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
Investigative bronchoscopy was performed in a subset of participants in the Severe Asthma Research Program (SARP) to gain insights into the pathobiology of severe disease. We evaluated the safety aspects of this procedure in this cohort with specific focus on patients with severe asthma.
To prospectively evaluate changes in lung function and the frequency of adverse events related to investigative bronchoscopy.
Bronchoscopy was performed using a common Manual of Procedures. A subset of very severe asthma was defined by severe airflow obstruction, chronic oral corticosteroid use and recent asthma exacerbations. Subjects were monitored for changes in lung function and contacted by telephone for 3 days after the procedure.
436 subjects underwent bronchoscopy (97 normal, 196 not severe, 102 severe and 41 very severe asthma). Nine subjects were evaluated in hospital settings after bronchoscopy; seven of these were respiratory related events. Recent Emergency Department visits, chronic oral corticosteroid use and a history of pneumonia were more frequent in subjects who had asthma exacerbations after bronchoscopy. The fall in FEV1 following bronchoscopy was similar in the severe compared to milder asthma group. Pre-bronchodilator FEV1 was the strongest predictor of change in FEV1 after bronchoscopy with larger decreases observed in subjects with better lung function.
Bronchoscopy in severe asthma subjects was well tolerated. Asthma exacerbations were rare and reduction in pulmonary function after the procedure was similar to subjects with less severe asthma. With proper precautions, investigative bronchoscopy can be performed safely in severe asthma.
investigative bronchoscopy; safety; severe asthma; exacerbation
Asthma is a complex genetic disease with multiple genetic and environmental determinants contributing to the observed variability in response to common anti-asthma therapies. Asthma pharmacogenetic research has focused on multiple candidate genes including the β2-adrenergic receptor gene (ADRβ2) and its effect on individual responses to beta agonist therapy. At present, knowledge about the effects of ADRβ2 variation on therapeutic responses is evolving and should not alter current Asthma Guideline approaches consisting of the use of short acting beta agonists for as-needed symptom based therapy and the use of a regular long-acting beta agonist in combination with inhaled corticosteroid therapy for optimal control of asthma symptoms in those asthmatics who are not controlled on inhaled corticosteroid alone. This approach is based upon studies showing a consistent pharmacogenetic response to regular use of short acting beta agonists (SABA) and less consistent findings in studies evaluating long acting beta agonist (LABA). While emerging pharmacogenetic studies are provocative and should lead to functional approaches, conflicting data with responses to LABA therapy may be caused by factors that include small sample sizes of study populations and differences in experimental design that may limit the conclusions that may be drawn from these clinical trials at the present time.
Studies of asthma phenotypes have identified obesity as a component of a group characterized by a high proportion of adult-onset asthmatics. However, whether age of asthma onset modifies the association between obesity and asthma is unknown.
From the Severe Asthma Project (SARP), we defined age of asthma onset as early (before 12 years of age) and late-onset (12 and higher). Comparisons of body mass index (BMI) categories were done within age of onset groups and obesity was also compared across these groups. Multivariable logistic regression analysis was done to evaluate the association between BMI categories with healthcare utilization and respiratory symptoms and multivariable linear regression for the association between duration of asthma and weight gain (BMI change/yr). An interaction between obesity and age of asthma onset was included in the multivariable analyses.
The study population consisted on 1,049 subjects of which the median age for asthma onset was 10 years (IQR 4 – 25); 48% were late-onset (≥ 12) and 52% were early-onset (<12). Compared to late-onset obese asthmatics, early-onset obese asthmatics had more airway obstruction, bronchial hyperresponsiveness, and higher OR of ever having 3 or more oral steroid tapers preceding/year or ICU admissions for asthma/preceding year (Interactions between obesity and age of asthma onset were respectively p=0.055 and p=0.02). In early-onset, but not in late-onset asthmatics, there was a significant association between increasing BMI and duration of asthma, after adjusting for confounders. The interaction between asthma duration and age of asthma onset was p < 0.01.
