The Proceedings of the American Thoracic Society 1:364-367 (2004)
© 2004 The American Thoracic Society
Asthma Steroid Pharmacogenetics
A Study Strategy to Identify Replicated Treatment Responses
Scott T. Weiss,
Stephen L. Lake,
Eric S. Silverman,
Edwin K. Silverman,
Brent Richter,
Jeffrey M. Drazen and
Kelan G. Tantisira
Channing Laboratory, Brigham and Women's Hospital and Harvard Medical School, Boston; and Pulmonary Division, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts
Correspondence and requests for reprints should be addressed to Scott T. Weiss, M.D., M.S., Director, Respiratory, Genetic, and Environmental Epidemiology, Channing Laboratory, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115. E-mail: restw{at}channing.harvard.edu
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ABSTRACT
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Asthma treatment with inhaled steroids demonstrates significant between-person variability. Genetic variation could contribute to this response to inhaled glucocorticosteroids. Difficulties in performing genetic association studies are well known. We designed a test and validation strategy to assess steroid pathway candidate genes. One hundred thirty-one single nucleotide polymorphisms in 14 candidate genes in the steroid pathway were genotyped in an 8-week clinical trial of 470 adults with moderate to severe asthma. We then validated our findings in a second population of individuals with childhood asthma in a 4-year clinical trial of inhaled corticosteroids and a third population of adults with asthma. One gene, corticotrophin-releasing hormone receptor 1 (CRHR1, NM_004382
[GenBank]
), demonstrated multiple single nucleotide polymorphism associations within each of the three populations. The approach of a test and multiple replication populations is a valuable strategy in asthma pharmacogenetics, which can insure valid association findings.
Key Words: association studies • asthma • drug treatment • pharmacogenetics • steroids
Inhaled glucocorticosteroids are the most commonly used (1–3) controller therapy for asthma. Variation in treatment response to this medication has been well identified. Because within-person variation in treatment response to inhaled corticosteroid is highly repeatable (4), it is possible that there could be a genetic basis for variation in treatment response.
We hypothesized that sequence variants in the genes in the steroid pathway contribute to the variation in inhaled steroid treatment response. This hypothesis was tested using a pathway candidate gene association study in adults with asthma and the results were then replicated in a second association study using a 4-year clinical trial of an inhaled steroid (budesonide) in children (termed "CAMP" for the Childhood Asthma Management Program) (5) and a third association study using an Asthma Clinical Research Network (ACRN) trial.
There are a number of problems with candidate gene studies (Table 1). Of the problems noted in this table, the major problems are the small sample size of most studies and multiple comparisons, both of which lead to inconsistent results across studies. To minimize these problems, we set up an experimental design in which we would first test our candidate genes in one population and then replicate any positive findings in both a second and third population. The three populations were chosen to maximize the generalizability of our results. This study has recently been reported, and provided here is a summary of our general approach (6).
Using this study design strategy, we found that one of these genes, corticotrophin-releasing hormone receptor 1 (CRHR1, NM_004382
[GenBank]
), was associated with treatment response in all three populations.
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STUDY POPULATIONS
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DNA samples from three clinical trials were used to test our hypothesis. All patients or their legal guardians provided consent for the study protocol and for ancillary genetic testing. The Adult Study was a multicenter 8-week clinical trial used to compare the effect of once-daily high dose inhaled flunisolide versus standard inhaled corticosteroid therapy. Patients were randomized in a 4:1 flunisolide versus other therapy ratio. Because the change in the FEV1 in both treatment groups was the same (p = 0.30), we used the combined results in our analyses. The age range of study subjects was from 16 to 75 with a slight (58%) female preponderance. Inclusion criteria included a history of asthma and documentation within the previous year of at least 12% improvement in FEV1 with administration of the short acting ß-agonist, albuterol. In addition, all subjects were required to have been using inhaled steroids at randomization. Exclusion criteria included a history of significant pulmonary disease other than asthma, smoking in excess of 10 pack-years, recent upper respiratory infection, and recent asthma exacerbations requiring hospitalization or oral prednisone usage. Subjects were followed by phone contact on a weekly basis, and had follow-up spirometry at 4 and 8 weeks. 88% of the subjects in this trial were self-reported as Caucasian. Based on the initial and 8-week FEV1, we defined the steroid response phenotype as the percent change in FEV1.
