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1.  Genome-Wide Association Study Identifies Novel Pharmacogenomic Loci For Therapeutic Response to Montelukast in Asthma 
PLoS ONE  2015;10(6):e0129385.
Genome-wide association study (GWAS) is a powerful tool to identify novel pharmacogenetic single nucleotide polymorphisms (SNPs). Leukotriene receptor antagonists (LTRAs) are a major class of asthma medications, and genetic factors contribute to variable responses to these drugs. We used GWAS to identify novel SNPs associated with the response to the LTRA, montelukast, in asthmatics.
Using genome-wide genotype and phenotypic data available from American Lung Association - Asthma Clinical Research Center (ALA-ACRC) cohorts, we evaluated 8-week change in FEV1 related to montelukast administration in a discovery population of 133 asthmatics. The top 200 SNPs from the discovery GWAS were then tested in 184 additional samples from two independent cohorts.
Twenty-eight SNP associations from the discovery GWAS were replicated. Of these, rs6475448 achieved genome-wide significance (combined P = 1.97 x 10-09), and subjects from all four studies who were homozygous for rs6475448 showed increased ΔFEV1 from baseline in response to montelukast.
Through GWAS, we identified a novel pharmacogenomic locus related to improved montelukast response in asthmatics.
PMCID: PMC4470685  PMID: 26083242
2.  Genetic predictors associated with improvement of asthma symptoms in response to inhaled corticosteroids 
To date, genome-wide association studies (GWASs) of inhaled corticosteroid (ICS) response in asthmatic patients have focused primarily on lung function and exacerbations.
We hypothesized that GWAS analysis could identify novel genetic markers predicting a symptomatic response to ICSs.
We analyzed differences in asthma symptoms in response to ICSs in 124 white children from the Childhood Asthma Management Program (CAMP) trial using scores from diary cards. Of the 440,862 single nucleotide polymorphisms (SNPs) analyzed, the top 100 ranked SNPs were pursued for replication initially in subjects from the pediatric Childhood Asthma Research and Education trials (77 white children) and then in subjects from the adult Asthma Clinical Research Network (110 white adults) and Leukotriene Modifier or Corticosteroid or Corticosteroid-Salmeterol trials (110 white adults).
The lowest P value for GWAS analysis in the CAMP trial was 8.94 × 10−8 (rs2388639). Of the 60 SNPs available in the Childhood Asthma Research and Education Network trials, rs1558726 (combined P = 1.02 × 10−5), rs2388639 (combined P = 8.56 × 10−9), and rs10044254 (combined P = 9.16 × 10−8) independently replicated. However, these 3 SNPs were not additionally replicated in the adult asthmatic patients of the remaining trials. rs10044254 lies in the intronic region of F-box and leucine-rich repeat protein 7 (FBXL7) and is associated with decreased expression in immortalized B cells derived from CAMP participants.
We have identified a novel SNP, rs10044254, associated with both decreased expression of FBXL7 and improved symptomatic response to ICSs in 2 independent pediatric cohorts. Our results suggest that there might be a specific genetic mechanism regulating symptomatic response to ICSs in children that does not carry over to adults.
PMCID: PMC4112383  PMID: 24486069
Asthma; child; glucocorticoid; pharmacogenomics; polymorphism
4.  Obesity in Children with Poorly-Controlled Asthma: Sex Differences 
Pediatric pulmonology  2012;48(9):847-856.
Obesity increases asthma risk, and may alter asthma severity. In adults, sex appears to modify the effect of obesity on asthma. Among children, the effect of sex on the relationship between obesity and asthma severity remains less clear, particularly when considering race.
To determine how obesity affects disease characteristics in a diverse cohort of children with poorly controlled asthma, and if obesity effects are altered by sex.
We analyzed 306 children between 6–17 years of age with poorly controlled asthma enrolled in a 6-month trial assessing lansoprazole for asthma control. In this secondary analysis, we determined associations between obesity and symptom severity, spirometry, exacerbation risk, airway biomarkers, bronchial reactivity and airflow perception. We used both a multivariate linear regression and longitudinal mixed-effect model to determine if obesity interacted with sex to affect asthma severity.