Asthmatics are differentially affected by obesity, based on whether they developed asthma early (<12 years) or later in life. These results highlight the need to understand obesity as a comorbidity that affects specific clinical phenotypes and not all asthma subjects alike.
Severe; asthma; obesity; SARP
Evidence suggests that variation in the length of the poly-C repeat in the 3′ untranslated region (3′UTR) of the β2-adrenergic receptor gene (ADRB2) may contribute to interindividual variation in β-agonist response. However, methodology in previous studies limited the assessment of the effect of sequence variation in the context of poly-C repeat length. The objectives of this study were to design a novel genotyping method to fully characterize sequence variation in the ADRB2 3′UTR poly-C repeat in asthma patients treated with inhaled corticosteroid and long-acting β2-adrenergic agonist (ICS/LABA) combination therapy, and to analyze the effect of the poly-C repeat polymorphism on clinical response.
In 2,250 asthma patients randomized to treatment with budesonide/formoterol or fluticasone/salmeterol in a six-month study (AstraZeneca study code: SD-039-0735), sequence diversity in the ADRB2 poly-C repeat region was determined using a novel sequencing-based genotyping method. The relationship between the poly-C repeat polymorphism and the incidence of severe asthma exacerbations, and changes in pulmonary function and asthma symptoms from baseline to the average during the treatment period, were analyzed.
Poly-C repeat genotypes were assigned in 97% (2,192/2,250) of patients. Of the 13 different poly-C repeat alleles identified, six alleles occurred at a frequency of >5% in one or more population in this study. The repeat length of these six common alleles ranged from 10 to 14 nucleotides. Twelve poly-C repeat genotypes were observed at a frequency of >1%. No evidence of an association between poly-C repeat genotype and the incidence of severe asthma exacerbations was observed. Patients’ pulmonary function measurements improved and asthma symptoms declined when treated with ICS/LABA combination therapy regardless of poly-C repeat genotype.
The extensive sequence diversity present in the poly-C repeat region of the ADRB2 3′UTR did not predict therapeutic response to ICS/LABA therapy.
Asthma; β2-agonist; Inhaled corticosteroid; Genotype; Polymorphism; β2-adrenergic receptor; 3′ untranslated region; Poly-C repeat
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 β2-agonists for optimal asthma management.
Clinical trial registered with www.clinicaltrials.gov (NCT00156819, NCT00046644, and NCT00073840).
pharmacogenetics; asthma; β2-agonist
The T helper 2 (Th2) inflammatory pathway, including the Th2-activating cytokine interleukin 33 and its receptor interleukin 1 receptor-like 1 have been strongly implicated in asthma susceptibility (Moffatt MF, et al NEJM 2010). However, the role of Th2 pathway genetic variation in asthma progression and severity is not well understood. Our research group recently developed a clustering algorithm based on comprehensive phenotype information to assign subjects with asthma in the Severe Asthma Research Program (SARP) to 5 primary clusters; 3 of which represent increasing severe allergic asthma (Moore WC, et al AJRCCM, 2010). We hypothesized that common and potentially deleterious rare variation in this pathway would be associated with severe asthma based on SARP cluster designation.
To evaluate common variants (minor allele frequency or MAF >5%), 419 SARP non-Hispanic white participants with a cluster assignment were genotyped for 182 single nucleotide polymorphisms (SNPs) in Th2 pathway genes using whole-genome SNP data. Individual SNPs and a cumulative model of significant SNPs were evaluated using contingency tables with a chi-square test for trend and ordinal regression models adjusted for age, sex, and principal components. Rare (MAF <5%) amino acid changes and splice site alterations in this pathway were tested for association with asthma severity outcomes in 20 SARP subjects with whole exome sequence data.
Individual Th2 pathway variants were associated with overall SARP cluster assignment, and allergic clusters of increasing severity (1, 2, and 4), including GATA3 polymorphism rs1244186 (P = 0.005). In an 18-SNP additive model, an increasing number of Th2 pathway risk genotypes were highly associated with severe allergic asthma (P = 3.9 × 10−6). For example, in cluster 4, the percentage of subjects with at least 9 risk genotypes was 83% compared to 35% in cluster 1. Additionally, there was evidence that subjects with rare variants in this pathway were more likely to report allergy symptoms (P = 0.006), especially in the fall (P = 0.003), compared to subjects with no rare variants.
