Abstract
Background
Allergic rhinitis (AR) is an inflammatory disorder of the upper airway. T-helper (Th)2 cytokines seems to have major roles behind the scene of unpleasant symptoms resulted from AR. Expression of interleukin (IL)-4 and its receptor could be affected by single nucleotide polymorphisms (SNPs). This study assessed the effect of 4 genetic variants within genes of IL-4 and IL-4R in AR.
Methods
Allele frequencies of one IL-4R variant ( rs1801275 ) and three SNPs of IL-4 ( rs2243248 , rs2243250 , and rs2070874 ) were investigated in 98 patients with AR, compared to a group of controls, using PCR sequence-specific-primers (PCR-SSP) method.
Results
Homozygosity for the C allele of rs2243250 in IL-4 was significantly overrepresented in the patient group. CC genotype in rs2070874 significantly was correlated with AR. GG/CC/CC and TT/TT/TT ( rs2243248 , rs2243250 , and rs2070874 ) haplotypes in the IL-4 gene had a significant negative correlation with AR.
Conclusion
SNPs in IL-4 are associated with AR and could change the clinical picture of the disease in patients.
1
Introduction
Allergic rhinitis (AR) is a common inflammatory disorder of the upper airway , which has a major impact on the quality of life of the patients by unpleasant symptoms such as sneezing, nasal congestion, nasal pruritus, rhinorrhea and obstruction of the nasal passages. It has also a systemic effect on peripheral blood, bone marrow and lungs . AR is a known risk factor of comorbid conditions such as asthma, rhinosinusitis, nasal polyposis, and sleep disorders .
Although pathogenesis of AR has been extensively studied, it has remained largely unknown yet. Imbalance between T-helper (Th)1/Th2 immune response results in allergen-specific immunoglobulin production and binding of immunoglobulin (Ig)E to mast cells . Further passage of allergens through the upper airway and their interaction with the IgE results in release of several mediators, chemokines and cytokines from mast cells and consequently accumulation of eosinophil T cells, mast cells and basophils in the nasal mucosa . Furthermore, they release more mediators responsible for the late phase of allergic reactions and systemic component of AR . Behind this scene, genetic factors are major players affecting development, severity and treatment of AR . Several susceptibility loci were identified as a result of extensive studies. A better insight on underlying genetic component of AR provides new ways to develop novel therapies.
A variety of cytokines are released by eosinophils, T cells, mast cells and basophils during the course of the disease; they are emphasized to play a major role in the pathogenesis of AR . Cytokines released by Th2 cells such as interleukin (IL)-4 and IL-5 are predominantly involved in IgE-mediated allergic inflammation . IL-4, a B cell growth factor, plays a central role in the development of allergic reaction by advancing immunoglobulin isotype class switching from IgM to IgE and modulating the differentiation of T cells to Th2 cells . It also increases antigen presenting capacity of B cells by inducing expression of major histocompatibility complex (MHC) class II molecules, CD80, CD86, CD40 and surface IgM . IL-4 can increase the adhesion of inflammatory cell to the endothelial cells by over expression of vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells . IL-4 signals through two types of receptors to drive its effects. Both of these receptors recruit IL-4 receptor α chain (IL-4Rα), a 140-kDa binding chain. IL-4Rα is also a component of IL-13 receptor complex . Expression of these molecules can be altered by several single nucleotide polymorphisms (SNPs), identified on their genes, 5q31 and 16p12, on which genes of IL-4 and IL-4R are located, have been associated with allergic reactions .
Associations of IL-4 SNPs in different diseases have been investigated so far . The aim of this study was to evaluate the effect of functional SNPs in a group of patients with AR. In this study, we aimed to investigate the effect of 4 SNPs found in the genes responsible for encoding IL-4 and IL-4R in patients with AR.
2
Patients and methods
2.1
Subjects
Ninety eight patients with AR who referred to the Children’s Medical Center, the Pediatrics Center of Excellence in Tehran, Iran, were recruited in this study as the patient group. The diagnosis of AR was established according to the standard criteria by the Allergic Rhinitis and its Impact on Asthma (ARIA) document. We also used this document to classify the severity of the disease in patients . Interview and medical files were means of collection of demographic and clinical data. Total serum IgE concentration was determined using enzyme-linked immunosorbent assay (ELISA) method (Life Diagnostics, Inc., PA). We determined allergen sensitivity of the patients by a commercially available skin prick test (SPT) kit, including allergens such as cockroach, tree pollen, grass pollen, weeds, mold, dust mite and animal dander (Stallergen, Paris, France). Results of the skin prick tests were evaluated according to European Academy of Allergy and Clinical Immunology (EAACI) criteria . One hundred and thirty nine healthy volunteers from the same ethnicity of the patients were recruited as the control group . Informed consents were obtained from all the participants. All the principles of the Declaration of Helsinki were applied in the study and the study was approved by the Ethical Committee of Tehran University of Medical Sciences.
