Published ahead of print on September 3, 2004, doi:10.1164/rccm.200404-524OC
© 2004 American Thoracic Society doi: 10.1164/rccm.200404-524OC
Genome-wide Linkage of Forced Mid-expiratory Flow in Chronic Obstructive Pulmonary DiseaseChanning Laboratory and Division of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital; Division of Pulmonary and Critical Care Medicine, Department of Medicine, Beth Israel Deaconess Medical Center; Harvard Medical School; Department of Biostatistics, Harvard School of Public Health, Boston, Massachusetts; and Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California at San Francisco, San Francisco, California Correspondence and requests for reprints should be addressed to Dawn L. DeMeo, M.D., M.P.H., Channing Laboratory, 181 Longwood Avenue, Boston, MA 02115. Email: redld{at}channing.harvard.edu
Familial aggregation of forced expiratory flow during the middle half of the FVC (FEF25–75%) and FEF25–75%/FVC has been observed in the Boston Early-Onset Chronic Obstructive Pulmonary Disease Study, but linkage results have not been reported for these phenotypes. An autosomal whole genome-wide linkage scan was performed in 72 pedigrees ascertained through a proband with severe, early-onset chronic obstructive pulmonary disease, and linkage analyses of FEF25–75% and FEF25–75%/FVC were performed using Sequential Oligogenic Linkage Analysis Routines. There was suggestive evidence for linkage of FEF25–75%/FVC with chromosome 2 (LOD 2.60 at 216 cM). In a smokers-only analysis, evidence for linkage was observed for postbronchodilator FEF25–75% with chromosome 12 (LOD 5.03 at 35 cM) and chromosomes 2 and 12 for FEF25–75%/FVC (LOD 4.12 at 221 cM and LOD 3.46 at 35 cM, respectively); in the smokers-only model, evidence for linkage also was robust for FEV1/FVC on chromosome 2 (LOD 4.13 at 229 cM) and FEV1 on chromosome 12 (LOD 3.26 at 36 cM). Our analyses provide evidence for linkage of FEF25–75% and FEF25–75%/FVC on chromosomes 2q and 12p. LOD scores of greater than two were also observed for chromosomes 16, 20, and 22 with the smokers-only analysis, which may suggest gene-by-smoking interactions in these regions.
Key Words: chronic obstructive pulmonary disease • genetics • linkage mapping • pulmonary disease • smoking Chronic obstructive pulmonary disease (COPD, MIM: 606963) is a complex human disease that is influenced by both genes and environment. Although cigarette smoking remains the predominant factor influencing disease development, the occasional development of COPD in nonsmokers and the variable development of COPD in smokers suggests that influences other than cigarette smoking are integral for disease susceptibility. Linkage to several genomic regions has been demonstrated for FEV1 and FEV1/FVC in extended pedigrees of individuals with early-onset COPD. Specifically, in the Boston Early-Onset COPD Study, significant evidence for linkage of FEV1/FVC to chromosome 2q was found, with a maximum LOD (logarithm of the likelihood for linkage) score of 4.42 at 222 cM (1, 2). Assessment of additional quantitative phenotypes may assist in further characterizing genetic risk for the development of COPD. Forced expiratory flow during the middle half of the FVC (FEF25–75%) and FEF25–75%/FVC have been demonstrated to be highly heritable, with heritability estimates similar to those of FEV1 and FEV1/FVC in the Boston Early-Onset COPD Study (3). Reduced measures of FEF25–75% and FEF25–75%/FVC in nonsmoking first-degree relatives of COPD probands compared with nonsmoking control subjects may suggest an independent genetic susceptibility to develop COPD that is accentuated in the presence of cigarette smoking (3). FEF25–75% and FEF25–75%/FVC are decreased in the presence of airflow obstruction, which may be associated with inflammation and/or fibrosis of the small airways. Alterations in FEF25–75% and FEF25–75%/FVC have also been interpreted as indicators of airway–parenchymal dysanaptic lung growth. Our current analysis presents autosomal genome-wide linkage results for FEF25–75% and FEF25–75%/FVC in pedigrees of probands with severe early-onset COPD. Because of the importance of cigarette smoking in the development of COPD, linkage analyses were extended to include stratified models of cigarette smoking to identify regions of linkage suggestive of gene-by-smoking interactions, potentially relevant to disease susceptibility.