Common Th2 pathway variants predict an increased likelihood of severe allergic asthma and rare variants were associated with increased seasonal allergy symptoms.
Interleukin 6 (IL6) belongs to a family of cytokines with both pro- and anti-inflammatory properties. The functional relationship between IL6 signaling and airway disease has not be well characterized; however, IL6 expression is increased during lung inflammation and injury. In this study, serum IL6 and soluble IL6R levels were assessed in non-Hispanic whites with asthma from the Severe Asthma Research Program. Correlations between serum IL6 and IL6R levels, lung function, phenotypic asthma clusters, and asthma severity were evaluated.
Serum IL6 and soluble IL6R was measured in 149 subjects with mild to severe asthma. Serum sIL6R levels were measured using the sIL-6R DuoSet (R&D Systems, Minneapolis, MN) ELISA kit and reported as ng/ml. Serum IL6 measurements were determined using the IL-6 ELISA kit (R&D Systems, Minneapolis, MN) and reported as pg/ml. Serum IL6 and sIL6R measurements were transformed to normalize distribution. The continuous variables analyzed included: % predicted FEV1 [ppFEV1], % predicted FVC [ppFVC], and FEV1/FVC. Serum samples were collected at Wake Forest. Phenotypic asthma clusters were derived as previously described (Am J Respir Crit Care Med. 2010;181:315–323).
Elevated serum IL6 was associated with lower ppFEV1 (P = 0.02) and lower ppFVC (P = 0.003), while elevated serum soluble IL6R was associated with lower ppFEV1 (P = 0.02) and lower ppFVC (P = 0.008). Increasing trends in serum IL6 were observed in atopic asthma Clusters 2 and 4 and the later onset fixed airways obstruction Cluster 5. The highest IL6 serum levels were observed in Cluster 3 characterized has having late onset asthma and elevated BMI. Serum IL6 levels were elevated in subjects with severe asthma (log IL6 = 0.33; N = 25) compared to subjects with mild/moderate asthma (log IL6 = 0.16; N = 69).
Serum IL6 and sIL6R levels are elevated in non-Hispanic white asthma subjects with lower lung function. Serum IL6 and sIL6R are potentially important biomarkers that may distinguish between non-severe and severe asthma and between atopic asthma Clusters.
Inhaled corticosteroids are the recommended first-line treatment for asthma but adherence to therapy is suboptimal. The objectives of this study were to compare the efficacy and safety of once-daily (OD) evening and twice-daily (BD) regimens of the novel inhaled corticosteroid fluticasone furoate (FF) in asthma patients.
Patients with moderate asthma (age ≥ 12 years; pre-bronchodilator forced expiratory volume in 1 second (FEV1) 40-85% predicted; FEV1 reversibility of ≥ 12% and ≥ 200 ml) were randomized to FF or fluticasone propionate (FP) regimens in a double-blind, crossover study. Patients were not permitted to have used any ICS for ≥ 8 weeks prior to enrolment and subsequently received doses of FF or FP 200 μg OD, FF or FP 100 μg BD and matching placebo by inhalation for 28 days each. Primary endpoint was Day 28 evening pre-dose (trough) FEV1; non-inferiority of FF 200 μg OD and FF 100 μg BD was assessed, as was superiority of all active treatment relative to placebo. Adverse events (AEs) and 24-hour urinary cortisol excretion were assessed.
The intent-to-treat population comprised 147 (FF) and 43 (FP) patients. On Day 28, pre-dose FEV1 showed FF 200 μg OD to be non-inferior (pre-defined limit -110 ml) to FF 100 μg BD (mean treatment difference 11 ml; 95% CI: -35 to +56 ml); all FF and FP regimens were significantly superior to placebo (p ≤ 0.02). AEs were similar to placebo; no serious AEs were reported. Urinary cortisol excretion at Day 28 for FF was lower than placebo (ratios: 200 μg OD, 0.75; 100 μg BD, 0.84; p ≤ 0.02).
