Association of the TNF-α, IL-2, and IL-2RB gene variants with susceptibility to psoriasis in a Turkish cohort

Introduction

Psoriasis (Ps) (OMIM 177900) is a chronic inflammatory dermatological disease manifested by red scaly and well-demarcated skin lesions developed due to the hyperproliferation of epidermal keratinocytes [1]. This proliferation is mediated by cytokines produced by activated tissue-resident immune cells, an infiltrate of T cells, dendritic cells, and cells of the innate immune system, as well as keratinocytes [2]. Studies on genome-wide association have found variants within or near a number of genes encoding cytokines, cytokine receptors, or elements of their signal transduction pathways, and this finding suggests that these cytokines play a role in the mechanism of pathogenesis of this disease [3].
Tumour necrosis factor-alpha (TNF-) (OMIM 191160) and interleukin 2 (IL-2) (OMIM 147680) have an impact on regulation of immune and inflammatory responses. The TNF- gene is located within the major histocompatibility complex (MHC) region on chromosome 6p21.3, a significantly polymorphic region [4]. Since genetic variations in the promoter region may modulate TNF- production, variants in the promoter region of the TNF- gene including –308 (rs1800629), –238 (rs361525), and –857 (rs1799724) in humans have been studied for their potential roles on gene transcription as well as their possible associations with certain inflammatory diseases.
IL-2 was originally described as a T cell growth factor; later it was reported that IL-2 induces T cell proliferation, survival, and differentiation of effectors (Th1/Th2). On the other hand, the main non-redundant function of IL-2 involves sustaining peripheral T cell tolerance, and the disorders of regulatory T cells are believed to be the basic cause of autoimmunity in the absence of IL-2. This cytokine can also sensitise previously activated T cells to go through apoptosis or activation-induced cell death, restricting the immune response [5]. Human IL-2 gene is encoded on chromosome 4q26 with well-defined variants, of which one –330 (rs2069762) has been described in the promoter region [6]. The IL-2 (–330) variant is associated with cytokine synthesis levels in vitro, with the TT and GT genotypes being related to low production and the GG genotype being associated with increased production [7]. It has been suggested that the IL-2 (–330) variant plays a role in high susceptibility to several inflammatory diseases and malignant neoplasms.
The IL-2 receptor, which plays a role in T-cell-mediated immune responses, is found in three distinct forms in terms of ability to bind IL-2. IL-2 Receptor B (IL-2RB) is located in the 22q13 chromosomal region. Its protein product belongs to the IL-2 signalling pathway, which is crucial for T cell activation [8]. There is a T/C polymorphic site located at the 889th nucleotide of IL-2RB mRNA [9]. Due to the relationship between Ps and immunity, we set out to test the association between TNF- (–308, –238, and –857), IL-2 (–330), and IL-2RB (+114) gene variants and Ps in a Turkish cohort.

Material and methods

Patient population

The study population consisted of 74 unrelated patients with Ps (40 males and 34 females; mean age: 42.31 ±13.94 years) and 74 (38 males and 36 females; mean age: 40.84 ±13.65 years) unrelated healthy controls. Patients were diagnosed and followed at the Department of Dermatology at Gaziantep University, Gaziantep, Turkey. All subjects were from similar ethnic background and the same geographic area. Clinical diagnoses were made by dermatologists, and all patients had at least two skin lesions. The participants were well informed about the nature of this study and their participation was voluntary. Clinical data including family history, age at onset (early onset: ≤ 40 years, late onset > 40 years) were collected. Disease severity was quantified by the Psoriasis Area Severity Index (PASI) [10]. Informed, written consent was obtained from all participants, and the design of the study was approved by the local Ethics Committee (TF.09.25).

Genotyping analysis

The DNA of the participants was isolated from peripheral blood mononuclear cells using the salting out method [11]. TNF- (–308, –238, and –857), IL-2 (–330), and IL-2RB (+114) gene variants were genotyped by polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) analysis, as described previously [9, 12]. The primer sequences, restriction enzymes, and laboratory conditions related to these variants are listed in Table 1.

Statistical analysis

All data were analysed using SPSS software version 14.0 for Windows (SPSS Inc., Chicago, IL, USA). The statistical significance of the differences between the patient and the control groups was estimated by Pearson’s 2 analysis [13]. Odds ratio (OR) and 95% confidence interval (CI) were also calculated. OR (95% Cl) was adjusted for age and sex. The data were analysed for appropriateness between the observed and expected genotypes as well as for Hardy-Weinberg Equilibrium (HWE), as described elsewhere. All analyses were two-tailed, and differences were interpreted as statistically significant when p < 0.05.

Results

Clinical and demographical characteristics

Complete demographical information and clinical parameters for the study population are shown in Table 2. A total of 148 subjects from the Turkish population were recruited in this study. There were 40 (54.1%) males and 34 (45.9%) females in the patient group. Demographic features such as gender, age, PASI, disease onset, arthritis, and family history of Ps were analysed. The genotype distribution of TNF- (–308, –238, and–857), IL-2 (–330), and IL-2RB (+114) variants in patients and the controls, as well as their associations with risk of Ps, are shown in Table 3.

