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1.  Predicting Inhaled Corticosteroid Response in Asthma with Two Associated SNPs 
The pharmacogenomics journal  2012;13(4):306-311.
Inhaled corticosteroids are the most commonly used controller medications prescribed for asthma. Two single-nucleotide polymorphisms (SNPs), rs1876828 in CRHR1 and rs37973 in GLCCI1, have previously been associated with corticosteroid efficacy. We studied data from four existing clinical trials of asthmatics who received inhaled corticosteroids and had lung function measured by forced expiratory volume in one second (FEV1) before and after the period of such treatment. We combined the two SNPs rs37973 and rs1876828 into a predictive test of FEV1 change using a Bayesian model, which identified patients with good or poor steroid response (highest or lowest quartile, respectively) with predictive performance of 65.7% (p = 0.039 vs. random) area under the receiver-operator characteristic curve in the training population and 65.9% (p = 0.025 vs. random) in the test population. These findings show that two genetic variants can be combined into a predictive test that achieves similar accuracy and superior replicability compared with single SNP predictors.
doi:10.1038/tpj.2012.15
PMCID: PMC3434304  PMID: 22641026
Pharmacogenetics; Asthma; Glucocorticoids; Predictive Modeling
2.  A polymorphism in the thyroid hormone receptor gene is associated with bronchodilator response in asthmatics 
The pharmacogenomics journal  2012;13(2):130-136.
A pro-asthmatic culture milieu and β2-agonist (isoproterenol) were previously shown to regulate the expression of select transcription factors (TFs) within human airway epithelial and smooth muscle cells. This study tests 1116 single nucleotide polymorphisms (SNPs) across 98 of these TF genes for association with bronchodilator response (BDR) in asthma patients. Genotyping was conducted using the Illumina HumanHap550v3 Beadchip in 403 non-Hispanic White asthmatic children and their parents. SNPs were evaluated for association with BDR using family and population-based analyses. Forty-two SNPs providing p values < 0.1 in both analyses were then genotyped in three adult asthma trials. One SNP 5’ of the thyroid hormone receptor beta gene was associated with BDR in the childhood population and two adult populations (p value = 0.0012). This investigation identified a novel locus for inter-individual variability in BDR and represents a translation of a cellular drug-response study to potential personalization of clinical asthma management.
doi:10.1038/tpj.2011.56
PMCID: PMC3349771  PMID: 22212731
Bronchodilator response; transcription factor; association; thyroid hormone receptor β; asthma; pharmacogenetics
3.  Effect of Vitamin D and Inhaled Corticosteroid Treatment on Lung Function in Children 
Rationale: Low vitamin D levels are associated with asthma and decreased airway responsiveness. Treatment with inhaled corticosteroids improves airway responsiveness and asthma control.
Objectives: To assess the effect of vitamin D levels on prebronchodilator FEV1, bronchodilator response, and responsiveness to methacholine (PC20, provocative concentration of methacholine producing a 20% decline in FEV1) in patients with asthma treated with inhaled corticosteroids.
Methods: We measured 25-hydroxyvitamin D levels in the serum of children with persistent asthma at the time of enrollment in the Childhood Asthma Management Program. We divided subjects into the vitamin D sufficiency (>30 ng/ml), insufficiency (20–30 ng/ml), and deficiency (<20 ng/ml) groups. Covariates included age, treatment, sex, body mass index, race, history of emergency department visits, hospitalizations, and season that vitamin D specimen was drawn. Our main outcome measures were change in prebronchodilator FEV1, bronchodilator response, and PC20 from enrollment to 8–12 months.
Measurements and Main Results: Of the 1,024 subjects, 663 (65%) were vitamin D sufficient, 260 (25%) were insufficient, and 101 (10%) were deficient. Vitamin D–deficient subjects were more likely to be older, African American, and have a higher body mass index compared with the vitamin D–sufficient and insufficient subjects. In the inhaled corticosteroid treatment group, prebronchodilator FEV1 increased from randomization to 12 months by 140 ml in the vitamin D–deficient group and prebronchodilator FEV1 increased by 330 ml in the vitamin D insufficiency group and by 290 ml in the vitamin D sufficiency group (P = 0.0072), in adjusted models.
