Prevalence and CCR3-T51C genotype– phenotype correlation of bronchial asthma among basic education school children: an observational study

Background Bronchial asthma (BA) is a chronic inflammatory disorder identified by different endotypes and phenotypes. Chemokine receptor 3 (CCR3) is one of the essential chemokine receptors that have a crucial role in asthma development by activating the migration of eosinophils through eotaxin production. We aimed to determine asthma prevalence among school children and to investigate the association between CCR3-T51C gene polymorphisms and the symptom-based clinical asthma phenotypes. Methods This study employed a hybrid design, conducted at a single center in Egypt from 2020 to 2021, to explore the relationship between asthma, its clinical phenotypes, and the CCR3-T51C gene polymorphism. Initially, a cross-sectional analysis was performed, utilizing a modified version of the International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire

migration of eosinophils, owing to the expression of the eotaxin family.These receptors are located on the surface of white blood cells (particularly eosinophils and basophils), T helper-2 lymphocytes, mast cells, and airway epithelial cells.These receptors are encoded by the CCR3 gene located on the short arm of chromosome 3 (3p21.3)[15].
The binding of CCL11 or related chemokines to CCR3 causes actin polymerization, chemotaxis, intracellular calcium flux in eosinophils, and toxic reactive oxygen species (ROS) release [3].Preventing eosinophil activation and migration may offer a novel treatment approach to inflammatory diseases.The CCR3 receptor is a promising drug target for developing antagonists [13,16].
It has been shown that asthma is closely related to total IgE serum levels [17].In recent years, researchers have identified CCR3 + and CD123 + HLA-DR − cells in peripheral blood mononuclear cells (PBMCs) as basophils [18], the primary effector cells of allergies such as allergic asthma and rhinitis [19].Moreover, patients with allergic asthma have phenotypically different basophil populations in their blood, which exhibit a robust response to IgE-mediated activation.Further research has shown that bronchial biopsies of patients with asthma and post-mortem lung tissues of those who died from asthma have a significantly higher concentration of basophil [20].
In the context of medical treatment, it is imperative to consider the biological background of individual patients concerning the "driving mechanisms" of inflammation as a strong predictor of their response to treatment [21].Omalizumab, a recombinant humanized monoclonal antibody that attaches to circulating IgE antibodies, is a viable treatment for uncontrolled allergic asthma despite conventional pharmacological therapies [22].
Evidence indicates that asthma is primarily a clinical syndrome with complex and overlapping phenotypes resulting from the interaction between genetic and environmental variables [23].Thus, the current study aims to estimate bronchial asthma prevalence among schoolaged children and to test associations between asthma and its clinical phenotypes with the CCR3-T51C gene polymorphisms.

Study design and population
The study was designed to be conducted in two consequential phases.Phase I: A cross-sectional design in the primary and preparatory schools in El Manzala City, Dakahliya Governorate, Egypt, during the period from 2020 to 2021 to explore asthma prevalence among school-aged children.
Six hundred school-aged children (6-16 years old) were included.An initial sample size of 544 participants with childhood asthma was considered based on Zedan et al. prevalence of 7.7% [5], with a two-sided 5% significance level with 95% power and 5% precision.A ten percent non-response rate was considered.Taking into consideration a ten percent non-response rate, a total sample of 600 participants was assumed.Figure 1 represents a flowchart of the study participants.
We used a modified form of the ISAAC questionnaire (ISAAC phase three) along with ISAAC phase two and environmental questionnaire [24].All questions were translated from English to Arabic and then back-translated to confirm their syntax.Patients were asked to fill out questionnaires to collect data on the patient's age, sex, birth weight, and smoking exposure.The questionnaire also inquired about any diagnoses of respiratory and allergic conditions and their symptoms experienced within the past 12 months, the most common clinical asthma symptoms, and the family history of respiratory and allergic diseases.Written questionnaires were used.
Phase II: A case-control study on randomly selected 60 asthmatic children and age and sex-matched 100 healthy controls to test the association between asthma and its clinical phenotypes with the CCR3-T51C gene polymorphisms.Patients were enrolled from the pulmonology and allergy unit outpatient clinics, at Mansoura University Children's Hospital, Egypt.The control subjects also underwent assessment at the same clinics to verify the absence of asthma or any pulmonary diseases.Asthma and its severity were diagnosed according to the Global Initiative for Asthma (GINA) Guideline 2020 [25].Asthmatic patients with comorbidities were excluded.
The study was approved by the Institutional Research Board of Medical Faculty at Mansoura University, Fig. 1 The flowchart of the study participants Egypt, with the code number MS.18.08.238.Informed consent was obtained from caregivers of the study participants.

