INTRODUCTION
Asthma has become a disease with increasing prevalence, morbidity and mortality [1–3]. In developed countries, the prevalence of asthma was found to be between 4% and 23% by the international study of asthma and allergies in childhood (ISAAC) study [4]. In Türkiye, the cumulative prevalence of asthma varies between 13.7% and 15.3% in childhood prevalence studies conducted within the ISAAC study [5].
Inflammation in asthma is a T-helper type 2 (Th2) reaction. Inhaled allergens encounter antigen presenting cells (APC), and after activation, naive T cells differentiate into helper T2 (Th2) cells. B lymphocyte transforms into plasma cells with interleukin-4 (IL-4) and IL-13 released from this cell, and antigen-specific immunoglobulin (Ig) E production begins. IgE circulating in the blood binds to basophils, mast cells, eosinophils, and macrophages. As a result of this binding, mediators (such as leukotrienes, prostaglandins, serotonin) are released to the periphery and cause an early phase reaction. The late phase reaction develops after 6–8 h. These events, called remodeling, cause the irreversible change of the airway. In addition, among the most important mediators playing a role in the pathogenesis of bronchial asthma are cysteinyl leukotrienes [6–9].
Although asthma is considered as a multifactorial disease, when we look at the mechanism of inflammation in asthma, IL-4 and leukotriene C4 synthase (LTC4S) are involved in many pathways. It has long been known that the risk of asthma is elevated in children of asthmatic parents. There are many studies on asthma genetics today, but asthma genetics is still not clearly understood. We could not find any study in the literature in which IL-4 and LTC4S genes were screened by NGS (next generation sequencing) analysis in pediatric patients with asthma. The basis of the NGS method is to create a library with many DNA fragments by cutting DNA with enzymatic reactions and multiplying the DNA fragments that make up the library. Due to the development of NGS platforms, great progress has begun to be made in understanding the molecular basis of many diseases. In addition to whole genome and whole exome sequencing with NGS technologies, many genes can be sequenced simultaneously for diseases with genetic heterogeneity in the etiology with targeted NGS panels. By revealing the genetic etiology that causes diseases, more accurate genetic counseling can be provided to patients, and family members at risk can be quickly screened [10–13].
AIM
We aim to reveal the genetic change responsible for the etiology by performing an NGS analysis in asthmatic patients in the pediatric age group in this study.
MATERIAL AND METHODS
This is a prospective study conducted on 50 patients followed up with asthma at an outpatient clinic of pediatrics in a university hospital. Volunteers who signed the written participant consent form were included in the study. Verbal and written information about the study was provided to the asthmatic patients and their families.
According to the GINA criteria, the patients were diagnosed with asthma. In addition, all of the diagnosed patients had at least 1 major or 2 minor risk factors according to the modified asthma predictive index (mAPI) and more than three wheezing attacks in the last 1 year. Detailed history, physical examination and treatment arrangements were made for all children diagnosed with asthma, and they were followed up in the outpatient clinic of pediatrics. The GINA 2020 guideline was used to assess the control status and/or severity of the disease.
Again, according to this guideline, asthma severity was determined according to the treatment that controlled the asthma. Asthma severity was classified as mild asthma, moderate asthma, and severe asthma. The new classification is shown in Table 1. Asthma patients whose relationship with these genetic mutations were evaluated and acute urticaria, allergic rhinitis, food allergy, atopic dermatitis and allergic conjunctivitis diseases in these asthmatic patients were diagnosed and followed up.
TABLE 1
Determination of asthma severity according to treatments that controlled asthmatic patients [2]
Demographic characteristics, risk factor status, background information, family history and laboratory findings of the asthmatic patients included in the study were recorded . IL-4 and LTC4S genes were analyzed by the NGS method.
STATISTICAL ANALYSIS
The data of our study were evaluated using the statistical program SPSS 17.0 (SPSS Inc., Chicago, Illinois, USA). The Mann-Whitney U test was used for pairwise comparisons and the Kruskal-Wallis test was used for more than two comparisons. The relationship between two categorical (nominal or graded) variables was examined by cross-tab analysis. Obtained p-values less than 0.05 were considered as significant.
