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Human Molecular Genetics Advance Access originally published online on May 6, 2005
Human Molecular Genetics 2005 14(12):1671-1677; doi:10.1093/hmg/ddi175
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

Molecular properties and pharmacogenetics of a polymorphism of adenylyl cyclase type 9 in asthma: interaction between ß-agonist and corticosteroid pathways

Kelan G. Tantisira1,2,{dagger}, Kersten M. Small3,{dagger}, Augusto A. Litonjua1,2, Scott T. Weiss1 and Stephen B. Liggett3,*

1Channing Laboratory and 2Pulmonary Division, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA and 3Department of Medicine and the Cardiopulmonary Research Center, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA

* To whom correspondence should be addressed at: Department of Medicine and the Cardiopulmonary Research Center, University of Cincinnati College of Medicine, 231 Albert Sabin Way, ML 0564, Cincinnati, OH 45267-0564, USA. Tel: +1 5135580484; Fax: +1 5135580835; Email: stephen.liggett{at}uc.edu

Received March 9, 2005; Revised April 22, 2005; Accepted April 29, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In asthma, the response to ß-agonists acting at ß2-adrenergic receptors (ß2AR) displays extensive interindividual variation. One effector for airway ß2AR, adenylyl cyclase type 9 (AC9), was considered a candidate locus for predicting ß-agonist efficacy in the absence and presence of corticosteroid treatment. One non-synonymous AC9 polymorphism has been identified, which results in substitution of Met for Ile at amino acid 772. Under standard culture conditions in stably transfected cells, we found decreased catalytic activity of Met772. However, cells cultured in the presence of glucocorticoid expressing Met772 had a significantly increased albuterol-stimulated adenylyl cyclase response (~80%) when compared with those expressing Ile772 (~20%, P=0.02). An equivalent increase in ß2AR expression was observed in both lines due to glucocorticoid, but AC9 expression was unaffected. The hypothesis that Met772-AC9 is associated with an improved albuterol bronchodilator response in asthmatics was investigated in 436 asthmatic children who were followed for 4 years and randomized to receive placebo or the inhaled corticosteroid budesonide. Met772 carriers on budesonide showed a significant improvement in forced expiratory volume in 1 s (P=0.005). Moreover, a highly significant interaction (P=0.002) was found for budesonide treatment and the AC9 polymorphism. These in vitro and human association studies are consistent with this AC9 polymorphism altering albuterol responsiveness in the context of concomitant inhaled corticosteroid administration, which is a common asthma regimen. The Met772-AC9 polymorphism represents one of most likely several multi-gene polymorphisms along the receptor-relaxation axis, which together may provide for a composite pharmacogenetic index for asthma therapy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Asthma is a chronic inflammatory disease of the airways characterized by contraction of airway smooth muscle due to actions of multiple local bronchoconstrictive substances (1Go). The syndrome affects approximately 300 million individuals worldwide and is a substantial international health care burden (2Go). The inflammatory component of asthma is typically treated with corticosteroids, whereas the bronchoconstriction is treated with ß-agonist bronchodilators, which as a class are the most prescribed therapeutic for asthma treatment worldwide (3Go). However, the clinical response to ß-agonists in the treatment of asthma displays a high degree of interindividual variation that is not readily reconciled by clinical characteristics or baseline lung function. A significant fraction of patients appear to obtain no objective or subjective improvement with ß-agonists, particularly when administered on a regularly scheduled regimen (4Go). Indeed, a recent analysis has estimated that ~60% of the population variance in the forced expiratory volume in 1 s (FEV1) response to albuterol can be attributed to genetic variation (4Go). We have thus sought the genetic basis of ß-agonist responsiveness by examining the key genes in the early portion of the signal transduction pathway.

ß-Agonists evoke bronchodilation by binding to airway smooth muscle ß2-adrenergic receptors (ß2AR). These receptors, members of the superfamily of seven-transmembrane (7-TM) receptors, carry out signal transduction by activating the heterotrimeric stimulatory guanine nucleotide binding protein (Gs), whose {alpha} subunit activates the effector adenylyl cyclase (5Go). Human airway smooth muscle expresses several isoforms of adenylyl cyclase, including types 5, 6 and 9 (6Go). Their activation by G{alpha}s results in the catalytic conversion of ATP to cAMP, which subsequently activates the cAMP-dependent protein kinase PKA. This kinase phosphorylates multiple proteins in airway smooth muscle resulting in smooth muscle relaxation (7Go).