Regardless of sex, BMI>95th percentile did not affect asthma control, exacerbation risk or airway biomarkers. Sex changed the effect of obesity on lung function (sex*obesity FEV1%, interaction p-value <.01, sex*obesity FEV1/FVC, interaction p-value=.03). Obese males had significantly worse airflow obstruction compared to non-obese males, while in females there was no obesity effect on airflow obstruction. In females, obesity was associated with significantly greater FEV1 and FVC, and a trend toward reduced airway reactivity.
Obesity did not affect asthma control, airway markers or disease stability; however obesity did affect lung function in a sex-dependent manner. In males, obesity associated with reduced FEV1/FVC, and in females, obesity associated with substantially improved lung function.
PMCID: PMC3578966  PMID: 23143849
Asthma; Obesity; Children; Sex; Body Mass Index; Spirometry
5.  A genome-wide association study of bronchodilator response in asthmatics 
The pharmacogenomics journal  2013;14(1):41-47.
Reversibility of airway obstruction in response to β2-agonists is highly variable among asthmatics, which is partially attributed to genetic factors. In a genome-wide association study of acute bronchodilator response (BDR) to inhaled albuterol, 534,290 single nucleotide polymorphisms (SNPs) were tested in 403 white trios from the Childhood Asthma Management Program using five statistical models to determine the most robust genetic associations. The primary replication phase included 1397 polymorphisms in three asthma trials (pooled n=764). The second replication phase tested 13 SNPs in three additional asthma populations (n=241, n=215, and n=592). An intergenic SNP on chromosome 10, rs11252394, proximal to several excellent biological candidates, significantly replicated (p=1.98×10−7) in the primary replication trials. An intronic SNP (rs6988229) in the collagen (COL22A1) locus also provided strong replication signals (p=8.51×10−6). This study applied a robust approach for testing the genetic basis of BDR and identified novel loci associated with this drug response in asthmatics.
PMCID: PMC3706515  PMID: 23508266
pharmacogenetics; asthma; bronchodilator response; genome-wide association study; albuterol
6.  Polygenic Heritability Estimates in Pharmacogenetics: Focus on Asthma and Related Phenotypes 
Pharmacogenetics and genomics  2013;23(6):324-328.
While accurate measures of heritability are needed to understand the pharmacogenetic basis of drug treatment response, these are generally not available, since it is unfeasible to give medications to individuals for which treatment is not indicated. Using a polygenic linear mixed modeling approach, we estimated lower-bounds on asthma heritability and the heritability of two related drug-response phenotypes, bronchodilator response and airway hyperreactivity, using genome-wide SNP data from existing asthma cohorts. Our estimate of the heritability for bronchodilator response is 28.5% (se 16%, p = 0.043) and airway hyperresponsiveness is 51.1% (se 34%, p = 0.064), while we estimate asthma genetic liability at 61.5% (se 16%, p < 0.001). Our results agree with previously published estimates of the heritability of these traits, suggesting that the LMM method is useful for computing the heritability of other pharmacogenetic traits. Furthermore, our results indicate that multiple SNP main-effects, including SNPs as yet unidentified by GWAS methods, together explain a sizable portion of the heritability of these traits.
PMCID: PMC3767309  PMID: 23532052
Asthma; Pharmacogenetics; Heritability; Bronchodilator Response; Airway Hyperresponsiveness
7.  ALOX5 Polymorphism Associates with Increased Leukotriene Production and Reduced Lung Function and Asthma Control in Children with Poorly Controlled Asthma 
Identification of risk factors for reduced asthma control could improve the understanding and treatment of asthma. A promoter polymorphism in the 5-lipoxygenase gene affects gene expression and response to asthma therapy but its impact on disease control remains unclear.
We sought to determine if the ALOX5 promoter SP1 tandem repeat polymorphism was associated with changes in cysteinyl leukotriene production, lung function, airway inflammation and asthma control score.
We analyzed 270 children 6-17 years old with poorly controlled asthma enrolled in a 6-month clinical trial (NCT00604851). In secondary analysis, we associated the ALOX5 promoter SP1 tandem repeat polymorphism genotype (rs59439148) with asthma outcomes using both additive and recessive genetic models. We evaluated FEV1 percent predicted, symptom control, exhaled nitric oxide and urinary LTE4 levels.
14.8% (40/270) of all children (and 28% (38/135) of African Americans) carried 2 non-5 repeat variant alleles of rs59439148. Children who were homozygous for variant alleles had significantly higher urinary LTE4 levels (38 versus 30 nmol/mol creatinine, p=.0134), significantly worse FEV1% predicted (84 versus 91, p=.017), and a trend toward worse asthma control. FEV1% predicted values were significantly negatively correlated with urinary LTE4 (r = -0.192, p=.009).
