Introduction

Acne is a chronic inflammatory disease of pilosebaceous unit widely affecting adolescents and young adults [19]. It is a multifactorial disease and several pathogenetic factors have been identified including sebum overproduction, abnormal follicular keratinization, Propionibacterium acnes (P. acnes) proliferation, inflammation, delayed-type immune response, external factors, and genetics [17, 19, 23, 30, 46]. The genetic influence on the pathogenesis of the disease has been well documented in twins [9, 17, 18, 42], but there are only few studies investigating the relevant genetic elements [5, 22, 29, 34, 35, 39].

It is suggested that acne vulgaris is likely to be a genuine inflammatory disease [30, 46]. Tumor necrosis factor-alpha (TNF-alpha) is one of the main proinflammatory cytokines that play a central role in initiating and regulating the cytokine cascade during an inflammatory response [8]. It has an important role in the pathogenesis of acne as in the other inflammatory skin diseases [21, 30, 41]. Factors affecting its production may possibly influence the degree of inflammatory response and hence may account for the clinical severity of acne. The TNF-alpha gene is located on chromosome 6 (6p21.3) between HLA-B and DR within the class III region of the major histocompatibility complex [13]. Several single-nucleotide polymorphisms (SNPs) in the TNF gene promoter have been identified, some of which may regulate TNF-alpha expression. One of them represents a guanine (G) to adenine (A) transition at position -308 (TNFA-308 G/A), and has been examined in several inflammatory diseases [6, 7, 14, 15, 28, 40, 44]. The aim of our study was to investigate for the first time whether TNFA-308 G/A polymorphism might be involved in the pathogenesis of acne and whether there is a relationship between this polymorphism and severity of the disease.

Materials and methods

Subjects

We examined 113 patients with acne (90 women, 23 men) and 114 healthy control subjects (45 men, 69 women). The diagnosis of acne was based on a thorough physical examination. The clinical grade of acne was assessed based on the Global Acne Grading System [16]. According to the system, patients were divided into four categories namely mild, moderate, severe, and very severe acne. The control group was chosen from healthy individuals without any systemic and dermatologic disease. The study was approved by the local ethic committee and informed consent was obtained from each individual before sample collection.

Analysis of TNFA -308 G/A polymorphism

Venous blood samples were collected in ethylenodiaminotetra acetic acid (EDTA) containing tubes. DNA was extracted from whole blood using a genomic DNA purification kit (MBI Fermentas, Vilnius, Lithuania). Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) assay was used to determine TNFA -308 G/A polymorphism. The oligonucleotide primers used to determine the -308 G/A polymorphism within the TNF-alpha gene that were described previously [10]. The primers, forward 5′-AGGCAATAGGTTTTGAGGGCCAT-3′,s reverse 5′-TCCTCCCTGCTCCGATTCCG-3′ were used to amplify the TNF-alpha gene. PCR was performed in a 25 μl volume with 100 ng DNA, 100 μm dNTPs, 20 pmol of each primer, 1.5 mM MgCl2, 1x PCR buffer with (NH4)SO4 (MBI Fermentas, Vilnius, Lithuania,), 10% DMSO, and 2U Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania). Amplification was performed on an automated thermal cycler (Techne Flexigene, Cambridge, UK). PCR conditions were 2 min for initial denaturation at 95°C; 35 cycles at 95°C for 45 s for denaturation, 1 min at 60°C for annealing and 90 s at 72°C for extension, followed by 7 min at 72°C for final extension. The PCR products were 107 bp. After amplification, PCR products were digested by restriction endonuclease 10U NcoI (MBI Fermentas, Vilnius, Lithuania) for 14 h at 37°C. The genotyping of the TNF-alpha gene was determined by fragment separation at 120 V for 40–50 min on a 3.5% agarose gel containing 0.5 μg/ml ethidium bromide. A 50 bp marker (50 bp DNA Ladder, MBI Fermentas) was used as a size standard for each gel lane. The gel was visualized under UV light using a gel electrophoresis visualizing system (Vilber Lourmat). The NcoI restricted products of TNFA-308 G/A; GG, GA, and AA genotypes had band sizes of 87bp/20 bp, 107bp/87bp/20 bp, and 107 bp, respectively. Selected samples from each gel were repeated to confirm the results. Genotyping was based upon independent scoring of the results by two reviewers who were unaware of case/control status.

