Interleukin 9: a candidate gene for asthma

Abstract

Asthma is a complex heritable inflammatory disorder of the airways associated with clinical signs of atopy and bronchial hyperresponsiveness. Recent studies localized a major gene for asthma to chromosome 5q31-q33 in humans. Thus, this segment of the genome represents a candidate region for genes that determine susceptibility to bronchial hyperresponsiveness and atopy in animal models. Homologs of candidate genes on human chromosome 5q31-q33 are found in four regions in the mouse genome, two on chromosome 18, and one each on chromosomes 11 and 13. We assessed bronchial responsiveness as a quantitative trait in mice and found it linked to chromosome 13. Interleukin 9 (IL-9) is located in the linked region and was analyzed as a gene candidate. The expression of IL-9 was markedly reduced in bronchial hyporesponsive mice, and the level of expression was determined by sequences within the qualitative trait locus (QTL). These data suggest a role for IL-9 in the complex pathogenesis of bronchial hyperresponsiveness as a risk factor for asthma.

Figure 1

Synteny and linkage homology for bronchial responsiveness implicate IL-9 candidate. (A) The likelihood ratio χ² statistic curve on mouse chromosome 13 for bronchial responsiveness in 24 BXD RI strains that are derived from the hyporesponsive C57BL/6J and the hyperresponsive DBA/2J progenitor strains. The log₁₀ transformed mean value for the APTI phenotype measured for each RI line was used to generate the likelihood ratio χ² statistic curve. The permutation test of Churchill and Doerge (10) was used to assess significance after adjusting on a comparison basis for the multiple comparisons performed on chromosome 13. The threshold for significant linkage was 9.1 for the χ² statistic after this multiple comparisons adjustment. (B) Illustration of the genetic map of human chromosome 5q31-q33 and syntenic regions in the mouse. The region of human chromosome 5q31-q33 demonstrating significant evidence for linkage with BHR is homologous to portions of mouse chromosomes 11, 13, and 18 which contain numerous candidate genes including chromosome 11: IL-4 (IL4), IL-5 (IL5), granulocyte macrophage stimulating factor (CSF2); IL-13 (IL13), immune regulatory factor 1 (IRF1); γ amino butyric acid receptor A1 (GABRA1); chromosome 13: IL-9 (IL9), and chromosome 18: adrenergic receptor β 2 (ADRB2); glucocorticoid receptor 1 (GRL1); fibroblast growth factor acidic (FGFA); platelet-derived growth factor receptor (PDGFR); colony stimulating factor receptor 1 (CSF1R); early growth response element 1 (EGR1). Relative map positions in cM are illustrated to the right of each candidate in the mouse (centromere representing 0 cM).

Figure 2

Reduced abundance of the IL-9 candidate in hyporesponsive B6 mice. (A) RNA were isolated from B6 and D2 splenocyte in vitro after 0, 12, 24, 48, and 72 h of Con A stimulation. Southern blots of RT-PCR for IL-9, IL-4, IL-5, and interferon-γ are shown. Actin RNA were used as an internal control. The symbols (+) and (−) designate with and without reverse transcriptase, respectively. (B) Western blot analysis of cytokines from the lungs of hyporesponsive B6, intermediate (B6D2)F₁, and hyperresponsive D2 mice demonstrating reduced IL-9 levels in B6 mice.

Figure 3

Comparison of expression for surrounding loci in B6 and D2 mice. Analyses of B6 and D2 splenocytes 24 h after Con A stimulation by RT-PCR for expression of surrounding loci on chromosome 13. The glioblastoma oncogene homolog 3(Gli3) is 8 cM from the centromere, dihydrofolate reductase (Dhfr) is located at 51 cM, and calcium modulating ligand is located within the linked QTL at 35 cM (Caml).

Figure 4

Characterization of the molecular defect at the B6 IL-9 locus. (A) Illustration of the IL-9 locus with an intragenic polymorphic dinucleotide repeat (see arrows) within intron 3 which is 3 dinucleotides larger in the B6 gene than in the D2 gene. The black line represents the chromosome, the dark grey represents the untranslated regions, and the open boxes are the exons. (B) Quantification of cloned transcripts obtained from hnRNA in (B6D2)F₁ splenocyte.