|Year : 2020 | Volume
| Issue : 2 | Page : 132-136
Association between the interleukin-5 receptor alpha-subunit G-80A polymorphism and immunological parameters in asthmatic children
Raghdah Maytham Hameed1, Haidar Abdul Amir Najim Abood2, Mohanad Mohsin Ahmed1
1 Department of Medical Microbiology, University of Karbala, Karbala, Iraq
2 Department of Pharmacology, University of Karbala, Karbala, Iraq
|Date of Submission||16-Feb-2020|
|Date of Acceptance||25-Feb-2020|
|Date of Web Publication||17-Jun-2020|
Ms. Raghdah Maytham Hameed
Department of Medical Microbiology, University of Karbala, Karbala
Source of Support: None, Conflict of Interest: None
Introduction: The expression of human interleukin-5 (IL-5) receptor alpha (Rα)-subunit presented on eosinophils, mast cells, and basophils. Hence, a polymorphism in IL-5 Rα may be implicated in the development of asthma and effect on immunological parameters (IL-5 level, absolute eosinophil count, and total serum immunoglobulin E [IgE]) production. Methods: A total of 85 children, including 59 males and 26 females with asthma with ages between 1 and 16 years, attended the Respiratory Clinic at Karbala Pediatric Hospital, with a nonasthmatic children group who had the same age and gender. Restriction fragment length polymerase chain reaction was performed to determine IL-5 Rα G-80A genetic polymorphisms. The total IgE level was measured using the EUROIMMUN IgE ELISA kit and serum IL-5 levels using Elabscience ELISA kit. The absolute eosinophil count was measured by five differential automated hematology analyzers and confirmed by the examination of peripheral blood smear. Results: There were no statistically significant differences in GG, AG, and AA genotypes frequency of IL-5 Rα G-80A between asthmatic patients and controls (P = 0.437, 0.160, and 0.106, respectively). The results showed no statistically significant correlation between immunological parameters (IL-5 level, absolute eosinophil count, and total serum IgE) and IL-5 Rα G-80A genotypes (P = 0.649, 0.06, and 0.552 respectively). Conclusions: Asthma in children is not associated with IL-5 Rα G-80A polymorphism. IL-5 Rα G-80A polymorphism has not any impact on IL-5 levels, eosinophil count, and total serum IgE.
Keywords: Absolute eosinophil count, asthma, interleukin-5, total serum immunoglobulin E
|How to cite this article:|
Hameed RM, Abood HA, Ahmed MM. Association between the interleukin-5 receptor alpha-subunit G-80A polymorphism and immunological parameters in asthmatic children. Biomed Biotechnol Res J 2020;4:132-6
|How to cite this URL:|
Hameed RM, Abood HA, Ahmed MM. Association between the interleukin-5 receptor alpha-subunit G-80A polymorphism and immunological parameters in asthmatic children. Biomed Biotechnol Res J [serial online] 2020 [cited 2021 Sep 28];4:132-6. Available from: https://www.bmbtrj.org/text.asp?2020/4/2/132/286838
| Introduction|| |
Asthma is one of the most common diseases in the world, resulting in a substantial burden of disease. It is highly prevalent chronic inflammatory diseases of the airways, with differences in etiology, immunologic mechanisms, clinical presentation, pathogenesis, comorbidities, prognosis, and response to treatment, arising from not fully understood heterogenic gene–environment interactions, while environmental factors are important in the development of asthma, genetic factors could have a critical role in the expression of the disease, but the genetic background of bronchial asthma is complex, and it is likely that multiple genes contribute to its development both directly and through gene–gene interactions.,,,
Many previous studies have shown an association among single-nucleotide polymorphism (SNP) cytokine gene and cytokine receptor gene and asthma susceptibility in different ethnic populations.,,
The human interleukin-5 (IL-5) receptor is a heterodimer consisting of a unique alpha-subunit and a beta-subunit, expression of human IL-5 receptor alpha (Rα)-subunit presented on eosinophils, mast cells, and basophils, whereas the beta-subunit is more widely expressed. IL-5 Rα-subunit expression may be implicated in the development of allergic diseases. Polymorphisms in IL-5 Rα might be among the genetic risk factors for asthma development, especially in atopic populations., In addition, IL-5 Rα has a regulatory pathway in human eosinophils and their gene has a role for controlling eosinophils in the peripheral blood.,
The gene for the IL-5 Rα subunit is a protein-coding gene located on chromosome 3 in the region 3p24–3p26. The alternative splicing of the IL-5 Rα gene which contains 14 exons can yield several alpha-IL-5 receptor isoforms., The G-80A polymorphisms in IL-5 Rα are located in the promoter region (regulatory gene region).