2.2
Genotyping
Peripheral blood was collected from all the participants in EDTA-treated tubes. “Salting out” method was used to isolate genomic DNA from peripheral blood. Allele frequencies for one IL-4R variant (a replacement of adenosine with guanine at the site of + 1902 of gene which is referred to as rs1801275 ) and three SNPs in IL-4 (replacement of thymine with guanine at the site of − 1098 referred to as rs2243248 and replacement of cytosine with thymine at the site of − 590 and − 33 referred to respectively as rs2243250 and rs2070874 ) were determined using PCR sequence-specific-primers (PCR-SSP) methods by Heidelberg cytokine gene polymorphism SSP kit (Heidelberg University, Heidelberg, Germany). PCR was performed in a final volume of 519.9 μL containing 329 μL of H2O, 50 μL of genomic DNA, 2.9 μL Taq DNA Polymerase, 138 μL of Master Mix and each primer using a Techne Felexigene thermal cycler (Rosche, Cambridge, UK). Amplification conditions included an initial denaturation at 94°C for 2 min; denaturation 94°C for 10 s; annealing + extension at 65°C, for 1 min (ten cycles); denaturation at 94°C for 10 s; annealing at 61°C for 50 s; and extension at 72°C for 30 s (20 cycles). A part of the β-globin gene or C-reactive protein (CRP) gene was included in each of the primer mixes as positive controls. To visualize PCR products, 2% agarose gel electrophoresis was done on an ultraviolet transilluminator.
2.3
Statistics
All the statistical analyses were performed using SPPS version 18 software (SPSS Inc., Chicago, Illinois, USA). p value of less than 0.05 was considered statistically significant. To compare numeric variables such as IgE levels, eosinophil count, and eosinophil percentage within genotypic subgroups we used one way ANOVA test. Odds ratios (OR) with 95% confidence intervals (CI) for genotypes were calculated by comparing the frequencies among patients and controls and chi-square test and Fisher’s test were used to determine the p value.
2
Patients and methods
2.1
Subjects
Ninety eight patients with AR who referred to the Children’s Medical Center, the Pediatrics Center of Excellence in Tehran, Iran, were recruited in this study as the patient group. The diagnosis of AR was established according to the standard criteria by the Allergic Rhinitis and its Impact on Asthma (ARIA) document. We also used this document to classify the severity of the disease in patients . Interview and medical files were means of collection of demographic and clinical data. Total serum IgE concentration was determined using enzyme-linked immunosorbent assay (ELISA) method (Life Diagnostics, Inc., PA). We determined allergen sensitivity of the patients by a commercially available skin prick test (SPT) kit, including allergens such as cockroach, tree pollen, grass pollen, weeds, mold, dust mite and animal dander (Stallergen, Paris, France). Results of the skin prick tests were evaluated according to European Academy of Allergy and Clinical Immunology (EAACI) criteria . One hundred and thirty nine healthy volunteers from the same ethnicity of the patients were recruited as the control group . Informed consents were obtained from all the participants. All the principles of the Declaration of Helsinki were applied in the study and the study was approved by the Ethical Committee of Tehran University of Medical Sciences.
2.2
Genotyping
Peripheral blood was collected from all the participants in EDTA-treated tubes. “Salting out” method was used to isolate genomic DNA from peripheral blood. Allele frequencies for one IL-4R variant (a replacement of adenosine with guanine at the site of + 1902 of gene which is referred to as rs1801275 ) and three SNPs in IL-4 (replacement of thymine with guanine at the site of − 1098 referred to as rs2243248 and replacement of cytosine with thymine at the site of − 590 and − 33 referred to respectively as rs2243250 and rs2070874 ) were determined using PCR sequence-specific-primers (PCR-SSP) methods by Heidelberg cytokine gene polymorphism SSP kit (Heidelberg University, Heidelberg, Germany). PCR was performed in a final volume of 519.9 μL containing 329 μL of H2O, 50 μL of genomic DNA, 2.9 μL Taq DNA Polymerase, 138 μL of Master Mix and each primer using a Techne Felexigene thermal cycler (Rosche, Cambridge, UK). Amplification conditions included an initial denaturation at 94°C for 2 min; denaturation 94°C for 10 s; annealing + extension at 65°C, for 1 min (ten cycles); denaturation at 94°C for 10 s; annealing at 61°C for 50 s; and extension at 72°C for 30 s (20 cycles). A part of the β-globin gene or C-reactive protein (CRP) gene was included in each of the primer mixes as positive controls. To visualize PCR products, 2% agarose gel electrophoresis was done on an ultraviolet transilluminator.