FF 200 μg OD in the evening is an efficacious and well tolerated treatment for asthma patients and is not inferior to the same total BD dose.
Asthma; fluticasone furoate; inhaled corticosteroid; once daily; efficacy; safety
Indacaterol is an inhaled, once-daily long-acting β2-agonist bronchodilator for regular use in patients with chronic obstructive pulmonary disease (COPD). As indacaterol is the first once-daily β2-agonist to be developed, it is relevant to evaluate its bronchodilator efficacy, safety, and tolerability.
Data were pooled from three randomized, double-blind, clinical studies in patients with moderate-to-severe COPD treated with indacaterol 150 μg qd (n = 627) or placebo (n = 1021). Bronchodilator efficacy was assessed as trough (24-hour post-dose) forced expiratory volume in 1 second (FEV1) after 12 weeks (primary endpoint in individual studies) and FEV1 measured serially post-dose. Rescue use of albuterol was monitored.
At week 12, indacaterol increased trough FEV1 by 160 mL compared with placebo (P < 0.001), exceeding the 120 mL level prespecified as clinically important. FEV1 during the first 12-hour post-dose at week 12 averaged 210 mL higher with indacaterol than with placebo (P < 0.001). Patients receiving indacaterol recorded 53% of days without use of rescue albuterol, compared with 38% of days in the placebo group (P < 0.001). Adverse events (mostly mild or moderate) were reported for 52% and 46% of patients receiving indacaterol and placebo, respectively, and serious adverse events for 4% and 5%. Worsening of COPD was the most frequent adverse event (10% indacaterol; 15% placebo). Indacaterol had little effect on pulse or blood pressure or measures of systemic β2-adrenoceptor activity (blood glucose, serum potassium, and corrected QT interval).
Indacaterol was an effective bronchodilator and was well tolerated, with a good safety profile over 12 weeks of treatment. It should prove a useful treatment for patients with moderate-to-severe COPD.
chronic obstructive pulmonary disease; tolerability; inhaled corticosteroids
Patients with severe asthma have increased granulocytes in their sputum compared to patients with mild to moderate asthma.
We hypothesized that inflammatory granulocytes in sputum may identify specific asthma severity phenotypes and are associated with different patterns of inflammatory proteins in sputum supernatants.
This hypothesis was tested in 242 asthmatics enrolled in the Severe Asthma Research Program who provided sputum samples for cell count, differential cell determinations, cell lysates for Western blot, and supernatant analyses by inflammatory protein microarrays and ELISAs. ANOVA and multiple linear regression models tested mediator associations.
Stratified by sputum granulocytes, < or ≥2%eosinophils and < or ≥40%neutrophils, subjects with both increased eosinophils and neutrophils had the lowest lung function, increased symptoms and healthcare utilization. Subjects with elevated eosinophils with or without increased neutrophils had significantly increased FeNO, serum eosinophils and greater frequency of daily β-agonist use. Microarray data, stratified by granulocytes revealed 25–28 inflammatory proteins increased >2-fold in sputa with ≥40% neutrophils. Microarray analyses stratified by severity of asthma, identified 6–9 proteins increased >2-fold in sputa in subjects with severe asthma compared to nonsevere asthma. ELISA data, stratified by sputum granulocytes, showed significant increases in BDNF, IL-1β, and MIP-3α/CCL20 for those with ≥40%neutrophils; these mediators demonstrated positive associations with neutrophil counts.
Combined increased sputum eosinophils and neutrophils identified asthmatics with the lowest lung function and worse asthma control, increased symptoms and healthcare requirements. Inflammatory protein analyses of sputum supernatants found novel mediators increased in asthmatics, predominantly associated with increased sputum neutrophils.
asthma phenotypes; protein microarrays; BDNF; CXCL13; TNFSF14; CCL20; CCL18
COPD; Genetics; Association analysis; Consortium
Asthma is a heterogeneous disease that is caused by the interaction of genetic susceptibility with environmental influences. Genome-wide association studies (GWAS) represent a powerful approach to investigate the association of DNA variants with disease susceptibility. To date, few GWAS for asthma have been reported.
