|Year : 2020 | Volume
| Issue : 1 | Page : 45-50
The correlation between total immunoglobulin-E levels and interleukin-13 gene polymorphism in asthmatic children treated with inhaled corticosteroids or montelukast
Saad Hashim Abood1, Aqeel Mahdi Hussein2, Mohanad Mohsin Ahmed1, Haidar Abdul Amir Najim Abood3
1 Department of Medical Microbiology, University of Kerbala, Kerbala, Iraq
2 Consultant Pediatrician, Kerbala Teaching Hospital for Pediatrics (KTHP), Kerbala, Iraq
3 Department of Pharmacology, University of Kerbala, Kerbala, Iraq
|Date of Submission||24-Dec-2019|
|Date of Acceptance||01-Jan-2020|
|Date of Web Publication||17-Mar-2020|
Mr. Saad Hashim Abood
Department of Medical Microbiology, University of Kerbala, Kerbala
Source of Support: None, Conflict of Interest: None
Background: The aim of this research was to study the possible correlation between single-nucleotide polymorphism of interleukin (IL)-13-1112 C/T with specific parameters in asthmatic children (total serum immunoglobulin-E [TSIgE] levels and asthma severity) treated with montelukast or inhaled corticosteroid. Methods: The study included one hundred asthmatic patients attending Karbala Teaching Hospital for children and similar numbers of healthy unrelated age-matched controls from the same locality of Iraq. TSIgE levels were determined by ELISA technique. DNA was extracted and processed by the allele-specific polymerase chain reaction technique for characterization of genetic variants of IL-13-1112 C/T gene polymorphisms. Results: Iraqi children with asthma showed a lower frequency of the IL-13-1112 CC genotype (13% vs. 16%) for control cases with a higher frequency of the heterozygous IL-13-1112 CT genotype (78% vs. 75%). IL-13-1112 showed significantly negative association with asthma in the dominant, codominant, and overdominant models of inheritance. On the other hand, comparing genotypes of subgroups related to gender, asthma severity, and asthma control showed a nonsignificant difference (P > 0.05). Homozygous wild types of IL-13-1112 TT might be considered protective to Iraqi asthma children. Conclusion: The IL-13 gene may be associated with the level of control in treated asthmatic patients. There is no correlation between TSIgE and the SNP.
Keywords: Asthma severity, interleukin-13 1112C/T, level of control, total serum immunoglobulin-E
|How to cite this article:|
Abood SH, Hussein AM, Ahmed MM, Najim Abood HA. The correlation between total immunoglobulin-E levels and interleukin-13 gene polymorphism in asthmatic children treated with inhaled corticosteroids or montelukast. Biomed Biotechnol Res J 2020;4:45-50
|How to cite this URL:|
Abood SH, Hussein AM, Ahmed MM, Najim Abood HA. The correlation between total immunoglobulin-E levels and interleukin-13 gene polymorphism in asthmatic children treated with inhaled corticosteroids or montelukast. Biomed Biotechnol Res J [serial online] 2020 [cited 2020 Jul 8];4:45-50. Available from: http://www.bmbtrj.org/text.asp?2020/4/1/45/280861
| Introduction|| |
Asthma in childhood is a heterogeneous disease with variable clinical manifestations and different phenotypes, which depend on the gender, age, genetic background, and environmental influences of the patients. In Iraq, asthma prevalence is about 15.8% among children under 5 years old. Eosinophils play an important role in key asthma processes such as airway hyperresponsiveness, mucus hypersecretion, and airway remodeling. Asthma is considered a Type I hypersensitivity reaction where it is produced from a combination of allergens with immunocytes, which released special proteins (cytokines) and antibodies such as immunoglobulin type E (IgE) that result in asthma symptoms. The rise in total serum immunoglobulin-E (TSIgE) levels appears in patients with atopic diseases including asthma. Total serum immunoglobulin (IgE) test is usually performed to aid in the diagnosis of allergic diseases.
