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. 2012 Jul;8(7):e1002824.
doi: 10.1371/journal.pgen.1002824. Epub 2012 Jul 5.

Genome-wide Association Analysis in Asthma Subjects Identifies SPATS2L as a Novel Bronchodilator Response Gene

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Free PMC article

Genome-wide Association Analysis in Asthma Subjects Identifies SPATS2L as a Novel Bronchodilator Response Gene

Blanca E Himes et al. PLoS Genet. .
Free PMC article

Abstract

Bronchodilator response (BDR) is an important asthma phenotype that measures reversibility of airway obstruction by comparing lung function (i.e. FEV(1)) before and after the administration of a short-acting β(2)-agonist, the most common rescue medications used for the treatment of asthma. BDR also serves as a test of β(2)-agonist efficacy. BDR is a complex trait that is partly under genetic control. A genome-wide association study (GWAS) of BDR, quantified as percent change in baseline FEV(1) after administration of a β(2)-agonist, was performed with 1,644 non-Hispanic white asthmatic subjects from six drug clinical trials: CAMP, LOCCS, LODO, a medication trial conducted by Sepracor, CARE, and ACRN. Data for 469,884 single-nucleotide polymorphisms (SNPs) were used to measure the association of SNPs with BDR using a linear regression model, while adjusting for age, sex, and height. Replication of primary P-values was attempted in 501 white subjects from SARP and 550 white subjects from DAG. Experimental evidence supporting the top gene was obtained via siRNA knockdown and Western blotting analyses. The lowest overall combined P-value was 9.7E-07 for SNP rs295137, near the SPATS2L gene. Among subjects in the primary analysis, those with rs295137 TT genotype had a median BDR of 16.0 (IQR = [6.2, 32.4]), while those with CC or TC genotypes had a median BDR of 10.9 (IQR = [5.0, 22.2]). SPATS2L mRNA knockdown resulted in increased β(2)-adrenergic receptor levels. Our results suggest that SPATS2L may be an important regulator of β(2)-adrenergic receptor down-regulation and that there is promise in gaining a better understanding of the biological mechanisms of differential response to β(2)-agonists through GWAS.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Study overview.
(A) Primary GWAS was conducted using subjects from CAMP, LOCCS, LODO, Sepracor, ACRN, and CARE cohort. Samples genotyped on Illumina platforms (i.e. CAMP/LOCCS/LODO/Sepracor) were pooled and analyzed first. Results from samples genotyped on Affymetrix platforms were analyzed separately and then combined to obtain the primary GWAS results. 1000GP imputed data was utilized to expand the primary GWAS association results. (B) Replication of the top (i.e. P-value<1E-04) SNPs from the primary GWAS were attempted in two independent populations: SARP and DAG. (C) The SPATS2L gene was selected for functional validation based on nominal replication of association results in SARP and analysis of publicly available resources.
Figure 2
Figure 2. Region of association near SPATS2L SNPs to BDR.
The x-axis denotes position along Chromosome 2. The y-axis denotes −Log10(P) corresponding to 1000GP imputed data P-values. LD between the SNP with the lowest P-value (rs295137) to each SNP in the plot is denoted in colors and was computed according to 1000GP June 2010 CEU data. Plot was created using LocusZoom .
Figure 3
Figure 3. Summary of BDR by genotype of the SNP (rs295137) near SPATS2L with lowest P-value among all subjects in the primary populations.
The TT genotype was associated with higher BDR (median 16.0; inter-quartile range (IQR) = [6.2, 32.4]), than the TC genotype (median 11.2; IQR = [5.2, 22.6]) or the CC genotype (median 10.3; IQR = [4.1, 21.7]).
Figure 4
Figure 4. Effect of siRNA-mediated SPATS2L knockdown on β2-adrenergic receptor levels in HASM cells.
(A) Knockdown efficiency of two SPATS2L siRNAs. Quantitative real-time PCR was done on RNAs extracted from HASM cells transfected with control non-targeting (NT) or SPATS2L-specific siRNAs. (B) Western blot analysis of β2AR protein in SPATS2L knockdown HASM cells. Three independent experiments (siRNA transfection and Western blot analysis) were done in HASM cells. The upper panel is a representative of the three Western blots. Quantification of the β2AR protein amount (normalized against the control β-actin protein) from three Western blots is shown in the lower panel.

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