RESULTS
A total of 50 patients, 32 (64%) male and 18 (36%) female, were included in the study. The mean age of the patients was 7.12 ±3.8 years (range: 1–16 years), and the mean age of symptom onset was 2.73 ±3.2 years (range: 3 months–13 years). It was observed that 38 (76%) of the patients were delivered by cesarean section and 12 (24%) by normal vaginal delivery.
Genetic variation was detected in 31 (62%) of 50 patients included in our study. Variation in only the IL-4 gene was found in 6 (12%) of our patients, variation only in the LTC4S gene in 19 (38%), and variation in both IL-4 and LTC4S genes in 6 (12%) patients. The demographic findings of the cases with variation and whether they have other allergic disease diagnoses are shown in Tables 2 and 3.
TABLE 2
Demographic findings of patients with mutation and association with other allergic diseases
TABLE 3
Mutation rates observed in other allergic diseases
DEMOGRAPHIC, CLINICAL AND LABORATORY CHARACTERISTICS OF THE PATIENTS WITH VARIATION IN THE IL-4 GENE
The number of cases diagnosed in this group was 12; of these patients, 10 (83%) were male and 2 (17%) were female. The mean age of the patients was 10 ±4.3 years and the mean age of symptom onset was 3.7 ±4.1 years. In cases with variation in the IL-4 gene, the presence of humidity at home (p = 0.041), the presence of urticaria diagnosis (p = 0.011) and the hemoglobin value (p = 0.008) were found to be statistically significantly higher than the cases without genetic variation. In addition, it was found that the probability of acute urticaria occurrence in individuals carrying variation in the IL-4 gene was 5.6 times higher than in non-carriers (OR = 5.6; CI: 1.380–22.724) (χ2 = 6.445; p = 0.011).
GENETIC CHARACTERISTICS OF THE PATIENTS WITH VARIATION IN THE IL-4 GENE
In cases 2, 27, 42, 45, 46, 47 and 50, heterozygous variation of c.-33C>T (rs2070874) in intron 1 of the IL-4 gene and heterozygous variation of c.361-9C>A (rs2243290) in intron 3 were detected. Three cases were evaluated as mild asthma phenotype and 4 cases as moderate asthma phenotype.
In case 4, c.-33C>T (rs2070874) homozygous variation in intron 1 of the IL-4 gene and c.361-9C>A (rs2243290) homozygous variation in intron 3 were detected. This case was evaluated as moderately severe asthma phenotype.
In case 8, c.23G>A (rs201210927) homozygous variation in exon 4 of the IL-4 gene and c.360+18 C>A (rs71645916) homozygous variation in intron 3 were detected. This case was evaluated as severe asthma phenotype. In this case, all of the allergic diseases evaluated were present together.
In cases 12 and 18, c.23G>A (rs201210927) heterozygous variation in exon 4 of the IL-4 gene and c.360+18 C>A (rs71645916) heterozygous variation in intron 3 were detected. Case 12 was evaluated as moderate asthma phenotype and case 18 was evaluated as mild asthma phenotype.
In case 16, c.361-9C>A (rs2243290) heterozygous variation in intron 3 of the IL-4 gene and c.360+18 C>A (rs71645916) heterozygous variation in intron 3 were found. This case was evaluated as moderate asthma phenotype.
DEMOGRAPHIC, CLINICAL AND LABORATORY CHARACTERISTICS OF THE PATIENTS WITH VARIATION IN THE LTC4S GENE
The number of cases diagnosed in this group was 25; of these patients, 15 (60%) were male and 10 (40%) were female. The mean age of the patients was 7 ±3.8 years and the mean age of symptom onset was 3 ±3.2 years. Having siblings with asthma was found to be statistically significantly more common in patients with variation in the LTC4S gene compared to those without (p = 0.088).
GENETIC CHARACTERISTICS OF THE PATIENTS WITH VARIATION IN THE LTC4S GENE
In cases 2, 5, 6, 12, 15, 19, 21, 24, 27, 30, 33, 36, 38, 40, 43, 44 and 50 in intron 4 of the LTC4S gene, c.312-16 T>C (rs2306795) heterozygous variation was detected. Seven cases had mild asthma phenotype and 10 cases had moderate asthma phenotype. Allergic rhinitis was diagnosed in 12 (71%) of the cases in this group.