Data collection
After validating asthma symptoms, the children with asthma were sub-divided into three groups based on their predominant symptoms: (1) cough asthma phenotype children, (2) wheezy asthma phenotype children (wheezes are defined as creaking rattling and jingling) [26], and (3) cough and wheezy asthma phenotype where both cough and wheezes are predominant symptoms [27].
All study participants had entire history taking and physical examinations to validate asthma symptoms.The patients' data included age, gender, consanguinity, family history of allergic diseases, residence, nutritional history, and paternal smoking, as well as clinical asthma phenotypes.

Availability of data and materials
Due to the sensitive nature of the clinical data collected in our study, including potentially identifiable patient information, direct access to the dataset is not feasible in order to uphold patient confidentiality and comply with data protection regulations.

Laboratory analysis
Six ml of blood was withdrawn in EDTA tubes for complete blood count and absolute and relative eosinophil counts.The absolute eosinophil count was calculated manually, and an automated cell counter determined the peripheral relative eosinophil count (%).
The total serum IgE levels were measured by collecting 2 ml of untreated blood in test tubes, which were then analyzed using Abia IgE total kits (Cat No: DK.048.01.3) by the accurate ELISA technique, AB Diagnostic Systems (GmbH, Sportfliegerstraße 4, Berlin, Germany).

Single nucleotide polymorphism (SNP) genotyping examination
DNA was extracted from blood samples using QIAamp DNA extraction kits (QIAGEN Inc., Germany, Cat No: 51104).The extracted DNA was then amplified by PCR using the sense primer 5′-CTT TGG TAC CAC ATC CTA CCA-3′ and antisense primer 5′-TGA GAG GAG CTT ACA CAT GC-3′.PCR-RFLP was achieved using Taq polymerase and an attached buffer containing MgCl2 (with a final concentration of 1.5 mM).The process included an initial denaturation at 95 ºC for 5 min, followed by 45 cycles of denaturation for 30 s, and a final extension at 72 ºC for 10 min [28].The amplified DNA was then digested by the N1a III restriction enzyme.Agarose gel electrophoresis was performed to verify the PCR products, and the CCR3-T51C gene polymorphism was photographed.

Statistical analysis
Statistical analysis was conducted using IBM SPSS software, version 26 (Social Package for Statistical Sciences, IBM Corporation; Armonk, NY, USA).Categorical data were presented as numbers and percentages, and a chisquare test was used to examine their association.For continuous data, normality was checked using the Kolmogorov-Smirnov test, and the independent t-test was utilized to compare means between the two groups Median (minimum-maximum) was used to present continuous variables.Mann-Whitney test was utilized to compare the two groups.The Kruskal-Wallis test was used for comparing more than two medians.P values < 0.05 were considered statistically significant.

Identification of the baseline socio-demographic characteristics and distribution of the allergic diseases among the studied children
Of 600 children, 92% were aged between 7 and 16 years.The majority of the children were male (56%).Seventytwo children had a birth weight < 2.5 kg and NICU admission (12%), while 16% were exposed to smoke.The overall asthma prevalence was 12% (n = 72) among school-aged children.We found that 12% of the children had a history of wheezes, with 66.7% of them experiencing it in the last year.Forty-eight had a history of wheezing during exercise, night cough, and skin allergy (Table 1).

Comparisons between asthmatic and healthy children regarding the demographic and clinical characteristics
Median age of asthmatic patients was 9.17 ± 3.28 years old, with 61.7% males and 38.3% females, as shown in Table 2. Additionally, 61.7% of the patients lived in rural areas.However, no significant differences were detected between patients and control groups in terms of age, gender, residence, and consanguinity (p = 0.417, 0.671, 0.151, and 0.304, respectively).The prevalence of a positive family history of allergic disease was significantly higher among children with asthma in comparison to those in the control group (35% vs. 13%) (p = 0.001).
Asthmatic patients had a significantly lower rate of breastfeeding in comparison to controls (58.3% vs. 86%) (p < 0.001).Significantly higher proportions of paternal smoking and crowding index > 2/room were observed among asthmatic children (46.7% and 35, respectively) compared to the control group (at 12% for both) (p < 0.001) (Table 2).

