Association of cytokine gene polymorphisms with psoriasis in cases from the nile delta of egypt.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease with an immunogenetic background. This work was planned to check for the association of polymorphisms related to cytokine genes TNF-α(-308) (G/A), IL-10(-1082) (G/A), IL-6(-174) (G/C), and IL-1Ra (VNTR) with psoriasis in cases from Egypt.
MATERIALS AND METHODS: This work included 46 cases with psoriasis recruited from the Dermatology Departments, University Hospitals, Nile Delta region of Egypt. They included 14 males and 32 females with an age mean ± SD of 46.68 ± 12.16 years and range of 15-70 years. Their genotypes were compared to 98 healthy controls of matched age and sex from the same locality. Genotyping was done through deoxyribonucleic acid amplification using PCR with sequence specific primers for polymorphic alleles.
RESULTS: Compared to controls, cases showed significant higher frequency of certain genotypes including IL-6(-174) CC (P < 0.001, OR = 6.7), IL-10(-1082) GG (P < 0.05, OR = 5.1), and TNF-α(-308) GG (P < 0.05, OR = 3.7). TNF-α(-308) GG and IL-10(-1082) GG genotypes were higher among cases with plaque subtype of moderate severity. Combined heterozygosity for IL-10 GA, IL-6 GC with TNF GA showed a significant low frequency among studied cases.
CONCLUSION: Genetic polymorphisms related to IL6, IL10, and TNF-α genes showed a particular pattern of association with psoriasis that may have a potential impact on disease counseling and management.

Introduction

Psoriasis is a chronic, T lymphocyte-mediated inflammatory disorder that affects the skin, joints, and tendons in up to 4.8% of worldwide population.[1] The immune system particularly involving Th1 cells play a dominant role in the initiation and maintenance of psoriasis.[2]

Recently, IL-6 is assumed to play an important role in the pathophysiology of psoriasis.[3] A polymorphism in the 5’ flanking region of the IL-6 gene on chromosome seven at position-174 has been reported that corresponds to its serum level and is associated with immune and inflammatory disorders.[4]

IL-10 is an anti-inflammatory and immunosuppressive substance that plays a role in the regulation of immune responses including psoriasis.[5-7] The gene for IL-10 is in chromosome region 1q31-q32. It includes multiple polymorphisms of which the promoter polymorphism -1082 is associated with multiple immune and inflammatory disorders.[8]

The pro-inflammatory cytokine IL-1 is constitutively expressed by keratinocytes in vivo and has been shown to be expressed in psoriatic lesional skin.[9] One of the important polymorphisms at the IL-1 gene cluster is that of IL-IRa-VNTR caused by variable numbers of an 86 bp tandem repeat located in the second intron of the gene. The most common alleles have been termed allele one (A1, four repeats), allele two (A2, two repeats) in addition to other three alleles.[10]

Tumor necrosis factor alpha (TNF-α) is a potent inflammatory cytokine with a potential important role in psoriasis.[11] TNF-α gene is located within the highly polymorphic major histocompatibility complex (MHC) region on chromosome 6p21.3.[12] Many studies have shown that single nucleotide polymorphism (SNP) at position -308 G/A is associated with different inflammatory conditions.[13]

This study aims at testing the association of four types of genetic polymorphisms that are related to cytokine genes of TNF-α at position -308, IL-6 at position -174, IL-10 at position -1082 in addition to IL-1Ra VNTR with psoriasis among affected cases from the Nile Delta region of Egypt.

Materials and Methods

This study included 46 cases with psoriasis recruited from the Department of Dermatology, University Hospitals, Nile Delta Region of Egypt. They included 14 (30.43%) males and 32 (69.56%) females with a mean age ± SD of 46.68 ± 12.16 years and range of 15–70 years. Cases were classified into early-onset psoriasis ≤30 years (17 cases, 37%) and late-onset psoriasis >30 years (29 cases, 63%). These cases were categorized into cases with moderate disease (36 cases, 78.3%) and cases with severe disease (10 cases, 21.7%) according to the National Psoriasis Foundation-Psoriasis Score (NPF-PS).[14] Case genotypes were compared to 98 healthy unrelated volunteers from the same locality having no family history of inflammatory and immune disorders. They included 52 males and 46 females of a mean age of 44.9 ± 6.7 years.