Conclusion and Clinical Relevance
Carrying two copies of a minor variant ALOX5 promoter SP1 tandem repeat allele contributes to increased cysLT exposure as determined by urinary LTE4 levels, reduced lung function, and potentially worse asthma control. ALOX5 promoter SP1 tandem repeat genotype may be a risk factor for worse asthma outcomes.
PMCID: PMC3633142  PMID: 23600541
Asthma; Children; ALOX5; 5-Lipoxygenase; Leukotriene; Asthma Control; FEV1; Exhaled Nitric Oxide; Genetic Association
8.  Nutrigenetic Response to Omega-3 Fatty Acids in Obese Asthmatics (NOOA): Rationale and Methods 
Contemporary clinical trials  2013;34(2):326-335.
Uncontrolled asthma is a major cause of hospitalizations and emergency room visits. Factors including obesity, African ancestry and childhood are associated with increased asthma severity. Considering the high morbidity caused by asthma, relatively few classes of drugs exist to control this common disease. Therefore, new therapeutic strategies may be needed to reduce asthma’s impact on public health. Data suggest that a high fat diet that is deficient in omega-3 fatty acids could promote both obesity and excessive inflammation, resulting in greater asthma severity. Small trials with supplemental omega-3 fatty acids have been conducted with encouraging but inconsistent results. The variability in response seen in past trials may be due to the past subjects’ genetics (specifically ALOX5 rs59439148) or their particular asthma phenotypes. Therefore, the “Nutrigenetic response to Omega-3 Fatty acids in Obese Asthmatics (NOOA)” trial is currently underway and was designed as a randomized, double-blind, placebo controlled intervention study to determine if supplemental omega-3 fatty acids improves symptoms among obese adolescents and young adults with uncontrolled asthma. Here we report the design and rationale for the NOOA trial. Participants were given either 3.18g daily of eicosapentaenoic acid and 822mg daily docosahexaenoic acid, or matched control soy oil, for 24 weeks. Change in the asthma control questionnaire score was the primary outcome. Secondary outcomes included spirometry, impulse oscillometry, exacerbation rate, airway biomarkers, systemic inflammation, leukotriene biosynthesis and T-lymphocyte function. NOOA may lead to a new therapeutic treatment strategy and greater understanding of the mechanistic role of diet in the pathogenesis of asthma.
PMCID: PMC3638234  PMID: 23298524
Asthma Control; Obesity; Children; Nutrigenetics; Omega-3 Fatty Acids
9.  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.
PMCID: PMC3434304  PMID: 22641026
Pharmacogenetics; Asthma; Glucocorticoids; Predictive Modeling
10.  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.
PMCID: PMC3349771  PMID: 22212731
Bronchodilator response; transcription factor; association; thyroid hormone receptor β; asthma; pharmacogenetics
11.  Genomewide Association between GLCCI1 and Response to Glucocorticoid Therapy in Asthma 
The New England journal of medicine  2011;365(13):1173-1183.
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.
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.
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.
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; number, NCT00000575.)
PMCID: PMC3667396  PMID: 21991891
12.  Absorption of Montelukast is Transporter Mediated: a Common Variant of OATP2B1 is Associated with Reduced Plasma Concentrations and Poor Response 
Pharmacogenetics and genomics  2009;19(2):129-138.
(i) determine if montelukast undergoes carrier mediated uptake; (ii) classify the carrier protein(s) responsible for uptake; (iii) identify specific transporters that mediate transport of montelukast; (iv) evaluate whether variation in the gene encoding the transport protein(s) influences the pharmacokinetics and pharmacodynamics of montelukast.
In vitro permeability studies of montelukast are conducted using Caco-2 cell culture, a standard model of human intestinal drug transport. In vivo plasma concentrations of montelukast in an asthmatic population are determined by HPLC, and genotyping of transport proteins is by LightTyper analysis.