Comparisons of respiratory inflammatory biomarkers across the groups
The median absolute eosinophil count was significantly lower in the asthmatic group (0.32 K/UL) than in the control group (5.7 K/UL) (p < 0.001).Conversely, asthmatic patients had significantly higher median relative eosinophil count (4.18) and total IgE (59.75 IU/ml) compared to controls (2.85 and 5.7 IU/ml) (p = 0.012 and < 0.001, respectively) (Table 3).

Respiratory inflammatory biomarkers among different asthmatic phenotypes and healthy children
Median absolute eosinophil counts were significantly lower in patients with cough (0.21 K/UL), wheezy (0.29 K/UL), and cough and wheezy (0.38 K/UL) in comparison to controls (5.7 K/UL) (p < 0.001 for all).Patients with wheezy and cough and wheezy had significantly higher median relative eosinophil counts of 4.07 and 5.11, respectively than controls (2.85) (p = 0.049 and 0.006, respectively).However, no significant difference was detected between the cough group and control (p = 0.73).Total IgE was significantly higher among patients with cough (111.25 IU/ml), wheezy (78.80 IU/ml), and cough and wheezy (29.8 IU/ml) compared to the control group (5.70 IU/ml; p < 0.001 for all) (Table 4).

The associations between the CCR3-T51C genotypes and asthma with its clinical phenotypes
As regards the CCR3-T51C polymorphisms, the TT genotype was slightly less frequent in patients than in controls (63.3% vs. 64%; p = 0.422) but without achieving a statistically significant difference.In addition, the T allele was slightly more frequent in the asthmatic children than in controls, without a significant difference (76.7% vs. 74%; p = 0.59) (Table 5).

Association between the CCR3-T51C genotypes and different asthma phenotypes based on laboratory inflammatory biomarkers
There was no statistically significant association between the CCR3-T51C genotypes and serum inflammatory biomarkers among cough, wheezy, and cough and wheezy groups (p > 0.05), except for the cough group with the CT genotype had a significantly lower serum relative eosinophil count than those with wheezy (1.7 vs. 4.62; p = 0.016) (Table 6).

