Psoriasis is a common autoimmune inflammatory skin disease with a multifactorial genetic basis 1. It affects ∼4.8% of the world’s population 2.
The hypothesis of immunological disorder in psoriasis is described by abnormal keratinocyte proliferation mediated through T lymphocytes-induced inflammation 3. Autoimmune disorders and inflammatory reactions are currently segregated into cell-mediated Th1 or Th2 categories. Psoriasis is associated with an overexpression of proinflammatory cytokines produced by Th1 cells and the relative underexpression of Th2 cytokines 4.
Tumor necrosis factor-α (TNF-α) has been shown to play a fundamental role in the pathogenesis of psoriasis 5. This role may be mediated through several pathogenic mechanisms, including the expression of adhesion molecules on the surface of endothelial cells, keratinocyte, and dendritic cells that promote leukocyte migration 6. TNF-α and interferon-γ stimulate the production of TNF-α by epidermal cells that may, together with interleukin-8 (IL-8), be involved in autocrine stimulation of keratinocyte proliferation 7. Recently, several TNF-α inhibitors have either been approved or are undergoing clinical trials for the treatment of psoriasis 8.
Genetic factors are likely to play a fundamental role in the manifestation of psoriasis 9. The results of family, twin, and human leukocyte antigen (HLA) allotype studies as well as genome-wide scans in affected families all point to a genetic basis for psoriasis susceptibility, which is complex and likely to reflect the action of a number of genes 10.
Many studies have attempted to identify genetic polymorphisms in psoriasis that act as markers of disease susceptibility 10,11. The main genes analyzed are the major histocompatibility complex HLA-Cw*0602 11 and the genes encoding TNF 12, various interleukins such as IL-1, IL-6, and IL-10 13, subunit p40 common to IL-12 and IL-23, a subunit of the IL-23 receptor 14, IL-13 and IL-15 15, SNF313 (a gene implicated in protein ubiquitination) 16, and transforming growth factor, 17 as well as the promoter of the gene encoding interferon 18.
The TNF-α gene is located on the short arm of chromosome 6, very close to the major histocompatibility complex B. This region is highly polymorphic, with up to 44 polymorphisms reported 19.
In psoriasis, the most widely studied polymorphisms are substitution of guanine by adenine at positions 238 and 308 (−238G→A, −308G→A), which have been associated with the severity of psoriasis 20, substitution of cytosine by thymine at position 857 (−857C→T), which has been associated with a greater risk of psoriatic arthritis, and substitution of thymine by cytosine at position 1031(−1031T→C) 21.
This work aimed to investigate the genetic polymorphisms in the TNF-α308 promoter in a sample of Egyptian patients and to establish possible allelic and genetic differences between them and healthy controls and its relation to TNF-α in serum.
Patients and methods
Thirty patients with psoriasis vulgaris attending the Dermatology Outpatient Clinic of Menoufiya University Hospital were recruited for this case–control study during the period from October 2012 to July 2013. The study was approved by the Committee of Human Rights in Research of Menoufiya University. Twenty age-matched and sex-matched healthy volunteers were included as controls. An informed consent was signed by all participants.
Patients of both sexes with psoriasis vulgaris were included in this study.
Patients receiving any topical or systemic treatment for psoriasis in the last 2 weeks before blood sampling, and participants (patients or controls) with a personal or a family history, or association of any autoimmune diseases were excluded from the study.
Each patient in the study was subjected to the following:
- Full clinical assessment.
- Assessment of the psoriasis area and severity index (PASI) scores to determine the degree of severity of psoriasis 22.
- Blood sampling for:
- detection of TNF-α serum level;
- detection of the TNF-α gene polymorphism.
Control individuals were subjected to blood sampling for the detection of TNF-α serum level and the TNF-α gene polymorphism.
A volume of 10 ml of venous blood was withdrawn from the cubital vein of all participants under aseptic conditions. A volume of 5 ml was transferred slowly into a plain tube for determination of the serum TNF-α level. The other 5 ml was transferred slowly into a vaccumated EDTA tube for isolation of white blood cells for genotyping.
Detection of serum TNF-α level
Serum levels of TNF-α were measured using an enzyme linked immunosorbent assay following the protocol provided by the manufacturer (Anogen, Mississauga, Ontario, Canada).