Permeability of montelukast has an activation energy of 13.7±0.7 kcal/mol, consistent with carrier mediated transport. Permeability is saturable at high concentrations of montelukast and follows Michaelis-Menten kinetics. Permeability is subject to competition by sulfobromophthalein, estrone 3-sulfate, pravastatin, taurocholic acid, and cholic acid (p<0.05, % of control; 72±7 – 86±7) and is inhibited by 5–10% citrus juice (p<0.05, maximal inhibition % of control; 31±2). An MDCKII cell line expressing OATP2B1 (coded for by the SLCO2B1 gene) displays significantly increased permeability of montelukast (p<0.05, % of control; 140±20). A nonsynonymous polymorphism in SLCO2B1, (rs12422149; SLCO2B1{NM_007256.2}:c.935G>A) associates with significantly reduced plasma concentration in subjects measured on the morning after an evening dose (p<0.025, square root mean transformed plasma concentration ± SE; c.[935G>A]+[935G]=3±1, c.[935G]+[935G]=7.0±0.9) and differential response as assessed by change in baseline Asthma Symptom Utility Index scores following one month of therapy (delta mean Asthma Symptom Utility Index; c.[935G>A]+[935G] = 0.02±0.01, p=1.0; c.[935G]+[935G] = 1.0±0.3, p<0.0001).
Together these observations suggest that the genetics of SLCO2B1 may be an important variable in determining the pharmacokinetics and the pharmacodynamics of montelukast.
PMCID: PMC2641037  PMID: 19151602
Asthma; human OATP2B1 protein; intestinal absorption; leukotriene antagonists; membrane transport proteins; montelukast; pharmacodynamics; pharmacokinetics; single nucleotide polymorphism
13.  Pharmacogenetics of asthma 
Purpose of review
Patient response to the asthma drug classes, bronchodilators, inhaled corticosteroids and leukotriene modifiers, are characterized by a large degree of heterogeneity, which is attributable in part to genetic variation. Herein, we review and update the pharmacogenetics and pharmaogenomics of common asthma drugs.
Recent findings
Early studies suggest that bronchodilator reversibility and asthma worsening in patients on continuous short-acting and long-acting β-agonists are related to the Gly16Arg genotype for the ADRB2. More recent studies including genome-wide association studies implicate variants in other genes contribute to bronchodilator response heterogeneity and fail to replicate asthma worsening associated with continuous β-agonist use. Genetic determinants of the safety of long-acting β-agonist require further study. Variants in CRHR1, TBX21, and FCER2 contribute to variability in response for lung function, airways responsiveness, and exacerbations in patients taking inhaled corticosteroids. Variants in ALOX5, LTA4H, LTC4S, ABCC1, CYSLTR2, and SLCO2B1 contribute to variability in response to leukotriene modifiers.
Identification of novel variants that contribute to response heterogeneity supports future studies of single nucleotide polymorphism discovery and include gene expression and genome-wide association studies. Statistical models that predict the genomics of response to asthma drugs will complement single nucleotide polymorphism discovery in moving toward personalized medicine.
PMCID: PMC2754311  PMID: 19077707
asthma; genes; personalized medicine; polymorphisms; response heterogeneity
14.  Association of corticotropin releasing hormone receptor 2 (CRHR2) genetic variants with acute bronchodilator response in asthma 
Pharmacogenetics and genomics  2008;18(5):373-382.
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.
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.
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.
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.
PMCID: PMC3208318  PMID: 18408560
Asthma; genetics; corticotrophin releasing hormone receptor 2; CRHR2; bronchodilator response; polymorphism; β2 adrenergic receptor agonist
15.  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.
PMCID: PMC3572953  PMID: 23457522
16.  Asthma in the Elderly: Current Understanding and Future Research Needs 
Asthma in the elderly (AIE) is under diagnosed and under treated and there is a paucity of knowledge. The National Institute on Aging convened this workshop to identify what is known, what gaps in knowledge remain and suggest research directions needed to improve the understanding and care of AIE. Asthma presenting at an advanced age often has similar clinical and physiologic consequences as seen with younger individuals but co-morbid illnesses and the psychosocial effects of aging may affect the diagnosis, clinical presentation and care of asthma in this population. At least two phenotypes exist among elderly asthma; those with long-standing asthma have more severe airflow limitation and less complete reversibility than those with late-onset asthma. Many challenges exist in the recognition and treatment of asthma in the elderly. Furthermore, the pathophysiological mechanisms of AIE are likely to be different from those seen in young asthmatics and these differences may influence the clinical course and outcomes of asthma in this population.
