Rationale: To date, most studies aimed at discovering genetic factors influencing treatment response in asthma have focused on biologic candidate genes. Genome-wide association studies (GWAS) can rapidly identify novel pharmacogenetic loci.
Objectives: To investigate if GWAS can identify novel pharmacogenetic loci in asthma.
Methods: Using phenotypic and GWAS genotype data available through the NHLBI-funded Single-nucleotide polymorphism Health association-Asthma Resource Project, we analyzed differences in FEV1 in response to inhaled corticosteroids in 418 white subjects with asthma. Of the 444,088 single nucleotide polymorphisms (SNPs) analyzed, the lowest 50 SNPs by P value were genotyped in an independent clinical trial population of 407 subjects with asthma.
Measurements and Main Results: The lowest P value for the GWAS analysis was 2.09 × 10−6. Of the 47 SNPs successfully genotyped in the replication population, three were associated under the same genetic model in the same direction, including two of the top four SNPs ranked by P value. Combined P values for these SNPs were 1.06 × 10−5 for rs3127412 and 6.13 × 10−6 for rs6456042. Although these two were not located within a gene, they were tightly correlated with three variants mapping to potentially functional regions within the T gene. After genotyping, each T gene variant was also associated with lung function response to inhaled corticosteroids in each of the trials associated with rs3127412 and rs6456042 in the initial GWAS analysis. On average, there was a twofold to threefold difference in FEV1 response for those subjects homozygous for the wild-type versus mutant alleles for each T gene SNP.
Conclusions: Genome-wide association has identified the T gene as a novel pharmacogenetic locus for inhaled corticosteroid response in asthma.
polymorphism; genome; pharmacogenomics; glucocorticoid
The National Heart, Lung, and Blood Institute Severe Asthma Research Program (SARP) has characterized over the past 10 years 1,644 patients with asthma, including 583 individuals with severe asthma. SARP collaboration has led to a rapid recruitment of subjects and efficient sharing of samples among participating sites to conduct independent mechanistic investigations of severe asthma. Enrolled SARP subjects underwent detailed clinical, physiologic, genomic, and radiological evaluations. In addition, SARP investigators developed safe procedures for bronchoscopy in participants with asthma, including those with severe disease. SARP studies revealed that severe asthma is a heterogeneous disease with varying molecular, biochemical, and cellular inflammatory features and unique structure–function abnormalities. Priorities for future studies include recruitment of a larger number of subjects with severe asthma, including children, to allow further characterization of anatomic, physiologic, biochemical, and genetic factors related to severe disease in a longitudinal assessment to identify factors that modulate the natural history of severe asthma and provide mechanistic rationale for management strategies.
asthma; remodeling; inflammation; bronchoscopy; imaging
Rationale: Recent studies suggest that people with asthma of different racial backgrounds may respond differently to various therapies.
Objectives: To use data from well-characterized participants in prior Asthma Clinical Research Network (ACRN) trials to determine whether racial differences affected asthma treatment failures.
Methods: We analyzed baseline phenotypes and treatment failure rates (worsening asthma resulting in systemic corticosteroid use, hospitalization, emergency department visit, prolonged decrease in peak expiratory flow, increase in albuterol use, or safety concerns) in subjects participating in 10 ACRN trials (1993–2003). Self-declared race was reported in each trial and treatment failure rates were stratified by race.
Measurements and Main Results: A total of 1,200 unique subjects (whites = 795 [66%]; African Americans = 233 [19%]; others = 172 [14%]; mean age = 32) were included in the analyses. At baseline, African Americans had fewer asthma symptoms (P < 0.001) and less average daily rescue inhaler use (P = 0.007) than whites. There were no differences in baseline FEV1 (% predicted); asthma quality of life; bronchial hyperreactivity; or exhaled nitric oxide concentrations. A total of 147 treatment failures were observed; a significantly higher proportion of African Americans (19.7%; n = 46) experienced a treatment failure compared with whites (12.7%; n = 101) (odds ratio = 1.7; 95% confidence interval, 1.2–2.5; P = 0.007). When stratified by treatment, African Americans receiving long-acting β-agonists were twice as likely as whites to experience a treatment failure (odds ratio = 2.1; 95% confidence interval, 1.3–3.6; P = 0.004), even when used with other controller therapies.
Conclusions: Despite having fewer asthma symptoms and less rescue β-agonist use, African-Americans with asthma have more treatment failures compared with whites, especially when taking long-acting β-agonists.
asthma; long-acting β-agonist; African Americans; race; treatment failure
Rationale: Severe asthma (SA) remains poorly understood. Mast cells (MC) are implicated in asthma pathogenesis, but it remains unknown how their phenotype, location, and activation relate to asthma severity.