Conclusions: In children with asthma treated with inhaled corticosteroids, vitamin D deficiency is associated with poorer lung function than in children with vitamin D insufficiency or sufficiency.
doi:10.1164/rccm.201202-0351OC
PMCID: PMC3480528  PMID: 22798322
asthma; vitamin D; lung function; forced expiratory volume; children
4.  ITGB5 and AGFG1 variants are associated with severity of airway responsiveness 
BMC Medical Genetics  2013;14:86.
Background
Airway hyperresponsiveness (AHR), a primary characteristic of asthma, involves increased airway smooth muscle contractility in response to certain exposures. We sought to determine whether common genetic variants were associated with AHR severity.
Methods
A genome-wide association study (GWAS) of AHR, quantified as the natural log of the dosage of methacholine causing a 20% drop in FEV1, was performed with 994 non-Hispanic white asthmatic subjects from three drug clinical trials: CAMP, CARE, and ACRN. Genotyping was performed on Affymetrix 6.0 arrays, and imputed data based on HapMap Phase 2, was used to measure the association of SNPs with AHR using a linear regression model. Replication of primary findings was attempted in 650 white subjects from DAG, and 3,354 white subjects from LHS. Evidence that the top SNPs were eQTL of their respective genes was sought using expression data available for 419 white CAMP subjects.
Results
The top primary GWAS associations were in rs848788 (P-value 7.2E-07) and rs6731443 (P-value 2.5E-06), located within the ITGB5 and AGFG1 genes, respectively. The AGFG1 result replicated at a nominally significant level in one independent population (LHS P-value 0.012), and the SNP had a nominally significant unadjusted P-value (0.0067) for being an eQTL of AGFG1.
Conclusions
Based on current knowledge of ITGB5 and AGFG1, our results suggest that variants within these genes may be involved in modulating AHR. Future functional studies are required to confirm that our associations represent true biologically significant findings.
doi:10.1186/1471-2350-14-86
PMCID: PMC3765944  PMID: 23984888
Asthma; Airway hyperresponsiveness; Genome-wide association study; ITGB5; AGFG1
5.  Corticosteroid use and bone mineral accretion in children with asthma: effect modification by vitamin D 
Background
The adverse effects of corticosteroids on bone mineral accretion (BMA) have been well documented. Vitamin D insufficiency, a prevalent condition in the pediatric population, has also been associated with decreased bone mineral density (BMD).
Objective
To determine whether children with asthma who have lower vitamin D levels are more susceptible to the negative effects of corticosteroids on BMD over time.
Methods
Children aged 5–12 years with mild-to-moderate asthma who participated in the Childhood Asthma Management Program were followed for a mean of 4.3 years. Total doses of inhaled and oral corticosteroids (OCS) were recorded, serum 25-hydroxyvitamin D3 levels were measured at the beginning of the trial and serial DEXA scans of the lumbar spine were performed. Annual BMA rates were defined as: [(BMD at 4 years follow-up − BMD at baseline)/4 years].
Results
BMA was calculated for 780 subjects. In boys, baseline vitamin D levels significantly modified the relationship between OCS and BMA (vitamin D x OCS interaction, p=0.023). Stratification by vitamin D levels showed a decrease in BMA with increased use of OCS in vitamin D insufficient boys only (p<0.001). Compared to vitamin D sufficient boys, vitamin D insufficient boys exposed to more than 2 courses of oral corticosteroids per year had twice the decrease in BMA rate (relative to boys who were OCS-unexposed).
Conclusions
Vitamin D levels significantly modified the effect of oral corticosteroids on bone mineral accretion in boys. Further research is needed to examine whether vitamin D supplementation in children with poorly controlled asthma may confer benefits to bone health.
doi:10.1016/j.jaci.2012.04.005
PMCID: PMC3387323  PMID: 22608570
Asthma; vitamin D; bone mineral density; corticosteroids
6.  Genomewide association study of the age of onset of childhood asthma 
BACKGROUND
Childhood asthma is a complex disease with known heritability and phenotypic diversity. Although an earlier onset has been associated with more severe disease, there has been no genome-wide association study of the age of onset of asthma in children.
