METHODS

Study population

This cross-sectional study was conducted at the Pediatric Allergy Department of Trakya University between May 2017 and May 2019 and included 102 patients and 86 healthy controls.
The patient group included three subgroups: EW (n = 42; median age 41 months), MW (n = 41; median age 39 months), and asthma (n = 19; median age 72 months). Asthma diagnoses were based on the Global Initiative for Asthma (GINA) guidelines22. Wheezing phenotypes in young children were discriminated, as proposed by the European Task Force4. The control group included 86 (median age 20 months) non-allergic, unrelated, healthy outpatients with no history of recurrent wheezing and no individual or family history of allergic illness (e.g., asthma, atopic dermatitis, allergic rhinitis, allergic rhinoconjunctivitis, and food allergy). The inclusion criteria were the presence of ≥ 4 EW or MW episodes in a year for children up to five years of age or having asthma for children over five years of age. Patients with any other chronic disease or wheezing-associated diseases, such as cystic fibrosis, ciliary dyskinesia, anatomic abnormalities, and immunodeficiency disorder, and preterm-born children were excluded from the study.
Study forms were completed by pediatric allergists during patient visits at our pediatric allergy outpatient clinic. These forms included demographic data (i.e., age and gender) tobacco exposure, wheezing or asthma exacerbations per year, history of atopic dermatitis, presence of allergic rhinitis symptoms (i.e., nasal congestion, nasal itching, rhinorrhea, and sneezing), parental asthma, or other allergic diseases.

Laboratory tests

Serum total IgE levels in the patient group were measured by chemiluminescent immunometric assay (AU5800; Beckman Coulter Inc., CA, USA). Complete blood count analysis was performed by an automatic analyzer (UniCell DxH 800 Coulter Cellular Analysis System; Beckman Coulter Inc.) within one hour of blood sampling. The original manufacturer’s kits were utilized for the laboratory tests.

Skin tests

Aeroallergen and food allergen sensitivity were determined using a skin prick test. The following aeroallergens, common in Turkey, and food allergens were used: Dermatophagoides pteronyssinus and farinae, Felis domesticus, Canis familiaris, Blattella germanica, Dactylis glomerata, Festuca pratensis, Poa pratensis, Lolium perenne, Phleum pratense), Corylus avellana, Betula verrucosa, Alnus glutinosa, Artemisia vulgaris, Chenopodium album, Plantago lanceolata, Parietaria judaica, Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Penicillium, milk, egg white, egg yolk, wheat, hazelnuts, peanuts, walnuts, almond, and cacao (ALK-Abelló, Madrid, Spain). Histamine was used as a positive control and saline as a negative control. Skin reactions were evaluated 15 minutes after the skin test. A positive reaction was defined as wheal diameter ≥ 3 mm.

Genetic analyses

DNA isolations and real-time PCR studies were carried out in the Genetic Diseases Diagnosis Center’s Molecular Genetics Laboratory of Trakya University Medical Faculty.

SNPs selection

We searched PubMed and open-access databases10-12 for the keywords “arginase, asthma genetics, atopy, asthma severity, SNP, bronchodilator response.” SNPs in ARG1 and ARG2 were found associated with an increased risk of atopic sensitization, risk of asthma, asthma severity, and bronchodilator/steroid response in asthma in previous studies13,16,20. These were selected for investigation in our study.

Genomic DNA isolation

Peripheral blood samples from both the patient and control groups were collected using tubes containing EDTA. Genomic DNA isolation from peripheral blood samples was performed using a PureLink™ Genomic DNA Mini Kit (Invitrogen™, Catalog number: K182001; ThermoFisher Scientific) according to the optimized protocol specified in the manufacturer’s manual. Quality control and the purity of the isolated genomic DNA samples were determined using a DNA spectrophotometer (NanoDrop 2000C; ThermoFisher Scientific Inc., MA, USA). High-quality samples that had A260/280 values of 1.8–2.0 were included in the study, while low-quality samples were re-isolated from the blood samples.

Genotyping with real-time PCR

The real-time polymerase chain reaction (RT-PCR) method was used for allelic discrimination for the genotyping. The discrimination of the genotypes in the SNPs was performed using a PCR machine (Applied Biosystems StepOnePlusTM Real-Time PCR system; ThermoFisher Scientific) and TaqManAssays®(ThermoFisher Scientific). The RT-PCR reaction was performed according to the manufacturer’s recommended protocols for six SNPs in theARG1 gene and six SNPs in the ARG2 gene, respectively (rs2781668, rs3756780, rs2781659, rs2781665, rs2781667, and rs2246012 and rs3759757, rs742869, rs3742879, rs17249437, rs3742880, and rs6573788) (see Figure 1).
Each PCR reaction was prepared in a 20 µl total volume of 10 µl 2xPrecision MasterMix, 1 µL TaqManAssay, 4 µL RNAse/DNAse free water, and 5 µL genomic DNA. The PCR conditions were 95 °C for eight minutes for enzyme activation, 95 °C for 10 s for denaturation, and 60 °C for 60 s for the first extension in 10 cycles followed by 95 °C for 10 s for a second denaturation and 68 °C for 60 s for the second extension in 35 cycles. The ROXTM channel and VICTMchannel of the RT-PCR machine were coded for the wild-type probe or mutant-type probe with sequences determined by the TaqManAssay® manufacturer. According to the signals from the canals, the SNPs were considered either homozygous mutant, homozygous wild, or heterozygous. Fluorogenic data for each of the 12 polymorphisms were collected through the ROX and VIC channels at the end of each cycle of the second extension and determined individually.

Haplotype analysis

After determining the homozygote and heterozygote genotypes for the selected SNPs in ARG1 and ARG2 , 18 haplotypes forARG1 and 31 haplotypes for ARG2 were constituted. The GVSc names and H19 localizations of the selected SNPs and haplotypes in theARG1 and ARG2 genes are shown in Figure 2.

Ethical considerations

This study was approved by Trakya University’s ethical committee (approval number 2016/264). Written informed consent was obtained from either parents or children more than seven years old.

Statistical analyses

Mean, standard deviation, median, minimum, maximum, and number (percentage) values were used for the descriptive statistics. Variable distribution was checked using the one-sample Kolmogorov–Smirnov test. Kruskal–Wallis and Mann–Whitney U tests with Bonferroni correction were used for a comparison of the quantitative data. Comparisons of genotype and haplotype frequencies and other categorical data were performed using Pearson’s chi-squared test or Fisher’s exact test when the sample size was small. The Hardy–Weinberg equilibrium test was used to calculate genotype frequencies. P -values < 0.05 were considered statistically significant. IBM SPSS Statistics for Windows, Version 20.0 (Armonk, NY: IBM Corp) was used for the statistical analyses.