Objectives: To compare MC-related markers measured in bronchoscopically obtained samples with clinically relevant parameters between normal subjects and subjects with asthma to clarify their pathobiologic importance.
Methods: Endobronchial biopsies, epithelial brushings, and bronchoalveolar lavage were obtained from subjects with asthma and normal subjects from the Severe Asthma Research Program (N = 199). Tryptase, chymase, and carboxypeptidase A (CPA)3 were used to identify total MC (MCTot) and the MCTC subset (MCs positive for both tryptase and chymase) using immunostaining and quantitative real-time polymerase chain reaction. Lavage was analyzed for tryptase and prostaglandin D2 (PGD2) by ELISA.
Measurements and Main Results: Submucosal MCTot (tryptase-positive by immunostaining) numbers were highest in “mild asthma/no inhaled corticosteroid (ICS) therapy” subjects and decreased with greater asthma severity (P = 0.002). In contrast, MCTC (chymase-positive by immunostaining) were the predominant (MCTC/MCTot > 50%) MC phenotype in SA (overall P = 0.005). Epithelial MCTot were also highest in mild asthma/no ICS, but were not lower in SA. Instead, they persisted and were predominantly MCTC. Epithelial CPA3 and tryptase mRNA supported the immunostaining data (overall P = 0.008 and P = 0.02, respectively). Lavage PGD2 was higher in SA than in other steroid-treated groups (overall P = 0.02), whereas tryptase did not differentiate the groups. In statistical models, PGD2 and MCTC/MCTot predicted SA.
Conclusions: Severe asthma is associated with a predominance of MCTC in the airway submucosa and epithelium. Activation of those MCTC may contribute to the increases in PGD2 levels. The data suggest an altered and active MC population contributes to SA pathology.
prostaglandin D2; chymase; carboxypeptidase A
Rationale: Exhaled nitric oxide (FeNO) is a biomarker of airway inflammation in mild to moderate asthma. However, whether FeNO levels are informative regarding airway inflammation in patients with severe asthma, who are refractory to conventional treatment, is unknown. Here, we hypothesized that classification of severe asthma based on airway inflammation as defined by FeNO levels would identify a more reactive, at-risk asthma phenotype.
Methods: FeNO and major features of asthma, including airway inflammation, airflow limitation, hyperinflation, hyperresponsiveness, and atopy, were determined in 446 individuals with various degrees of asthma severity (175 severe, 271 nonsevere) and 49 healthy subjects enrolled in the Severe Asthma Research Program.
Measurements and Main Results: FeNO levels were similar among patients with severe and nonsevere asthma. The proportion of individuals with high FeNO levels (>35 ppb) was the same (40%) among groups despite greater corticosteroid therapy in severe asthma. All patients with asthma and high FeNO had more airway reactivity (maximal reversal in response to bronchodilator administration and by methacholine challenge), more evidence of allergic airway inflammation (sputum eosinophils), more evidence of atopy (positive skin tests, higher serum IgE and blood eosinophils), and more hyperinflation, but decreased awareness of their symptoms. High FeNO identified those patients with severe asthma characterized by the greatest airflow obstruction and hyperinflation and most frequent use of emergency care.
Conclusions: Grouping of asthma by FeNO provides an independent classification of asthma severity, and among patients with severe asthma identifies the most reactive and worrisome asthma phenotype.
nitric oxide; severe asthma; phenotype; airway reactivity; exhaled breath
Rationale: The Severe Asthma Research Program cohort includes subjects with persistent asthma who have undergone detailed phenotypic characterization. Previous univariate methods compared features of mild, moderate, and severe asthma.
Objectives: To identify novel asthma phenotypes using an unsupervised hierarchical cluster analysis.
Methods: Reduction of the initial 628 variables to 34 core variables was achieved by elimination of redundant data and transformation of categorical variables into ranked ordinal composite variables. Cluster analysis was performed on 726 subjects.
Measurements and Main Results: Five groups were identified. Subjects in Cluster 1 (n = 110) have early onset atopic asthma with normal lung function treated with two or fewer controller medications (82%) and minimal health care utilization. Cluster 2 (n = 321) consists of subjects with early-onset atopic asthma and preserved lung function but increased medication requirements (29% on three or more medications) and health care utilization. Cluster 3 (n = 59) is a unique group of mostly older obese women with late-onset nonatopic asthma, moderate reductions in FEV1, and frequent oral corticosteroid use to manage exacerbations. Subjects in Clusters 4 (n = 120) and 5 (n = 116) have severe airflow obstruction with bronchodilator responsiveness but differ in to their ability to attain normal lung function, age of asthma onset, atopic status, and use of oral corticosteroids.
