|Year : 2019 | Volume
| Issue : 3 | Page : 156-161
Specific IL-5 snp is associated with high serum IL-5 levels and higher eosinophil counts among iraqi asthmatic children
Raghdah Maytham Hameed1, Mohanad Mohsin Ahmed1, Haidar Abdul Amir Najim Abood2
1 Department of Medical Microbiology, University of Kerbala, Kerbala, Iraq
2 Department of Pharmacology, University of Kerbala, Kerbala, Iraq
|Date of Submission||26-Jul-2019|
|Date of Decision||09-Aug-2019|
|Date of Acceptance||18-Aug-2019|
|Date of Web Publication||10-Sep-2019|
Ms. Raghdah Maytham Hameed
Department of Medical Microbiology, University of Kerbala, Kerbala
Source of Support: None, Conflict of Interest: None
Objective: Interleukin-5 (IL-5) is a cytokine known to play major role in the regulation of eosinophil formation, maturation, recruitment, and survival. Hence, an increased production of IL-5 may be contributed to the pathogenesis of eosinophil-dependent inflammatory diseases such as asthma. Methods: A total of 85 children, including 59 males and 26 females with asthma with ages between 1 year and 16 years, attended the Respiratory Clinic at Karbala pediatric hospital, with a nonasthmatic children group which have the same age and gender. Restriction fragment length polymerase chain reaction was performed to determine IL-5 C-703T genetic polymorphisms. Total immunoglobulin E (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: The TT genotype of IL-5 C-703T polymorphism was associated with asthmatic children (P: 0.033). In addition, TT genotype was associated with increase of serum IL-5 levels and high absolute eosinophil count (P = 0.008 and 0.021, respectively). Whereas, CT genotype of IL-5 C-703T was found to be associated with high total serum IgE (P < 0.001). Conclusions: Asthma in children is associated with IL-5 C-703T polymorphism. In addition, IL-5 C-703T polymorphism has impact on IL-5 levels and eosinophil count. TT genotype of IL-5 C703T consider a risk factor for mild asthma in Iraqi asthmatic children.
Keywords: Bronchial asthma, eosinophil, interleukin 5, total IgE
|How to cite this article:|
Hameed RM, Ahmed MM, Abood HA. Specific IL-5 snp is associated with high serum IL-5 levels and higher eosinophil counts among iraqi asthmatic children. Biomed Biotechnol Res J 2019;3:156-61
|How to cite this URL:|
Hameed RM, Ahmed MM, Abood HA. Specific IL-5 snp is associated with high serum IL-5 levels and higher eosinophil counts among iraqi asthmatic children. Biomed Biotechnol Res J [serial online] 2019 [cited 2019 Nov 20];3:156-61. Available from: http://www.bmbtrj.org/text.asp?2019/3/3/156/266571
| Introduction|| |
Asthma is one of the most common chronic, noncommunicable diseases in children and adults. It refers to an abnormality of airway function, specifically to wide variations in airflow limitation over short periods of time. Asthma is characterized by airway inflammation rich in eosinophils. The eosinophil is well recognized as a central effector cell in the inflamed asthmatic airway. Eosinophils induce airway hyperresponsiveness and mucus accumulation and play a critical role in airway remodeling. The activation of eosinophils regulates their growth, and survival was done by interleukin 5 (IL-5).
IL-5 is produced by both hematopoietic and nonhematopoietic cells, including T-cells, granulocytes, and natural helper cells. In humans, IL-5 is a very selective cytokine as a result of the restricted expression of their receptor on eosinophils and basophils.
The IL-5 gene is a potential candidate gene in the pathogenesis of asthma. It was suggested to play a role in blood eosinophilia associated with atopic dermatitis. The polymorphisms in the IL-5 genes may contribute to the susceptibility to atopic bronchial asthma and could determine the clinical course of the disease.
Genetic factors could have a critical role in the expression of asthma. However, the genetic background of bronchial asthma is complex and still far from being completely understood. Further, it is likely that multiple genes contribute its development both directly and through gene–gene interactions. Many candidate genes that could potentially contribute to the susceptibility to the disease have not been investigated to date, and not all of the polymorphisms of the candidate genes have been tested for a possible association with the disease. These circumstances make it necessary to examine new polymorphisms of asthma candidate genes regarding their contribution to the disorder and to its clinical manifestation.
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 in the populations and among the bronchial asthma patients and investigate the correlation between specific single nucleotide polymorphisms (SNPs) IL-5 C-703T with IL-5 levels and with eosinophil count in asthmatic children.
| Methods|| |
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 American Thoracic Society criteria for asthma. Their ages were ranged between 1 year and 16 years. The control groups included 85 children (51 males and 34 females) were randomly selected from the hospitals and the local community, that have the same ages and sex of the patients.
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.
Serum and whole blood were collected from each participant; sera were used to determine the total serum 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 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 = (WBC × Eosinophils %)/100
Genotyping of interleukin-5 C-703T polymorphism
The genomic DNA was extracted from the nucleated cells of study groups under the aseptic condition and according to the protocol of ReliaPrep™ Blood gDNA Miniprep System, promega, USA. The extracted DNA was safely stored at −20°C for later use.