Polymorphisms of the ß2AR gene have been associated with the acute and chronic bronchodilation response to ß-agonists (8Go–11Go). However, within such stratified populations, there is still evidence for interindividual variation, suggesting contributions of polymorphisms in other genes. It is generally accepted that the rate limiting component in the ß2AR pathway is either the receptor itself or the effector, adenylyl cyclase (6Go,12Go). Gs is thought to be in relative excess, probably because it is shared by over approximately hundred 7-TM receptors, and is expressed in virtually all cell-types. As such, a genetic variation imposing a significant effect in Gs function would have broad physiologic consequences. Indeed, the coding region of G{alpha}s appears to be devoid of non-synonymous polymorphisms (13Go). A rare coding mutation of G{alpha}s is responsible for the Albright hereditary osteodystrophy syndrome (14Go). This has prompted our examination of the next component in the pathway, the adenylyl cyclases. An additional aspect to ß-agonist therapy is the apparent improvement in ß2AR function in vitro, and ß-agonist responsiveness in vivo, that occurs in the presence of corticosteroids (15Go). One mechanism of such interaction is the increase in cellular ß2AR expression that occurs with corticosteroids (15Go). In addition, studies utilizing direct stimulators of adenylyl cyclase have revealed increased catalytic activity in response to chronic corticosteroid treatment of cultured cells (16Go,17Go), a phenomenon whose mechanism remains obscure.

In the current work, we examine the in vitro and in vivo phenotypes of a non-synonymous polymorphism of adenylyl cyclase type 9 (AC9). We previously identified this polymorphism by resequencing the AC9 coding regions where we identified only one non-synonymous single nucleotide polymorphism (A2316G, GenBank accession no. DQ008441), which results in a substitution of Ile for the more common Met at amino acid 772 (18Go). For simplicity, the polymorphisms and the two different encoded proteins are heretofore referred to as either Met772 or Ile772. The substitution lies in the C1b region of the catalytic domain of the enzyme. In the current work, we examine signal transduction of the two AC9s in a transfected lung cell line in the absence and presence of corticosteroid treatment, examining adenylyl cyclase activities in response to ß2AR stimulation. In addition, in a large placebo-controlled clinical trial of the corticosteroid budesonide in pediatric asthmatics, potential associations between the AC9 polymorphism and ß-agonist responsiveness were sought. Given the previously reported association of SNPs and haplotypes of the ß2AR gene with the degree of bronchodilator response (9Go,10Go,19Go), we also examined the influence of these SNPs and haplotypes on bronchodilator response in relation to Met772 status. Taken together, these results show that in vitro Met772 polymorphism of AC9 imparts enhanced ß2AR signal transduction in a corticosteroid-specific manner, and in human asthma the polymorphism is associated with enhanced ß-agonist bronchodilator response under circumstances when corticosteroids are co-administered.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
AC9-Met772 phenotype in A431 cells
Stably transfected cell lines were screened by western blots to identify lines expressing equivalent levels of wild-type (WT) and Met772-AC9. Two lines expressing each genotype were chosen for further study. The signaling characteristics of WT and Met772-AC9 are shown in Fig. 1. Basal adenylyl cyclase activities, as well as those stimulated by the partial agonist albuterol, were not different between the lines. The most prominent feature was the decreased catalytic activity of Met772 as assessed by stimulation with NaF (which stimulates by activating G{alpha}s) and MnCl2 (which directly activates adenylyl cyclase). Met772 activities under these conditions were decreased when compared with WT. (Of note, AC9 is not stimulated by forskolin.) The endogenous ß2AR expression, as determined by 125I-CYP radioligand binding, was not different between the two lines (WT=51±6.2 versus Met772=66±8.9 fmol/mg). We next explored the phenotypes under conditions of 24 h of corticosteroid exposure. As expected, ß2AR expression increased in both lines (to 103±7.8 and 109±9.0 fmol/mg, respectively); however, the expression of WT AC9 or the variant was not changed by dexamethasone treatment (Fig. 2). The effects of dexamethasone treatment on basal and albuterol-stimulated adenylyl cyclase activities are shown in Fig. 3. As can be seen, the corticosteroid increased albuterol-stimulated activities to a greater extent in the Met772 cells compared with WT (24±1.3 to 43±3.4 versus 27±2.1 to 33±2.5 pmol/min/mg, respectively, N=5, P=0.02). This was also the case when one considered the fold-increase in over basal activities promoted by albuterol. In the presence of dexamethasone, these values were 4.9±0.60- versus 3.2±0.36-fold, respectively, P=0.006. Using another set of clonal cell lines, these values were 4.6±0.45-fold for Met772 and 2.9±0.11-fold for WT AC9 (P=0.02). Taken together, albuterol responsiveness was found to be greater in cells expressing the Met772-AC9 when compared with WT AC9, but only in the presence of corticosteroid.