GWAS was performed on a population of severe or difficult-to-treat asthmatics to identify genes that are involved in the pathogenesis of asthma.
292,443 SNPs were tested for association with asthma in 473 TENOR cases and 1,892 Illumina general population controls. Asthma-related quantitative traits (total serum IgE, FEV1, FVC, and FEV1/FVC) were also tested in identified candidate regions in 473 TENOR cases and 363 phenotyped controls without a history of asthma to further analyze GWAS results. Imputation was performed in identified candidate regions for analysis with denser SNP coverage.
Multiple SNPs in the RAD50-IL13 region on chromosome 5q31.1 were associated with asthma: rs2244012 in intron 2 of RAD50 (P = 3.04E-07). The HLA-DR/DQ region on chromosome 6p21.3 was also associated with asthma: rs1063355 in the 3’ UTR of HLA-DQB1 (P = 9.55E-06). Imputation identified several significant SNPs in the TH2 locus control region (LCR) 3’ of RAD50. Imputation also identified a more significant SNP, rs3998159 (P = 1.45E-06), between HLA-DQB1 and HLA-DQA2.
This GWAS confirmed the important role of TH2 cytokine and antigen presentation genes in asthma at a genome-wide level and the importance of additional investigation of these two regions to delineate their structural complexity and biologic function in the development of asthma.
Asthma; GWAS; RAD50; IL13; HLA-DQB1; TENOR
Asthma is a heterogeneous clinical disorder. Methods for objective identification of disease subtypes will focus on clinical interventions and help identify causative pathways. Few studies have explored phenotypes at a molecular level.
We sought to discriminate asthma phenotypes based on cytokine profiles in bronchoalveolar lavage (BAL) samples from mild-moderate and severe asthmatics.
Twenty five cytokines were measured in BAL samples of 84 patients (41 severe, 43 mild-moderate) using bead-based multiplex immunoassays. The normalized data were subjected to statistical and informatics analysis.
Four groups of asthmatic profiles could be identified on the basis of unsupervised analysis (hierarchical clustering) that were independent of treatment. One group, enriched in severe asthmatics, showed differences in BAL cellular content, reductions in baseline pulmonary function and enhanced response to methacholine provocation. Ten cytokines were identified that accurately predicted this group. Classification methods for predicting methacholine sensitivity were developed. The best model analysis predicted hyper-responders (HR) with 88% accuracy in 10 trials using a 10-fold cross validation. The cytokines that contributed to this model were IL-2, IL-4, and IL-5. Based on this classifier, three distinct HR Classes were identified that varied in BAL eosinophil count and PC20 methacholine.
Cytokine expression patterns in BAL can be used to identify distinct types of asthma and identify distinct subsets of methacholine HR. Further biomarker discovery in BAL may be informative.
Asthma; inflammation; cytokines; phenotypes; ELISA; hierarchical clustering; bioinformatics; class discovery
The "Th2 hypothesis for asthma" asserts that an increased ratio of Th2:Th1 cytokine production plays an important pathogenic role in asthma. Although widely embraced, the hypothesis has been challenged by various empirical observations and has been described as overly simplistic. We sought to establish whether CD3+CD28-mediated and antigen-independent accumulation of type 1 and type 2 T cells differs significantly between nonasthmatic and asthmatic populations.
An ex vivo system was used to characterize the regulation of IFN-γ-producing (type 1) and IL-13-producing (type 2) T cell accumulation in response to CD3+CD28 and IL-2 stimulation by flow cytometry.
IL-13-producing T cells increased in greater numbers in response to antigen-independent stimulation in peripheral blood lymphocytes from female atopic asthmatic subjects compared with male asthmatics and both male and female atopic non-asthmatic subjects. IFN-γ+ T cells increased in greater numbers in response to either antigen-independent or CD3+CD28-mediated stimulation in peripheral blood lymphocytes from atopic asthmatic subjects compared to non-asthmatic subjects, regardless of gender.