TNF-α (–308, –238, and –857)

Genotype distribution of TNF- (–308) and (–238) variants was found to be significantly different between the patients and the controls. The AA genotype of TNF- (–308) was significantly more common in the patients than in the controls (p = 0.013, OR = 0.475, 95% CI: 0.400-0.565). TNF- (–238) AA genotype was significantly higher in the patients (p = 0.028, OR = 0.479, 95% CI: 0.403-0.589), while the GG genotype was lower in the patients group, compared to the controls (p = 0.016, OR = 3.306, 95% CI: 1.295-8.440). The analysis of the genotype distribution for TNF- (–857) variant did not show a statistically significant difference between the groups. The genotype frequencies of the TNF- (–857) variants were in HWE in both the patients and the controls (p = 0.129, p = 0.17, respectively). However, genotype distribution of TNF- (–308) variant in the controls and TNF- (–238) in the patients did not conform to HWE (p = 0.017, p = 0.001 respectively).

IL-2 (–330) and IL-2RB (+114)

The genotype distribution of the IL-2 (–330) variant was statistically different between the patients and the controls. IL-2 (–330) variant GG and TT genotype was more common in the patients (p = 0.037, OR = 2.716, 95% CI: 1.061-6.949; p = 0.009, OR = 3.257, 95% CI: 1.387-7.646, respectively), while IL-2 (–330) GT genotype was increased in control subjects (p = 0.001, OR = 5.130, 95% CI: 1.926-3.667). There was no significant difference for the IL-2RB (+114) genotype distribution between the patients and healthy controls. Statistical calculation for IL-2RB (+114) indicated that the patient and the control groups were not in HWE, (p = 0.029, p = 0.0000, respectively). The observed and expected frequencies of the variation for IL-2 (–330) in patients were not in HWE (p = 0.0001).

Clinical findings according to TNF-α, IL-2, and IL-2RB genotypes

In addition, the relationship between the genotype distribution of TNF-, IL-2, and IL-2RB gene variants and clinical features including arthritis, family history, disease onset, and PASI were also investigated in the patient group (Tables 4-7). TNF- (–308, –238, and –857) variant genotype distribution did not show any difference according to clinical features (p > 0.05). IL-2 (–330) GG genotype frequency was significantly lower (p = 0.021) and the TT genotype frequency was significantly higher (p = 0.014) among patients with psoriatic arthritis in comparison with Ps patients. IL-2RB (+114) TC genotype frequency was significantly lower and TT genotype frequency was significantly higher in patients with positive family history of Ps, compared to the patients with negative family history (p = 0.017, p = 0.014, respectively). Also, IL-2RB (+114) CC genotype was significantly high among patients with late-onset Ps in comparison to the early onset Ps group (p = 0.009). The frequency of IL-2 (–330) TT genotype was significantly higher in mild Ps patients than in moderate-severe patients (p = 0.043).

Discussion

Ps is among the most common chronic inflammatory skin diseases, estimated to affect 1-3% of the general population. The pathogenesis of this disease has not been established clearly. However, it is thought that Ps is a genetic disease related to inflammation, which is mediated and sustained by numerous factors of the immune system.
The skin is an essential lymphoid organ, bearing an effective immunological surveillance system helped by antigen-presenting cells, cytokine-synthesizing keratinocytes, epidermotropic T cells, dermal capillary endothelial cells, lymph nodes, mast cells, tissue macrophages, granulocytes, fibroblasts, and non-Langerhans cells. Communication among these cells is achieved by cytokine secretion, and they respond through stimulation by bacteria, chemicals, ultraviolet light, and other irritating factors. Ps is regarded as a T-helper-1 (Th1) disease because an increase in Th1 pathway cytokines (interferon-, TNF-, IL-2, and IL-12 has been detected in the Ps plaques) [14]. In present study, we aimed to investigate the association between TNF-(–308, –238 and –857), IL-2 (–330), and IL-2RB (+114) variants and Ps.