A heterozygous variation of c.230-12 T>C (rs188919983) was detected in intron 3 of the LTC4S gene in cases 4, 10 and 20. Two cases had mild asthma phenotype and 1 case had moderate asthma phenotype.
In cases 16, 17 and 32, c.312-16 T>C (rs2306795) heterozygous variation in LTC4S gene intron 4 and c.59-10 C>A (rs145738814) variation in intron 1 were detected. Three cases were evaluated as moderate asthma phenotype. All 3 patients in this group were exposed to cigarettes and stove heating at home.
In case 28, c.-33 C>T (rs2070874) heterozygous variation in LTC4S gene intron 1 and c.361-9 C>A (rs2243290) heterozygous variation in intron 3 were detected. This case had severe asthma phenotype.
In case 49, a heterozygous variation of c.59-10 C>A (rs145738814) was detected in intron 1 of the LTC4S gene. The case had mild asthma phenotype.
Finally, mutations in the LTC4S gene were observed in 51% of patients diagnosed with allergic rhinitis. Mutations in the IL-4 gene were found in 44% of the patients with urticaria, and a mutation in the LTC4S gene was observed in 50% of the patients with urticaria.
DISCUSSION
The first systematic classification of asthma genetics was made by Cooke and Vander Veer in 1916 [14]. As a result of studies, genes were associated with asthma: cytokines, β2-adrenergic receptor gene, signaling proteins and receptor genes of transcription factors involved in Th1 and Th2 cell differentiation. IL-4, IL4RA, LTC4S, IFNG, IFNGR1, STAT6, ADAM, GATA3, TBX21, IL-13, IL-13 receptor, TLR4, CD14 and FCER1B genes are some genes associated with asthma [15–18]. According to our review of the literature, no study has yet been conducted in which the IL-4 or LTC4S gene has been screened by NGS analysis. Therefore, our study is the first such study.
Interleukin-4 is a cytokine that plays an important role in the regulation of immune system responses. Variation in the IL-4 gene may lead to atopy. Depending on the variation in the IL-4 receptor gene, IgE production and the incidence of atopic asthma increase. However, the genes related to this mechanism have not been fully determined yet. As a result of defining these factors, explaining their interactions and detecting genetic variants, new treatment strategies can be developed in the treatment of the disease [19–21].
When the studies on IL-4 in the literature are examined, Kabesh et al. [22] identified C-589T, C-33T and VE6523 polymorphisms, Hosseini-Farahabadi et al. [23] -590C>T polymorphism, Li et al. [24] C-589T polymorphism and Zhang et al. [25] found the 590 C>T polymorphism to be associated with asthma. Miyake et al., in a 2013 study in Japan on the relationship between polymorphisms in IL-4 and asthma, identified rs2243290 polymorphism as significantly associated with asthma risk [26].
In our study, as a result of NGS analysis of the IL-4 gene, variation in the IL-4 gene was detected in 24% (n = 12) of the cases. The variation c.-33C>T (rs2070874) in intron 1 and c.361-9C>A (rs2243290) in intron 3 were associated with asthma. In studies in the literature, IL-4 rs2243290 variation has been associated with asthma as in our study [27]. Variation of c.360+18 C>A (rs71645916) in intron 3, which has been described before, although there are no data on asthma in the literature, was detected in cases 8, 12, 16 and 18. In addition, c.23G>A (rs201210927) variation in exon 4 was detected in cases 8, 12 and 18.
When the clinical and laboratory characteristics of the cases with variation in the interleukin-4 gene and the cases without variation were compared, the presence of humidity in the home of the patients with variation in the IL-4 gene (p = 0.041) and the presence of urticaria diagnosis (p = 0.011) were statistically significant higher compared to the patients with no such variation. It was observed that the probability of coexistence of acute urticaria in individuals carrying the variation in the IL-4 gene was 5.6 times higher than in non-carriers (OR = 5.6; CI: 1.380–22.724) (χ2 = 6.445; p = 0.011); thus a relationship was found between variation in the IL- 4 gene and urticaria. In addition, it can be hypothesized that moisture has a mutagenic effect on the IL-4 gene, but more comprehensive studies are needed.