We demonstrate that T cells from asthmatics are programmed for increased accumulation of both type 2 and type 1 T cells. Gender had a profound effect on the regulation of type 2 T cells, thus providing a mechanism for the higher frequency of adult asthma in females.
Severe asthma causes the majority of asthma morbidity. Understanding mechanisms that contribute to the development of severe disease is important.
The goal of the Severe Asthma Research Program is to identify and characterize subjects with severe asthma to understand pathophysiologic mechanisms in severe asthma.
We performed a comprehensive phenotypic characterization (questionnaires, atopy and pulmonary function testing, phlebotomy, exhaled nitric oxide) in subjects with severe and not severe asthma.
A total of 438 subjects with asthma were studied (204 severe, 70 moderate, 164 mild). Severe subjects with asthma were older with longer disease duration (P < .0001), more daily symptoms, intense urgent health care utilization, sinusitis, and pneumonia (P ≤ .0001). Lung function was lower in severe asthma with marked bronchodilator reversibility (P < .001). The severe group had less atopy by skin tests (P = .0007), but blood eosinophils, IgE, and exhaled nitric oxide levels did not differentiate disease severity. A reduced FEV1, history of pneumonia, and fewer positive skin tests were risk factors for severe disease. Early disease onset (age < 12 years) in severe asthma was associated with longer disease duration (P < .0001) and more urgent health care, especially intensive care (P = .002). Later disease onset (age ≥ 12 years) was associated with lower lung function and sinopulmonary infections (P ≤ .02).
Severe asthma is characterized by abnormal lung function that is responsive to bronchodilators, a history of sinopulmonary infections, persistent symptoms, and increased health care utilization.
Lung function abnormalities in severe asthma are reversible in most patients, and pneumonia is a risk factor for the development of severe disease.
Severe asthma; definition; bronchodilator response; pathophysiology; phenotype; pneumonia
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
β-agonist treatment of asthma displays substantial interindividual variation, which has prompted polymorphism discovery and characterization of β2-adrenergic (β2AR) signaling genes. β2AR function undergoes desensitization during persistent agonist exposure due to receptor phosphorylation by G-protein coupled receptor kinases (GRKs). GRK5 was found to be highly expressed in airway smooth muscle, the tissue target for β-agonists. The coding region is polymorphic at codon 41, where Gln can be substituted by Leu (minor allele), but almost exclusively in those of African descent. In transfected cells, GRK5-Leu41 evoked a greater degree of agonist-promoted desensitization of adenylyl cyclase compared to GRK5-Gln41. Consistent with this functional effect, agonist-promoted β2AR phosphorylation was greater in cells expressing GRK5-Leu41, as was the rate of agonist-promoted receptor internalization. In studies with mutated β2AR lacking PKA-phosphorylation sites, this phenotype was confirmed as being GRK-specific. So, GRK5-Leu41 represents a gain-of-function polymorphism that evokes enhanced loss-of-function of β2AR during persistent agonist exposure, and thus may contribute to β-agonist variability in asthma treatment of African-Americans.
Polymorphism; tachyphylaxis; β-agonist; kinases; desensitization; asthma
Variation in ADAM33 has been shown to be important in the development of asthma and altered lung function. This relationship however, has not been investigated in the population susceptible to COPD; long term tobacco smokers. We evaluated the association between polymorphisms in ADAM33 gene with COPD and lung function in long term tobacco smokers.
Caucasian subjects, at least 50 year old, who smoked ≥ 20 pack-years (n = 880) were genotyped for 25 single nucleotide polymorphisms (SNPs) in ADAM33. COPD was defined as an FEV1/FVC ratio < 70% and percent-predicted (pp)FEV1 < 75% (n = 287). The control group had an FEV1/FVC ratio ≥ 70% and ppFEV1 ≥ 80% (n = 311) despite ≥ 20 pack years of smoking. Logistic and linear regressions were used for the analysis. Age, sex, and smoking status were considered as potential confounders.