Detection of the TNF-α gene polymorphism
Peripheral blood mononuclear cells isolation was performed using a lymphofot solution (BiotestAG, Dreieich, Germany). DNA extraction was performed using QIA Amp (R) DNA Blood Mini Kits (Qiagen, Hilden, Germany). Genotyping of the TNF-α gene polymorphism was carried out using allele-specific polymerase chain reaction to detect the G→A transition polymorphism of position −308 of the TNF-α gene. Three primers were used for the allele-specific polymerase chain reaction: the forward primer (F: CTGCATCCCCGTCTTTCTCC) was used in combination with either R1 primer (ATAGGTTTTGAGGGGCATCG) for the G allele or R2 primer (ATAGGTTTTGAGGGGCATCA) for the A allele. The amplification reaction was performed in 25 μl final volume(10 μl DNA template+15 μl master mix containing 2.5 μl,10× PCR, 2 μl MgCl 25 mmol/l, 0.5 μl dNTPs, 10 mmol/l, and 1.0 μl of each forward and reverse primer 20 mmol/l and 7.5 μl distilled water). PCR amplification was performed using Perkin Elmar thermal Cycler 2400 (Foster, California, USA). PCR conditions were as follows: first initial denaturation at 96°C for 5 min, 35 cycles, 94°C for 30 s (denaturation), 58°C for 30 s (annealing), 72°C for 1 min (extraction), and then a final extraction step at 72°C for 5 min.
The computer software package SPSS 15.0 (SPSS Inc., Chicago, Illinois, USA) was used in the analysis. For continuous quantitative data, mean/median (as a measure of central tendency), SD, minimum, and maximum (as measures of variability) were presented. Frequency and percentages were presented for categorical data. Independent t-test, Mann–Whitney test, and Kruskal–Wallis test were used to estimate differences in quantitative variables. The χ 2-test was used to estimate differences in qualitative variables. Odds ratio with a 95% confidence interval was used to compare cases with controls for genotype or allele frequencies. P-value less than 0.05 was considered significant.
Patient group comprised 13 male (43.3%) and 17 female (56.7%) patients; their ages ranged between 12 and 63 years, mean 35.93±14.34 years. In terms of onset of psoriasis, 15 patients had early-onset psoriasis (onset before 30 years of age) and the other 15 patients had late-onset psoriasis (onset after 30 years of age). The duration of illness ranged between 1 and 21 years, mean 7.93±6.30 years. The PASI score ranged between 3.1 and 29.1, with a mean of 10.4±5.7. Twenty sex-matched and age-matched healthy volunteers were chosen as control participants; there were 12 male and eight female patients, age range 13–63 years, mean 36.35±16.11 years.
TNF-α serum levels
Compared with the controls, TNF-α serum levels in psoriatic patients were highly significantly elevated (3.98±0.65) and (6.85±1.12) pg/ml, respectively (P=0.001) (Fig. 1). In terms of the relation between TNF-α serum levels and the clinical parameters studied (Tables 1 and 2), we observed a positive significant correlation between TNF-α serum levels and disease severity (PASI score) (P<0.03). Patients with a positive family history were found to have significantly high TNF-α serum levels than those with a negative family history (P<0.001).
TNF-α-308 gene polymorphism
The distributions of genotype and allele frequencies of analyzed DNA are shown in (Figs 2–4). Frequencies of the TNF-α gene in psoriasis showed a significant elevation in the polymorphisms of the TNF-α promoter −308 (GG) and a decrease in the (AA) genotype compared with the controls (40 vs. 10% and 16.7 vs. 35%), respectively (P=0.05) (Fig. 3). We observed a significantly higher frequency of the G allele and a lower frequency of the A allele in cases than in the controls (61.7 vs. 40% and 38.3 vs. 59.5%), respectively (odds ratio=2.37, 95% confidence interval=1.06–5.3, P=0.03; Fig 4).
On plotting TNF-α genotypes against the clinical parameters studied, the result showed that the TNF-α-308 (GG) genotype was associated with the severity of psoriasis (high values of the PASI score), and in patients with a positive family history (P=0.02, for both), no other significant association could be detected for other clinical parameters (all P’s>0.05) (Table 3).
On comparing TNF-α serum levels of different TNF-α-308 genotypes (AA, GA, and GG), the TNF-α-308GG genotype was found to be associated with high serum TNF-α levels (P<0.001) (Table 3).