PMCID: PMC3164961  PMID: 21872730
Aging; airway; allergy; asthma; elderly; immune mechanisms; immunosenescence
Genes involved in the regulation of catecholamine function may be important in obesity because of the role catecholamines play in energy expenditure and lipolysis. To determine if common single nucleotide polymorphisms (SNPs) in β1 (ADRB1), β2 (ADRB2), β3 (ADRB3) and α2a adrenergic receptor (ADRA2A) genes associate with obesity and metabolic alterations, we recruited 74 healthy African American and 161 Caucasian males and females (age: 18–49y) to participate in this case-control genetic association study. Genotypes were determined by PCR and RFLP. Associations between genotype and body mass index (BMI), percent body fat (by measuring skin fold thickness in 7 different sites), fasting (12-hour) plasma glucose, insulin, potassium concentrations, glycated hemoglobin (Hgb AIC) and insulin resistance (homeostasis model assessment score (HOMA-IR)) were performed. Among Caucasians, the ADRB1 Arg389→Gly variant associated with insulin concentrations and HOMAIR: mean ± SD values for insulin and HOMAIR in Arg389 homozygotes and carriers of the Gly were 10 ± 7.0 and 12 ± 9.4 μIU/ml (p=0.02) and 2.1 ± 1.7 and 2.6 ± 2.2 (p=0.057), respectively. Systolic blood pressure was higher in Caucasian for carriers of the ADBR1 Ser49 compared to Gly49 homozygotes 124 ± 12.6 vs. 119 ± 11.3 mm Hg, respectively (p = 0.02). Subsequent analysis revealed that these associations were attributable to a higher BMI among obese participants. The ADRA2A G1780A SNP associated with BMI and percent body fat in African Americans (p=0.05). Interactions were detected between ADRA2A C-1291G and ADRB2 Gln27→Glu variants for obesity in African Americans; and between ADRA2A C-1291G SNP and ADBR1 haplotype for obesity in Caucasians. We conclude that common SNPs in adrenergic receptor genes may be important susceptibility loci for obesity and related alterations. Because of the limited size of our populations, our results should be interpreted with caution and should be replicated in larger populations.
PMCID: PMC2040233  PMID: 17512307
association study; obesity; adrenergic receptor polymorphisms; insulin resistance; gene-gene interactions
18.  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.
PMCID: PMC3390407  PMID: 22792082
19.  Influence of Leukotriene Pathway Polymorphisms on Response to Montelukast in Asthma 
Rationale: Interpatient variability in montelukast response may be related to variation in leukotriene pathway candidate genes.
Objective: To determine associations between polymorphisms in leukotriene pathway candidate genes with outcomes in patients with asthma receiving montelukast for 6 mo who participated in a clinical trial.
Methods: Polymorphisms were typed using Sequenom matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass array spectrometry and published methods; haplotypes were imputed using single nucleotide polymorphism–expectation maximization (SNP-EM). Analysis of variance and logistic regression models were used to test for changes in outcomes by genotype. In addition, χ2 and likelihood ratio tests were used to test for differences between groups. Case-control comparisons were analyzed using the SNP-EM Omnibus likelihood ratio test.
Measurements: Outcomes were asthma exacerbation rate and changes in FEV1 compared with baseline.
Results: DNA was collected from 252 participants: 69% were white, 26% were African American. Twenty-eight SNPs in the ALOX5, LTA4H, LTC4S, MRP1, and cysLT1R genes, and an ALOX5 repeat polymorphism were successfully typed. There were racial disparities in allele frequencies in 17 SNPs and in the repeat polymorphism. Association analyses were performed in 61 whites. Associations were found between genotypes of SNPs in the ALOX5 (rs2115819) and MRP1 (rs119774) genes and changes in FEV1 (p < 0.05), and between two SNPs in LTC4S (rs730012) and in LTA4H (rs2660845) genes for exacerbation rates. Mutant ALOX5 repeat polymorphism was associated with decreased exacerbation rates. There was strong linkage disequilibrium between ALOX5 SNPs. Associations between ALOX5 haplotypes and risk of exacerbations were found.
Conclusions: Genetic variation in leukotriene pathway candidate genes contributes to variability in montelukast response.
PMCID: PMC2662939  PMID: 16293801
antiinflammatory; montelukast; pharmacodynamic; pharmacogenetic
20.  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 (NCT00156819, NCT00046644, and NCT00073840).