PCR amplification

Deoxyribonucleic acid (DNA) was extracted promptly using DNA extraction and purification kit (Gentra Systems, USA). Three SNPs of promoter sites TNF-α -308 (G/A), IL-10 -1082 (G/A), and IL-6 -174 (G/C) as well as IL-1Ra intron 2-VNTR were analyzed as previously described.[15-18] For SNPs related to TNF-α, IL-6, and IL-10 genes, PCR with sequence-specific primers was used in two reactions employing a common forward and two reverse primers. For IL-1Ra VNTR polymorphism, a single PCR reaction employing a forward, and a reverse primer was used. All primers, Taq polymerase, dNTP, and MgCl2 were purchased from QiaGene (USA). The assay was performed in Techne-Genius thermal-cycler (England). Briefly, 100–500 ng of genomic DNA was added to 25 μL of reaction mixture containing 1 μM of each common/specific primer, 200 μM of each dNTP, and 1 U of Taq DNA polymerase [Table 1]. We were careful to use master mixes for multiple cases and also for different polymorphisms at the same sitting with concomitant use of negative and positive controls to obtain accurate subject genotyping. Positive amplification with specific band size was detected by UV light after electrophoresis on 2% agarose gel stained with ethidium bromide [Figure 1].

Table 1

Primer sequences and PCR conditions of the studied cytokines genes

Figure 1

PCR amplification products for detection of polymorphisms related to cytokine genes showing: Panel 1; IL-10 -1082 (allele G above and A below), Panel 2: IL-6 at position -174 (allele G above and C below), Panel 3: TNF-α at position -308 (allele G above and A below), and Panel 4: IL-1Ra (allele A1 and A2). Positive amplification of one allele indicates homozygosity for that allele while positive amplification of both alleles indicates heterozygosity of both alleles

Statistical analysis

Data were processed and analyzed using the Statistical Package of Social Science (SPSS, version 10.0). The frequency of studied allelic polymorphisms among cases was compared to that of controls and tested for positive association using Fisher's exact test and odds ratio (OR) with a minimum level of significance of P < 0.05.

Results

Compared to controls, all cases have shown a significant higher frequency of IL-6-174 CC (P < 0.001, OR = 6.7, 95% CI = 2.3-18.9), IL-10-1082GG (P < 0.05, OR = 5.1, 95% CI = 1.7-15.6) and TNF-α-308GG (P < 0.05, OR = 3.7, 95% CI = 1.2-11.2) genotypes, with a significant lower frequency of IL-6-174GC, IL-10-1082GG, and TNF-α-308GG genotypes (P < 0.05 for each). In addition, IL-6-174 C allele frequency was significantly higher among all cases compared to controls (P < 0.05, OR = 1.8, 95% CI = 1.1-2.9) with a significant lower frequency of the G allele (P < 0.05). Furthermore, analysis of cases-subgroups showed that TNF-α-308 GG and IL-10-1082 GG genotypes were higher among cases with plaque type of the disease and cases with moderate severity whereas IL-6-174 CC phenotypes were associated with all cases-subgroups [Tables 2–5].

Table 2

Frequency of IL-10-1082 (G/A) genotype and allelic polymorphisms among psoriasis cases compared to controls with their statistical significance

Table 5

Frequency of IL-1Ra VNTR genotype and allelic polymorphisms among psoriasis cases compared to controls with their statistical significance

On the other hand, all cases as well as case-subgroups showed no significant difference in IL-1Ra VNTR genotype and allele frequencies compared to controls (P > 0.05).

Analysis of combined genotypes showed a significantly higher frequency of combined homozygous genotype of IL-10 GG with other homozygous genotypes of TNF-a GG and IL-6 CC. On the other hand, combined heterozygosity for IL-6-174 GC with TNF-α-308 GA showed a significant low frequency among all cases. Additionally, no significant difference was found comparing the frequencies of different studied genotypes and alleles related to cases-subgroups of age, sex, and parental consanguinity (data not shown).