Conclusions: Five distinct clinical phenotypes of asthma have been identified using unsupervised hierarchical cluster analysis. All clusters contain subjects who meet the American Thoracic Society definition of severe asthma, which supports clinical heterogeneity in asthma and the need for new approaches for the classification of disease severity in asthma.
asthma phenotype; definition; cluster analysis; severe asthma
Rationale: Bronchial thermoplasty (BT) is a bronchoscopic procedure in which controlled thermal energy is applied to the airway wall to decrease smooth muscle.
Objectives: To evaluate the effectiveness and safety of BT versus a sham procedure in subjects with severe asthma who remain symptomatic despite treatment with high-dose inhaled corticosteroids and long-acting β2-agonists.
Methods: A total of 288 adult subjects (Intent-to-Treat [ITT]) randomized to BT or sham control underwent three bronchoscopy procedures. Primary outcome was the difference in Asthma Quality of Life Questionnaire (AQLQ) scores from baseline to average of 6, 9, and 12 months (integrated AQLQ). Adverse events and health care use were collected to assess safety. Statistical design and analysis of the primary endpoint was Bayesian. Target posterior probability of superiority (PPS) of BT over sham was 95%, except for the primary endpoint (96.4%).
Measurements and Main Results: The improvement from baseline in the integrated AQLQ score was superior in the BT group compared with sham (BT, 1.35 ± 1.10; sham, 1.16 ± 1.23 [PPS, 96.0% ITT and 97.9% per protocol]). Seventy-nine percent of BT and 64% of sham subjects achieved changes in AQLQ of 0.5 or greater (PPS, 99.6%). Six percent more BT subjects were hospitalized in the treatment period (up to 6 wk after BT). In the posttreatment period (6–52 wk after BT), the BT group experienced fewer severe exacerbations, emergency department (ED) visits, and days missed from work/school compared with the sham group (PPS, 95.5, 99.9, and 99.3%, respectively).
Conclusions: BT in subjects with severe asthma improves asthma-specific quality of life with a reduction in severe exacerbations and healthcare use in the posttreatment period.
Clinical trial registered with www.clinialtrials.gov (NCT00231114).
asthma; Alair Bronchial Thermoplasty System; bronchial thermoplasty; bronchoscopic procedure; Asthma Quality of Life
Rationale: As the sole nitrogen donor in nitric oxide (NO) synthesis and key intermediate in the urea cycle, arginine and its metabolic pathways are integrally linked to cellular respiration, metabolism, and inflammation.
Objectives: We hypothesized that arginine (Arg) bioavailability would be associated with airflow abnormalities and inflammation in subjects with asthma, and would be informative for asthma severity.
Methods: Arg bioavailability was assessed in subjects with severe and nonsevere asthma and healthy control subjects by determination of plasma Arg relative to its metabolic products, ornithine and citrulline, and relative to methylarginine inhibitors of NO synthases, and by serum arginase activity. Inflammatory parameters, including fraction of exhaled NO (FeNO), IgE, skin test positivity to allergens, bronchoalveolar lavage, and blood eosinophils, were also evaluated.
Measurements and Main Results: Subjects with asthma had greater Arg bioavailability, but also increased Arg catabolism compared with healthy control subjects, as evidenced by higher levels of FeNO and serum arginase activity. However, Arg bioavailability was positively associated with FeNO only in healthy control subjects; Arg bioavailability was unrelated to FeNO or other inflammatory parameters in severe or nonsevere asthma. Inflammatory parameters were related to airflow obstruction and reactivity in nonsevere asthma, but not in severe asthma. Conversely, Arg bioavailability was related to airflow obstruction in severe asthma, but not in nonsevere asthma. Modeling confirmed that measures of Arg bioavailabilty predict airflow obstruction only in severe asthma.
Conclusions: Unlike FeNO, Arg bioavailability is not a surrogate measure of inflammation; however, Arg bioavailability is strongly associated with airflow abnormalities in severe asthma.
asthma; arginine; arginase; nitric oxide; methylarginine
Rationale: Airway inflammation is common in severe asthma despite antiinflammatory therapy with corticosteroids. Lipoxin A4 (LXA4) is an arachidonic acid–derived mediator that serves as an agonist for resolution of inflammation.