PMCID: PMC3056223  PMID: 20851928
pharmacogenetics; asthma; β2-agonist
21.  Genotyping the GGGCGG Tandem Repeat Promoter Polymorphism in the 5-Lipoxygenase Enzyme Gene (ALOX5) by Pyrosequencing Assay 
Aims: Efficient genotyping methods for many biologically significant repeat genetic polymorphisms, particularly in GC-rich regions of the genome, are limited. In particular, a short tandem repeat polymorphism [GGCGGG] in the promoter region of ALOX5 has been implicated as an important marker for inflammatory diseases. We developed a pyrosequencing assay to genotype the ALOX5 short tandem repeat polymorphism using pyrosequencing technology that will make assessing this important genetic marker in large, diverse populations more accessible than using current methods. Materials and Methods: We used a nested polymerase chain reaction approach to amplify DNA for pyrosequencing. Population allele frequencies were assessed in two cohorts of previously collected human DNA samples with 188 and 1032 samples, respectively. Sixteen genetic samples with known genotypes were used to confirm the accuracy of the method. Results and Discussion: Genotypes were 100% concordant with samples of known genotype. Genotype frequencies in European American, Hispanic, and African American agreed with previously published results (wild-type homozygotes 66%, 64%, and 19%, respectively). The method presented here will facilitate both genetic association and pharmacogenomic research on this polymorphism in large samples that are ethnically and/or racially admixed.
PMCID: PMC2998028  PMID: 19473080
22.  ARG1 Is a Novel Bronchodilator Response Gene 
Rationale: Inhaled β-agonists are one of the most widely used classes of drugs for the treatment of asthma. However, a substantial proportion of patients with asthma do not have a favorable response to these drugs, and identifying genetic determinants of drug response may aid in tailoring treatment for individual patients.
Objectives: To screen variants in candidate genes in the steroid and β-adrenergic pathways for association with response to inhaled β-agonists.
Methods: We genotyped 844 single nucleotide polymorphisms (SNPs) in 111 candidate genes in 209 children and their parents participating in the Childhood Asthma Management Program. We screened the association of these SNPs with acute response to inhaled β-agonists (bronchodilator response [BDR]) using a novel algorithm implemented in a family-based association test that ranked SNPs in order of statistical power. Genes that had SNPs with median power in the highest quartile were then taken for replication analyses in three other asthma cohorts.
Measurements and Main Results: We identified 17 genes from the screening algorithm and genotyped 99 SNPs from these genes in a second population of patients with asthma. We then genotyped 63 SNPs from four genes with significant associations with BDR, for replication in a third and fourth population of patients with asthma. Evidence for association from the four asthma cohorts was combined, and SNPs from ARG1 were significantly associated with BDR. SNP rs2781659 survived Bonferroni correction for multiple testing (combined P value = 0.00048, adjusted P value = 0.047).
Conclusions: These findings identify ARG1 as a novel gene for acute BDR in both children and adults with asthma.
PMCID: PMC2556451  PMID: 18617639
pharmacogenetics; asthma; bronchodilator agents
23.  The arginine-16 β2-adrenoceptor polymorphism predisposes to bronchoprotective subsensitivity in patients treated with formoterol and salmeterol 
The relationship between β2-adrenoceptor polymorphisms and bronchoprotective response with long-acting β2-adrenoceptor agonists is unknown.
We retrospectively analysed data from six placebo-controlled randomized studies in corticosteroid treated asthmatics where formoterol or salmeterol were administered over a 1–2-week period, with prior 1–2 week washout, assessing the primary end point of methacholine PD20 and adenosine monophosphate PC20, following first and last dose, expressed as doubling dose difference from placebo.
There was no significant heterogeneity between the different studies. Patients who had homozygous or heterozygous genotypes containing the arginine-16 polymorphism (Arg16-Arg16 or Arg16-Gly16) had greater bronchoprotective subsensitivity compared with the homozygous glycine-16 genotype (Gly16-Gly16), amounting to a mean doubling dose difference of 1.49 (95% CI 0.50, 2.48), after the last dose. Subsensitivity of response was greater with formoterol than salmeterol after the last dose in all genotypes, especially with the arginine-16 polymorphism, amounting to a doubling dose difference of 3.00 (95% CI 1.01, 4.99) between formoterol and salmeterol.
Our retrospective analysis showed that the arginine-16 polymorphism was associated with subsensitivity of response for bronchoprotection, which was greater for formoterol than salmeterol. A prospective study will be required in order to further evaluate these findings, particularly to assess whether these differences are mirrored by exacerbations.