Five SNPs in ADAM33 were associated with COPD (Q-1, intronic: p < 0.003; S1, Ile → Val: p < 0.003; S2, Gly → Gly: p < 0.04; V-1 intronic: p < 0.002; V4, in 3' untranslated region: p < 0.007). Q-1, S1 and V-1 were also associated with ppFEV1, FEV1/FVC ratio and ppFEF25–75 (p values 0.001 – 0.02). S2 was associated with FEV1/FVC ratio (p < 0.05). The association between S1 and residual volume revealed a trend toward significance (p value < 0.07). Linkage disequilibrium and haplotype analyses suggested that S1 had the strongest degree of association with COPD and pulmonary function abnormalities.
Five SNPs in ADAM33 were associated with COPD and lung function in long-term smokers. Functional studies will be needed to evaluate the biologic significance of these polymorphisms in the pathogenesis of COPD.
The asthmatic response to the common cold is highly variable and early characteristics that predict worsening of asthma control following a cold have not been identified.
In this prospective multi-center cohort study of 413 adult subjects with asthma, we used the mini-Asthma Control Questionnaire (mini-ACQ) to quantify changes in asthma control and the Wisconsin Upper Respiratory Symptom Survey-21 (WURSS-21) to measure cold severity. Univariate and multivariable models examined demographic, physiologic, serologic, and cold-related characteristics for their relationship to changes in asthma control following a cold.
We observed a clinically significant worsening of asthma control following a cold (increase in mini-ACQ of 0.69 ± 0.93). Univariate analysis demonstrated season, center location, cold length, and cold severity measurements all associated with a change in asthma control. Multivariable analysis of the covariates available within the first 2 days of cold onset revealed the day 2 and the cumulative sum of the day 1 and 2 WURSS-21 scores were significant predictors for the subsequent changes in asthma control.
In asthmatic subjects the cold severity measured within the first 2 days can be used to predict subsequent changes in asthma control. This information may help clinicians prevent deterioration in asthma control following a cold.
asthma; asthma control; common cold
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: The comprehensive evaluation of gene variation, haplotype structure, and linkage disequilibrium is important in understanding the function of β2-adrenergic receptor gene (ADRβ2) on disease susceptibility, pulmonary function, and therapeutic responses in different ethnic groups with asthma.
Objectives: To identify ADRβ2 polymorphisms and haplotype structure in white and African American subjects and to test for genotype and haplotype association with asthma phenotypes.
Methods: A 5.3-kb region of ADRβ2 was resequenced in 669 individuals from 429 whites and 240 African Americans. A total of 12 polymorphisms, representing an optimal haplotype tagging set, were genotyped in whites (338 patients and 326 control subjects) and African Americans (222 patients and 299 control subjects).
Results: A total of 49 polymorphisms were identified, 21 of which are novel; 31 polymorphisms (frequency > 0.03) were used to identify 24 haplotypes (frequency > 0.01) and assess linkage disequilibrium. Association with ratio (FEV1/FVC)2 for single-nucleotide polymorphism +79 (p < 0.05) was observed in African Americans. Significant haplotype association for (FEV1/FVC)2 was also observed in African Americans.
Conclusions: There are additional genetic variants besides +46 (Gly16Arg) that are important in determining asthma phenotypes. These data suggest that the length of a poly-C repeat (+1269) in the 3′ untranslated region of ADRβ2 may influence lung function, and may be important in delineating variation in β-agonist responses, especially in African Americans.
asthma; β2-adrenergic receptor; β-agonist therapy; DNA polymorphisms; pharmacogenomics
Changes in pulmonary function are important in determining asthma outcome. Genetic factors may influence airway obstruction in asthma. We performed a genomewide screen in 200 families of probands objectively diagnosed with asthma in the 1960s to identify chromosomal regions related to changes in pre- and postbronchodilator lung function (FEV1, VC, and FEV1%VC) and assess influences of early-life smoke exposure. Smoking (pack-years), age, sex, and height were covariates in variance component analyses. Significant evidence for linkage of pre- and postbronchodilator FEV1%VC was obtained for chromosome 2q32 (LOD,4.9, increasing to 6.03 with additional fine-mapping markers, and 3.2, respectively). Linkage existed for chromosome 5q for pre- and postbronchodilator VC (likelihood of disease [LOD], 1.8 and 2.6, respectively). Results for pre- and postbronchodilator FEV1 were less significant (LOD, 1.5 and 1.6, chromosomes 11p and 10q, respectively). Results were not affected by passive smoke exposure. There is significant evidence for linkage of FEV1%VC to chromosome 2q32 in families of probands with asthma, 35 cM proximal from linkage previously observed in families of probands with early-onset chronic obstructive pulmonary disease. Thus, there may be multiple genes on chromosome 2q that are important in determining presence and degree of airflow limitation in families ascertained for obstructive airway disease.