OBJECTIVE
To identify genetic variants associated with earlier onset of childhood asthma.
METHODS
We conducted the first genome-wide association study (GWAS) of the age of onset of childhood asthma among participants in the Childhood Asthma Management Program (CAMP), and used three independent cohorts from North America, Costa Rica, and Sweden for replication.
RESULTS
Two SNPs were associated with earlier onset of asthma in the combined analysis of CAMP and the replication cohorts: : rs9815663 (Fisher’s P value=2.31 × 10−8) and rs7927044 (P=6.54 × 10−9). Of these two SNPs, rs9815663 was also significantly associated with earlier asthma onset in an analysis including only the replication cohorts. Ten SNPs in linkage disequilibrium with rs9815663 were also associated with earlier asthma onset (2.24 × 10−7 < P < 8.22 ×10−6). Having ≥1 risk allele of the two SNPs of interest (rs9815663 and rs7927044) was associated with lower lung function and higher asthma medication use during 4 years of follow-up in CAMP.
CONCLUSIONS
We have identified two SNPs associated with earlier onset of childhood asthma in four independent cohorts.
doi:10.1016/j.jaci.2012.03.020
PMCID: PMC3387331  PMID: 22560479
Asthma; pediatrics; age of onset; asthma genetics; C1orf100; genome-wide association study; pediatric asthma
7.  Integrative systems biology approaches in asthma pharmacogenomics 
Pharmacogenomics  2012;13(12):1387-1404.
In order to improve therapeutic outcomes, there is a tremendous need to identify patients who are likely to respond to a given asthma treatment. Pharmacogenomic studies have explained a portion of the variability in drug response and provided an increasing list of candidate genes and SNPs. However, as phenotypic variation arises from a network of complex interactions among genetic and environmental factors, rather than individual genes or SNPs, a multidisciplinary, systems-level approach is required in order to understand the inter-relationships among these factors. Systems biology, which seeks to capture interactions between genetic factors and other variables, offers a promising approach to improved therapeutic outcomes in asthma. This aritcle will review and update progress in the pharmacogenomics of asthma and then discuss the application of systems biology approaches to asthma pharmacogenomics.
doi:10.2217/pgs.12.126
PMCID: PMC3553555  PMID: 22966888
asthma; genes; GWAS; network medicine; pharmacogenomics; SNP; systems biology
8.  Genome-wide Association Identifies the T Gene as a Novel Asthma Pharmacogenetic Locus 
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.
doi:10.1164/rccm.201111-2061OC
PMCID: PMC3381232  PMID: 22538805
polymorphism; genome; pharmacogenomics; glucocorticoid
9.  Genomewide Association between GLCCI1 and Response to Glucocorticoid Therapy in Asthma 
The New England journal of medicine  2011;365(13):1173-1183.
BACKGROUND
The response to treatment for asthma is characterized by wide interindividual variability, with a significant number of patients who have no response. We hypothesized that a genomewide association study would reveal novel pharmacogenetic determinants of the response to inhaled glucocorticoids.
METHODS
We analyzed a small number of statistically powerful variants selected on the basis of a family-based screening algorithm from among 534,290 single-nucleotide polymorphisms (SNPs) to determine changes in lung function in response to inhaled glucocorticoids. A significant, replicated association was found, and we characterized its functional effects.
RESULTS
We identified a significant pharmacogenetic association at SNP rs37972, replicated in four independent populations totaling 935 persons (P = 0.0007), which maps to the glucocorticoid-induced transcript 1 gene (GLCCI1) and is in complete linkage disequilibrium (i.e., perfectly correlated) with rs37973. Both rs37972 and rs37973 are associated with decrements in GLCCI1 expression. In isolated cell systems, the rs37973 variant is associated with significantly decreased luciferase reporter activity. Pooled data from treatment trials indicate reduced lung function in response to inhaled glucocorticoids in subjects with the variant allele (P = 0.0007 for pooled data). Overall, the mean (± SE) increase in forced expiratory volume in 1 second in the treated subjects who were homozygous for the mutant rs37973 allele was only about one third of that seen in similarly treated subjects who were homozygous for the wild-type allele (3.2 ± 1.6% vs. 9.4 ± 1.1%), and their risk of a poor response was significantly higher (odds ratio, 2.36; 95% confidence interval, 1.27 to 4.41), with genotype accounting for about 6.6% of overall inhaled glucocorticoid response variability.