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  		Figure 1. Characteristics of WT and Met772-AC9 in transfected A431 cells. The results from adenylyl cyclase studies performed in membranes shown from cells expressing the same levels of transfected WT and Met772-AC9. MnCl2- and NaF-stimulated activities were lower in Met772 cells when compared with WT. Results are from five experiments. *P<0.05 compared with WT.

 


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  		Figure 2. Effects of corticosteroid treatment of transfected A431 cells on ß2AR and AC9 expression. (A) Results from 125I-CYP radioligand studies show that cells expressing WT and Met772-AC9 both displayed an equivalent increase in ß2AR expression after 12 h of exposure to dexamethasone (dex). Results are from five experiments. *P<0.01 compared with the absence of dexamethasone. (B) A representative western blot of AC9 shows that dexamethasone treatment did not increase expression of either AC9 allelic variants in these cells.

 


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  		Figure 3. Effects of corticosteroid treatment of transfected A431 cells on albuterol-stimulated adenylyl cyclase activities. The increase in albuterol-stimulated activities from dexamethasone (dex) treatment was greater for Met772 expressing cells versus WT, compared as either the absolute activities (P=0.02) or the fold-increase over basal activities (P=0.006). Asterisk represents levels of significance as defined.

 
AC9-Met772 associates with enhanced bronchodilation
The relationship between this AC9 polymorphism and ß-agonist promoted bronchodilation was assessed by genotyping a subset of patients who had been enrolled in the CAMP study (Materials and Methods). The baseline clinical characteristics of these patients stratified by genotype is shown in Table 1. There were no differences in these parameters between those with and without Met772. In particular, bronchodilator response, airway hyperresponsiveness (as manifested by PC20), FEV1 (%predicted), FVC (%predicted) and FEV1/FVC, were not different among the groups at randomization.


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  		Table 1. Baseline characteristics of patients stratified by the AC9 polymorphism
 
The results from the analysis of primary outcome variable, the ß-agonist bronchodilator response over a 4 year follow-up period, are shown in Table 2. The univariate models revealed no discernable differences in bronchodilator response over time for the overall cohort. Moreover, although there was no significant difference in the bronchodilator response over time in individuals with varying genotypes on placebo (P=0.21), the Met772 carriers on the inhaled corticosteroid budesonide demonstrated ~2% point increase in bronchodilation response when compared with wild-type (6.76±0.69 versus 8.72±0.54, P=0.005). There was a significant interaction noted for the use of inhaled steroids in combination with Met772 carrier status (interaction P=0.002). Even after adjusting for age, gender and baseline bronchodilator response, those on inhaled corticosteroids with the variant genotype still demonstrated significant increases in bronchodilator response when compared with wild-type (P=0.04), whereas those on placebo did not. The interaction P-value for use of budesonide and Met772 is 0.03 with this analysis. These relationships are further demonstrated graphically by plotting average bronchodilator response over the 4 year follow-up period by genotype and treatment status (Fig. 4). Analysis of the stratification SNPs revealed no evidence of population stratification for the entire cohort or for either treatment group alone; all P-values were>0.05 for the highest versus the lowest mean bronchodilator quartiles (data not shown).


