Genetics and airway expression of interleukin-13 receptors in asthma

Abstract

The primary objectives of this thesis were to examine the influence of polymorphisms in the IL-13RA1 and the IL13RA2 genes on the genetic susceptibility to asthma, and to evaluate the expression of IL-13 receptor subunits in the airways.

Using solid-phase chemical cleavage of mismatches (SP-CCM), we screened the coding regions of IL13RA1 and IL13RA2, as well as the IL13RA1 promoter region for polymorphisms.  We identified a novel T>G substitution at -281 in the IL13RA1 promoter, as well as two previously identified non-amino acid altering polymorphisms in IL13RA1, a C>T transition at 1050 in the coding region and an A>G substitution at 1365 in the proximal 3’ UTR.  No common variants in the coding region of IL13RA2 were found.  In view of the detrimental effects of IL-13 on the airways, we hypothesised that the -281T>G SNP in the IL13RA1 promoter region, that regulates IL-13Rα1 levels, and the 1365A>G SNP in the IL13RA1 3’ UTR, that regulates IL-13Rα1 mRNA stability, may confer susceptibility to asthma and atopy. To test this hypothesis, we completed a large scale genetic association study of the IL13RA1 –281T>G and the IL13RA1 1365A>G polymorphisms with asthma and asthma-associated phenotypes by genotyping 341 asthma-enriched Caucasian families with at least two affected sibs and 180 non-asthmatic control subjects.  TDT analysis showed  no association of the – 281T>G and the 1365A>G polymorphisms with risk of asthma, or asthma with the presence of atopy, raised serum total IgE, PC₂₀≤16 mg/ml, or PC₂₀<4 mg/ml.  Case-control studies showed no significant association of the -281T>G and the 1365A>G variants with asthma.  In the genotype-phenotype association studies, a borderline association between the IL13RA1 -281T to 1365A haplotype and raised serum total IgE levels in adult female asthmatics was found (odds ratio versus G-G haplotype=2.8, odds ratio versus G-A haplotype=1.07, P=0.049).  No other correlations of IL13RA1 genotypes and 2-allele haplotypes with asthma-related traits were found.  These findings demonstrate that neither of the IL13RA1 polymorphisms constituted risk factors for the development of asthma or associated  phenotypes.

Using RT-PCR analysis, basal expression of mRNA for IL-13Rα1, IL-13Rα2 and IL-4Rα was detected in primary bronchial epithelial cells, primary bronchial fibroblasts and the bronchial epithelial cell lines 16-HBE, NCBI-H292 and A549.  Immunohistochemistry demonstrated that the columnar cells of the bronchial epithelium are the major sites of IL-13Rα1, IL-13Rα2 and IL-4Rα immunoreactivity in the airway mucosa.  Using flow cytometry and confocal laser scanning microscopy, we have shown the presence of a large intracellular pool of IL-13Rα2 with diffuse cytoplasmic distribution in primary bronchial epithelial cells and primary bronchial fibroblasts.

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