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Airflow limitation in COPD patients is not fully reversible. However, there may be large variability in bronchodilator responsiveness (BDR) among COPD patients, and familial aggregation of BDR suggests a genetic component. Therefore we investigated the association between six candidate genes and BDR in subjects with severe COPD. A total of 389 subjects from the National Emphysema Treatment Trial (NETT) were analyzed. Bronchodilator responsiveness to albuterol was expressed in three ways: absolute change in FEV1, change in FEV1 as a percent of baseline FEV1, and change in FEV1 as a percent of predicted FEV1. Genotyping was completed for 122 single nucleotide polymorphisms (SNPs) in six candidate genes (EPHX1, SFTPB, TGFB1, SERPINE2, GSTP1, ADRB2). Associations between BDR phenotypes and SNP genotypes were tested using linear regression, adjusting for age, sex, pack-years of smoking, and height. Genes associated with BDR phenotypes in the NETT subjects were assessed for replication in 127 pedigrees from the Boston Early-Onset COPD (EOCOPD) Study. Three SNPs in EPHX1 (p = 0.009 – 0.04), three SNPs in SERPINE2 (p = 0.004 – 0.05) and two SNPs in ADRB2 (0.04 – 0.05) were significantly associated with BDR phenotypes in NETT subjects. BDR. One SNP in EPHX1 (rs1009668, p = 0.04) was significantly replicated in EOCOPD subjects. SNPs in SFTPB, TGFB1, and GSTP1 genes were not associated with BDR. In conclusion, a polymorphism of EPHX1 was associated with bronchodilator responsiveness phenotypes in subjects with severe COPD.
Chronic obstructive pulmonary disease (COPD) is characterized by airflow limitation that is not fully reversible; however, COPD patients are often treated with bronchodilator medications (1). There is large variability in bronchodilator responsiveness (BDR) among COPD patients, which has been related to various factors such as age (2), smoking (3), baseline lung function (4), and eosinophil biomarkers in bronchoalveolar lavage fluid (5). These findings suggest that differences in disease characteristics of COPD subjects may be associated with interindividual variation in pharmacological response to bronchodilator medications.
A previous paper found significant familial aggregation of BDR in the Boston Early-Onset COPD Study (6), suggesting the effect of genetic factors. Recent studies revealed that polymorphisms in hemopoeitic cell kinase (HCK) (7) and β2-adrenergic receptor genes (8) may be associated with BDR in patients with COPD. However, the genetic determinants of BDR in COPD have not been definitively established.
Glutathione s-transferase pi 1 (GSTP1), microsomal epoxide hydrolase (EPHX1), transforming growth factor-β1 (TGFB1), serpin peptidase inhibitor clade E member 2 (SERPINE2), surfactant protein B (SFTPB) and β2-adrenergic receptor (ADRB2) are six candidate genes previously associated with COPD susceptibility in at least two studies (9) (10) (11) (12) (13). ADRB2 has been associated with BDR in subjects with asthma and COPD (14) (8). We hypothesized that genetic variants in these potential COPD susceptibility genes may explain some of the variability in bronchodilator responsiveness phenotypes. This may improve our understanding of COPD by identifying subsets of patients showing small or large bronchodilator responsiveness influenced by a particular COPD susceptibility gene. Therefore, we investigated the association between six candidate genes and BDR in two populations of subjects with severe COPD.
The current analysis included 389 non-Hispanic white subjects in the National Emphysema Treatment Trial (NETT) (15). Subjects enrolled in NETT had severe airflow obstruction (FEV1 ≤ 45% predicted), hyperinflation, and bilateral emphysema on high-resolution chest CT. Spirometry was performed according to American Thoracic Society (ATS) standards before and after administration of 2 puffs (180 mcg) of inhaled albuterol (16). CT analysis on NETT subjects has been described previously (17).
Genes associated with BDR phenotypes in the NETT subjects were examined in participants in the Boston Early-Onset COPD Study. Enrollment of subjects and phenotyping in the Boston Early-Onset COPD Study have been described previously (18). Spirometry was performed in accordance with ATS specifications before and after inhalation of 2 puffs (180mcg) of albuterol using a spacer device. Extended pedigrees were ascertained through probands under 53 years old with COPD with FEV1<40% predicted. The current analysis included 949 subjects in 127 pedigrees.