Discussion

Psoriasis is a genetically heterogeneous disorder involved with multiple genetic and environmental interactions. On the basis of its genetic framework, disease severity and locations (e.g., nails, joints, and palmoplantar) may differ between individuals and populations.[19]

Once at the inflamed skin site, the activated T lymphocytes encounter the initiating antigen, and release T-helper type 1 cytokines, which play a central role in the phenotypic expression of psoriasis.[2021]

In this study, certain cytokine gene polymorphisms were presented in a significant higher frequency among Egyptian psoriatic cases compared to controls. These genotypes included IL-6-174 CC, IL-10-1082 GG, and TNF-α-308 GG. The latter two markers were particularly noted to be high among cases of the plaque type and cases with moderate severity of the disease. On the other hand, IL-6-174 GC and TNF-α-308 GA genotypes showed a significant lower frequency among psoriasis cases compared to controls. Regarding the allelic frequencies of studied cytokine genes, only IL-6-174 C allele has shown significant higher frequency among cases in contrast to IL-6-174 G allele that showed a significantly lower frequency compared to controls.

Although the serum levels of studied cytokine were not determined, it is expected that these cases mostly had lower levels of IL-6 and TNF-α with higher levels of IL-10 since the IL-6 C allele and TNF-α G allele were found to be associated with a lower plasma level, whereas the IL-10 G allele is associated with higher plasma levels.[422]

This is probably supported by the previous observation that high levels of IL-10 in skin lesions, synovial fluid, and sera of patients with psoriasis have an influence on disease susceptibility in patients with psoriasis.[2324] In addition, systemically administered TNF-α has also caused an improvement in some cases with psoriasis.[2526]

Other studies have also reported a decreased frequency of TNF-α -308A allele,[27] with a trend for increased frequency of G allele in early-onset psoriasis.[2829] However, other investigators reported no difference in the distribution of TNF-α alleles from control subjects.[3031]

Other studies among English population also showed that polymorphisms in the genes encoding for IL-10 were probably contributing to susceptibility to psoriasis.[32] On the other hand, Kingo et al. demonstrated that the IL-10 haplotype has a role in determining severity and course of plaque type of psoriasis among Estonian population.[33] Other authors demonstrated an increase in frequency of the heterozygous IL-10 GA genotype, and a corresponding lower frequency of both GG and AA genotypes in the subset of patients with late-onset psoriasis. The result is only of borderline statistical significance.[30] Researchers from South Carolina have also reported no significant differences in the genotype distribution with respect to age of onset of psoriasis, gender, or between patients with early-onset psoriasis and controls with a borderline result in comparing patients with late-onset psoriasis with controls from South Carolina. Cases with late-onset psoriasis had a higher frequency of the heterozygous GA genotype and lower frequencies of both GG and AA genotypes.[282934] On the other hand, Chang et al. found no significantly different allelic, genotypic, and haplotypic frequencies in patients with PsA compared to controls among Chinese cases from Taiwan.[35]

An association of IL-1Ra VNTR allele A2 was previously reported with a variety of epithelial-related chronic inflammatory diseases including alopecia areata, lichen sclerosus, systemic lupus erythematosus, ulcerative colitis, and scleroderma areata.[3637] Tarlow et al. have found that the frequency of allele A2 was raised in the cohort with early-onset psoriasis and significantly decreased in the late-onset cohort compared with controls among English population.[510] In contrast, this study showed that there was no significant difference in the frequencies of all genotypes and alleles related to IL-1Ra VNTR polymorphism in Egyptian psoriasis cases compared to controls. These results are supported by other studies among cases in Taiwan and in Newfoundland, Canada stating that the IL-1Ra genetic polymorphisms did not appear to be associated with susceptibility to psoriasis vulgaris and psoriatic arthritis.[3538]

We can come to the conclusion that cytokine gene polymorphisms especially related to IL-10, TNF-α, and IL-six genes can be considered genetic markers for disease susceptibility and/or severity among Egyptian cases with psoriasis with potential impact on family counseling and disease management.

Table 3

Frequency of IL-6-174 (G/C) genotype and allelic polymorphisms among psoriasis cases compared to controls with their statistical significance

Table 4

Frequency of TNF-α-308 (G/A) genotype and allelic polymorphisms among psoriasis cases compared to controls with their statistical significance