Functional Variant in the Autophagy-Related 5 Gene Promotor is Associated with Childhood Asthma

Rationale and Objective: Autophagy is a cellular process directed at eliminating or recycling cellular proteins. Recently, the autophagy pathway has been implicated in immune dysfunction, the pathogenesis of inflammatory disorders, and response to viral infection. Associations between two genes in the autophagy pathway, ATG5 and ATG7, with childhood asthma were investigated. Methods: Using genetic and experimental approaches, we examined the association of 13 HapMap-derived tagging SNPs in ATG5 and ATG7 with childhood asthma in 312 asthmatic and 246 non-allergic control children. We confirmed our findings by using independent cohorts and imputation analysis. Finally, we evaluated the functional relevance of a disease associated SNP. Measurements and Main Results: We demonstrated that ATG5 single nucleotide polymorphisms rs12201458 and rs510432 were associated with asthma (p = 0.00085 and 0.0025, respectively). In three independent cohorts, additional variants in ATG5 in the same LD block were associated with asthma (p,0.05). We found that rs510432 was functionally relevant and conferred significantly increased promotor activity. Furthermore, Atg5 expression was increased in nasal epithelium of acute asthmatics compared to stable asthmatics and non-asthmatic controls. Conclusion: Genetic variants in ATG5, including a functional promotor variant, are associated with childhood asthma. Thes

Characteristics of the GCPCR population.

a<p>Indicates sample passing quality control.</p>b<p>Indicates significant differences (p<0.05) with non-allergic control children.</p

Asthma-associated rs510432 SNP G variant allele confers enhanced promotor activity.

<p>A. ATG5 promotor fragments generated from genomic DNA isolated from human peripheral blood mononuclear cells. B, C. Luciferase activity of promotor assay vectors. In the mutant the corresponding sites of the two strands of the DNA were mutated from A to G. Promotor activities (corrected for transfection efficiency) are presented as fold increase relative to empty vector (PGL4.20). The fold ratio of empty PGL4.20 Firefly Luciferase plasmid to PGL4.73 Renilla transfection control vector was normalized to 1. Mean ± SD, n = 3 independent experiments.</p

ATG5 expression enhanced in nasal mucosal samples from children with acute asthma.

<p>Data are presented as normalized expression from Affymetrix array data using (A) 202511_s_at and (B) 210639_s_at <i>ATG5</i> probesets. Intensities were normalized to the median expression level of the control samples.</p

LD plot and identification of haplotype block in the <i>ATG5</i> gene.

<p>The position of the 6 SNPs within the <i>ATG5</i> gene are shown above the plot. <i>D’</i> values, indicating extent of LD between SNPs, are noted on the squares. Higher color intensity of the squares indicates higher LD between SNPs. The inverted black triangle represents a single haplotype block (estimated by Gabriel’s 90% bounds on <i>D’</i>³⁰).</p

Identification of association between asthma and <i>ATG5</i> SNPs.

<p>Negative log₁₀ p value of the associations in GCPCR, CAMP, CARE, and CINCY cohorts are presented as well as the meta analysis (METAL). Black circles represent genotyped SNPs and grey triangles represent imputed SNPs. The dotted line represents significance (p = 0.007 for GCPCR and METAL after correction for multiple comparisons, p = 0.05 for CAMP, CARE, CINCY).</p

Genotyped SNPs and their minor allele frequencies (MAF) in the GCPCR cohort.

a<p>p = 0.00085; OR = 0.52, 95% CI, 0.36–0.77.</p>b<p>p = 0.0025; OR = 1.47, 95% CI, 1.14–1.88.</p