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  		Table 2. Longitudinal bronchodilator response in CAMP, stratified by Met772 genotypea
 


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  		Figure 4. Interaction between corticosteroid treatment, AC9 genotype and ß-agonist bronchodilation in asthmatics. Results are from the clinical cohort as described in Materials and Methods, showing the average bronchodilator response over the 4 year follow-up period. Met772 patients experienced an improvement in bronchodilator response only if they were being treated with budesonide (P=0.006). The interaction P-value for genotype and budesonide treatment is 0.002.

 
The potential for epistasis between ß2AR SNPs and haplotypes with the Met772 variant was examined, using eight ß2AR SNPs previously reported (9Go,10Go). No significant interactions between any individual ß2AR SNP and the Met772 variant was noted (data not shown). However, the power to detect such an association was relatively low. For instance, in the Caucasian children who carried Met772, and who were on inhaled corticosteroids, only three were homozygous for the SNP at position 523. Although we have previously shown that haplotypes within the ß2AR gene may have more power to detect phenotypic outcomes (9Go), we focused the remainder of our analyses on the potential for effect modification of the ß2AR haplotypes by Met772 on bronchodilator outcomes.

Using multivariable models in ‘haplo.score’ (20Go), we evaluated global P-values of association between ß2AR haplotypes and mean bronchodilator response, with and without stratification by Met772 status and treatment group assignment. For the entire cohort, ß2AR haplotypes were nominally associated with mean bronchodilator response (P=0.05). After subsetting by AC9 genotype status alone, ß2AR haplotypes in the Ile772 group were not associated with bronchodilator response (P=0.34). However, in those individuals with at least one Met772 allele, a modest association with ß2AR haplotypes and bronchodilator response was observed (P=0.03). Most notably, the global association of ß2AR haplotypes with bronchodilator response was particularly strong for the Met772 subgroup who were randomized to inhaled corticosteroids (P<0.001).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the current work, we assessed the effects of a coding, non-synonymous, polymorphism of AC9 on ß2AR signaling in the absence and presence of corticosteroids in transfected cells in vitro, and in pediatric asthma patients who participated in a large clinical trial of inhaled corticosteroids. We considered this as a possible pharmacogenetic locus for ß-agonists in the treatment of asthma because of the relatively high allele frequency, the critical role that adenylyl cyclase plays in ß2AR signal transduction, and our previous in vitro studies showing an altered phenotype of Met772 compared with its allelic counterpart. In a transfected human embryonic kidney cell line (HEK-293), we noted a decrease in NaF- and MnCl2-stimulated adenylyl cyclase activities in membranes derived from Met772 expressing cells compared with the wild-type (Ile772) (18Go). These results were consistent with the location of the substituted amino acid, in that it lies within the C1b region of the protein. In addition, the full ß2AR agonist isoproterenol had lower maximal stimulation in Met772 HEK-293 cells. However, the basal levels of activity in these Met772 cells were also lower, such that the net stimulation, or the fold-stimulation over basal, evoked by ß2AR activation was the same between Met772 and WT AC9. In the current work, we utilized A431 cells, which are a lung epidermoid carcinoma cell line. This line was chosen, in part, because we observed an increase in ß2AR expression when these cells were exposed to dexamethasone, which we did not find with the HEK-293 cells. Given that altered expression of proteins, such as the ß2AR, is part of the corticosteroid treatment effect in asthma, we considered the A431 line to be preferable as a model system. When cells were cultured under standard conditions, NaF- and MnCl2-stimulated adenylyl cyclase activities were decreased in the Met772 lines, consistent with our previous findings. The partial agonist albuterol was utilized to stimulate ß2AR in the current in vitro studies, since albuterol is ß2AR (when compared with ß1AR) selective, and was the same agent used in the clinical trial. Albuterol-stimulated activities did not differ between WT and Met772 expressing cells in the absence of corticosteroid treatment. However, the increase in albuterol-stimulated activities evoked by exposure of the cells to dexamethasone was clearly higher for Met772 cells. Given that ß2AR expression increases were equivalent, and AC9 expression did not change with steroid treatment, we do not have a ready explanation for the phenotype. However, there is a large number of modifying proteins which can affect G-protein and adenylyl cyclase function (21Go,22Go), and thus it is reasonable to consider that an interaction between a partially defective catalytic unit and altered expression of such proteins due to corticosteroids might result in the phenotype observed.