CONCLUSIONS
A functional GLCCI1 variant is associated with substantial decrements in the response to inhaled glucocorticoids in patients with asthma. (Funded by the National Institutes of Health and others; ClinicalTrials.gov number, NCT00000575.)
doi:10.1056/NEJMoa0911353
PMCID: PMC3667396  PMID: 21991891
10.  Acceptance of Asthma Pharmacogenetic Study by Children and Adults 
Background
Pharmacogenetic testing may change clinical medicine by allowing clinicians to tailor medications based on a patient’s genetic makeup, however, these tests must first be validated in large, real-life populations of subjects that include children. A dearth of knowledge exists for whether pediatric populations are as willing as adult populations to provide samples for such studies.
Objective
(1) To assess whether pediatric and adult patients with persistent asthma are willing to provide specimens for DNA extraction and genetic studies. (2) To assess whether patients’ willingness to provide blood as compared to buccal smear specimens differ.
Methods
Of 644 patients ages 4–38 years who had three or more prescription fills for inhaled corticosteroids in one year, 60% (385) were randomized to the blood specimen group and 40% (259) were randomized to the buccal smear group in order to study acceptance of different biospecimen collection methods. Research assistants contacted subjects to obtain consent, perform a phone survey, and request a specimen.
Results
There were no baseline differences between subjects randomized to the blood specimen group versus buccal smear group with respect to age, gender, or number of dispensings of inhaled corticosteroids. Of 259 subjects in the buccal smear group, 30% (78) provided samples, and of 385 subjects in the blood specimen group, 16% (60) provided samples. Subjects randomized to the buccal smear group were more likely to provide specimens for genetic study compared to subjects randomized to the blood specimen group (RR 1.21; 95% CI 1.10 – 1.32), even after adjusting for age. Pediatric subjects were more likely to provide specimens for genetic study than adult subjects with 23% (113) of pediatric subjects providing samples and 15% (25) of adult subjects providing samples (p=0.03).
Conclusion
Children with asthma are as likely to participate in genetic studies as adults. Both children and adult subjects are more likely to provide buccal smear specimens rather than blood specimens for genetic study.
doi:10.4172/2153-0645.1000103
PMCID: PMC3614400  PMID: 23560243
pharmacogenetics; patient recruitment; children; buccal swab; blood specimen
11.  Integration of Mouse and Human Genome-Wide Association Data Identifies KCNIP4 as an Asthma Gene 
PLoS ONE  2013;8(2):e56179.
Asthma is a common chronic respiratory disease characterized by airway hyperresponsiveness (AHR). The genetics of asthma have been widely studied in mouse and human, and homologous genomic regions have been associated with mouse AHR and human asthma-related phenotypes. Our goal was to identify asthma-related genes by integrating AHR associations in mouse with human genome-wide association study (GWAS) data. We used Efficient Mixed Model Association (EMMA) analysis to conduct a GWAS of baseline AHR measures from males and females of 31 mouse strains. Genes near or containing SNPs with EMMA p-values <0.001 were selected for further study in human GWAS. The results of the previously reported EVE consortium asthma GWAS meta-analysis consisting of 12,958 diverse North American subjects from 9 study centers were used to select a subset of homologous genes with evidence of association with asthma in humans. Following validation attempts in three human asthma GWAS (i.e., Sepracor/LOCCS/LODO/Illumina, GABRIEL, DAG) and two human AHR GWAS (i.e., SHARP, DAG), the Kv channel interacting protein 4 (KCNIP4) gene was identified as nominally associated with both asthma and AHR at a gene- and SNP-level. In EVE, the smallest KCNIP4 association was at rs6833065 (P-value 2.9e-04), while the strongest associations for Sepracor/LOCCS/LODO/Illumina, GABRIEL, DAG were 1.5e-03, 1.0e-03, 3.1e-03 at rs7664617, rs4697177, rs4696975, respectively. At a SNP level, the strongest association across all asthma GWAS was at rs4697177 (P-value 1.1e-04). The smallest P-values for association with AHR were 2.3e-03 at rs11947661 in SHARP and 2.1e-03 at rs402802 in DAG. Functional studies are required to validate the potential involvement of KCNIP4 in modulating asthma susceptibility and/or AHR. Our results suggest that a useful approach to identify genes associated with human asthma is to leverage mouse AHR association data.
