematosus [26][27][28]. However, the results were not consistent, mainly due to differences of the studied populations, or insufficient sample size. TNF -308 A allele had shown to be a stronger transcriptional activator than the common TNF -308 G allele in vitro and patients with TNF -308 GA heterozygosity had increased TNF production [11,29].
The findings in this meta-analysis should be interpreted with caution because of several limitations. First, a relatively small number of studies were included and the sample sizes were still relatively small, which may not provide sufficient power to estimate the association between TNF -308 G/A polymorphism and acne vulgaris risk. Second, the included publications were limited to Caucasian and Asian populations, so future work should examine other populations. Third, although no obvious publication bias was identified, potential bias cannot be completely ruled out. Fourth, there had been some differences in severity of cases between the included studies (Table 1), in which mild, moderate and severe acne occupied different proportion, although the diffrences were not statistically significant. Nonetheless, it was well acknowledged that many other factors, such as gene-environment interactions may affect the risk of acne vulgaris.

Conclusions
To the best of our knowledge, this is the first meta-analysis to assess the relationship between the TNF -308 G/A polymorphism  and acne vulgaris risk. Our results suggest that the -308 G/A polymorphism may be a potential risk factor for acne vulgaris, especially in Caucasian population. However, further studies are still needed to warrant and validate the association between TNF -308 G/A gene polymorphism with acne vulgaris risk.