Analysis of Association between Asthma and IL23-Receptor R381Q Gene Variant

BACKGROUND

Asthma is a common chronic inflammatory disease of the airways. Th17 andTh2 derived cytokines (such as IL-17, IL-4, IL-5, IL-13) are involved in pathophysiology of asthma. IL-23 is a pro-inflammatory cytokine mainly produced by dendritic cells and monocytes and active macrophages and is need for differentiation of naive CD4⁺ T cells into Th17 cells. IL-23 identified as a major factor in promoting inflammatory diseases such as inflammatory bowel disease, rheumatoid arthritis, psoriasis and asthma.

Gene has a polymorphism that results in arginine (R) to glutamine (Q) substitution in position of 381 (R381Q).

It is shown that, this amino acid substitution in IL-23R which makes R381Q variant, reduces Th17 cell differentiation by limiting IL-23–induced IL-17A production. Genome-wide association studies(GWAS) have shown that the IL23R R381Q gene variant protects against psoriasis, Crohn’s disease and ankylosing spondylitis.

The aim of this study was to determine whether IL23R R381Q gene variant increases or decreases susceptibility to asthma.

METHOD:

PCR-RFLP applied to determine R381Q variant of IL-23R in all subjects. PCR products were chosen at random for sequencing.

RESULT:

Significantly differences in the frequency of the R381Q SNP were identified between the asthma patients and healthy controls in Isfahan population (P value= 0.004).

CONCLUSION:

The present study suggested that R381Q polymorphism in IL-23 receptor may be a predisposing or protective allele for asthma in Isfahan population.

Key words

asthma , TH2, TH17, polymorphism, single nucleotide polymorphism, IL-23R R381Q

Introduction

It is well proved that dominant pathological features of asthma including extreme eosinophilic infiltration, airway remodeling, and airway inflamation are mediated by IL-4, IL-5, and IL-13 (Th2 cytokines) from Th2 and inflammatory mediators that secreted by activated mast cells (10-12), Other studies suggested that IL-17A and IL-17F also infiltrate neutrophil in to the airway and bronchus of animal models of asthma (13,14). IL-17A expression in the airways of asthmatic patients is correlated with the severity of asthma (15-17). Also IL-17A stimulate involved cells in asthma, such as epithelial cells, fibroblasts, and smooth muscles, and induce the secretion of a diversity of cytokines and chemokines, which are significant for neutrophil recruitment (18).Therefore IL-23/IL-23R signaling plays a crucial role in differentiation and maintenance of Th17 cells [28].

IL-23 has been recognized as a new IL-12 family cytokine which is consist of p19 and a p40 subunits (7). In spite of a structural resemblance between IL-12 and IL-23, it is obvious that IL-23, rather than IL-12, plays pathogenic roles in chronic inflammatory diseases such as inflammatory bowel diseases, arthritis, and psoriasis, (3,4). In other study has showed that IL-23 is vital for the maintenance of Th17 cells (8). In addition, IL-23 is required for effector function of Th17 cells (9). These results indicate that IL-23-Th17 axis plays a very important role in the development of inflammatory diseases. Other studies have shown that IL-23 can cause eosinophilic inflammation in the airways.

IL-23 signals through its heterodimeric IL-23R complex [6]. This complex composed of the IL-23R subunit and the IL-12Rb1 subunit . Binding of IL-23 to IL-23R complex leads to phosphorylation and dimerization of STAT3, and consequently expression of IL-23-dependent gene.

The IL-23R protein is comprised of three domain that includ an extracellular domain, a single transmembrane domain, and a cytoplasmic domain.(3) The Arg381Gln variant identified in the GWAS for a subunit of the IL-23R located on chromosome 1p31.

Polymorphisms in the IL-23R chain may effect on IL-23 responses. The frequency of R381Q polymorphism occurs at a range of 0-17 % depending on the population. The R381Q allele confers protection against IBD (19), psoriasis (20), ankylosing spondylitis (21), and graft versus host disease after bone marrow transplantation (22).

The aim of this study was to determine whether IL23R R381Q gene variant increases or decreases susceptibility to asthma.

Considering that these variants in asthma not studied so far, so we will check it. Maybe this survey can help to better understanding the immunopathogenesis process of asthma.

Materials and Methods

Study population

The subjects studied were asthmatic patients attending the Allergy Clinic of the Amin hospital of Isfahan. A full verbal and written explanation of the study was given to all patients interviewed and they gave informed consent and participated in this study. Informed consent for subjects younger than 18 years old was

given by their parents.

Each patient was questioned regarding asthmatic symptoms and underwent a physical examination by physician. Asthma was diagnosed in subjects according to the criteria of the Global Initiative for Asthma (GINA).

This case-control study was done with, 209 patients ,and 200 sex and age matched controls . These controls did not have any of the symptoms or personal or family history of allergic and respiratory disease in their previous history or past physical check-up . Both of these groups, were living in the same area of (the) Isfahan province in Iran. The average ages of the patients with asthma and healthy controls were 42.84 years and 43.2 years, respectively

This study was approved by the Committee of Ethics, the medical science of Isfahan.

PCR-RFLP

In this study we were used to Genomic DNA Isolation Kit (Genet Bio, south korea) that is designed for the rapid preparation of genomic DNA from up to 100µl of a blood sample .

For performing PCR, the primers were designed by Primer3.the sequence of the primer pairs included 5′- CTTTTCTGGCAGGGTCATTTTG-3′(forward primer(22bp) ) and 5′-AAGTTGTTTCCTGGGGTAGTTGTG-3′(reverse primer(24bp) ).

The PCR conditions were optimized for R381Q SNP as follows: 95°C for 5 min; 30 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min. PCR amplification was carried out in a programmable PCR in an Eppendorf thermal cycler (Eppendorf, Germany).