doi:10.1371/journal.pone.0056179
PMCID: PMC3572953  PMID: 23457522
12.  Genome-wide association study reveals class I MHC–restricted T cell–associated molecule gene (CRTAM) variants interact with vitamin D levels to affect asthma exacerbations 
Background
It has recently been shown that vitamin D deficiency can increase asthma development and severity and that variations in vitamin D receptor genes are associated with asthma susceptibility.
Objective
We sought to find genetic factors that might interact with vitamin D levels to affect the risk of asthma exacerbation. Methods: We conducted a genome-wide study of gene–vitamin D interaction on asthma exacerbations using population-based and family-based approaches on 403 subjects and trios from the Childhood Asthma Management Program. Twenty-three polymorphisms with significant interactions were studied in a replication analysis in 584 children from a Costa Rican cohort. Results: We identified 3 common variants in the class I MHC–restricted T cell–associated molecule gene (CRTAM) that were associated with an increased rate of asthma exacerbations based on the presence of a low circulating vitamin D level. These results were replicated in a second independent population (unadjusted combined interaction, P =.00028–.00097; combined odds ratio, 3.28–5.38). One variant, rs2272094, is a nonsynonymous coding polymorphism of CRTAM. Functional studies on cell lines confirmed the interaction of vitamin D and rs2272094 on CRTAM expression. CRTAM is highly expressed in activated human CD8+ and natural killer T cells, both of which have been implicated in asthmatic patients.
Conclusion
The findings highlight an important gene-environment interaction that elucidates the role of vitamin D and CD8+ and natural killer T cells in asthma exacerbation in a genome-wide gene-environment interaction study that has been replicated in an independent population. The results suggest the potential importance of maintaining adequate vitamin D levels in subsets of high-risk asthmatic patients.
doi:10.1016/j.jaci.2011.09.034
PMCID: PMC3360942  PMID: 22051697
Gene-environment interaction; genome-wide association study; vitamin D; asthma exacerbation
13.  Genome-Wide Association Analysis in Asthma Subjects Identifies SPATS2L as a Novel Bronchodilator Response Gene 
PLoS Genetics  2012;8(7):e1002824.
Bronchodilator response (BDR) is an important asthma phenotype that measures reversibility of airway obstruction by comparing lung function (i.e. FEV1) before and after the administration of a short-acting β2-agonist, the most common rescue medications used for the treatment of asthma. BDR also serves as a test of β2-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 FEV1 after administration of a β2-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 β2-adrenergic receptor levels. Our results suggest that SPATS2L may be an important regulator of β2-adrenergic receptor down-regulation and that there is promise in gaining a better understanding of the biological mechanisms of differential response to β2-agonists through GWAS.
Author Summary
Bronchodilator response (BDR) is an important asthma phenotype that measures reversibility of airway obstruction by comparing lung function before and after the administration of short-acting β2-agonists, common medications used for asthma treatment. We performed a genome-wide association study of BDR with 1,644 white asthmatic subjects from six drug clinical trials and attempted to replicate these findings in 1,051 white subjects from two independent cohorts. The most significant associated variant was near the SPATS2L gene. We knocked down SPATS2L mRNA in human airway smooth muscle cells and found that β2-adrenergic receptor levels increased, suggesting that SPATS2L may be a regulator of BDR. Our results highlight the promise of pursuing GWAS results that do not necessarily reach genome-wide significance and are an example of how results from pharmacogenetic GWAS can be studied functionally.