The previous study mentioned the effect of racial and ethnic specificities in the frequency distributions of the IL-5 and their receptor. This is the first study in Iraq to detect the association between polymorphism of the IL-5 Rα in the populations and among the bronchial asthma patients and investigate the correlation between specific SNPs IL-5 Rα G-80A with IL-5 levels and with eosinophil count in asthmatic children.
| Methods|| |
The study protocol was approved by the Ethical Committee in the Kerbala Health Directorate at October 14, 2018. In addition, verbal approval was obtained from the patients and/or their parents before taking the sample. Health measures and safety were taken when sampling.
The study included a total of 85 consecutive asthmatic children (59 males and 26 females) attending the respiratory clinic at Karbala Pediatric Hospital in the period extending from October 2018 to December 2018. All children had the American Thoracic Society criteria for asthma. Their ages ranged between 1 year and 16 years. The control groups included 85 children (51 males and 34 females) who were randomly selected from the hospitals and the local community and had the same ages and sex of the patients.
Immunological parameters measurement
Serum and whole blood were collected from each participant; sera were used to determine the total serum immunoglobulin E (IgE) levels for all samples by BioTek EL × 800 automated immunoassay analyzer (BioTek, USA) using EUROIMMUN total IgE ELISA kit (LOT NO. A180417AC), whereas serum IL-5 was used for asthmatic children only by BioTek EL × 800 automated immunoassay analyzer (BioTek, USA) using Elabscience ELISA kit (LOT NO. 4CN1IPT94C). Further, whole blood was used for total and differential white blood cells' count that was measured by Sysmex XN-350 five differential automated hematology analyzer (Sysmex, Japan). Differential leukocyte count was displayed in the percentage. Therefore, the absolute eosinophil count was measured by the following equation: absolute eosinophil count = (white blood cell × eosinophils %)/100.
Genotyping of interleukin5 receptor G-80A polymorphism
The nucleotide sequences of the forward and reverse primer used for polymerase chain reaction (PCR) are 5-AAT-GGC-TAT-CTG-GAC-GAG-AG-3 and 5-TTA-GAG-GCG-GTT-CTT-CAC-TC-3, respectively. A PCR mixture included 10 pmol of each specific primer, 12.5 μl of Green Master Mix (Go Taq® Promega), and 3 μl of DNA.
The amplification was reformed by including the reaction mix for 30 cycles in a thermo cycler. Each cycle consisted of denaturation of DNA at 94°C for 45 s followed by annealing at 57°C for 1 min and extension at 72°C for 45 s with initial delay for 5 min at 94°C at the beginning of the first cycle and 5 min delay at 72°C at the end of the last cycle.
Amplification products were digested with appropriate restriction enzymes (Acs I, sibenzyme or Apo I, Bio Lab) at optimal temperatures (60°C) for 15 min. The reaction mixture included 1 μg of PCR solution, 2.5 μl of restriction buffer, and 0.2 μl of the restriction enzyme. The reaction products were separated on 2% agarose gels with ethidium bromide at 70 V for 55 min and visualized in the ultraviolet light. The wild-type allele is not distinguished (206 bp), while the mutant allele is distinguished into two bands at 154 bp and 52 bp.
Data were analyzed using the Statistical Package for the Social Sciences version 21 for Windows (GraphPad Software, San Diego, California, USA). The results were expressed as mean ± standard deviation and the comparisons between the two mean were performed using t-test. Chi-square was used to compare two categorical variables. P < 0.05 was considered to indicate the statistical significance and highly significant if P < 0.001.
Genotypes of IL-5 Rα G-80A were presented as percentage frequencies, and statistically significant differences between their distributions in asthmatic patients and controls were assessed by one-tailed Fisher's exact probability (P). In addition, the odds ratio (OR) were also estimated to define the association between a genotype with the disease.
Allele frequencies of genes were calculated by direct gene counting methods, while a significant departure from Hardy–Weinberg equilibrium (HWE) was estimated using HWE calculator for two alleles, which is available on online Online Encyclopedia for Genetic Epidemiology (OEGE) studies http://www.oege.org/software/hwe-mr-calc.shtml.
| Results|| |
[Table 1] shows the demographic and biochemical profile of asthmatics patients and healthy controls. Frequencies of genotypes and alleles in the study participants are shown in [Table 2]. [Table 3] presents the expected frequencies of genotypes of the IL-5 Rα SNP using HWE. Further, [Table 4] shows the comparison of immunological parameters (serum IL-5 levels, absolute eosinophil count, and total serum IgE) according to the IL-5 Rα G-80A genotypes in asthmatic patients, while [Table 5] shows a comparison between IL-5 Rα G-80A genotypes and asthma severity in asthmatic children.