In the clinical study, we observed no association between the Met772 and the albuterol bronchodilator response in children with asthma who were randomized to the placebo group. However, in those receiving the inhaled corticosteroid budesonide, Met772 was associated with an increased bronchodilator response to albuterol when compared with those with the WT AC9 genotype. It is recognized that this effect on FEV1 is relatively small; however, these patients had minimal baseline obstruction (FEV1 ~94% predicted). Nevertheless, the increase in bronchodilator response amounted to ~30% in the Met772 group, which was statistically significant, as was the interaction between genotype and corticosteroid use. These clinical results are even more compelling when taken together with the in vitro data, which also showed a greater increase in the albuterol-stimulated adenylyl cyclase response with Met772 under conditions of corticosteroid exposure. There are several validated promoter SNPs in the human AC9 gene which could be in linkage disequilibrium with the coding SNP utilized in the current study. However, the in vitro data generated are entirely consistent with the asthma study, suggesting that this coding SNP contributes to the clinical phenotype. It is attractive to consider whether SNPs of the ß2AR gene, which we and others have shown to be associated with certain ß-agonist phenotypes (8Go–11Go), act in an additive or other epistatic fashion with the AC9-Met772 polymorphism. Our analyses of the current cohort evaluating whether SNPs at nucleotide positions 46 (or 79) and 523 in the coding exon of the ß2AR gene significantly modify the association of the AC9-Met772 polymorphism yielded no evidence for epistasis. However, haplotypic analyses yielded more insight into these relationships. We found a strong interaction among ß2AR haplotype, AC9-Met772, corticosteroid use and the albuterol bronchodilator response. To our knowledge, this represents the first reported two-gene, two-drug interaction in asthma pharmacogenetic studies to date. The magnitude of this interaction, or whether this epistasis is additive or potentially synergistic, could not be ascertained using the analytical technique employed, which was necessitated by the small sample sizes of subjects when multiply stratified.

In conclusion, the AC9-Met772 polymorphism is associated with a glucocorticoid-specific upregulation of the bronchodilatory response both in transfected lung cells and in childhood asthmatics. That this response is specific to treatment with glucocorticoids lends further insight into the mechanisms behind the molecular interactions of the corticosteroid and ß-adrenergic pathways. Albuterol remains the most commonly employed medication in the acute therapy of asthma (and is often used for chronic control as well), and inhaled glucocorticoids are prescribed as long-term ‘controller’ agents for asthma. Thus, our findings may have relevance in the community management of asthma, since this is a common two-drug treatment regimen. Additional studies, though, will be necessary to determine whether acting on this pharmacogenomic information, such as utilizing alternative regimens, will improve outcome. Along with polymorphisms of the ß2AR gene, the polymorphism in the AC9 gene represents a second pharmacogenomic locus that has predictive potential for ß-agonist responsiveness. This lends credence to the approach of ‘working-through’ the polymorphisms of genes within a specific pathway (in this case from the receptor to the effector, and in the future genes involved in smooth muscle relaxation) in order to ascertain potential variants that predict clinical efficacy. Ultimately, these can be ranked and a composite pharmacogenetic index can be calculated to provide the greatest predictive power for clinical guidance of therapy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Transfections and cell culture
The WT cDNA sequence for the human AC9 gene considered is that of GenBank accession no. DQ005545. With the adenine of the initiator codon being considered nucleotide 1, we have previously described a polymorphism consisting of an A>G transition at nucleotide 2316 of the cDNA, which results in substitution of Ile with Met at amino acid position 772 (GenBank accession no. DQ008441) (18Go). The cDNAs for Ile772 (also referred to as WT) and Met772 coding regions were generated from human lymphocyte RNA, subcloned into pCNDA3, and transfections of A431 cells carried out as described earlier (18Go). Selection of positive clones was carried out in media containing 400 µg/ml G418. Expression of AC9 was determined in individual clonal lines by western blots (as described subsequently). A431 cells were grown in monolayers in Dulbecco's modified Eagle's medium with 4 mM L-glutamate, 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin, at 37° in a 5% CO2 atmosphere. In some studies, cells at 90% confluency in the absence of serum were treated with 500 nM dexamethasone for 12 h and then utilized as described subsequently.