Discussion
Our hybrid study design comprised two phases: the cross-sectional analysis for detecting asthma prevalence, and the case-control study for investigating the genetic association reveals that 12% of school-aged children in El Manzala City, Dakahlia, Egypt, have asthma.Through the analysis of serum biomarkers, asthmatic children have shown a significantly higher eosinophil count and total serum IgE level compared to controls.Moreover, no significant differences existed in CCR3-T51C genotypes or alleles between asthmatic children and controls.
The study findings reported that most of the studied children were aged between 7 and 16 years.Additionally, most of the subjects evaluated were male, comprising over half of the total population.Asthma prevalence differs by sex across the lifespan, where boys have higher rates than girls before puberty, but this reverses in adolescence [29].
According to a study using an ISAAC questionnaire, the Sohag Governorate found that 12.5% of children aged 6 to 12 years old in public primary schools had asthma, which is consistent with our result [30].On   the other hand, this percentage was greater than in the Nile Delta area of Egypt in 2009; overall asthma prevalence was 7.7%, with a slightly higher prevalence in males (50.7%) [6].Another survey in Cairo demonstrated that the asthma prevalence rate among school children aged 3-15 years was 8.2% [31].Meanwhile, a study conducted in 1996 by Khallaf et al. revealed an asthma prevalence rate of 4.8% in Egypt [32].It is worth noting that the disparities in prevalence rates may be attributed to differences in populations and environmental factors, leading to varying degrees of allergen exposure.Also, this study revealed that 12% of the children exhibited previous wheezes, and 66.7% them had experienced wheezing during the last year.A previous nationwide survey of school-aged children indicated that 26.5% of them had persistent wheezing, and 14.7% experienced wheezing in the last year of the disease [33].Similarly, another national study focused on primary school children aged 8 to 12 years, which found that the prevalence of wheezing was 16.83%, which increased to 26.74% over the past 12 months [34].The findings of these studies are crucial and provide insight into the prevalence of this disease, highlighting the need for continued research to better understand and manage the disease.
Additionally, this study shows that only 8% of children suffered from night cough last year and wheezed during   exercise.Also, Kajbaf et al. reported that 7.2% and 3.4% of Iranian primary school students had night coughs and exercise-induced wheezing, respectively [35].Exercise-induced wheezing may be caused by factors such as dryness of the mucosal lining, increased osmolality that triggers mast cell degranulation, and rapid airway rewarming after exercise, which can lead to vascular congestion, enhanced permeability, and edema [36].This study revealed no notable differences in age, sex, residence, and consanguinity between asthmatic and control groups.However, a positive family history of asthma was significantly more common in asthmatic patients as compared to the control group.This is consistent with a previous study that documented a higher positive family history of atopy among asthmatic cases compared to controls.Also, it was found that 88.6% of responsive and 80% of resistant asthma patients had positive family histories, respectively, while controls had 0% [37].Children with a family history of asthma have a 4.2 times higher risk of developing asthma (95% CI 3.91-4.50)than those without [30].
Furthermore, it was observed that asthmatic patients were less likely to have been breastfed and more artificially fed than the control group.This is consistent with Zedan et al., who revealed that only 51.7% of asthmatic cases received exclusive breastfeeding versus 86% of controls [38].It is worth noting that breastfeeding exclusively for 6 months may prevent asthma or atopic diseases [39].
In the current study, asthmatic children had a higher rate of paternal smoking, and a crowding index of more than two children per room than controls.Prior research has established that students who are exposed to smoking and reside in houses with high crowding indices have a higher prevalence of asthma than the control group [38,40].This could be due to the decrease in Forkhead/ winged helix transcription factor (FoxP3) levels and tumor growth factor-β caused by passive smoking, which is linked with T-reg cells, and the increase in interleukin-17A and interleukin-23, which are connected with Th17 cell [41].Besides, the study by Islam et al. suggests that overcrowding enhances respiratory infection transmission and elevates the risk of asthma [42].
This study demonstrated that asthmatic children exhibit a lower absolute eosinophil count but a higher relative eosinophil count in comparison to controls.Likewise, patients with asthma have higher eosinophil counts in their blood [43], suggesting a pivotal role for eosinophils in asthma development and that an increased eosinophil count can lead to irreversible changes in lung function and airway remodeling [44].Basophil populations in the blood of individuals with allergic asthma respond to IgE-mediated activation [45].
Eosinophil accumulation in the bronchial wall is the primary mechanism of action.Inflammatory cells' secretory granules stain dark pink in eosin and hematoxylin preparations.These inflammatory cells have secretory granules that stain dark pink in eosin and hematoxylin preparations.The content of these granules can be released locally through different mechanisms.When activated, human eosinophils undergo cytolysis in the tissues, leading to the extracellular release of membranebound granules [15].
These granulocytes release the eosinophil cationic protein and eosinophil peroxidase, which can damage surrounding tissues.Around two-thirds of patients with asthma have an allergic component, and 50% of those with severe asthma have an allergic component.The most significant known contributing factor to the development of asthma is an increase in IgE production due to exposure to environmental allergens (atopy), especially when sensitization occurs in early life [46].On the other hand, the expression of the high-affinity IgE receptor, Fc ƐRI, on mast cells and basophils has been shown to be sensitive to the presence of IgE or cytokines previously [47].