The PCR products were digested by the Hpy188I restriction enzyme (New England Biolab). The RFLP was performed in a total volume of 20 µl containing 10µl PCR product, 7.8µl distillated water, 2µl related buffer and 0.2 Restriction enzyme . The RFLP conditions were optimized in 37°c for 3 hours. The restriction-digested fragments were separated on 2 % agarose gels and stained with DNA green viewer and visualized under UV illumination.ELISATotal IgE was determined by enzyme-linked immunosorbent assay (ELISA) using Euroimmun IgE kit in serum. (made in Germany)

Peripheral blood smear samples checked for eosinophil count using light microscopy.[CS1]

Statistical Analyses

The SPSS 20 software package (SPSS company, Chicago, IL, USA) was used to carry out statistical analyses.The chi square test was first applied to compare the frequency distribution of gender, and smoking status between cases and controls. In addition, this test was used to compare the genotype distributions, allele frequencies between two groupe of patients and controls. Association between this polymorphism and asthma was expressed(declared) as odds ratios (OR) estimates with 95% confidence intervals (95% CI). Logistic regression analysis was used to predict(prophesy) the relation of the IL23R rs11209026 G>A polymorphism with susceptibility to asthma. Furthermore age, eosinophils count and total serum IgE levels between two groups were compared by means of Independent T-Test and also to eliminate confounding factors between the two groups, we used analysis of covariance (ANCOVA) method. Pvalue <0.05 was considered significant in all of these tests. In addition to having a normal distribution for the analysis IgE levels were change to log10 values.

RESULTS

In the present study, we analyzed IL23R rs11209026 G>A polymorphism in 209 patients with asthma and 200 controls from normal population. Selected characteristics of two groups and the relationship with asthma are shown in Table1. According to this table the matching found on two variables, age and gender, was sufficient and there were no major variances in mean the two variables distribution between patients and controls. Compared with the controls, the cases had higher eosinophils count and total serum IgE levels (P value = 0.000). In contrast smoking status was not statistically different between two groups (Pvalue =0.726).

Table 1. frequency distributions of selected variables and characteristics of the study population in case and control

Table 2. Genotype and allele frequencies of IL23R rs11209026 G>A polymorphism

The frequency of GA and G/G genotypes in patients was 3.8 % and 96.2% respectively and the frequency of A and G alleles was 1.9%and 98.1%, respectively. The multivariate logistic regression analysis was applied to investigate the association between the IL23R rs11209026 G>A polymorphism genotypes and asthma. As shown in Table 3, there is an association between G/A genotype and asthma (adjusted OR = 0.274, 95%, CI = 0.114-0.656, P value =0.004 , crude OR = 0.266, 95%, CI = 0.118-0.604, P value =0.001 ) .

Table 3. Adjusted Odds Ratios with 95% Confi dence Interval (CI) in IL23R R381Q genotypes with adjustment for age, gender,smoke statuse, eosinophil count and total IgE.

DISCUSSION

Interelukin-23 (IL-23) is an IL12-related cytokine, which plays an important role in the regulation of cell-mediated immune responses (Kastelein et al. 2007). IL-23 stimulates the differentiation and proliferation of Th17 cells that are critical mediators of inflammation in several mouse models of autoimmune disease and inflammatory disease (Kikly et al. 2006[e2]).

In this study, for the first time, we analyzed association between asthma and IL23R R381Q gene variant.

To date, there have been no studies evaluating the frequency of the IL-23R Arg381Gln polymorphism in world. We undertook this study to determine the presence and prevalence of this gene mutation in asthmatic patients.

The study of F.Burda et al on gastric cancer in Romanian population showed that no significant association between IL23R R381Q polymorphism and gastric cancer susceptibility(P=0.97, OR=0.95, 95%CI=0.41-2.27[e3]).

Several studies have done for association between IL23R R381Q and various inflammatory diseases such as inflammatory bowel diseas(IBD),crohn^,s disease(CD), ulcerative colitis(UC), psoriasis, multiple sclerosis(MS) and ankylosing spondylitis(AS) in different population. Their findings were concordant. In this study, we analyzed association between asthma and IL23R R381Q gene variant. Significant association was found between the frequency of IL23R R381Q genotype and asthma.

Judy et al in 2007 by study on population of Jewish and non-Jewish shown that this variant to protect against progress in CD in both non-Jewish and Jewish case-control cohorts. The frequency of A allele in the non-Jewish CD patients was 1.9% and control 7[e4]%. In our study, we found a similar frequency of A allele in asthmatic patients(1.9%) with CD patients and 7% for controls.

P.L. Lakatos et al in 2008 showed that the frequency of IL23R R381Q in UC patients was about 3% and in controls 6%.although, allele frequency in patients was lower than control, but the difference did not reach significance(OR=0.55, 95% CI= 0.22-1.34). [5,8–11[e5]]

Paola Di Megilo et al showed that IL23R R381Q polymorphism protect against inflammatory diseases[e6].

All previous studies have supported protective role of the R381Q polymorphism in IL23R gene against inflammatory diseases. Of course, the allele frequencies of this polymorphism in different disease and population were divers. This diversity may be due to the difference in sample size, ethnic and geographic diversity.

CONCLUSION:

We confirmed that IL2 R R381Q associated with asthma in this population. Further studies are needed to confirm the protective role of this polymorphism in asthma.

[CS1]

[e2]Sequence variant in the gene

[e3]THE IL23R R381Q GENE POLYMORPHISM

[e4]Il23r and ibd

[e5]IL23R R381Q AND CD PATIENTS

[e6]Il23r r381q protect against