Both studies were approved by institutional review boards at participating centers. All subjects provided written informed consent.
Genotyping was done for 122 single nucleotide polymorphisms (SNPs) in six candidate genes (19 in EPHX1, 5 in SFTPB, 21 in TGFB1, 64 in SERPINE2, 7 in GSTP1, 6 in ADRB2) including upstream and downstream chromosomal regions. We used pairwise linkage disequilibrium (LD)-tagging in Tagger with (19) minimum minor allele frequency of 0.10 and r2-threshold of 0.9, based on genotype data from Caucasian (CEU) trios in Phase II of the HapMap Project (20). Additional SNPs were also genotyped, based on previously reported genetic association analyses of COPD-related phenotypes (9) (10) (11). SNPs were genotyped using TaqMan (Applied Biosystems, Foster City, CA) or Sequenom (San Diego, CA) assays as previously reported (21) (22).
Response to albuterol was expressed in three ways: absolute change in FEV1 [BDRABS = postbronchodilator FEV1 − prebronchodilator FEV1], change in FEV1 as a percent of baseline FEV1 [BDRBASE = ((postbronchodilator FEV1 − prebronchodilator FEV1) / prebronchodilator FEV1) × 100], and change in FEV1 as a percent of predicted FEV1 [BDRPRED = ((postbronchodilator FEV1 − prebronchodilator FEV1) / predicted FEV1) × 100].
In NETT, associations between the three BDR phenotypes and SNP genotypes were tested using linear regression, adjusting for age, sex, and pack-years of smoking, assuming additive genetic models. Models for BDRABS were additionally adjusted for height. Analyses were conducted using SAS 9.1 (SAS Institute, Cary, NC). Association between haplotypes and BDR phenotypes in NETT subjects was tested using haplo.stats (23). In the Boston Early-Onset COPD Study, family-based association analysis was performed using PBAT software version 3.5 (24) assuming additive genetic models, adjusting for age, sex, and pack-years of smoking. Models for BDRABS were additionally adjusted for height. A p-value ≤ 0.05 was used to define a statistically significant result.
The mean age of NETT subjects was 67.4 years, and mean baseline pre-bronchodilator FEV1 was 24.8% predicted. Mean BDRABS was 0.09 L (range -0.17 to 0.45), 13.3% (range -16.5 to 67.9) for BDRBASE, and 3.2% (range -4.4 to 15.2) for BDRPRED (Table 1). Correlation between BDRABS and BDRBASE was 0.91, between BDRABS and BDRPRED was 0.94, and between BDRBASE and BDRPRED was 0.94 in NETT subjects. All subjects in NETT were required to abstain from smoking for 4 months prior to initial evaluation (25). The mean age of probands in the Boston Early-Onset COPD Study was 48.1 years, and the mean baseline pre-bronchodilator FEV1 was 19.2% predicted (Table 1). Sixteen out of 127 early-onset COPD probands (13%) were current smokers. Mean BDR phenotypes of probands in the Boston Early-Onset COPD Study were similar to those of NETT subjects (Table 1).
As shown in Table 2, two SNPs in EPHX1 (rs3753661 p = 0.02, rs3766934 p = 0.02) and two SNPs in SERPINE2 (rs6712954 p = 0.047, rs7588220 p = 0.01) were significantly associated with BDRABS. Three SNPs in EPHX1 (rs3753661 p = 0.01, rs3766934 p = 0.009, rs1009668 p = 0.04 (Figure 1)) and two SNPs in SERPINE2 (rs3795877 p = 0.04, rs7588220 p = 0.01) were associated with BDRBASE. One SNP in EPHX1 (rs3766934 p = 0.04), 3 SNPs in SERPINE2 (rs6712954 p = 0.04, rs3795877 p = 0.03, rs7588220 p = 0.004) and 2 SNPs in ADRB2 (rs1042717 p = 0.04, rs1042718 p = 0.045) were associated with BDRPRED. The Arg16Gly (rs1041713) and Gln27Glu (rs1041714) polymorphisms in ABRD2 were not associated with BDR phenotypes. SNPs rs3753661 and rs3766934 of EPHX1 were in complete LD (r2 = 1.0). The three significant SNPs in SERPINE2 were not in LD (r2< 0.1). SNPs in SFTPB, TGFB1, and GSTP1 were not associated with BDR phenotypes.