doi:10.1371/journal.pgen.1002824
PMCID: PMC3390407  PMID: 22792082
14.  Thymic Stromal Lymphopoietin Gene Promoter Polymorphisms Are Associated with Susceptibility to Bronchial Asthma 
Thymic stromal lymphopoietin (TSLP) triggers dendritic cell–mediated T helper (Th) 2 inflammatory responses. A single-nucleotide polymorphism (SNP), rs3806933, in the promoter region of the TSLP gene creates a binding site for the transcription factor activating protein (AP)–1. The variant enhances AP-1 binding to the regulatory element, and increases the promoter–reporter activity of TSLP in response to polyinosinic-polycytidylic acid (poly[I:C]) stimulation in normal human bronchial epithelium (NHBE). We investigated whether polymorphisms including the SNP rs3806933 could affect the susceptibility to and clinical phenotypes of bronchial asthma. We selected three representative (i.e., Tag) SNPs and conducted association studies of the TSLP gene, using two independent populations (639 patients with childhood atopic asthma and 838 control subjects, and 641 patients with adult asthma and 376 control subjects, respectively). We further examined the effects of corticosteroids and a long-acting β2-agonist (salmeterol) on the expression levels of the TSLP gene in response to poly(I:C) in NHBE. We found that the promoter polymorphisms rs3806933 and rs2289276 were significantly associated with disease susceptibility in both childhood atopic and adult asthma. The functional SNP rs3806933 was associated with asthma (meta-analysis, P = 0.000056; odds ratio, 1.29; 95% confidence interval, 1.14–1.47). A genotype of rs2289278 was correlated with pulmonary function. Moreover, the induction of TSLP mRNA and protein expression induced by poly(I:C) in NHBE was synergistically impaired by a corticosteroid and salmeterol. TSLP variants are significantly associated with bronchial asthma and pulmonary function. Thus, TSLP may serve as a therapeutic target molecule for combination therapy.
doi:10.1165/rcmb.2009-0418OC
PMCID: PMC3159073  PMID: 20656951
asthma; TSLP; bronchial epithelial cells; combination therapy; genetic polymorphisms
15.  The Pharmacogenetics and Pharmacogenomics of Asthma Therapy 
The Pharmacogenomics Journal  2011;11(6):383-392.
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.
doi:10.1038/tpj.2011.46
PMCID: PMC3298891  PMID: 21987090
asthma; pharmacogenetics; pharmacogenomics
16.  Regulatory Haplotypes in ARG1 Are Associated with Altered Bronchodilator Response 
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).
doi:10.1164/rccm.201005-0758OC
PMCID: PMC3056223  PMID: 20851928
pharmacogenetics; asthma; β2-agonist
17.  Genetic Influences on Asthma Susceptibility in the Developing Lung 
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.
doi:10.1165/rcmb.2009-0412OC
PMCID: PMC3159089  PMID: 20118217
asthma susceptibility; lung development; developmental gene expression
18.  Chromosome 17: Association of a large inversion polymorphism with corticosteroid response in asthma 
Pharmacogenetics and genomics  2008;18(8):733-737.
A 900-KB inversion exists within a large region of conserved linkage disequilibrium (LD) on chromosome 17. CRHR1 is located within the inversion region and associated with inhaled corticosteroid response in asthma. We hypothesized that CRHR1 variants are in LD with the inversion, supporting a potential role for natural selection in the genetic response to corticosteroids. We genotyped 6 single nucleotide polymorphisms (SNPs) spanning chr17:40,410,565–42,372,240, including 4 SNPs defining inversion status. Similar allele frequencies and strong LD were noted between the inversion and a CRHR1 SNP previously associated with lung function response to inhaled corticosteroids. Each inversion-defining SNP was strongly associated with inhaled corticosteroid response in adult asthma (p-values 0.002–0.005). The CRHR1 response to inhaled corticosteroids may thus be explained by natural selection resulting from inversion status or by long-range LD with another gene. Additional pharmacogenetic investigations into to regions of chromosomal diversity, including copy number variation and inversions, are warranted.
doi:10.1097/FPC.0b013e3282fe6ebf
PMCID: PMC3225071  PMID: 18622266
CRHR1; tau haplotype; MAPT; inversion; asthma; corticosteroid; pharmacogenetics
19.  Association of corticotropin releasing hormone receptor 2 (CRHR2) genetic variants with acute bronchodilator response in asthma 
Pharmacogenetics and genomics  2008;18(5):373-382.