Statistical analysis

All statistical tests were carried out using SPSS 11.5 for Windows. The distribution of genotypes and Hardy–Weinberg equilibrium were tested with Chi-square (χ2) test for quality of fit. The z-approximation test was used to compare two independent proportions of patients and control subjects. P values smaller than 0.05 were considered statistically significant. The strength of association was estimated by calculating the odds ratios (ORs) and 95% confidence intervals (95% CIs) from the 2 × 2 table data.

Results

Of the 113 patients, 32 suffered from mild acne whereas 51 had moderate, and 30 had severe acne. The main age was 30.6 ± 13.2 for the control subjects and 22.0 ± 7.4 for the patients.

All patients and control subjects were genotyped at the TNFA-308 locus. The distribution of genotypes was in Hardy–Weinberg equilibrium in all groups. Genotype frequencies for the patients and controls are presented in Table 1. The distribution of the GG, GA, and AA genotypes was 58.4, 38.1, and 3.5%, respectively, in acne patients. Genotypes in healthy controls were as follows: GG 86.8%, GA 13.2%, and AA 0%. The difference in the distributions of the TNFA genotypes between the patient and control group was statistically significant. The GA genotype frequency was significantly increased in acne patients (P < 0.001, OR 4.054, 95% CI 2.090–7.865) compared with healthy controls.

Table 1 Distribution of TNFA genotypes in acne patients and control subjects
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The severity of acne was not associated with TNFA genotypes (P = 0.463) as shown in Table 2. GG, GA, and AA genotype distributions were similar between male and female patients (P = 0.518). There was also no male/female difference in the distribution of GG and GA genotypes (P = 0.602) in the controls (Table 3).

Table 2 Distribution of TNFA genotypes in patients according to severity of acne
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Table 3 Distribution of TNFA genotypes in patients and controls according to gender
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Table 4 shows a comparison of the distribution for TNFA genotypes of our control subjects with those of other populations from Turkey and different countries. When the genotype frequencies of our controls were compared with those of other populations from Turkey [6, 7, 26], Iran [25], and Korea [28], there were no significant differences. However, when the genotype frequencies of our controls were compared to those of European people from Italy [32], and England [37], genotype distributions differed; GA genotype frequency was found to be decreased in our controls, while GG genotype frequency was found to be decreased in European populations (P < 0.001).

Table 4 Distribution of TNFA genotypes in different populations, and comparison of genotype frequencies of our control subjects with those of some previous reports
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Discussion

During the development of an acne vulgaris lesion, the earliest morphological change in the pilosebaceous unit is abnormal follicular keratinization. Follicular hyperkeratosis and increased sebum production result in the development of microcomedones, changes in follicular milieu, and intensive growth of P. acnes. P. acnes secretes several proinflammatory products including lipases, proteases, hyaluronidases, and chemotactic factors [2]. The chemotactic factors produced by P. acnes attract cells of the immune system such as neutrophils, monocytes, and lymphocytes [19, 21, 27, 30]. Microcomedones or comedones may then later develop into inflammatory lesions as a result of CD4 + T-cell activation and migration, cytokine production by keratinocytes, macrophages, and neutrophils recruited to the site, hormonal factors and enhanced sebum production [19, 27].

Proinflammatory cytokines (IL-1 alpha, IL-8, and TNF-alpha) are the main responsible mediators of inflammatory acne [30, 46]. It has been shown that P. acnes stimulates cytokine production from lymphocytes, monocytes, and keratinocytes. Both intact P. acnes and isolated cellular factors induce production of proinflammatory cytokines, including TNF-alpha, IL-1alpha, granulocyte/macrophage colony stimulating factor (GM-CSF) [21], IL-1beta, and IL-8 [24, 41].