When the genetic characteristics of the patients with variation in the IL-4 gene were examined, the asthma clinical status of the case with c.-33C>T in intron 1 and c.361-9C>A homozygous variation in intron 3 was worse than the cases with heterozygous variation. The clinical status of case 8, with c.23G>A homozygous variation in exon 4 and c.360+18 C>A in intron 3, was quite serious. In this case, all the allergic diseases evaluated were observed together and were resistant to asthma treatment. In conclusion, the clinical picture of the asthmatic patient with homozygous variation in the IL-4 gene is more serious than in heterozygous patients. This suggests that the IL-4 gene variants c.-33C>T in intron 1, c.360+18 C>A in intron 3, and c.361-9C>A and c.23G>A in exon 4 may be associated with asthma.
Leukotriene C4 is a cysteinyl leukotriene (CysLT), a potent family of inflammatory mediators. LTC4S enzyme is required for LTC4 production. LTC4S is an 18 kDa integral membrane enzyme of the 5-lipoxygenase/LTC4 synthase pathway and is considered as the most important and only processed enzyme for the formation of cysteinyl leukotrienes [28]. One of the enzymes that is the subject of pharmacogenetic studies in asthma is LTC4S [29]. In our study, as a result of NGS analysis, variation in LTC4S gene was found in 50% (n = 25) of asthmatic patients. In our study, the variants intron 4 c.312-16 T>C (rs2306795), intron 3 c.230-12 T>C (rs188919983), intron 1 c.59-10 C>A (rs145738814), c.-33 C>T (rs2070874) in intron 1 and c.361-9 C>A (rs2243290) in intron 3 were identified.
When the clinical and laboratory characteristics of the cases with variation in the LTC4S gene and the cases without variation were compared, the presence of sibling asthma was found to be statistically significantly more frequent in patients with variants in the LTC4S gene (p = 0.088). However, 2 of our 3 patients with the c.312-16 T>C variant in intron 4 and the c.59-10 C>A variant in intron 1 were observed to be siblings. The high rate of sibling asthma diagnosis and detection of the same variation in sibling cases 16 and 17 suggested that genetic transmission was high in the LTC4S gene, but more comprehensive studies are needed.
When we analyzed the genetic characteristics of the cases with variation in the LTC4S gene, the c.312-16 T>C variant was detected in intron 4 in 34% (n =17) of all cases. In our study, a high rate of c.312-16 T>C genetic change in intron 4 was associated with asthma.
Recent studies on the pathogenesis and treatment of asthma provide new perspectives on the management of the disease. In particular, the effect of nicotine addiction management on the course of the disease in individuals with asthma and chronic obstructive pulmonary disease shows how genetic and environmental factors play a role in controlling the disease. The role of reactive oxygen species in the pathogenesis of diseases such as asthma, atopic dermatitis and allergic rhinitis contributes to a better understanding of the genetic background of these diseases. In addition, studies with biological agents such as mepolizumab emphasize the importance of targeted therapy approaches in the treatment of asthma. The examination of the immune response in pediatric burn patients also sheds light on our investigation of the role of systemic inflammation and the immune system [27, 30–33]. In this context, the determination of genetic changes in the IL-4 and LTC4S genes in asthma patients in our study will contribute to a better understanding of the genetic mechanisms related to the pathogenesis of asthma and will open new horizons for future diagnosis, treatment and management strategies of the disease.
CONCLUSIONS
In our study, we found 9 different genetic changes that they may contribute to the pathogenesis of the disease in the presence of available data in patients with asthma. It is of great importance that our study was the first to perform NGS analysis of IL-4 and LTC4S genes in asthmatic patients. This study emphasized that all of the genetic changes that might occur in both of these genes in asthmatic patients were examined at the molecular level, and the prognosis, possible pathogenic effects and phenotypic reflections of these changes in the genes were examined on a patient basis.
In addition, the genetic changes detected in IL-4 and LTC4S genes in our study will contribute to studies on the diagnosis and prognosis of asthma. Moreover, we believe that this research will shed light on future individual pharmacogenetic studies on IL-4 antagonist (pascolizumab) [34], leukotriene antagonist (montelukast) and other treatment strategies.