Objective
Corticotropin - releasing hormone receptor 2 (CRHR2) participates in smooth muscle relaxation response and may influence acute airway bronchodilator response to short – acting β2 agonist treatment of asthma. We aim to assess associations between genetic variants of CRHR2 and acute bronchodilator response in asthma.
Methods
We investigated 28 single nucleotide polymorphisms in CRHR2 for associations with acute bronchodilator response to albuterol in 607 Caucasian asthmatic subjects recruited as part of the Childhood Asthma Management Program (CAMP). Replication was conducted in two Caucasian adult asthma cohorts – a cohort of 427 subjects enrolled in a completed clinical trial conducted by Sepracor Inc. (MA, USA) and a cohort of 152 subjects enrolled in the Clinical Trial of Low-Dose Theopylline and Montelukast (LODO) conducted by the American Lung Association Asthma Clinical Research Centers.
Results
Five variants were significantly associated with acute bronchodilator response in at least one cohort (p-value ≤ 0.05). Variant rs7793837 was associated in CAMP and LODO (p-value = 0.05 and 0.03, respectively) and haplotype blocks residing at the 5’ end of CRHR2 were associated with response in all three cohorts.
Conclusion
We report for the first time, at the gene level, replicated associations between CRHR2 and acute bronchodilator response. While no single variant was significantly associated in all three cohorts, the findings that variants at the 5’ end of CRHR2 are associated in each of three cohorts strongly suggest that the causative variants reside in this region and its genetic effect, although present, is likely to be weak.
doi:10.1097/FPC.0b013e3282fa760a
PMCID: PMC3208318  PMID: 18408560
Asthma; genetics; corticotrophin releasing hormone receptor 2; CRHR2; bronchodilator response; polymorphism; β2 adrenergic receptor agonist
20.  Genome Wide Association Study to predict severe asthma exacerbations in children using random forests classifiers 
BMC Medical Genetics  2011;12:90.
Background
Personalized health-care promises tailored health-care solutions to individual patients based on their genetic background and/or environmental exposure history. To date, disease prediction has been based on a few environmental factors and/or single nucleotide polymorphisms (SNPs), while complex diseases are usually affected by many genetic and environmental factors with each factor contributing a small portion to the outcome. We hypothesized that the use of random forests classifiers to select SNPs would result in an improved predictive model of asthma exacerbations. We tested this hypothesis in a population of childhood asthmatics.
Methods
In this study, using emergency room visits or hospitalizations as the definition of a severe asthma exacerbation, we first identified a list of top Genome Wide Association Study (GWAS) SNPs ranked by Random Forests (RF) importance score for the CAMP (Childhood Asthma Management Program) population of 127 exacerbation cases and 290 non-exacerbation controls. We predict severe asthma exacerbations using the top 10 to 320 SNPs together with age, sex, pre-bronchodilator FEV1 percentage predicted, and treatment group.
Results
Testing in an independent set of the CAMP population shows that severe asthma exacerbations can be predicted with an Area Under the Curve (AUC) = 0.66 with 160-320 SNPs in comparison to an AUC score of 0.57 with 10 SNPs. Using the clinical traits alone yielded AUC score of 0.54, suggesting the phenotype is affected by genetic as well as environmental factors.
Conclusions
Our study shows that a random forests algorithm can effectively extract and use the information contained in a small number of samples. Random forests, and other machine learning tools, can be used with GWAS studies to integrate large numbers of predictors simultaneously.
doi:10.1186/1471-2350-12-90
PMCID: PMC3148549  PMID: 21718536
21.  Expression analysis of asthma candidate genes during human and murine lung development 
Respiratory Research  2011;12(1):86.