2.3
Statistics
All the statistical analyses were performed using SPPS version 18 software (SPSS Inc., Chicago, Illinois, USA). p value of less than 0.05 was considered statistically significant. To compare numeric variables such as IgE levels, eosinophil count, and eosinophil percentage within genotypic subgroups we used one way ANOVA test. Odds ratios (OR) with 95% confidence intervals (CI) for genotypes were calculated by comparing the frequencies among patients and controls and chi-square test and Fisher’s test were used to determine the p value.
3
Results
3.1
Frequencies of gene polymorphisms and haplotypes
SNP in IL-4R was not associated with the risk of AR. However, homozygosity for the C allele (CC genotype) of rs2243250 in IL-4 was associated with 7.17 fold (95%CI: 3.16-16.6) increase in risk of having AR. CC genotype in rs2070874 increased the risk of AR to a 3.94 fold (95%CI: 2.15-7.27) ( Table 1 ). Frequencies for possible haplotypes in patients group and healthy controls are also shown in Table 2 .
| Gene | SNPs | Allele/ Genotype | Controls N(%) | Patients N(%) | Odds Ratio (95% CI) | p value |
|---|---|---|---|---|---|---|
| IL-4R | rs1801275 | A | 242(87.7) | 165(84.18) | 0.75(0.43-1.31) | 0.341 |
| G | 34(12.3) | 31(15.82) | 1.34(0.77-2.33) | 0.341 | ||
| AA | 106(76.8) | 70(71.4) | 0.75(0.4-1.42) | 0.432 | ||
| GA | 30(21.7) | 25(25.51) | 1.23(0.64-2.37) | 0.603 | ||
| GG | 2(1.5) | 3(3.1) | 2.15(0.29-18.75) | 0.651 | ||
| IL-4 | rs2243248 | G | 84(30.2) | 54(27.55) | 0.88(0.57-1.34) | 0.598 |
| T | 194(69.8) | 142(72.45) | 1.14(0.74-1.74) | 0.598 | ||
| GG | 1(0.7) | 2(2.6) | 3.73(0.26-105.71) | 0.285 | ||
| GT | 82(59) | 38(50) | 0.7(0.38-1.27) | 0.260 | ||
| TT | 56(40.3) | 36(47.4) | 1.33(0.73-2.44) | 0.390 | ||
| rs2243250 | C | 149(53.6) | 133(67.86) | 1.58(1.07-2.32) | 0.02 | |
| T | 129(46.4) | 73(32.14) | 0.63(0.43-0.93) | 0.02 | ||
| CC | 10(7.2) | 35(35.7) | 7.17(3.16-16.6) | < 0.001 | ||
| TC | 129(92.8) | 63(64.3) | 0.14(0.06-0.32) | < 0.001 | ||
| TT | 0(0) | 0(0) | – | – | ||
| rs2070874 | C | 200(71.9) | 166(84.69) | 2.16(1.32-3.55) | 0.001 | |
| T | 78(28.1) | 30(15.31) | 0.46(0.28-0.76) | 0.001 | ||
| CC | 61(43.9) | 74(75.5) | 3.94(2.15-7.27) | < 0.001 | ||
| TC | 78(56.1) | 19(19.4) | 0.19(0.1-0.36) | < 0.001 | ||
| TT | 0(0) | 5(5.1) | 2.49(2.13-2.92) | 0.011 |
| Gene | SNPs | Haplotypes | Controls (n=139) N(%) | Patients (n=98) N(%) | Odds Ratio (95% CI) | p value |
|---|---|---|---|---|---|---|
| IL-4 | rs2243248, rs2243250, rs2070874 | TT/TT/CC | 51(18.3) | 34(17.35) | 0.93(0.56-1.55) | 0.874 |
| GG/CC/CC | 83(30) | 35(17.86) | 0.51(0.32-0.82) | 0.004 | ||
| TT/TT/TT | 76(27.3) | 13(6.63) | 0.19(0.1-0.36) | < 0.001 | ||
| TT/CC/CC | 65(23.4) | 86(43.88) | 2.56(1.69-3.88) | < 0.001 | ||
| TT/CC/TT | 2(0.7) | 8(4.08) | 5.87(1.14-40.47) | 0.019 | ||
| GG/TT/TT | 1(0.3) | 5(2.55) | 7.25(0.82-165.33) | 0.046 | ||
| GG/CC/TT | 0(0) | 1(0.51) | 2.43(2.18-2.7) | 0.413 | ||
| GG/TT/CC | 0(0) | 14(7.14) | 2.53(2.26-2.83) | < 0.001 |
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