Regulatory haplotypes in ARG1 are associated with altered bronchodilator response

Abstract

Rationale: β₂-agonists, the most common treatment for asthma, have a wide interindividual variability in response, which is partially attributed to genetic factors. We previously identified single nucleotide polymorphisms in the arginase 1 (ARG1) gene, which are associated with β₂-agonist bronchodilator response (BDR).

Objectives: To identify cis-acting haplotypes in the ARG1 locus that are associated with BDR in patients with asthma and regulate gene expression in vitro.

Methods: We resequenced ARG1 in 96 individuals and identified three common, 5' haplotypes (denoted 1, 2, and 3). A haplotype-based association analysis of BDR was performed in three independent, adult asthma drug trial populations. Next, each haplotype was cloned into vectors containing a luciferase reporter gene and transfected into human airway epithelial cells (BEAS-2B) to ascertain its effect on gene expression.

Measurements and main results: BDR varied by haplotype in each of the three populations with asthma. Individuals with haplotype 1 were more likely to have higher BDR, compared to those with haplotypes 2 and 3, which is supported by odds ratios of 1.25 (95% confidence interval, 1.03-1.71) and 2.18 (95% confidence interval, 1.34-2.52), respectively. Luciferase expression was 50% greater in cells transfected with haplotype 1 compared to haplotypes 2 and 3.

Conclusions: The identified ARG1 haplotypes seem to alter BDR and differentially regulate gene expression with a concordance of decreased BDR and reporter activity from haplotypes 2 and 3. These findings may facilitate pharmacogenetic tests to predict individuals who may benefit from other therapeutic agents in addition to β(2)-agonists for optimal asthma management. Clinical trial registered with www.clinicaltrials.gov (NCT00156819, NCT00046644, and NCT00073840).

Figures

Figure 1.

Odds ratios (OR) indicate greater likelihood of a high β-agonist BDR for haplotype 1. (A) Summary OR = 1.25 for haplotype 1 versus haplotype 2. (B) Summary OR = 2.18 for haplotype 1 versus haplotype 3. Boxes represent the point estimate for each study, the width of which is proportional to the standard error. The summary OR is represented as a diamond, the width of which is proportional to the standard error. Horizontal lines represent 95% confidence intervals.

Figure 2.

BEAS-2B cells express ARG1 and the ARG1 promoter displays enhanced luciferase reporter activity. (A) Western blot of BEAS-2B cells extract reveals a band at 34.7 kD consistent with endogenous expression. Cells transfected with human ARG1 show enhancement of this signal at the .expected molecular weight. (B) BEAS-2B cells were transfected with LUC2 empty vector or ARG1(Hap1)-LUC2, and luciferase activity determined. P < 0.001 versus LUC2 empty vector. Results were from four experiments

Figure 3.

The three most common haplotypes in the promoter region of the ARG1 gene differentially regulate the expression of a luciferase reporter gene. Haplotypes were cloned 5′ of a luciferase reporter gene and transfected into the human airway epithelial cell line BEAS-2B. (A) Expression was different based on haplotype. Shown are results from 10 experiments. *P < 0.001 versus haplotype 1; #P = 0.039 versus haplotype 2. (B) β2-agonist (isoproterenol) had equivalent effects in minor down-regulation of the promoter haplotypes. Results were from six experiments.