Objectives: Airway levels of LXA4, as well as the expression of lipoxin biosynthetic genes and receptors, in severe asthma.
Methods: Samples of bronchoalveolar lavage fluid were obtained from subjects with asthma and levels of LXA4 and related eicosanoids were measured. Expression of lipoxin biosynthetic genes was determined in whole blood, bronchoalveolar lavage cells, and endobronchial biopsies by quantitative polymerase chain reaction, and leukocyte LXA4 receptors were monitored by flow cytometry.
Measurements and Main Results: Individuals with severe asthma had significantly less LXA4 in bronchoalveolar lavage fluids (11.2 ± 2.1 pg/ml) than did subjects with nonsevere asthma (150.1 ± 38.5 pg/ml; P < 0.05). In contrast, levels of cysteinyl leukotrienes were increased in both asthma cohorts compared with healthy individuals. In severe asthma, 15-lipoxygenase-1 mean expression was decreased fivefold in bronchoalveolar lavage cells. In contrast, 15-lipoxgenase-1 was increased threefold in endobronchial biopsies, but expression of both 5-lipoxygenase and 15-lipoxygenase-2 in these samples was decreased. Cyclooxygenase-2 expression was decreased in all anatomic compartments sampled in severe asthma. Moreover, LXA4 receptor gene and protein expression were significantly decreased in severe asthma peripheral blood granulocytes.
Conclusions: Mechanisms underlying pathological airway responses in severe asthma include lipoxin underproduction with decreased expression of lipoxin biosynthetic enzymes and receptors. Together, these results indicate that severe asthma is characterized, in part, by defective lipoxin counterregulatory signaling circuits.
severe asthma; lipoxins; eicosanoids
Rationale: One-quarter to one-third of individuals with asthma smoke, which may affect response to therapy and contribute to poor asthma control.
Objectives: To determine if the response to an inhaled corticosteroid or a leukotriene receptor antagonist is attenuated in individuals with asthma who smoke.
Methods: In a multicenter, placebo-controlled, double-blind, double-dummy, crossover trial, 44 nonsmokers and 39 light smokers with mild asthma were assigned randomly to treatment twice daily with inhaled beclomethasone and once daily with oral montelukast.
Measurements and Main Results: Primary outcome was change in prebronchodilator FEV1 in smokers versus nonsmokers. Secondary outcomes included peak flow, PC20 methacholine, symptoms, quality of life, and markers of airway inflammation. Despite similar FEV1, bronchodilator response, and sensitivity to methacholine at baseline, subjects with asthma who smoked had significantly more symptoms, worse quality of life, and lower daily peak flow than nonsmokers. Adherence to therapy did not differ significantly between smokers and nonsmokers, or between treatment arms. Beclomethasone significantly reduced sputum eosinophils and eosinophil cationic protein (ECP) in both smokers and nonsmokers, but increased FEV1 (170 ml, p = 0.0003) only in nonsmokers. Montelukast significantly increased a.m. peak flow in smokers (12.6 L/min, p = 0.002), but not in nonsmokers.
Conclusions: In subjects with mild asthma who smoke, the response to inhaled corticosteroids is attenuated, suggesting that adjustments to standard therapy may be required to attain asthma control. The greater improvement seen in some outcomes in smokers treated with montelukast suggests that leukotrienes may be important in this setting. Larger prospective studies are required to determine whether leukotriene modifiers can be recommended for managing asthma in patients who smoke.
antiasthmatic agents; smoking adverse effects; corticosteroids; leukotrienes
Background: Severe asthma has been associated with severe exacerbations, lower lung function and greater tissue inflammation. Previous studies have suggested that mutations in interleukin-4 receptor α (IL4Rα) are associated with lower lung function, higher IgE, and a gain in receptor function. However, an effect on exacerbations and tissue inflammation has not been shown.
Hypothesis: Allelic substitutions in IL4Rα are associated with asthma exacerbations, lower lung function, and tissue inflammation, in particular to mast cells and IgE.
Methods: Two well-characterized cohorts of subjects with severe asthma were analyzed for five single nucleotide polymorphisms (SNPs) in IL4Rα. These polymorphisms were compared with the history of severe asthma exacerbations and lung function. In the primary (National Jewish) cohort, these polymorphisms were also compared with endobronchial tissue inflammatory cells and local IgE.