Analysis of the results of TNF-α-308 allele frequencies in relation to the clinical parameters studied showed that the TNF-α-308 G allele frequency was elevated compared with the TNF-α-308 A allele in patients with a positive family history (P=0.005), early onset of psoriasis (P=0.03), and those with nail changes (P=0.04) (Table 4).
TNF-α is a proinflammatory cytokine that has been implicated as a key cytokine in the pathogenesis of psoriasis 23.
In the current study, the serum TNF-α level in psoriatic patients was significantly higher than that in the controls. Our results are in agreement with those of many authors 24,25. In addition, Nakamura et al. 26 recorded a higher mean TNF-α serum level in patients with pustular psoriasis and palmoplantar pustulosis than controls. Moreover, TNF-α and its receptors play important roles in the development and persistence of psoriatic plaques 27. In contrast to our finding is that of Tigalonova et al. 28, who reported that the serum level of TNF-α did not differ between the psoriasis and the control groups.
The present study found a significant positive correlation between serum levels of TNF-α and PASI score (P=0.03); this result is in agreement with that of some investigators 26,29 and in disagreement with that of others 25.
The search for genetic factors in psoriasis has led to the identification of several psoriasis susceptibility loci (PSORS) 30. Of particular interest is the study of polymorphisms in the TNF-α gene. There are several polymorphisms in the promoter region of the TNF-α gene. The most common polymorphisms are two G to A transitions in the promoter at positions −238 and −308. These polymorphisms may affect cytokine production 31.
In this study, we observed a higher frequency of the TNF-α-308GG genotype in psoriatic patients than in the controls; this observation was reported among 375 German psoriatic patients 21 and in 46 cases with psoriasis recruited from the Nile Delta of Egypt 32. Also, Gallo et al. 33 observed a high frequency of the TNF-α-308GG genotype in psoriatic patients than in the controls (83.3 vs.78.9%) in their Spanish study, although the difference was not statistically significant.
However, some studies have found no significant associations between TNF-α-308 promoter polymorphisms and psoriasis 12,21,34,35. The different results can be attributed to differences in population or race, and insufficient sample size both for patients and for controls.
The highest PASI scores were found for the TNF-α-308GG genotype in our psoriatic patients. Also, Settin et al. 32 reported a high frequency of the TNF-α-308GG genotype in psoriatic cases with moderate severity, and two published studies 33,36 concluded that the TNF-α-238GG genotype is more frequent among individuals with more severe forms of psoriasis.
The TNF-α gene polymorphism is associated with increased TNF-α production in carriage of the TNF-α-857T genotype. This has been reported in many diseases such as psoriatic arthritis 21, acute uveitis 15, and gastric B cell lymphoma 16. Possibly, the association observed between TNF-α serum level and the TNF-α-308GG genotype in our psoriatic patients might also be explained by an increased TNF production.
Settin et al. 32, in their analysis of cases-subgroups, reported no significant difference in the TNF-α-308GG genotype related to age and sex of their studied cases, similar to of our results. Moreover, we did not find any significant difference among the TNF-α-308 genotypes (GG, AG, and AA) related to other clinical parameters studied (onset of disease, nail changes, and koebnerization), except for those patients with a positive family history of psoriasis.
With respect to allele counts, our investigation found a higher number of G alleles and lower number of A alleles in patients than in controls (P=0.03). Similar studies have also reported a decreased frequency of the TNF-α −308A allele 37, with a trend toward increased frequency of the G allele in early-onset psoriasis 38, similar to our result. However, other investigators reported no difference in the distribution of TNF-α alleles from control participants 13. Furthermore, in a meta-analysis published by Li et al. 39 on polymorphisms of TNF-α at positions −238 and −308, the authors concluded that the presence of a wild-type G allele might play a protective role in psoriasis.
On the basis of our results, we can conclude that polymorphisms of TNF-α at position −308 in psoriasis vulgaris could play an active role in the pathophysiology of this disease and might contribute toward its predisposition. Also, our findings support the concept that promoter polymorphisms of the TNF-α gene might modify certain aspects of psoriasis, such as its severity, age of onset, and associated nail changes.
To provide more conclusive evidence of the role of the TNF-α gene in psoriasis, a large number of patient and family studies are needed. Further investigation of polymorphisms of the TNF gene region and other proteins implicated in the pathophysiology of psoriasis will be helpful in elucidating its pathogenesis and identification of other possible therapeutic targets.
Conflicts of interest
There are no conflicts of interest.
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