Despite the availability of several classes of asthma medications and their overall effectiveness, a significant portion of patients fail to respond to these therapeutic agents. Evidence suggests that genetic factors may partly mediate the heterogeneity in asthma treatment response. This review discusses important findings in asthma pharmacogenetics and pharmacogenomics studies conducted to date, examines limitations of these studies and finally, proposes future research directions in this field. The focus will be on the three major classes of asthma medications: β-adrenergic receptor agonists, inhaled corticosteroids and leukotriene modifiers. Although many studies are limited by small sample sizes and replication of the findings is needed, several candidate genes have been identified. High-throughput technologies is also allowing for large-scale genetic investigations. Thus, the future is promising for a personalized treatment of asthma, which will improve therapeutic outcomes, minimize side effects and lead to a more cost-effective care.

Asthma is the leading serious pediatric chronic illness in the United States, affecting 7.1 million children. The prevalence of asthma in children under 4 years of age has increased dramatically in the last 2 decades. Existing evidence suggests that this increase in prevalence derives from early environmental exposures acting on a pre-existing asthma-susceptible genotype. We studied the origins of asthma susceptibility in developing lung in rat strains that model the distinct phenotypes of airway hyperresponsiveness (Fisher rats) and atopy (brown Norway [BN] rats). Postnatal BN rat lungs showed increased epithelial proliferation and tracheal goblet cell hyperplasia. Fisher pups showed increased lung resistance at age 2 weeks, with elevated neutrophils throughout the postnatal period. Diverse transcriptomic signatures characterized the distinct respiratory phenotypes of developing lung in both rat models. Linear regression across age and strain identified developmental variation in expression of 1,376 genes, and confirmed both strain and temporal regulation of lung gene expression. Biological processes that were heavily represented included growth and development (including the T Box 1 transcription factor [Tbx5], the epidermal growth factor receptor [Egfr], the transforming growth factor beta-1-induced transcript 1 [Tgfbr1i1]), extracellular matrix and cell adhesion (including collagen and integrin genes), and immune function (including lymphocyte antigen 6 (Ly6) subunits, IL-17b, Toll-interacting protein, and Ficolin B). Genes validated by quantitative RT-PCR and protein analysis included collagen III alpha 1 Col3a1, Ly6b, glucocorticoid receptor and Importin-13 (specific to the BN rat lung), and Serpina1 and Ficolin B (specific to the Fisher lung). Innate differences in patterns of gene expression in developing lung that contribute to individual variation in respiratory phenotype are likely to contribute to the pathogenesis of asthma.

Rationale: Animal models demonstrate that aberrant gene expression in utero can result in abnormal pulmonary phenotypes.

Objectives: We sought to identify genes that are differentially expressed during in utero airway development and test the hypothesis that variants in these genes influence lung function in patients with asthma.

Methods: Stage 1 (Gene Expression): Differential gene expression analysis across the pseudoglandular (n = 27) and canalicular (n = 9) stages of human lung development was performed using regularized t tests with multiple comparison adjustments. Stage 2 (Genetic Association): Genetic association analyses of lung function (FEV1, FVC, and FEV1/FVC) for variants in five differentially expressed genes were conducted in 403 parent-child trios from the Childhood Asthma Management Program (CAMP). Associations were replicated in 583 parent-child trios from the Genetics of Asthma in Costa Rica study.

Measurements and Main Results: Of the 1,776 differentially expressed genes between the pseudoglandular (gestational age: 7–16 wk) and the canalicular (gestational age: 17–26 wk) stages, we selected 5 genes in the Wnt pathway for association testing. Thirteen single nucleotide polymorphisms in three genes demonstrated association with lung function in CAMP (P < 0.05), and associations for two of these genes were replicated in the Costa Ricans: Wnt1-inducible signaling pathway protein 1 with FEV1 (combined P = 0.0005) and FVC (combined P = 0.0004), and Wnt inhibitory factor 1 with FVC (combined P = 0.003) and FEV1/FVC (combined P = 0.003).

Conclusions: Wnt signaling genes are associated with impaired lung function in two childhood asthma cohorts. Furthermore, gene expression profiling of human fetal lung development can be used to identify genes implicated in the pathogenesis of lung function impairment in individuals with asthma.