asthma; function; genes; linkage; lung
Single-nucleotide polymorphisms of the β2-adrenergic receptor gene and its 5′ promoter have been associated with differences in receptor function and desensitization. Linkage disequilibrium may account for inconsistencies in reported effects of isolated polymorphisms. Therefore, we have investigated the three most common homozygous haplotypes of the β2-adrenergic receptor (position 19 [Cys/Arg] of the 5′ leader cistron and positions 16 [Arg/Gly] and 27 [Gln/Glu] of the receptor) for putative differences in agonist-induced desensitization. Lymphocytes of well defined nonasthmatic, nonallergic subjects homozygous for the haplotype CysGlyGln, ArgGlyGlu, or CysArgGln were isolated. Desensitization of (−)-isoproterenol–induced cyclic adenosine monophosphate (cAMP) accumulation and β2-adrenergic receptor sequestration and downregulation were measured in relation to β2-adrenergic receptor-mediated inhibition of IFN-γ and interleukin-5 production. We observed that lymphocytes of individuals bearing the CysGlyGln haplotype were more susceptible to desensitization of the β-agonist–induced cAMP response than those of individuals with the ArgGlyGlu or CysArgGln haplotype. The haplotype-dependent desensitization of β-agonist–induced cAMP response was not associated with haplotype-dependent β2-adrenergic receptor sequestration or downregulation. In addition, our data suggest reduced inhibition, in lymphocytes of subjects with the CysGlyGln haplotype, of interleukin-5 production induced by treatment with antibodies to the T-cell receptor–CD3 complex and to costimulatory molecule CD28 (αCD3/αCD28). This is the first study demonstrating haplotype-related differences in agonist-induced β2-adrenergic receptor desensitization in primary human cells. This haplotype-related desensitization of the β2-adrenergic receptor in lymphocytes might have consequences regarding the regulation of helper T-cell type 2 inflammatory responses.
5′ leader cistron; cAMP; cytokine production; sequestration and downregulation; single-nucleotide polymorphism
Rationale: Increased oxidative stress and decreased superoxide dismutase (SOD) activity in the asthmatic airway are correlated to airflow limitation and hyperreactivity. We hypothesized that asthmatic individuals with higher levels of oxidative stress may have greater loss of SOD activity, which would be reflected systemically in loss of circulating SOD activity and clinically by development of severe asthma and/or worsening airflow limitation. Methods: To investigate this, serum SOD activity and proteins, the glutathione peroxidase/glutathione antioxidant system, and oxidatively modified amino acids were measured in subjects with asthma and healthy control subjects. Results: SOD activity, but not Mn-SOD or Cu,Zn-SOD protein, was lower in asthmatic serum as compared with control, and activity loss was significantly related to airflow limitation. Further, serum SOD activity demonstrated an inverse correlation with circulating levels of 3-bromotyrosine, a posttranslational modification of proteins produced by the eosinophil peroxidase system of eosinophils. Exposure of purified Cu,Zn-SOD to physiologically relevant levels of eosinophil peroxidase-generated reactive brominating species, reactive nitrogen species, or tyrosyl radicals in vitro confirmed that eosinophil-derived oxidative pathways promote enzyme inactivation. Conclusion: These findings are consistent with greater oxidant stress in asthma leading to greater inactivation of SOD, which likely amplifies inflammation and progressive airflow obstruction.
asthma; superoxide dismutase; glutathione; pulmonary functions; peroxidase