Results: In both cohorts, the presence of the minor alleles at E375A and Q551R, which were more common in African Americans, was associated with a history of severe exacerbations and lower lung function. In the National Jewish cohort, the C allele at E375A was associated with higher tissue mast cells and higher levels of IgE bound to mast cells. The significance for most of these associations remained when whites (the larger racial subgroup) were analyzed separately.
Conclusions: SNPs in IL4Rα, which are more common in African Americans, are associated with severe asthma exacerbations, lower lung function, and increased mast cell–related tissue inflammation. Further studies of the impact of these mutations in African Americans and on receptor function are indicated.
asthma; genetics; IL4Rα; exacerbations; mast cells; IgE
Rationale: Several studies suggest that patients with asthma who are homozygous for arginine at the 16th position of the β2-adrenergic receptor may not benefit from short-acting β-agonists.
Objectives: We investigated whether such genotype-specific effects occur when patients are treated with long-acting β-agonists and whether such effects are modified by concurrent inhaled corticosteroid (ICS) use.
Methods: We compared salmeterol response in patients with asthma homozygous for arginine at B16 (B16Arg/Arg) with those homozygous for glycine at B16 (B16Gly/Gly) in two separate cohorts. In the first, subjects were randomized to regular therapy with salmeterol while simultaneously discontinuing ICS therapy. In the second, subjects were randomized to regular therapy with salmeterol while continuing concomitant ICS.
Results: In both trials, B16Arg/Arg subjects did not benefit compared with B16Gly/Gly subjects after salmeterol was initiated. In the first cohort, compared with placebo, the addition of salmeterol was associated with a 51.4 L/min lower A.M. peak expiratory flow (PEF; p = 0.005) in B16Arg/Arg subjects(salmeterol, n = 12; placebo, n = 5) as compared with B16Gly/Gly subjects (salmeterol, n = 13; placebo, n = 13). In the second cohort, B16Arg/Arg subjects treated with salmeterol and ICS concurrently (n = 8) had a lower A.M. PEF (36.8 L/min difference, p = 0.048) than B16Gly/Gly subjects (n = 22) treated with the same regimen. In addition, B16 Arg/Arg subjects in the second cohort had lower FEV1 (0.42 L, p = 0.003), increased symptom scores (0.2 units, p = 0.034), and increased albuterol rescue use (0.95 puffs/d, p = 0.004) compared with B16Gly/Gly subjects.
Conclusions: Relative to B16Gly/Gly patients with asthma, B16Arg/Arg patients with asthma may have an impaired therapeutic response to salmeterol in either the absence or presence of concurrent ICS use. Investigation of alternate treatment strategies may benefit this group.
asthma; β-adrenergic receptor; β-agonists; pharmacogenetics; salmeterol
Rationale and Objectives: Severe asthma is characterized by increased airway inflammation that persists despite therapy with corticosteroids. It is not, however, merely an exaggeration of the eosinophilic inflammation that characterizes mild to moderate asthma; rather, severe asthma presents unique features. Although arachidonic acid metabolism is well appreciated to regulate airway inflammation and reactivity, alterations in the biosynthetic capacity for both pro- and antiinflammatory eicosanoids in severe asthma have not been determined.
Methods: Patients with severe asthma were identified according to National Heart, Lung, and Blood Institute Severe Asthma Research Program criteria. Samples of whole blood from individuals with severe or moderate asthma were assayed for biosynthesis of lipoxygenase-derived eicosanoids.
Measurements and Main Results: The counterregulatory mediator lipoxin A4 was detectable in low picogram amounts, using a novel fluorescence-based detection system. In activated whole blood, mean lipoxin A4 levels were decreased in severe compared with moderate asthma (0.4 [SD 0.4] ng/ml vs. 1.8 [SD 0.8] ng/ml, p = 0.001). In sharp contrast, mean levels of prophlogistic cysteinyl leukotrienes were increased in samples from severe compared with moderate asthma (112.5 [SD 53.7] pg/ml vs. 64.4 [SD 24.8] pg/ml, p = 0.03). Basal circulating levels of lipoxin A4 were also decreased in severe relative to moderate asthma. The marked imbalance in lipoxygenase-derived eicosanoid biosynthesis correlated with the degree of airflow obstruction.
Conclusions: Mechanisms underlying airway responses in severe asthma include underproduction of lipoxins. This is the first report of a defect in lipoxin biosynthesis in severe asthma, and suggests an alternative therapeutic strategy that emphasizes natural counterregulatory pathways in the airways.
biosynthesis; chromatography, eicosanoids; high-pressure liquid; inflammation mediators