Genetic variants altering cis-regulation of normal gene expression (cis-eQTLs) have been extensively mapped in human cells and tissues, but the extent by which controlled, environmental perturbation influences cis-eQTLs is unclear. We carried out large-scale induction experiments using primary human bone cells derived from unrelated donors of Swedish origin treated with 18 different stimuli (7 treatments and 2 controls, each assessed at 2 time points). The treatments with the largest impact on the transcriptome, verified on two independent expression arrays, included BMP-2 (t = 2h), dexamethasone (DEX) (t = 24h), and PGE2 (t = 24h). Using these treatments and control, we performed expression profiling for 18,144 RefSeq transcripts on biological replicates of the complete study cohort of 113 individuals (ntotal = 782) and combined it with genome-wide SNP-genotyping data in order to map treatment-specific cis-eQTLs (defined as SNPs located within the gene ±250 kb). We found that 93% of cis-eQTLs at 1% FDR were observed in at least one additional treatment, and in fact, on average, only 1.4% of the cis-eQTLs were considered as treatment-specific at high confidence. The relative invariability of cis-regulation following perturbation was reiterated independently by genome-wide allelic expression tests where only a small proportion of variance could be attributed to treatment. Treatment-specific cis-regulatory effects were, however, 2- to 6-fold more abundant among differently expressed genes upon treatment. We further followed-up and validated the DEX–specific cis-regulation of the MYO6 and TNC loci and found top cis-regulatory variants located 180 kb and 250 kb upstream of the transcription start sites, respectively. Our results suggest that, as opposed to tissue-specificity of cis-eQTLs, the interactions between cellular environment and cis-variants are relatively rare (∼1.5%), but that detection of such specific interactions can be achieved by a combination of functional genomic approaches as described here.

Author Summary

Population variation in normal gene expression has been convincingly shown to be under strong genetic control where the main genetic variants are located within close proximity to the gene itself (so called cis-acting). However, the extent to which controlled, environmental stimuli influences cis-regulation of gene expression is unclear. Here, we combine different functional genomic approaches and examine the role of common genetic variants on induced gene expression in a population panel of primary human cells derived from ∼100 unrelated donors treated under multiple conditions. Using these approaches, we find that the interaction between cellular environment and cis-variants are relatively rare, with only a small proportion of the identified genetic variants being specific to treatment. However, although treatment-specific genetic regulation of gene expression seems to be infrequent, we prove its existence by thorough validation of treatment-specific effects of the glucocorticoid-specific regulation of TNC expression. Taken together, these findings indicate that the regulatory landscape within a cell is very stable but, by combining functional genomic tools gene-environmental interactions of clinical importance, can be detected and possibly used as biomarkers in future pharmacogenomic studies.

Background

Pharmacogenetic studies of drug response in asthma assume that patients respond consistently to a treatment but that treatment response varies across patients, however, no formal studies have demonstrated this.

Objective

To determine the repeatability of commonly used outcomes for treatment response to asthma medications: bronchodilator response, forced expiratory volume in 1 second (FEV1), and provocative concentration of methacholine producing a 20% decline in FEV1 (PC20).

Methods

The Childhood Asthma Management Program (CAMP) was a multi-center clinical trial of children randomized to receiving budesonide, nedocromil, or placebo. We determined the intraclass correlation coefficient (ICC) for each outcome over repeated visits over four years in CAMP using mixed effects regression models. We adjusted for the covariates: age, race/ethnicity, height, family income, parental education, and symptom score. We incorporated each outcome for each child as repeated outcome measurements and stratified by treatment group.

Results

The ICC for bronchodilator response was 0.31 in the budesonide group, 0.35 in the nedocromil group, and 0.40 in the placebo group, after adjusting for covariates. The ICC for FEV1 was 0.71 in the budesonide group, 0.60 in the nedocromil group, and 0.69 in the placebo group, after adjusting for covariates. The ICC for PC20 was 0.67 in the budesonide and placebo groups and 0.73 in the nedocromil group, after adjusting for covariates.

Conclusion

The within treatment group repeatability of FEV1 and PC20 are high; thus these phenotypes are heritable. FEV1 and PC20 may be better phenotypes than bronchodilator response for studies of treatment response in asthma.

Aims

Bronchodilator response tests measure the effect of β2-agonists, the most commonly used short-acting reliever drugs for asthma. We sought to relate candidate gene SNP data with bronchodilator response and measure the predictive accuracy of a model constructed with genetic variants.

Materials & methods

Bayesian networks, multivariate models that are able to account for simultaneous associations and interactions among variables, were used to create a predictive model of bronchodilator response using candidate gene SNP data from 308 Childhood Asthma Management Program Caucasian subjects.

Results

The model found that 15 SNPs in 15 genes predict bronchodilator response with fair accuracy, as established by a fivefold cross-validation area under the receiver-operating characteristic curve of 0.75 (standard error: 0.03).