In order to assess replication of these associations, SNPs in EPHX1, SERPINE2 and ADRB2 were analyzed in the Boston Early-Onset COPD Study families. Among the significantly associated SNPs in the NETT subjects, one SNP downstream of EPHX1 (rs1009668) was associated with BDRPRED (p=0.04), and one SNP in intron 1 of SERPINE2 (rs7588220) was associated with BDRBASE (p=0.003) in the EOCOPD families. However, the direction of effect of SERPINE2 SNP rs7588220 was not consistent in the two studies. SNP rs7588220 is relatively rare (minor allele frequency = 0.02) in EOCOPD families, so there were only a small number of informative families in PBAT analysis (n=3). EPHX1 SNP rs1009668 was associated with reduced BDR in the two study samples. SNPs in ADRB2 were not associated with BDR phenotypes in EOCOPD families. However, rs1042713 (Arg16Gly) was associated with BDRPRED (p = 0.04) and BDRABS (p = 0.04) in subgroup with physician diagnosed asthma history in the EOCOPD families.
Because SNP rs1009668 was the only replicated SNP with BDR across both populations, we examined associations with FEV1 (% predicted) and chest CT emphysema. This SNP was not associated with FEV1 level, but it was associated with increased percent emphysema on chest CT scans at a threshold of -950 HU (β=0.04, p=0.009) in NETT subjects.
Haplotype analysis using haplo.stats in NETT subjects and using PBAT in EOCOPD families revealed associations similar to those found in the single SNP analysis in EPHX1 and SERPINE2 (Data not shown). In ADRB2, haplotypes of rs1042713 (Arg16Gly) and rs1042714 (Gln27Glu) were associated with BDRPRED in NETT (global p=0.046), but this was not replicated in EOCOPD families.
In this study, we examined SNPs in 6 COPD candidate genes including the ADRB2 gene, which has been shown to be associated with bronchodilator responsiveness, and found associations between bronchodilator responsiveness in NETT subjects and SNPs in the EPHX1, SERPINE2, ADRB2 genes. The association between one SNP in EPHX1 and bronchodilator responsiveness was replicated in extended pedigrees from the Boston Early-Onset COPD Study.
The product of EPHX1 is microsomal epoxide hydrolase, which is important for the metabolism of cigarette smoke by-products. In previous studies, the fast allele of EPHX1 (His139Arg) was found to be protective against COPD in a case-control study comparing NETT cases to control smokers (9), protective against upper lobe predominant emphysema in NETT (17), and associated with improved response to lung volume reduction surgery (LVRS), as measured by BODE score (22). The slow allele of EPHX1 (Tyr113His) has been associated with emphysema susceptibility (26) and reduced lung function (27). Haplotype analysis of His139Arg and Tyr113His revealed associations with rapid lung function decline in the Lung Health Study (28). In NETT, other SNPs in EPHX1 have been associated with exercise capacity, DLCO and response to LVRS (21) (22).
The replicated SNP, rs1009668, was chosen because it is downstream of EPHX1, but is actually located in exon 1 of an adjacent gene, KIAA0792. The function of this gene product is not known, but the gene has been shown to be expressed in adenocarcinoma of the lung (29). This SNP results in replacement of valine with methionine at amino acid position 622. In COPD patients, lower BDR may be related to more severe emphysema (4). This SNP (rs1009668), which was associated with lower BDR, was also associated with increased emphysema and may have a role in COPD pathogenesis. However, it is not clear whether this SNP association is due to linkage disequilibrium with a functional variant in EPHX1 or in KIAA0792.
A SNP in intron1 of SERPINE2 (rs7588220) was associated with BDR in both NETT subjects and Boston Early-Onset COPD Study families, although the directionality of association was not consistent. This gene was associated with COPD susceptibility in several COPD populations (11) (30). However, the role of this gene in pathophysiology of COPD has yet to be identified. It is possible that SERPINE2 variants could define a subset of COPD patients with differential bronchodilator responsiveness.