In this study, patients with asthma showed significantly increased total serum IgE levels compared to those without asthma, which aligns with previous findings by Zedan et al. [38].This suggests a potential role for the IgE-related mechanism in the up-regulation of neurokinin-1 receptor (NK1R) expression [48], where IgE at 1 μg/mL increases mean fluorescent intensity (MFI) of NK1R expression and NK1R mRNA expression in KU812 cells at 2 h following incubation, which was blocked by coincubation with anti-IgE antibody for 30 min.
Furthermore, it was observed that the cough, wheezy, and cough with wheezy phenotypes exhibited significantly lower absolute eosinophil counts compared to the control group, with no significant variance among the three.Conversely, the relative eosinophil count was notably higher in the wheezy and cough with wheezy phenotypic groups compared to the control group.Similarly, groups with cough-predominant asthma phenotype and wheezy phenotype showed a greater serum eosinophil count than the control group, with no significant difference between the cough and wheezy phenotypes [27].These findings suggest that different phenotypes of asthma have varying impacts on eosinophil counts, with cough and wheezy phenotypes presenting higher serum eosinophil counts than the control group.
CCR3 is the primary receptor for eotaxins, stimulating eosinophil migration, degranulation, and activation via G protein-dependent mechanisms [49].This study revealed no significant differences in the CCR3-T51C genotypes between asthmatic and control children; however, the TT genotype was more common among asthmatics.A previous study found no significant difference in CCR3-T51C genotype and allelic frequency between asthmatic cases and controls.However, asthmatic patients had a higher prevalence of the TT genotype [38], consistent with our study.According to a study conducted in Taiwan, the CCR3-T51C polymorphism was not found to be associated with asthma [50].However, it is likely that CCR3 + and CD123 + HLA-DR − cells play a role in AA and AR through SP and NK1R.The cells that express up-regulated SP in CCR3 + and CD123 + HLA-DR − granulocytes may be a subtype of basophils that is different from those found in PBMC.It is possible that this subtype may also include other types of cells.Previous research has shown that AA patients have unique basophil populations in their peripheral blood, and CCR3 + cells in blood granulocytes can include both eosinophils and basophils [45].Although there is no information available about CD123 + HLA-DR − cells in blood granulocytes, CD123 + granulocytes are composed of eosinophils, immature neutrophils, and basophils [48].
In Saudi Arabia, CCR3-T51C polymorphism was significantly correlated with increased asthma risk [51].However, the association was observed in the British population (odds ratio = 2.35, P < 0.01) and not in the Japanese [28].Conversely, a Japanese study found that the 64Ile and 51C variations in CCR2 and CCR3 genes are linked with cedar pollinosis.The haplotype 64Ile/780C/51C frequency was higher in individuals with pollinosis, suggesting that these genes are candidates for the condition [52].Differences in genetic variation may contribute to complex traits like asthma [12].
The effect of allergens on CCR3-T51C genotype expression in cough, wheezy, and cough-wheezy phenotypes has yet to be investigated.This study showed no statistically significant difference in laboratory biomarkers among the CCR3-T51C genotypes in cough, wheezy, and cough-wheezy groups, except for relative eosinophil count, which was significantly lower in CT cough phenotypes than the wheezy phenotype.CCR3 mRNA and protein expressions are increased in the bronchial mucosa of asthmatic patients and correlated with airway hyperresponsiveness [53].
The allergen extract affects eosinophil behavior in allergic asthma patients during late-phase airway inflammation [54].The bronchial HDME challenge increases sputum eosinophils and ECP in allergic asthma patients [55].Eosinophils express CCR3, which suggests that SP + CCR3 + granulocytes may be eosinophils [48,56].However, further studies are needed to confirm or disprove this association with other allergic sensitizations.This study is noteworthy for being the first to investigate the relationship between the CCR3-T51C gene polymorphism and asthma phenotype as well as various inflammatory biomarkers, such as absolute eosinophil count, relative eosinophil percentage, and IgE levels.Adding new genetic data about polymorphisms, from different ethnic groups, could be of great value in personalized medicine.
In conclusion, our study reveals that asthma affects 12% of the school-aged children.The CCR3-T51C genotype or allelic polymorphism frequency did not differ between asthmatics and controls; however, the TT genotype was more frequent in asthmatic children.Eosinophil count, serum IgE and gene polymorphism of CCR3-T51C appeared similar among different asthmatic phenotypes.

Table 1
Baseline socio-demographic data and distribution of allergic diseases among studied students

Table 2
Differences between patients and controls regarding the demographic data and clinical characteristics Data were expressed as n (%) and mean ± SD t Independent t-test, χ 2 chi-square test

Table 3
Comparison of inflammatory biomarkers between asthmatic and control groups Data were represented by median (min-max).IgE immunoglobulin E. Z Mann-Whitney

Table 4
Comparison of inflammatory biomarkers between three different asthma phenotypes and the control group Data were represented by median (min − max).Z Mann-Whitney test P1 comparison between the cough group and the control group, P2 comparison between the wheezy group and the control group, P3 comparison between cough and wheezy groups and the control group

Table 5
Distribution of the CCR3-T51C genotypes and alleles among asthmatic patients and controlsData were expressed as number (percent), CI confidence interval, OR odds ratio, r reference group, χ 2 chi-square test

Table 6
Associations of the CCR3-T51C genotypes with different asthma phenotypes in terms of laboratory inflammatory biomarkers Data were represented by median (Min-Max).KW Kruskal-Wallis test, Z Mann-Whitney test P1 comparison between cough and wheezy groups.P2 comparison between cough and cough and wheezy groups, P3 comparison between wheezy and cough and wheezy groups