The nucleotide sequences of the forward and reverse primer used for polymerase chain reaction (PCR) are 5-CAG-GGA-GAG-CCA-ATC-AGT-3 and 5-ATG-ATG-TCC-AGA-CTC-CAG-GAT-CT-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 60°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 (PstN I, sibenzyme or AlwNI, Bio Laboratory) at optimal temperatures (37°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 75 min and visualized in the ultraviolet light. The wild-type allele is distinguished to two bands at 160 and 18 bp, whereas the mutant allele is not distinguished.
Data were introduced into the Statistical Package for the Social Sciences (SPSS) version 21 (GraphPad Software, San Diego, California, USA) to do statistical analysis. Results were expressed as mean ± standard deviation. Comparisons between two means were performed using the t-test, while ANOVA was used to compare among the mean. The Chi-square test was used to compare between two categorical variables. In addition, the Pearson correlation was used to explain the relation between IL-5 levels with absolute eosinophil count and with total serum IgE levels. P < 0.05 was considered to indicate the statistical significance and highly significant if P < 0.001.
Genotypes of IL-5 C073T were presented as percentage frequencies, and significant differences between their distributions in asthmatic patients and controls were assessed using the 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 the Hardy–Weinberg equilibrium (HWE) was estimated using HWE calculator for two alleles, which is available on online OEGE-Online Encyclopedia for Genetic Epidemiology 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]. Expected frequencies of genotypes of the IL-5 SNP using HWE are presented in [Table 3]. Further, [Table 4] shows the comparison of clinical characteristics according to IL-5 C703T genotypes in asthmatic patients, whereas [Table 5] shows a comparison between IL-5 C-703T genotypes and asthma severity in asthmatic children. In addition, [Figure 1] and [Figure 2] show a correlation between IL-5 levels with absolute eosinophil count and with total serum IgE, respectively.
|Table 1: Demographic and biochemical profiles of the patients and controls|
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|Table 3: Expected frequencies of genotypes of the interleukin 5 single-nucleotide polymorphisms using the Hardy-Weinberg equilibrium|
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|Table 4: Comparison of clinical characteristics according to interleukin 5 C703T genotypes in asthmatic patients|
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|Table 5: Comparison between interleukin 5 C.703T genotypes and asthma severity in asthmatic children|
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|Figure 1: Scatterplot serum interleukin-5 pg/ml according to absolute eosinophil count. Pearson correlation coefficient: r = 0.201 (P = 0.039)|
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|Figure 2: Scatterplot serum interleukin-5 pg/ml according to total serum IgE. Pearson correlation coefficient: r = −0.76 (P = 0.246)|
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It was observed that no significant difference (P > 0.05) in demographic profiles between asthmatic patients and controls; only in the family history of asthma/allergic disease, there was a highly significant difference (P< 0.001) between patients and controls. 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, as shown in [Table 1].
The statistical analysis as shown in [Table 2] founded that in the asthmatic children, the frequency of genotype CC recorded OR = 0.771 with a confidence interval (CI) value between 0.409 and 1.452 under 95%; it showed a nonsignificant difference (P = 0.260) according to Fisher's exact test. The genotype CT recorded OR = 0.788 with CI between 0.429 and 1.444 under 95%; it showed a nonsignificant difference (P = 0.268) according to Fisher's exact test. The frequency of genotype TT recorded OR = 2.026 with CI between 0.947 and 4.335 under 95%; it showed a statistically significant difference (P = 0.045) according to Fisher's exact test. The allele frequency of C showed OR = 0.697 with CI range between 0.453 and 1.073 under 95%; it showed a nonsignificant difference (P = 0.063), whereas allele T showed OR = 1.434 with CI range between 0.932 and 2.207 under 95%; it showed a nonsignificant difference (P = 0.063) according to Fisher's exact test.
Genotypes of IL-5 C703T of asthmatic children and healthy controls were agreed with expected HWE (according to the web site OEGE-Online Encyclopedia for Genetic Epidemiology studies), as shown in [Table 3].
The study showed a higher correlation between IL-5 level and high absolute eosinophil count and TT genotype (P = 0.008 and 0.021, respectively) while serum IgE was found to have a higher correlation with CT genotype (P < 0.001), as presented in [Table 4].
By comparison of genotype frequencies, a significant negative association of genotypes CC IL-5 OR = 0.828, 95% CI = 0.319–2.151, P = 0.440 and CT IL-5 OR = 0.519, 95% CI = 0.209–1.289, P = 0.117 with mild asthma is shown in [Table 5]. Furthermore, a trend to a positive association of the genotype TT IL-5 with mild BA OR = 3.209, 95% CI = 0.975–10.567, P = 0.039 and a nonsignificant prevalence of the allele C and allele T IL-5 in patients with mild forms of the disease in comparison with that in individuals with moderate asthma OR = 0.612, 95% CI = 0.321–1.164, P = 0.090 and OR = 1.634, 95% CI = 0.859–3.111, P = 0.090, respectively) were observed.
| Discussion|| |
IL-5 is a key factor for eosinophilia, and therefore, it could be responsible for some of the tissue damage seen in asthma. The effects of IL-5 appear to be mainly in the circulation, inducing peripheral mobilization of eosinophils to the circulation.