A proinflammatory cytokine, TNF-alpha is a powerful inducer of the inflammatory response and a key regulator of innate immunity. It enhances major histocompatibility complex (MHC) class I molecule expression on activated T-cells, promotes IL-2-dependent T-cell proliferation, and is a cofactor in B-cell proliferation and immunoglobulin production. Inflammatory responses to TNF-alpha are mediated both directly and through stimulation of the expression of IL-1 and other proinflammatory cytokines [8]. In view of TNF-alpha plays a role in the acne pathogenesis, factors affecting its production may possibly influence the degree of inflammatory response and hence may account for the clinical severity of acne. The cytokine mRNA and protein levels depend on both genetic and environmental factors. Analysis of cytokine gene polymorphisms affecting the production of inflammatory mediators would lead the way to detect a genetic abnormality of cytokine regulation that may play a role in the pathophysiology of the disease. SNPs of certain genes of cytokines and/or cytokine receptors are associated with some human diseases suggesting their likely involvement in the pathogenesis [20]. Even if they do not have striking effects on the development of a disease, they might change penetrance of other important genes, modify disease manifestations and affect their severity.

The TNF-alpha gene is located on chromosome 6 (6p21.3) between HLA-B and DR within the class III region of the major histocompatibility complex [13]. This locus includes two closely linked genes that encode the cytokines TNF-alpha and lymphotoxin alpha (also known as TNF-beta). There are several polymorphisms in the promoter region of the TNF-alpha gene (-850, -863, -857, -575, -375, -308, -274, -238, -237, -162) [4, 45]. The most common polymorphisms are two G to A transition in the promoter at position -238 and -308. These polymorphisms may affect cytokine production [15, 43, 44]. The majority of studies investigating the functional significance of alpha-α promoter polymorphisms have focused on the biallelic SNP TNFA-308 G/A [14, 15, 40, 44]. TNFA-308 A allele has shown to be a stronger transcriptional activator than the common TNFA-308 G allele, in vitro [31, 44]. There are also some studies which show an increased production of TNF-alpha associated with TNFA-308 A allele [1, 11, 33]. Patients with TNFA-308 GA heterozygosity have increased TNF-alpha production [31, 43, 44]. However, in view of the chromosomal localization of the TNF gene, TNF-alpha expression may depend on polymorphisms in the TNF-alpha promoter region or a linkage association with the HLA genotype [15, 36, 43].

TNFA-308 G/A polymorphism have been examined in several autoimmune and inflammatory diseases [3, 6, 7, 12, 20, 28, 38, 40]. However, the results have varied, mainly due to differences in the origin of the studied populations, linkage disequilibrium with other MHC genes or insufficient sample size. This is the first report concerning TNFA-308 G/A polymorphism in acne patients. In the present study, we found that the TNFA-308 GA genotype frequency was statistically significantly increased in patients compared with the controls. However, we did not observe any statistical association between the genotype distributions and severity of acne. In view of acne is a multifactorial disease, it is possible that the severity of acne may be more readily influenced by other factors such as environmental and other genetic elements.

The relationship of acne and various genes has been previously investigated [5, 22, 29, 34, 35, 39]. It has been suggested that human cytochrome P450 1A1 gene (CYP1A1) [35], the steroid 21-hydroxylase gene (CYP21) [34], the epithelial mucin gene (MUC1) [5], and human cytochrome P450c17α gene (CYP17) [22] may be involved in the pathogenesis of acne. Androgen receptor polymorphism (CAG repeat lengths) [39] or Toll-like receptor (TLR)-2, and TLR4 polymorphisms [29] were also investigated in patients with acne, but no association has been shown.

The genotype frequencies of TNFA differ between ethnic groups [6, 20, 25, 28, 32, 37]. This is the first report investigating TNFA-308 G/A polymorphism in acne patients and there is no other study performed for detection of TNFA genotype frequencies in Turkish population with the larger sample size. For this reason, only data of the control subjects were compared with those of some previous studies, to evaluate the effect of ethnicity on our results. The distribution of genotypes of our control subjects was similar with those of some other populations from our country [6, 7, 26], Iran [25], and Korea [28], while it differed from European populations from Italy [32] and England [37], which had higher frequencies of GA genotypes (Table 4).

In summary, we have demonstrated an association between TNFA-308 G/A polymorphism and acne susceptibility. Even in the absence of a confirmed direct functional effect of the polymorphism, our results indicate that TNFA-308 G/A polymorphism may have a role in acne susceptibility in Turkish patients. Further studies with extended samples (from same and different populations) are necessary to confirm our results. In addition, further investigations of other polymorphisms of the TNF gene locus and its association with TNF production in acne patients may be helpful to clarify the pathogenesis of the disease.