In response to allergen presentation by airway dendritic cells, T-helper lymphocytes of the adaptive immune system control many aspects of the disease through secretion of cytokines (interleukin [IL]-4, IL-5, IL-13, IL-17, and IL-22), and these are counterbalanced by cytokines produced by Treg cells. Those cytokines are important and played a key role in regulating the chronic inflammation and changes of asthma and have involved in the evolution of new curative strategies in these diseases. ILs are a group of cytokines that were to be expressed by many of leukocytes such as T-cell, and ILs include proinflammatory cytokines. Of the most important ILs that play a major role in many allergic diseases as IL-13, that is chemical mediators have been implicated in the cascade of events that contribute to asthma and many chronic diseases, these ILs have a pleiotropic functions, the differential functions of ILs in the asthmatic lung are that IL-13 may be produced at higher quantities under allergic inflammatory conditions.
IL-13 is an important protein for humans, which is encoded by the IL-13 gene on chromosome 5q23-31. The secondary structural features of IL-13 are similar to that of IL-4. IL-13 is a central regulator in IgE synthesis, goblet cell hyperplasia, mucus hypersecretion, airway hyperresponsiveness, and fibrosis. It is a mediator of allergic inflammation and different diseases including asthma. Single-nucleotide polymorphisms in IL13 are associated with allergic phenotypes in several ethnically diverse populations. The genetic variations in IL-13 (IL-13 1112) are thought to play a role in the expression of inflammation and airway dysfunctions. Those variations in interleukins are called single nucleotide polymorphism (SNP), which was related to asthma and other serious diseases such as type 2 diabetes mellitus. SNP was utilized as a genetic marker in risk factor of asthma.,
The purpose of this research was to study the association between genetic variation of IL-13 gene and specific parameters in asthmatic children (TSIgE, asthma severity, and asthma control) treated with montelukast or inhaled corticosteroid (ICS). This study was conducted in an effort to better understand the development of asthma from the immunological and genetic perspective; this research addressed specific aims from the analytical study, which included an asthmatic and nonasthmatic children.
The study protocol was approved by the Ethical Committee in the Kerbala Health Directorate.
| Methods|| |
This was a case–control study that was conducted on 100 asthmatic patients and 100 nonasthmatic children as controls recruited during the time period from December 2017 to June 2018 from the Karbala Teaching Hospital for Children. They included 57% of males and 43% of females with a mean age of 8.1 years (standard deviation [SD] = 4.08). All patients had a diagnosis of asthma on the basis of the criteria developed by Urbano, level of severity, and assessment of control and also according to the guidelines. One hundred healthy unrelated subjects with a mean age of 8.09 years (SD = 4.09) from the same locality were used as the controls. Informed consent was taken from all participants before the study. Whole blood and sera were collected from each participant. Whole blood was used for eosinophil determination and for DNA extraction. Sera were used to determine TSIgE levels. DNA extraction was used to detect SNPs in IL-13 (IL-13-1112C/T) by the use of allele-specific polymerase chain reaction (AS-PCR). Data were statistically analyzed by specific software, the Statistical Package for the Social Sciences (SPSS) version 21 (GraphPad Software, San Diego, California, USA).
Genotyping of interleukin-13-1112 C/T polymorphisms
The genomic DNA was extracted from the nucleated cells of study groups under the aseptic condition and according to the protocol of gSYNC™ DNA extraction Kit, GS100 (100 Preparation Kit). Detection of the IL-13-1112 C/T gene polymorphism (rs1800925) was done by the AS-PCR technique as described. Beta-actin gene was used as an internal control. The sequences of primers and condition are shown in [Table 1], [Table 2], [Table 3], [Table 4].
|Table 4: Polymerase chain reaction conditions for genotyping of interleukin-13 gene position 1112 (C/T)|
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The products of PCR were resolved on 1.5% agarose gels The DNA 100 bp ladder (Bioneer, Korea) was also loaded on the agarose gel. The gel electrophoresis was performed using 70 V for 1 h and stained with ethidium bromide (Biotech, lot: K901R0KGA) for 15 min. The gel was documented with a gel documentation system (Cleaver, Scientific).
| Results|| |
Of patients, 57% were male and 43% were female, whereas of healthy controls, 55% were male and 45% were female. According to the age group, 43%, 30%, and 37% of patients and 31%, 42%, and 27% of healthy controls belonged to the age group of <5 years, 5–10 years, and >10 years, respectively.