PMCID: PMC1884417  PMID: 14678342
adenosine monophosphate PC20; bronchial hyper-responsiveness; formoterol; genotype; haplotype; long-acting β2-adrenoceptor agonist; methacholine PD20; salmeterol; subsensitivity; β2-adrenoceptor polymorphism
24.  Leukotriene C4 synthase polymorphisms and responsiveness to leukotriene antagonists in asthma 
Cysteinyl leukotrienes are important pro-inflammatory mediators in the pathogenesis of asthma, while leukotriene C4 synthase is a key enzyme in their biosynthesis. Our aim is to evaluate whether responsiveness to leukotriene receptor antagonists was determined by expression of the variant (C) or wild-type (A) polymorphism of this enzyme.
We carried out a retrospective analysis of 8 randomised, placebo-controlled trials performed in our department in mild-to-moderate asthmatics. In all trials, effect of leukotriene receptor antagonist was compared to placebo, where the primary outcome was bronchial hyperresponsiveness to adenosine monophosphate or methacholine. Secondary outcomes were forced expiratory volume in 1 second, exhaled nitric oxide and peripheral blood eosinophils.
For the primary outcome of attenuation of bronchial hyperresponsiveness by leukotriene receptor antagonist vs placebo, there were significant effects within each genotype on adenosine monophosphate (AMP) (n = 78): 2.21 and 2.07-fold improvements for AA and AC/CC, respectively; while for methacholine (n = 81) there were 1.39 and 1.36-fold improvements, respectively. There were no significant differences between genotypes (i.e. AA vs AC/CC): geometric mean fold-differences of 1.07 (95%CI 0.63–1.81) and 1.02 (95%CI 0.70–1.50) for AMP and methacholine, respectively. There were also no differences between genotypes for all secondary outcomes.
Polymorphisms of leukotriene C4 synthase did not determine responsiveness, in terms of attenuation of bronchial hyperresponsiveness, to leukotriene receptor antagonists in mild-to-moderate asthmatics. Further prospective large pharmacogenetic studies are required in more severe patients, where there may be greater improvements in pharmacodynamic outcome measures such as bronchial hyperresponsiveness and exhaled nitric oxide.
PMCID: PMC1884371  PMID: 12968987
asthma; bronchial hyperresponsiveness; leukotriene receptor antagonist
25.  Racial differences in propranolol enantiomer kinetics following simultaneous i.v. and oral administration  
1Racial differences in propranolol enantiomer kinetics following oral dosing were previously documented in our laboratory. The purpose of this study was to more completely describe propranolol kinetics in black and white subjects with the goal of gaining a better understanding of the mechanism(s) responsible for racial differences in oral propranolol kinetics.
2Twelve white and 13 black healthy males were included in the study. Poor metabolizers of dextromethorphan and mephenytoin were excluded. Subjects took oral propranolol 80 mg every 8 h for 16 doses and received an intravenous dose of radiolabelled propranolol with the 16th dose. Serum and urine samples were collected for 24 h after the 16th dose. Serum concentrations of R- and S-propranolol and urine concentrations of its three primary metabolites were determined by h.p.l.c.
3Apparent oral clearances of R- and S-propranolol were higher (P<0.05) in blacks than whites (R-propranolol: 5036±4175 ml min−1vs 2854±879 ml min−1; S-propranolol 3255±1723 ml min−1vs 2125±510 ml min−1; blacks vs whites).
4R- and S-propranolol clearances were higher in blacks than whites (R-propranolol 1069±316 ml min−1vs 841±161 ml min−1; S-propranolol 947±271 ml min−1vs 771±142 ml min−1; blacks vs whites, P<0.05).
5There were trends (P >0.05<0.10) toward higher side chain oxidation, 4-hydroxylation and R-propranolol glucuronidation in blacks compared with whites. Ethnic differences in the enantiomeric ratios of partial metabolic clearance values were not observed.
6We conclude the higher propranolol oral clearances in black subjects are explained by blacks having slightly higher hepatic metabolism via all three of its major metabolic pathways. Higher propranolol clearances among black subjects were also observed and we conclude this finding is explained largely by the higher hepatic metabolism, but also by slightly higher liver blood flow among black subjects.
PMCID: PMC2042685  PMID: 8877025
propranolol; racial differences; pharmacokinetics; drug metabolism; blacks; whites

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