CAMP is a multicenter, longitudinal, randomized, double-blinded clinical trial testing the safety and efficacy of inhaled budesonide versus nedocromil versus placebo in asthma. Trial design and methodology have been previously published (5). The mean duration of follow-up was 4 years (7). CAMP enrolled 1,041 children ages 5 to 12 years with mild to moderate chronic asthma over a 23-month period. Entry criteria included asthma symptoms and/or medication use for 6 months or more in the previous year. Eligibility for enrollment in the CAMP trial specified that children have chronic asthma as evidenced by one or more of the following historical findings for at least 6 months in the year before interview: (1) asthma symptoms at least twice a week, (2) use of an inhaled bronchodilator at least twice a week, and (3) daily asthma medication. Study participants were excluded from enrollment in CAMP if they needed prednisone bursts more than five times over the preceding year, had more than one hospitalization for asthma in the year preceding their initial interview, or had ever required intubation for asthma. Other exclusion criteria included: FEV1 less than 65% of predicted; other active pulmonary disease; severe chronic sinusitis or nasal polyposis; introduction of a change in allergy immunotherapy in the month before the interview; use of more than four sprays of nasal steroids daily (only beclomethasone allowed) at the time of randomization; and current use of antigastroesophageal reflux medication. All study participants demonstrated airway responsiveness to methacholine with provocative concentration of methacholine causing a 20% reduction in FEV1 (PC20) less than or equal to 12.5 mg/ml. Follow-up visits occurred every 4 months and spirometry was performed twice yearly. The replication sample subjects were the Caucasian CAMP children randomized to the steroid group. Our quantitative phenotype was the same as in the adult study.
Two completed trials conducted by the ACRN, the salmeterol or corticosteroids (8), and salmeterol with or without inhaled corticosteroids trials (9), had a common initial 6-week run-in period using four inhalations twice daily of triamcinolone prior to separate randomization to one of the two trials. Details regarding the entry criteria, run-in period, and randomization have been published with the primary trial results (8, 9). All patients met the American Thoracic Society definition of asthma and criteria for treatment with inhaled corticosteroids. Of the 339 subjects eligible for randomization, 336 had DNA available, forming the basis of our second replication sample.
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DEFINITION OF THE DRUG RESPONSE PHENOTYPE
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We defined steroid response phenotype similarly in all three studies. To closely parallel the Adult Study, we evaluated quantitative FEV1 change as a percent predicted at the 2-month visit in the CAMP study and at the end of the ACRN trial run-in periods.
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GENOTYPING OF CANDIDATE GENES
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Genotyping of the single nucleotide polymorphisms (SNPs) was performed via a SEQUENOM MassARRAY MALDI-TOF mass spectrometer (Sequenom, San Diego, CA) for analysis of unlabeled single-base extension minisequencing reactions. A semi-automated primer design program was used (Spectro DESIGNER; Sequenom). Our protocol implemented the very short extension method proposed by Sun and colleagues (10), whereby sequencing products are extended by only one base for three of the four nucleotides (due to the presence of dideoxynucleotides for three of the four nucleotides in the minisequencing reaction) and by several additional bases for the fourth nucleotide (specified in advance so as to represent one of the two alleles at a given SNP locus). This allowed for clearly delineated mass separation of the two allelic variants at a given locus. We addressed population stratification by stratifying our analysis by self-designated ethnic group. A set of random markers across the genome were also genotyped.
We tested the associations between individual SNPs and asthma phenotypes with standard analysis of covariance (ANCOVA) for quantitative traits using the assumption of an additive model. Although the use of conventional significance levels is liberal given concerns about multiple comparisons, the replication of effects across two separate populations at this level is unlikely to have occurred by chance alone.
The approach we used for genotyping and analyzing candidate genes for the pharmacogenetic response to inhaled corticosteroids is shown in Figure 1.
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Figure 1. General methodologic approach. After identifying candidate genes of interest, variants were identified via DNA sequencing and public databases. SNPs were selected with preference for known functional variants, allele frequencies over 10%, and no less than every 10 kb apart. Genotyping was performed initially on our Adult Study and haplotype-tagged SNPs were identified. Any gene with single allelic or haplotypic effects with significant (p < 0.05) effects were then genotyped in our pediatric population (Childhood Asthma Management Program [CAMP]). Replicated findings were retested in our second adult population (Asthma Clinical Research Network [ACRN]) before our final, multivariate analysis.
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POPULATIONS
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Clinical characteristics of our primary, Adult Study and replicate, CAMP and ACRN populations are shown in Table 2. The three populations differ in age, level of FEV1, disease severity, and length of follow-up.
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SINGLE SNP ANALYSIS
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Multiple SNPs in CRHR1 were significantly associated with steroid treatment response in the Adult Study and in CAMP (see supplementary data in Reference 6). Three haplotype-tagged SNPs, rs1876828, rs242939, and rs242941, were each associated with inhaled treatment response in both the Adult Study and CAMP. One of these three SNPs, rs1876828, was also associated with inhaled steroid treatment response in the ACRN population.