Background
Little is known about the role of most asthma susceptibility genes during human lung development. Genetic determinants for normal lung development are not only important early in life, but also for later lung function.
Objective
To investigate the role of expression patterns of well-defined asthma susceptibility genes during human and murine lung development. We hypothesized that genes influencing normal airways development would be over-represented by genes associated with asthma.
Methods
Asthma genes were first identified via comprehensive search of the current literature. Next, we analyzed their expression patterns in the developing human lung during the pseudoglandular (gestational age, 7-16 weeks) and canalicular (17-26 weeks) stages of development, and in the complete developing lung time series of 3 mouse strains: A/J, SW, C57BL6.
Results
In total, 96 genes with association to asthma in at least two human populations were identified in the literature. Overall, there was no significant over-representation of the asthma genes among genes differentially expressed during lung development, although trends were seen in the human (Odds ratio, OR 1.22, confidence interval, CI 0.90-1.62) and C57BL6 mouse (OR 1.41, CI 0.92-2.11) data. However, differential expression of some asthma genes was consistent in both developing human and murine lung, e.g. NOD1, EDN1, CCL5, RORA and HLA-G. Among the asthma genes identified in genome wide association studies, ROBO1, RORA, HLA-DQB1, IL2RB and PDE10A were differentially expressed during human lung development.
Conclusions
Our data provide insight about the role of asthma susceptibility genes during lung development and suggest common mechanisms underlying lung morphogenesis and pathogenesis of respiratory diseases.
doi:10.1186/1465-9921-12-86
PMCID: PMC3141421  PMID: 21699702
Asthma; Development; Expression; Genetics; Lung
22.  A Role for Wnt Signaling Genes in the Pathogenesis of Impaired Lung Function in 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.
doi:10.1164/rccm.200907-1009OC
PMCID: PMC2822972  PMID: 19926868
asthma; lung development; lung function; genetic variation; gene expression
23.  Global Analysis of the Impact of Environmental Perturbation on cis-Regulation of Gene Expression 
PLoS Genetics  2011;7(1):e1001279.
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.
doi:10.1371/journal.pgen.1001279
PMCID: PMC3024267  PMID: 21283786
24.  Transcriptomic Analysis of Human Lung Development 
Rationale: Current understanding of the molecular regulation of lung development is limited and derives mostly from animal studies.
Objectives: To define global patterns of gene expression during human lung development.
Methods: Genome-wide expression profiling was used to measure the developing lung transcriptome in RNA samples derived from 38 normal human lung tissues at 53 to 154 days post conception. Principal component analysis was used to characterize global expression variation and to identify genes and bioontologic attributes contributing to these variations. Individual gene expression patterns were verified by quantitative reverse transcriptase–polymerase chain reaction analysis.
Measurements and Main Results: Gene expression analysis identified attributes not previously associated with lung development, such as chemokine-immunologic processes. Lung characteristics attributes (e.g., surfactant function) were observed at an earlier-than-anticipated age. We defined a 3,223 gene developing lung characteristic subtranscriptome capable of describing a majority of the process. In gene expression space, the samples formed a time-contiguous trajectory with transition points correlating with histological stages and suggesting the existence of novel molecular substages. Induction of surfactant gene expression characterized a pseudoglandular “molecular phase” transition. Individual gene expression patterns were independently validated. We predicted the age of independent human lung transcriptome profiles with a median absolute error of 5 days, supporting the validity of the data and modeling approach.
Conclusions: This study extends our knowledge of key gene expression patterns and bioontologic attributes underlying early human lung developmental processes. The data also suggest the existence of molecular phases of lung development.
doi:10.1164/rccm.200907-1063OC
PMCID: PMC2797628  PMID: 19815808
microarrays; surfactant; principal component analysis
25.  Repeatability of Response to Asthma Medications 
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.
doi:10.1016/j.jaci.2008.10.015
PMCID: PMC2980870  PMID: 19064281
asthma; drug response; heritability; bronchodilator; pharmacogenetics

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