TNF-α (–308, –238, and –857) variants

TNF- is essential for local T cell proliferation; it is suggested that the development of Ps is mediated by TNF-, and the proliferation of local T cells depends on its local synthesis [15]. Some studies reported that psoriatic patients have higher serum levels of TNF- [4, 16]. The TNF- gene is highly polymorphic, and in previous studies it was found that sequence variation in the regulatory region of TNF- gene has been associated with various disorders. Two G versus A transitions in the promoter region at positions (–308) [17] and (–238) [18] have been reported to have an impact on TNF- expression. The TNF-(–308) A allele has been found to have greater potential as a transcriptional activator in vitro compared to the common G allele [19]. Therefore, the A allele frequency has been associated with the risk of autoimmune disorders [17, 20]. However, the reports of influence of TNF- (–238) on the transcriptional regulation of TNF have been conflicting [21].
There have been many studies to evaluate the possible association with between TNF- (–308 and–238) variants and Ps in different populations. Cabaleiro et al. [22] found that the frequency of the TNF- (–308 and –238) wild type genotype was greater in patients with Ps than in healthy controls in a Spanish population. Similarly, it was observed that TNF- (–238) wild type genotype (GG) had a higher prevalence in the patients compared to the controls [23]. But Arias et al. [24] reported that the frequency of TNF- (–238) GG genotype was decreased in patients but that of GA genotype was increased in patients. It was found that TNF- –238 A allele was more common in psoriatic patients [25, 26]. Also, it was reported that the frequency of TNF- (–238) A allele was significantly higher in patients with juvenile-onset Ps in a southwestern Chinese population [27]. In meta-analysis, data suggest that TNF- (–308) variant is associated with decreased risk of Ps, while TNF- (–238) is associated with increased risk of Ps [28]. Karam et al. [4] showed that TNF- (–308) G allele is more common in Ps patients than in controls, and they found that TNF- serum level was higher in patients with TNF- (–308) AA genotype. However, some studies have reported no association between TNF- (–308)/ (–238) variant and Ps in different populations [29-31].
We found that genotype distribution of TNF- (–308 and –238) variants was significantly different in the patients. The TNF- (–308) AA genotype and TNF- (–238) AA genotype carriers showed a significantly increased risk of developing Ps (Table 2). These findings are consistent with previous studies, which reported that TNF- (–308) A allele was associated with Ps. In the present study, TNF- (–238) variant GG genotype was less frequent in the patient group compared to the controls. There are few studies evaluating the association between TNF- (–857) variant and Ps. Gallo et al. found no association between TNF- (–857) variant and Ps [23]. However, Cabalerio et al. [22] reported that the frequency of TNF- (–857) mutant genotype was higher in Ps patients. In the present study there was no significant difference for genotype distribution of TNF- (–857) between the patients and the healthy controls.
Ps has a large spectrum of clinical features. Numerous population studies conducted to assess the possible association between some TNF- gene variants (–308 and –238) and psoriatic arthritis (PsA) resulted in controversial conclusions. Some researchers even established a strong association between these TNF- promoter variants and PsA, recommending that both variants could be used as biomarkers to anticipate the risk of the disease [32-34]. In the present study, genotype distribution of TNF- (–308, –238 and –857) variants according to clinical features including arthritis, family history, disease onset, and PASI did not show any statistical difference.

>IL-2 (–330) and IL-2RB (+114) variants

IL-2 plays a role in the chemical activity of T-cell-assisted immune responses, and as a growth factor it facilitates the cytolytic activity of natural killer cells. Mice lacking expression of IL-2, IL-2RA, and IL-2RB show autoimmunity [35]. The association of IL-2 and IL-2RB genes with several autoimmune diseases could imply a common pathogenic mechanism for these diseases. In previous studies it was reported that IL-2 levels were significantly elevated in the serum of psoriatic patients before treatment compared with those of controls [36, 37]. Warren et al. found that the IL-2 gene variant (rs2069778) is associated with early-onset Ps [38]. Kim et al. suggested that high IL-2 production influenced by IL-2 (–330) G allele could result in the Th1/Th2 cytokine balance to lean towards the Th1-type in Korean Ps, particularly late-onset Ps [39]. Also, Zhou et al. [40] showed that IL-2 (–330) GG and TG genotypes increased the risk in Ps patients who belonged to the northern Chinese Han population. Our results exhibit that GG and TT genotypes of IL-2 (–330) are significantly increased in patients with Ps compared with the control group (Table 3). Our data are consistent with those of Kim et al. and Zhou et al. Although it seems conflicting that genotypes which produce both high and low cytokines were found to be more common in the present study, this finding could be associated with the ethnic diversity of the study group.
In this study, the frequency of IL-2 (–330) GT genotype was significantly low in the patients with Ps compared to the controls. IL-2 (–330) GG genotype frequency was significantly low and that of TT genotype was significantly high, among patients with psoriatic arthritis, compared with Ps patients. The frequency of IL-2 (–330) TT genotype was significantly higher in mild Ps patients than in moderate-severe patients. Furthermore, the frequency of IL-2RB (+114) CC genotype was significantly high among the patients with late-onset Ps compared with the early onset Ps group. IL-2RB (+114) TC genotype frequency was significantly low and TT genotype frequency was significantly high in patients with positive family history of Ps compared with the patients who had a negative family history. Our results clearly showed that IL-2 and IL2RB variants are associated with clinical features in Ps.

Conclusions

Our data suggest a potential role of TNF-, IL-2RB, and IL-2 as a candidate gene for susceptibility to Ps in a Turkish population. This may have a potential impact on disease counselling and management. Further large and well-designed case-control studies in diverse ethnic groups with more detailed individual information that include well-matched controls concerning the effects of these gene variants are needed.

The authors declare no conflict of interests.

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