The current study showed a significant positive linear correlation between serum IL-5 levels and absolute eosinophil count in asthmatic children (P = 0.039). This result agrees with the previous study such as by Agache et al. who mentioned a robust relationship between blood eosinophilia and IL-5. In addition, Kuo et al. and Kouro and Takatsu, concluded that there was an increased expression of IL-5 of patients with asthma, and this expression may autoregulate eosinophil and increases eosinophil numbers. Other researchers found that the biologic effects of IL-5 were best characterized for eosinophils. This results because IL-5 is the key cytokine responsible for maturation, activation, proliferation, and survival of eosinophils. IL-5 is responsible for the maturation and release of eosinophils in the bone marrow. IL-5 is a critical regulator of eosinophilia and has effects on eosinophil progenitors. In addition, IL-5 acts on eosinophils at multiple functional levels and time points during their life span.
The study showed no significant correlation between IL-5 levels and total serum IgE in asthmatic children (P = 0.246). The result agrees with other previous results, who mentioned that IL-5 alone was not enough to induce IgE production.,, Deo et al. confirm the importance of IL-5 in eosinophilic inflammation; but, there was no correlation between IL-5 levels and IgE in asthma (P = 0.13). In contrast, another study showed that IL-5 production was associated within vivo total IgE levels in the infant.
The results suggested that TT genotype may be a risk factor for asthma in Iraqi children (OR = 2.026, P = 0.045). Further, there was a statistically significant difference in the frequency of TT genotype between asthmatic patients and controls (27.01% and 15.3%, respectively, P = 0.032). There was no prior evidence for an association of IL-5 common polymorphisms with asthma in Iraq. Thus, the association of C-703T with asthma in this study provides the first evidence for a possible contribution of a common polymorphism in the IL-5 gene with the increase of IL-5 levels, absolute eosinophil count, and total serum IgE levels in Iraq. The similar result was detected in Russians, who mentioned that polymorphisms in the IL-5 genes contribute to the susceptibility to atopic bronchial asthma and could determine the clinical course of the disease, but their data suggest a significant association of the C-703 allele of the IL-5 gene with atopic bronchial asthma. On the other hand, no previous international studies detected the association between IL-5 polymorphism and asthma such as Pereira et al. who mentioned that mutations of the IL-5 C703T gene coding region, its promoter, and receptor are unlikely to be common causes of an inherited predisposition to asthma in a study which included 30 asthmatic and 30 nonasthmatic participants. While in Korean children and Chinese population, the studies indicate that IL-5 T-746C polymorphism alone was associated with spirometric markers of asthma severity, whereas it was not associated with the presence of asthma.
Analysis of the HWE of IL-5 C703T 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 data suggest that genotype TT IL-5 is a risk factor for the mild course of atopic bronchial asthma (BA) (P = 0.039). This result disagrees with another previous study, which mentioned that TT genotype was a risk factor for a severe form of asthma. Taking into account the association of allele T-703C of IL-5 with BA, the significant prevalence of the genotype TT IL-5 in patients with mild asthma in comparison to those with moderate forms of the disorder appears identical; this may be due to difference in number of patients between mild and moderate in the study (56 and 29 patients, respectively).
The IL-5 level and absolute eosinophil count were higher in TT of IL-5 genotype compared with other genotypes (P = 0.008 and 0.021, respectively). Since the C-703T transition is located in the promoter region of IL-5, it could affect the expression of the gene and hence influence the serum level of IL-5. IL-5 is a cytokine which plays important roles in the pathophysiology of asthma though acts as a mediator of activation of eosinophils, influencing adhesion, membrane receptor expression, chemotaxis, and mediator synthesis. Taking into account that the association with asthma was found for the genotype TT of IL-5, it could be assumed that this variant of the gene was expressed at a higher level in comparison with that of CC, and CT genotype caused increased production of IL-5 and in turn increased absolute eosinophil count.
On the other hand, the result showed that a higher level of total serum IgE was highly associated with CT genotype (P < 0.001). The CT genotype of IL-5 has not had a statistically significant difference between patients and controls (P = 0.261) and the correlation between IL-5 and IgE level was a no significant negative correlation (r = −0.76, P = 0.246). Therefore, the result indicates that IL-5 expression did not have effect on IgE level. This result agrees with other previous studies,,, who said that IL-5 alone was not enough to induce IgE production.
| Conclusions|| |
The study demonstrated that asthma in children is associated with TT genotype of IL-5 C-703T polymorphism. Further, TT genotype of IL-5 C-703T polymorphism has an impact on IL-5 levels and eosinophil count but no impact on IgE level. TT genotype considers a risk factor for mild asthma.
We are primarily thankful to our patients who willingly participated in this study. We are thankful to Dr. Aqeel Mahdi Hussein, Kerbala health director in Iraq, for helping in the samples collection. We are also thankful to Dr. Alaa Saeed Taj Aldin, a hematologist at Al-Sader hospital in Iraq, for providing a helping in the examination of peripheral blood smear.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]