|Table 2: The statistical evaluations of interleukin-5 receptor alpha single-nucleotide polymorphism between patients and controls|
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|Table 3: Expected frequencies of genotypes of the interleukin-5 receptor alpha single-nucleotide polymorphism using Hardy-Weinberg equilibrium|
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|Table 4: Correlation between immunological parameters (serum interleukin-5, eosinophil count, and total serum immunoglobulin E) and interleukin-5 receptor alpha G-80A (GG and AG genotypes) in asthmatic patients|
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|Table 5: Comparison between Interleukin-5 receptor alpha G-80A genotypes and asthma severity in asthmatic children|
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It was observed that there was no statistically significant difference (P > 0.05) in demographic profiles between asthmatic patients and control. On the other hand, there was a highly significant difference (P < 0.001) in biochemical profiles (absolute eosinophil count and total serum IgE) between asthmatic children and controls [Table 1].
The statistical analysis as shown in [Table 2] founded that in the asthmatic children, the frequency of genotype GG recorded OR 0.905 with a confidence intervals (CI) value between 0.486 and 1.683 under 95% and it showed a nonsignificant difference (P = 0.437) according to Fisher's exact test. The genotype AG recorded OR 1.470 with CI between 0.765 and 2.826 under 95% and it showed a nonsignificant difference (P = 0.160) according to Fisher's exact test. The frequency of genotype AA recorded OR 0.352 with CI between 0.090 and 1.376 under 95% and it showed a nonsignificant difference (P = 0.106) according to Fisher's exact test. The allele frequency of G showed OR 1.108 with CI range between 0.663 and 1.851 under 95% and it showed a nonsignificant difference (P = 0.397), while allele A showed OR 0.902 with CI range between 0.540 and 1.507 under 95% and it showed a nonsignificant difference (P = 0.397) according to Fisher's exact test.
The results IL-5 Rα SNPs of asthmatic children and healthy controls were agreed with expected HWE (according to the website OEGE-studies) [Table 3].
The results showed no statistically significant correlation between immunological parameters (IL-5, eosinophil count, and IgE) and IL-5 Rα G-80A genotypes (P = 0.649, 0.06, and 0.552, respectively) [Table 4].
By the comparison of genotype frequencies, a negative association of genotypes GG IL-5 Rα OR = 1.176, 95% CI = 0.471–2.940, P = 0.453; AG IL-5Rα OR = 1.056, 95% CI = 0.412–2.704, P = 0.553; and AA IL-5Rα OR = 0.245, 95% CI = 0.021–2.828, P = 2.067 with mild asthma was found in [Table 5]. Furthermore, a nonsignificant prevalence of the allele G and allele A IL-5Rα in patients with mild forms of the disease in comparison with that in individuals with moderate asthma (OR = 1.302, 95% CI = 0.608–2.786, P = 0.312 and OR = 0.768, 95% CI = 0.359–1.644, P = 0.312, respectively) was observed.
| Discussion|| |
The genotypes of IL-5 receptor αlpha subunit G-80A (GG, AG, and AA) in the study were 61.2%, 35.3%, and 3.5%, respectively, of patients, while in healthy controls were 63.5%, 27.1%, and 9.4%, respectively. This is the first study of IL-5 Rα G-80A polymorphism in Iraq, which showed that IL-5 Rα G-80A polymorphism did not have any risk factor to develop asthma or increased asthma severity. Other international previous studies mentioned that interchangeable results explain the correlation between asthma and IL-5 Rα G-80A because of the existence of racial and ethnic specificities in the frequency distributions of the IL-5 receptor encoding polymorphic alleles. Some studies suggest that polymorphisms in IL-5Rα might be among the genetic risk factors for asthma development,, while another study said that IL-5 Rα receptor was unlikely to be a common cause of asthma.
The analysis of the HWE of IL-5 Rα G-80A revealed that asthmatic patient groups and control groups were in a good agreement with the equilibrium, and no significant variation between the observed and expected genotypes frequencies was observed.
The result showed no statistically significant differences in IL-5 level, absolute eosinophil count, and total serum IgE between IL-5Rα G-80A genotypes (P = 0.649, 0.06, and 0.552, respectively). These results agree with the previous study, which mentioned that the total IgE level did not show any significant difference according to the IL-5Rα polymorphism. On the other hand, the results disagree with another previous study, which showed that a functional polymorphism in IL-5Rα (−5993G > A, −5567C>G and − 5091G>A) may effect on the blood eosinophil counts as a result of differences in SNPs of IL-5 Rα between previous and present studies.
| Conclusions|| |
Asthma in children is not associated with IL-5 Rα G-80A polymorphism. IL-5 Rα G-80A polymorphism has not any impact on IL-5 levels, eosinophil count, and total serum IgE.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]