Body mass index was 18.04 ± 3.986 for patients and 17.54 ± 3.802 for healthy controls. The major characteristics of asthmatic patients were as follows: 59% were mild versus 41% were moderate in severity and 69% were well control versus 31% were not well control. Regarding the type of treatment, 75% were montelukast and 25% were receiving ICS. Highly significant (P < 0.001) differences were reported in TSIgE levels between patients and healthy controls [Table 5].
|Table 5: Distribution of total serum immunoglobulin-E between pateints and controlled|
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There were no significant differences in the distribution of the IL-13-1112 C/T SNPs between asthmatic patients and the control group (P < 0.05). Most of the asthmatic patients and healthy controls were found to carry the CT genotype (78% and 75%, respectively). In addition, the CC genotype was seen in 13% of asthmatic patients versus 16% os healthy controls, whereas TT genotype proportions were equal in both the groups. These results indicate that CT is a major genotype in the Iraqi population [Table 6] and [Table 7].
|Table 6: Distribution of Interleukin-13-1112 C/T genotype between patients and controls|
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|Table 7: Distribution of allele frequencies of Interleukin- 13-1112 (C>T) in asthma patients and controls|
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The analysis of demographic data showed significant association (P < 0.05) between gender and severity of asthma. Whereas, among females, the proportion of moderate asthma was slightly higher than mild asthma. Majority of male patients (68.4%) were found to have mild asthma. There was no significant difference (P > 0.05) between asthma severity and demographic data (age, weight, and height). Higher TSIgE levels were seen in moderate asthma in comparison to mild asthma; however, these differences were not statistically significant.
By comparison of IL-13-1112 C/T genotypes frequencies, only the CC genotype was seen in a higher proportion among mild asthma compared to moderate asthma [Table 8]. With respect to IL-13-1112C/T SNPs, the genotype CT was significantly higher in the well-controlled group of asthmatic patients, compared to the not well-controlled. These results indicate that certain genotypes have impact on the level of control of asthmatic patients. A statistically significant difference between well-controlled and the not well-controlled asthmatic patients (P < 0.05), where the TT genotype was found to be associated with well-controlled asthmatic patients. Moreover, another highly significant association was found between the CT genotype of IL-13-1112 C/T and well-controlled asthmatic patients.
|Table 8: Severity of asthmatic patients associated with their biomarkers|
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Hardy–Weinberg equilibrium (HWE) was used for the allele of IL-4 gene with no significant differences between observed and expected genotype frequencies. Genotypes of IL-13 gene allele of asthmatic children and healthy controls were not agreed with expected HWE thus, these genotypes were required for further analysis [Table 9].
|Table 9: Hardy-Weinberg distribution for the gene interleukin-13-1112 (C/T) genotypes in asthma and control|
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Polymerase chain reaction-based detection of single-nucleotide polymorphisms
Polymerase chain reaction amplification of interleukin-13 gene -1112 C/T showing the C and T alleles (both alleles are 396 bp in size) [Figure 1]. From each patient and control tubes, PCR runs are performed, the first have used primers specific for C allele, whereas the second has used primers specific to the T allele. (PCR products were visualized by gel electrophoresis using 1.5% agarose gel concentration, 70 V for 1 h). L: Ladder, Presence of one band for C lane and absence of this band for T (396 bp) lane refer to the genotype homogenous CC. Ladder 100(L).
|Figure 1: Polymerase chain reaction amplification of interleukin-13 gene -1112 C/T showing the C and T alleles (both alleles are 396 bp in size)|
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Association of level of control of asthma with study parameters
There was no significant difference in blood eosinophils and TSIgE between the well-controlled and not well-controlled asthmatic patients (P > 0.05) [Table 10]. However, there was a statistically significant difference between the well-controlled and the not well-controlled asthmatic patients (P = 0.009), where TT genotypes were found to be associated with well-controlled asthmatic patients. Moreover, another highly significant association was found between the CT genotype of IL-13-1112 C/T and well-controlled asthmatic patients.