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DISCUSSION
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Failure to demonstrate replication in genetic association studies has been attributed to variations in study design, including differing study populations and definitions of phenotypes (11, 12). Conversely, the replication of even modest effects in three populations with markedly differing clinical characteristics supports both the validity and generalizability of these findings. We have demonstrated that the CRHR1 gene has effects on the response to inhaled corticosteroids.
Corticotrophin-releasing hormone (CRH) has two primary receptors, CRHR1 and CRHR2. CRHR1 is the predominant receptor in the pituitary gland, mediating the release of adrenocorticotropic hormone (ACTH) (13, 14). In the CNS, CRHR1 is also the primary receptor of the catecholaminergic response to CRH (15–17). CRFR1 also has been reported to be the predominant receptor on the mast cell in rats (18). Thus, any alterations in the CRHR1 gene may have profound effects at multiple sites in the pathogenesis of inflammation in asthma. Altered immune system responses, in turn, can readily explain our noted differences in response to therapeutic interventions.
We used haplotype-tagged SNPs to increase the statistical efficiency of our analysis. It is unlikely, however, that the SNPs chosen are the actual disease modifying variants in the CRHR1 gene. Instead, the SNPs tagging the haplotypes are likely in linkage disequilibrium with those variants. CRHR1 is located on chromosome 17q21-22. It belongs to the family of Gs-protein–coupled receptors (19, 20) and contains 14 exons. CRHR1 has three known isoforms arising from alternative splicing (21, 22). Within the receptor, there are three separate regions that are important for optimal binding of CRH and activation of the receptor (23, 24). At least five potential glycosolylation and three potential phosphorylation sites exist throughout the gene (19, 20, 25). In addition, CRHR1 translation may be inhibited by an upstream regulator (26). Linkage disequilibrium between any of the alternative splice sites, the regulatory regions, or the upstream reporter and our haplotype could explain the variability noted in the response to inhaled steroids in our two populations.
In addition to the replication of the CRHR1 association in two disparate populations, our study has a number of other methodologic strengths. We selected our candidate genes specifically for their biologic plausibility. Genotyping of these candidates was done without knowledge of phenotypic status. Similarly, we created the pharmacogenetic response phenotypes within our populations with no knowledge of genotypic status. Because the populations were well characterized for enrollment in the two clinical trials, diagnostic bias is not a concern. However, the heterogeneity of the clinical response in both populations suggests that our hypothesis of alternative explanations for response within each population group is valid. From a statistical standpoint, our analysis incorporated both reasonably large sample sizes and adjustments for major potential confounders.
In conclusion, our findings of an association of the CRHR1 gene, as well as one specific haplotype within the CRHR1 gene with the degree of response to inhaled corticosteroids in two diverse populations of individuals with asthma, provide novel insights into the therapy of asthma. This genetic association with a therapeutic response to this class of commonly used medications may be the first step in the development of individualized therapy for asthma, providing a venue to decrease both morbidity and cost. These findings may also be of relevance to multiple other diseases whose therapeutic approaches include the use of corticosteroids.
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FOOTNOTES
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This work was supported by NIH U01 HL65899: The Pharmacogenetics of Asthma Treatment.
Conflict of Interest Statement: S.T.W. received a grant for $900,065 Asthma Policy Modeling Study from AstraZeneca from 1997–2003 and also was a Co-Investigator on a grant from Millennium Pharmaceuticals to pursue asthma genetics in China from 1996–2001 and received a total of $80,000 in salary support and received a grant of $50,000 from Pfizer to examine diabetes mellitus and its relationship to lung function between 2000–2003 and was also a consultant for Schering-Plough and received $5,000 from 1999–2000 and has been a co-investigator on a grant from Boehringer Ingelheim to investigate a COPD natural history model, which began in 2003, and has received no funds for his involvement in this project and has been a consultant for Variagenics on human subjects issues and received $5,000 in 2003 and has been a consultant to Genome Therapeutics in 2003 and received $1,500 and was a consultant for Merck Frost on asthma genetics in 2002 and received $2,000 and has been an advisor to the TENOR Study for Genentech and has received $5,000 for 2002–2003 and received a grant from Glaxo-Wellcome for $500,000 for genomic equipment from 2000–2003 and was a consultant for Roche Pharmaceuticals in 2000 and received no financial remuneration for this consultancy; S.L.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; E.S.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; E.K.S. received grant support and honoraria from GlaxoSmithKline for a study of COPD genetics and also received a $500 speaker fee from Wyeth for a talk on COPD genetics; B.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.M.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; K.G.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
(Received in original form September 23, 2004; )
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