Radioligand binding and western blots
Transfected A431 cells were washed three times with phosphate-buffered saline (PBS), detached by scraping in 5 mM Tris, pH 7.4, 2 mM ethylenediaminetetracetic acid (EDTA) buffer and centrifuged at 30 000g for 10 min. To determine endogenous ß2AR density, membrane pellets were resuspended in 75 mM Tris, pH 7.4, 12.5 mM MgCl2, 2 mM EDTA and radioligand binding with 125I-cyanopindolol was performed as described earlier (23Go). Non-specific binding was determined with 1 µM propranolol. Reactions were carried out for 2 h at 25°C, terminated by dilution and bound radioligand separated by vacuum filtration. AC9 expression in these transfected cell lines was assessed by quantitative immunoblotting so as to select lines expressing Ile772 or Met772 at the same levels for pharmacologic studies, and to ascertain whether corticosteroid treatment altered expression. Confluent cells were washed three times with PBS and then lysed in RIPA buffer (PBS containing 1% Igepal CA-630, 0.5% deoxycholate and 0.1% SDS) with protease inhibitors (10 µg/ml benzamidine, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml aprotinin and 5 µg/ml leupeptin). Western blots of these whole cell lysates were performed by enhanced chemiluminescence as previously described, using an isoform-specific polyclonal AC9 antibody (obtained from R. Premont, Duke University) at a dilution of 1:2000. Relative levels of AC9 expression were subsequently determined using ScanAnalysis software (BioSoft, Cambridge, UK).

Adenylyl cyclase activities
Cell membranes prepared as mentioned earlier were incubated with 30 mmol Tris, pH 7.4, 2 mM MgCl2, 0.8 mM EDTA, 120 µM ATP, 60 µM GTP, 2.8 mM phosphoenolpyruvate, 50 µg/ml myokinase, 4 U/ml pyruvate kinase, 100 µM cAMP and 1 µCi of [{alpha}-32P]ATP for 15 min as described earlier (24Go). The reactions were incubated in the presence of vehicle (basal), 10 µM albuterol, 1.8 mM MnCl2 or 10 mM NaF. [32P]cAMP was separated from [{alpha}-32P] by chromatography over alumina columns. The [3H]cAMP standard included in the stop buffer accounted for individual column recovery.

Patient population
The study cohort consisted of pediatric patients who participated in the Childhood Asthma Management Program (CAMP). The study was a multicenter, randomized, trial comparing regularly scheduled inhalation treatment with the corticosteroid budesonide, the putative mast-cell ‘stabilizer’ nedocromil, and placebo. The study design has been previously published (25Go). Briefly, children were enrolled between the ages of 5–12 years of age (mean age of asthma onset was 3.1 years) and followed for 4-6 years. Eighty-three percent of these children were classified as atopic, based on at least one positive skin test. Spirometry was performed twice yearly, before and after the administration of a standard dose of the ß-agonist bronchodilator albuterol. Once per year a methacholine challenge was performed to ascertain bronchial hyperresponsiveness. The current study utilized archived DNA from patients who participated in the CAMP DNA Ancillary Study. Because of the potential population stratification issues (the allele frequency of Met772 is ~0.30 in Caucasians and ~0.15 in African-Americans) and the small number of African-American children enrolled in CAMP (14%), the current study was confined to Caucasian children. Moreover, because the effects of nedocromil on bronchodilator response have not been biologically characterized, we excluded this treatment arm from the analysis. We focused our analysis, then, on 436 children, with follow-up through 48 months, the last time point in which nearly complete measures were obtained for the cohort. The subjects' parents provided informed consent, and the study was approved by the Institutional Review Boards of each participating center.