|Table 10: Level of control of asthmatic patient associated with their biomarkers|
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| Discussion|| |
It was observed that 49.0% from 100 asthmatic patients have high total serum IgE. This finding agreed with other previous studies., There was a significant difference in TSIgE between patient and controlled groups. Froidure A, and Heeringa JJ. and most of the studies found that children with allergic disease had increased numbers of IgE., While a previous study in Karbala showed that not all patients had high IgE levels. The role of IgE in allergic reactions, especially asthma, as a risk factor for the diagnosis of asthma patients. Many studies have shown that TSIgE levels reflect the severity of asthma in children.,,
IL-13 1112C/T gene in this study for genotypes CC, CT, and TT are 13%, 78%, and 9%, respectively, for patients and 16%, 75%, and 9%, respectively, for healthy group. In the Iraqi province of Thi Qar, Salman and Salih have found that the CT genotype frequency was similar to these research results. On the other hand, Haijun has found this gene (IL-13-1112C/T) and indicated that the SNPs predispose to asthma, whereas Li Hua has found that CC, CT, and TT genotypes are 68.3%, 28.25%, and 3.5%, respectively, for patents and 68.55, 28.1%, and 3.4%, respectively, for healthy control  that are nearly similar to T allele such as risk due to this allele higher than C allele. The data of the current study for IL13 1112 C/T genotyping is no significant difference between asthmatic patients and controls. Previous studies considered IL13111C/T as a risk factor in asthmatic patients,, especially the CT genotype that is higher than the CC and TT genotypes. Previous studies have shown that CC genotype is higher than CT and TT genotypes, in many Asian countries. The difference in the relationship between the IL-13 genotypes for the current study proves that most people who carry the genotype CT have an important relationship with other diseases as mentioned, which reflecting that these diseases are associated with IL-13 polymorphism. Asthma tends to be more common in children with insulin-dependent diabetes mellitus than in children without the disease. This indicates that the T helper 1 and TH2 diseases can coexist, indicating a common environmental denominator behind the disease processes. The patients are classified into two groups according to the asthma severity and response to treatments. Asthma severity is higher in mild asthma than moderate asthma, showing that this result is similar to Fleming et al. Al Musawi et al. found that moderate asthma is higher than mild asthma. Jabbar Rahi found that mild asthma was 21% whereas moderate asthma was 81%. The patients have received montelukast and the patients who have not responded well were shifted to ICS. Responding to the treatment protocol is considered as well-controlled, whereas the poor response to either protocol is considered not well-controlled, this may be caused by the sample size and/periods of the study. Asthma severity in children is associated with microbial exposures and associations differed based on atopic status. Despite considerable interest in monitoring asthma in children, there is a substantial lack of evidence, may be caused by the different response to treatment between mild to moderate asthma severity patients.
Bjermer et al. have found that the addition of montelukast, when uncontrolled, could provide equivalent control to salmeterol, whereas David has found that the 2-month montelukast is equivalent to ICS as first-line therapy and to long-acting beta-2-agonists as add-on, but is not proved equivalent at 2 years. Keith et al. have found that montelukast is effective for managing asthma symptoms in patients who are previously uncontrolled with ICS. Stelmach et al. have found that high doses of ICSs and montelukast decreased the serum IgE levels and perhaps long-term treatment with montelukast will be beneficial to asthma patients by decreasing IgE levels.
In the current study, there was a significant difference between well-controlled and not well-controlled asthmatic patients in the CC genotype distribution (8.6% and 22.5%, respectively) (P= 0.024) [Table 10]. Yang H. found that these data indicate that only the combined analyses of genetic alterations in the IL-13 pathway reveal its actual significance to the development of atopy and childhood asthma.
de Faria et al. have found that polymorphisms might be involved in the modulation of asthma severity, whereas Phipatanakul et al. have found that a component of corticosteroid nonresponsive pathobiology in adults with severe asthma may differ in children.
| Conclusion|| |
In conclusion the IL13 gene may be associated with the level of control in treated asthmatic patients. There is no correlation between total serum IgE and this SNP.
The authors are grateful to the staff of the Karbala Teaching Hospital for Children, Karbala University, Iraq, for their great help throughout this work.
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], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10]