Two previous studies have investigated the association between BDR and genetic polymorphisms in COPD. A recent publication reported an association of BDR and polymorphisms of the β2 adrenergic receptor gene (ADRB2) in 246 Japanese patients with COPD (8). Arg16 and Arg16-Gln27 haplotype was associated with decreased BDR. This gene has been studied as susceptibility gene for asthma (31) and COPD (12) (13), and also for direct role in drug response (32). Studies in asthma suggest the Arg16 allele is associated with greater acute bronchodilator responsiveness but decreased long-term response to regular use of short-acting β2 agonists (14).
In our study, there was association only with haplotypes of the codon 16 and 27 polymorphisms in NETT subjects, but this was not replicated in EOCOPD families. In the EOCOPD families, the codon 16 polymorphism was associated with BDR only in the subgroup of subjects with a history of physician-diagnosed asthma. This suggests that ADRB2 is not a major determinant of BDR in COPD, but may be relevant in patients with an asthmatic phenotype.
In another study (7), a polymorphism in the hemopoietic cell kinase (HCK) gene was associated with differential expression of Hck protein and myeloperoxidase release from polymorphonuclear leukocytes in 60 COPD patients. This polymorphism was associated with BDR in 487 subjects from the Lung Health Study, suggesting that this gene contributes to COPD pathogenesis and modifies BDR. Because this gene was not associated with COPD susceptibility, we did not include it in our candidate gene panel.
The present study has several limitations. First, because there is not a single optimal measurement for bronchodilator responsiveness, we analyzed bronchodilator phenotypes expressed in three ways. All three measurements have their advantages and limitations, so we investigated all three phenotypes and used a replication strategy in this study to limit the possibility of false positive results. Second, the replicated SNP association in EPHX1 was for a different, but highly correlated, BDR phenotype in our two study populations. Third, intra-individual variability in response to bronchodilator (33) may reduce power to detect genetic associations, yet we were still able to identify SNP-level replication for association between EPHX1 and BDR. Fourth, the LD-tagging approach yielded SNPs that may not be the specific functional variants. The associated SNPs were intronic or in an exon of a gene with unknown function. The actual causal variant or variants remain to be discovered. Fifth, multiple statistical comparisons are a potential concern in any complex disease genetics study. Though the optimal approach to adjust for multiple testing is not clear, we used replication of the results in another study sample to guard against false positive results.
The NETT subjects and the Boston Early-Onset COPD probands all have severe airflow obstruction. Thus the applicability of our findings to mild-to-moderate COPD patients may be limited. However, family members in the Boston Early-Onset COPD Study had a broad range of lung function values. Early-onset COPD subjects likely represent a unique subgroup of COPD patients. This could contribute to lack of replication of our associations in NETT.
In conclusion, polymorphisms of EPHX1, SERPINE2, and ADRB2 were associated with bronchodilator responsiveness phenotypes in one population of subjects with severe COPD. BDR in COPD patients may depend upon different disease subtypes, differences in drug metabolism, or other pharmacogenetic effects. This study revealed that EPHX1 -- which has been previously associated with COPD susceptibility, lung function and CT phenotypes – demonstrated replication in a second population. BDR may be partly explained by genetic factors. Future directions will include replication in additional populations, identification of the functional variant or variants, and determination whether there are subtypes of COPD with differential bronchodilator responsiveness.
Funding: This study was supported by NIH grants P01 HL083069, HL075478, HL71393, HL080242, HL072918, and the Alpha-1 Foundation. The National Emphysema Treatment Trial (NETT) was supported by contracts with the National Heart, Lung, and Blood Institute (N01HR76101, N01HR76102, N01HR76103, N01HR76104, N01HR76105, N01HR76106, N01HR76107, N01HR76108, N01HR76109, N01HR76110, N01HR76111, N01HR76112, N01HR76113, N01HR76114, N01HR76115, N01HR76116, N01HR76118, and N01HR76119), the Centers for Medicare and Medicaid Services, and the Agency for Healthcare Research and Quality.
Conflict of interest statement WK, CH, DD and JR have no conflicts of interest to disclose. ES received an honorarium from Bayer for a symposium at the ERS Meeting in 2005; an honorarium for a talk on COPD genetics in 2006, and grant support and consulting fees from GlaxoSmithKline for two studies of COPD; and he received an honorarium from Astra-Zeneca for a talk at the Lund Symposium in 2007 as well as consulting fees.
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