Genotyping
The AC9 SNP at cDNA nucleotide 2316 was genotyped via a SEQUENOM MassARRAY MALDI-TOF mass spectrometer (Sequenom, San Diego, CA, USA) for analysis of unlabeled single-base extension minisequencing reactions with a semiautomated primer design program (SpectroDESIGNER, Sequenom). The protocol implemented the very short extension method (26Go), whereby sequencing products are extended by only one base for three of the four nucleotides and by several additional bases for the fourth nucleotide (representing one of the alleles for a given SNP), permitting clearly delineated mass separation of the two allelic variants at a given locus. ß2AR SNPs at positions –709, –654, –47, 46, 79, 252, 491 and 523 (9Go) were determined using similar methods as previously described (10Go). To assess for possible population stratification, we also genotyped a random panel of 49 SNPs across the genome (27Go,28Go) (‘stratification SNPs’), obtained through the SNP Consortium (TSC) database. SNPs were chosen that are widely distributed throughout the genome and exclude promoters, genomic UTRs, exons and introns of known genes.

Statistical analysis
Bronchodilator response was defined as FEV1 after bronchodilator minus FEV1 before bronchodilator divided by FEV1 before bronchodilator multiplied by 100 [(post-FEV1–pre-FEV1)/(pre-FEV1)x100]. Longitudinal models of bronchodilator response, using repeated measures modeling with time as a linear effect and an unstructured covariance matrix, were used. These models evaluated change in bronchodilator response beginning at the 2 month follow-up visit, allowing for evaluation of change based on treatment type. Univariate and multivariable analyses adjusting for age, sex and bronchodilator response at baseline were performed, under the assumption of a dominant model. To test for interaction between steroid treatment and Ile772Met genotypic status, we evaluated treatment specific strata and included interaction terms in the models. A ‘mean bronchodilator’ response was also derived by taking all of the observations over the 4 years and dividing by the number of visits. This value was used as the primary outcome in the haplotypic assessment of interactions between the ß2AR locus and the AC9 polymorphism.

The potential for epistasis between the ß2AR gene and the Met772 variant was explored in relation to both individual SNPs and haplotypes. Eight ß2AR SNPs were evaluated, as previously reported (10Go); these were encoded in an additive fashion. Individual SNP analyses followed the within-treatment group repeated measures analyses outlined earlier with the addition of a SNP by Met772 interaction term. ß2AR haplotypes were analyzed using the ‘haplo.score’ (20Go) function from the haplo.stats package (Version 1.1.0. Mayo Clinic, Rochester, MN, USA). The global association of the ß2AR haplotypes with mean bronchodilator response over the 4 year trial period was evaluated for the entire cohort initially, then stratified by Met772 status. Finally, these analyses were further stratified by treatment arm and Met772 status.

Analysis of the stratification SNPs proceeded in two steps. First, for each individual SNP, an allelic {chi}2 test statistic was obtained in standard fashion, utilizing 2x2 contingency tables that allocated the totals of the wild-type and variant alleles for the highest and the lowest quartiles of mean bronchodilator response to each of four cells. An overall summary {chi}2 test statistic was then obtained by summing the individual {chi}2 test statistics, setting the degrees of freedom to the number of tests. This analysis was performed using SAS (Version 8, Cary, NC, USA).


   ACKNOWLEDGEMENTS
 
We thank all families for their enthusiastic participation in the CAMP Genetics Ancillary Study, supported by the National Heart, Lung and Blood Institute, N01-HR-16049. We also acknowledge the CAMP investigators and research team, for collection of CAMP Genetic Ancillary Study data. We would also like to acknowledge Esther Getz Moses for manuscript preparation. This work was also supported by National Institutes of Health grant U01 HL065899 from the National Heart, Lung and Blood Institute, and the State of Ohio sponsored Computational Medicine Center. All work on data from the CAMP Genetics Ancillary Study was conducted at the Channing Laboratory of the Brigham and Women's Hospital under appropriate CAMP policies and human subjects protections.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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