Evaluation of IL-4 and IL-13 Single Nucleotide Polymorphisms and Their Association With Childhood Asthma and Its Severity: A Hospital-Based Case-Control Study

Abstract

Background and objectives: Asthma is a common, chronic, atopic respiratory disease that is on the rise among children and adults worldwide. Various environmental, genetic, and biological interactions contribute to the surge in susceptibility to this disease. Interleukin (IL) genes, particularly IL-4 and IL-13, have been linked to asthma pathogenesis. The present study aims to investigate the genetic aberrations, specifically single nucleotide polymorphisms (SNPs) of IL-4 and IL-13, and their association with childhood asthma and its severity.

Methods: An unmatched hospital-based case-control study was conducted in a tertiary care hospital in Assam, India. The sample size was calculated to be 120 (60 cases and 60 controls) using the Epi Info software version 7.2 (Centers for Disease Control and Prevention, Atlanta, GA, USA), assuming a confidence interval of 95%, a power of the study at 80%, a ratio of control to cases as 1, a proportion of controls with exposure at 22%, and a proportion of cases with exposure at 46%. A total of 53 clinically diagnosed cases of childhood asthma in the age range of three to 12 years and 39 healthy controls free from respiratory diseases and having no history of asthma and/or allergy of the same age group attending a tertiary care hospital were included in the study. Children who never had asthma or allergies and who did not suffer from any upper or lower respiratory infections for the previous four weeks were considered controls. Prior informed consent and ethical clearance were obtained. Very seriously ill cases and controls were excluded from the study. The genetic investigation used polymerase chain reaction (PCR), followed by restriction fragment length polymorphism (RFLP), to discover SNPs in the IL-4 and IL-13 genes. Sequencing analysis was done for the cases with +2044 G>A of the IL-13 gene in relation to the severity of the disease. The difference in the proportions of specific SNPs between cases and controls was analyzed using the χ2 test (a p-value of <0.05 was considered significant).

Results: Both the rs2070874 and rs2243250 polymorphisms of IL-4 showed no statistically significant associations. The mutation of the IL-13 gene in 1111C>T was higher among cases than controls. Both genotypic and allelic distributions of the +2044G>A polymorphism of the IL-13 gene revealed a significant association (p<0.05) with the severity of the disease.

Conclusion: Genetic aberrations in SNPs of IL-4 and IL-13 are prevalent among the pediatric patients of the study region. The SNP +2044G>A of IL-13 is instrumental in disease manifestation and severity among the pediatric population of the study region.

Keywords: allele; childhood asthma; gene; interleukin 13; interleukin 4; mutation; single-nucleotide polymorphism.

Figure 1. Standardization of PCR

Lane 1 represents the 50 bp ladder. Lane 3 represents the PCR amplified product. PCR: Polymerase chain reaction

Figure 2. The PCR-RFLP gel electrophoresis for SNP C-33C>T of the IL-4 gene reveals an RFLP pattern obtained following digestion with restriction enzyme BsmA1.

Lane 1 represents the 50 bp ladder; lanes 2, 4, 5, and 6 represent the typical RFLP pattern (wild type); lanes 3 and 7 represent the RFLP pattern in heterozygous mutant allele; and lane 8 represents a typical RFLP Pattern in homozygous mutant allele. PCR: Polymerase chain reaction, RFLP: Restriction fragment length polymorphism, SNP: Single nucleotide polymorphism

Figure 3. The PCR-RFLP gel electrophoresis for SNP C-589C>T of the IL-4 gene reveals the RFLP pattern obtained following digestion with the restriction enzyme AVAII.

Lane 1 represents the 50 bp ladder; lanes 2 and 5 represent a typical RFLP pattern (wild type); lanes 4, 6, and 7 represent the RFLP pattern in the heterozygous mutant allele; and lane 3 represents the typical RFLP pattern in the homozygous mutant allele. PCR: Polymerase chain reaction, RFLP: Restriction fragment length polymorphism, SNP: Single nucleotide polymorphism

Figure 4. The PCR-RFLP gel electrophoresis for SNP -1111C>T of the IL-13 gene revealed an RFLP pattern obtained following digestion with the restriction enzyme HPY99I.

Lane 1 represents the 50 bp ladder; lanes 3 and 5 represent the typical RFLP pattern (wild type); lanes 4, 6, 7, 8, 9, 12, and 13 represent a typical RFLP pattern in the heterozygous mutant allele; and lanes 10 and 11 represent the typical RFLP pattern in the homozygous mutant allele. PCR: Polymerase chain reaction, RFLP: Restriction fragment length polymorphism, SNP: Single nucleotide polymorphism

Figure 5. The PCR-RFLP gel electrophoresis for SNP 4257G>A of the IL-13 gene reveals an RFLP pattern obtained following digestion with the restriction enzyme NlaIV.

Lane 1 represents the 50 bp ladder; lanes 5, 6, 9, 11, and 12 represent the typical RFLP pattern (wild type); lanes 2, 3, 7, 8, and 10 represent the typical RFLP pattern in a heterozygous mutant allele; lanes 4 and 13 represent a typical RFLP pattern in a homozygous mutant allele. PCR: Polymerase chain reaction, RFLP: Restriction fragment length polymorphism, SNP: Single nucleotide polymorphism

Figure 6. The PCR-RFLP gel electrophoresis for SNP +2044G>A of the IL-13 gene reveals an RFLP pattern obtained following digestion with the restriction enzyme NlaIV.

Lane 1 represents the 50 bp ladder; lane 2 represents the typical RFLP pattern (wild type); lanes 4, 5, 7, and 8 represent a typical RFLP pattern in a heterozygous mutant allele; and lanes 3 and 6 represent the typical RFLP pattern in a homozygous mutant allele. PCR: Polymerase chain reaction, RFLP: Restriction fragment length polymorphism, SNP: Single nucleotide polymorphism