Conclusion

Bayesian networks are an attractive approach to analyze large-scale pharmacogenetic SNP data because of their ability to automatically learn complex models that can be used for the prediction and discovery of novel biological hypotheses.

Background

Glucocorticoid function is dependent on efficient translocation of the glucocorticoid receptor (GR) from the cytoplasm to the nucleus of cells. Importin-13 (IPO13) is a nuclear transport receptor that mediates nuclear entry of GR. In airway epithelial cells, inhibition of IPO13 expression prevents nuclear entry of GR and abrogates anti-inflammatory effects of glucocorticoids. Impaired nuclear entry of GR has been documented in steroid-non-responsive asthmatics. We hypothesize that common IPO13 genetic variation influences the anti-inflammatory effects of inhaled corticosteroids for the treatment of asthma, as measured by change in methacholine airway hyperresponsiveness (AHR-PC20).

Methods

10 polymorphisms were evaluated in 654 children with mild-to-moderate asthma participating in the Childhood Asthma Management Program (CAMP), a clinical trial of inhaled anti-inflammatory medications (budesonide and nedocromil). Population-based association tests with repeated measures of PC20 were performed using mixed models and confirmed using family-based tests of association.

Results

Among participants randomized to placebo or nedocromil, IPO13 polymorphisms were associated with improved PC20 (i.e. less AHR), with subjects harboring minor alleles demonstrating an average 1.51–2.17 fold increase in mean PC20 at 8-months post-randomization that persisted over four years of observation (p = 0.01–0.005). This improvement was similar to that among children treated with long-term inhaled corticosteroids. There was no additional improvement in PC20 by IPO13 variants among children treated with inhaled corticosteroids.

Conclusion

IPO13 variation is associated with improved AHR in asthmatic children. The degree of this improvement is similar to that observed with long-term inhaled corticosteroid treatment, suggesting that IPO13 variation may improve nuclear bioavailability of endogenous glucocorticoids.

Background

With the recent development of microarray technologies, the comparability of gene expression data obtained from different platforms poses an important problem. We evaluated two widely used platforms, Affymetrix U133 Plus 2.0 and the Illumina HumanRef-8 v2 Expression Bead Chips, for comparability in a biological system in which changes may be subtle, namely fetal lung tissue as a function of gestational age.

Results

We performed the comparison via sequence-based probe matching between the two platforms. "Significance grouping" was defined as a measure of comparability. Using both expression correlation and significance grouping as measures of comparability, we demonstrated that despite overall cross-platform differences at the single gene level, increased correlation between the two platforms was found in genes with higher expression level, higher probe overlap, and lower p-value. We also demonstrated that biological function as determined via KEGG pathways or GO categories is more consistent across platforms than single gene analysis.

Conclusion

We conclude that while the comparability of the platforms at the single gene level may be increased by increasing sample size, they are highly comparable ontologically even for subtle differences in a relatively small sample size. Biologically relevant inference should therefore be reproducible across laboratories using different platforms.

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.

Rationale: T-bet (TBX21 or T-box 21) is a critical regulator of T-helper 1 lineage commitment and IFN-γ production. Knockout mice lacking T-bet develop airway hyperresponsiveness (AHR) to methacholine, peribronchial eosinophilic and lymphocytic inflammation, and increased type III collagen deposition below the bronchial epithelium basement membrane, reminiscent of both acute and chronic asthma histopathology. Little is known regarding the role of genetic variation surrounding T-bet in the development of human AHR.

Objectives: To assess the relationship between T-bet polymorphisms and asthma-related phenotypes using family-based association.

Methods: Single nucleotide polymorphism discovery was performed by resequencing the T-bet genomic locus in 30 individuals (including 22 patients with asthma). Sixteen variants were genotyped in 580 nuclear families ascertained through offspring with asthma from the Childhood Asthma Management Program clinical trial. Haplotype patterns were determined from this genotype data. Family-based tests of association were performed with asthma, AHR, lung function, total serum immunoglobulin E, and blood eosinophil levels.

Main Results: We identified 24 variants. Evidence of association was observed between c.−7947 and asthma in white families using both additive (p = 0.02) or dominant models (p = 0.006). c.−7947 and three other variants were also associated with AHR (log-methacholine PC20, p = 0.02–0.04). Haplotype analysis suggested that an AHR locus is in linkage disequilibrium with variants in the 3′UTR. Evidence of association of AHR with c.−7947, but not with other 3′UTR SNPs, was replicated in an independent cohort of adult males with AHR.

Conclusions: These data suggest that T-bet variation contributes to airway responsiveness in asthma.