Probands and Relative Enrollment The enrollment procedures for the Boston Early-Onset COPD Study have been described previously (4, 5). This analysis represents a subset of the original 585 participants from 72 pedigrees. For postbronchodilator analysis of FEV1 and FEV1/FVC (n = 562), 23 individuals were excluded from the original data, as they were lacking postbronchodilator values. For linkage analysis of FEF25–75% and FEF25–75%/FVC (n = 556 for prebronchodilator and postbronchodilator analyses), six additional individuals were excluded because of the measurement of these values outside the research study. Each participant completed a modified version of the 1978 American Thoracic Society-Division of Lung Diseases Epidemiology Questionnaire (6). Pack-years of cigarette smoking were calculated as the product of the duration of smoking in years and the average number of cigarettes smoked per day, divided by 20 to convert to packs of cigarettes. Spirometry testing was performed using a Survey Tach Spirometer (Warren E. Collins, Braintree, MA) (4) and in accordance with guidelines of the American Thoracic Society (7). The protocol was approved by the Human Research Committee of Partners Health Care and the Veterans' Administration Institutional Review Board.
Genotyping
Linkage Analysis As smoking represents the most important environmental exposure for the development of COPD, three models were considered for the linkage analysis of postbronchodilator phenotypes for FEV1, FEV1/FVC, FEF25–75%, and FEF25–75%/FVC: Model 1 was all subjects excluding any adjustment for smoking; model 2 was all subjects adjusting for intensity of cigarette smoking by including pack-years and pack-years2 as covariates, and model 3 was a smokers-only analysis including pack-years and pack-years2 as covariates. For the analysis of smokers only, the phenotypes of the nonsmokers were set to missing. We performed point-wise simulations in Sequential Oligogenic Linkage Analysis Routines using genotypes simulated for a fully informative unlinked marker. Simulations were assessed in 100,000 consecutive replicates; empirical p values were calculated by identifying the number of times LOD scores exceeded specific LOD score thresholds. To avoid false inference of linkage caused by deviations from multivariate normality, linkage analyses were repeated for chromosomes 2 and 12 using the multivariate "t" distribution option implemented in Sequential Oligogenic Linkage Analysis Routines (10). Additional detail about all methods is provided in an online supplement.
Demographics The age, smoking history, and postbronchodilator spirometry values for members of the pedigrees included in the genome scan linkage analysis are provided stratified by relationship to the proband (Table 1). The unadjusted values for these phenotypes are presented to convey the range of the phenotypes incorporated with covariates in the Sequential Oligogenic Linkage Analysis Routines analyses. Prebronchodilator and postbronchodilator percentage predicted values for this cohort have been presented in prior publications (1, 2). For FEV1, FEV1/FVC, FEF25–75%, and FEF25–75%/FVC, probands have the lowest values, followed by parents, older second-degree relatives, and then siblings.
Genome-wide Multipoint Variance Component Linkage Analysis for Prebronchodilator and Postbronchodilator FEF25–75% and FEF25–75%/FVC The estimated heritability for prebronchodilator FEF25–75% was 0.35; this heritability increased to 0.41 for postbronchodilator FEF25–75%, similar to our previously reported values (3). Significant covariates included in the linkage models for FEF25–75% were age, sex, height, pack-years, and pack-years2. Multipoint LOD scores greater than or equal to 1.00 were found for chromosomes 2 and 3 for both prebronchodilator and postbronchodilator FEF25–75% and chromosomes 1, 8, and 12 only for postbronchodilator FEF25–75% (Table 2). The highest LOD score was 1.80 at 60 cM on chromosome 8 for postbronchodilator FEF25–75%.
The estimated heritability of prebronchodilator and postbronchodilator FEF25–75%/FVC was 0.42 and 0.47, respectively, similar to our previously reported values (3). Significant covariates included in the linkage models were age, pack-years, and pack-years2. Multipoint LOD scores greater than or equal to 1.00 were observed for prebronchodilator FEF25–75%/FVC on chromosomes 2 and 4; multipoint LOD scores greater than 1.00 were noted for postbronchodilator FEF25–75%/FVC on chromosomes 2, 8, and 16 (Table 2).
Linkage Analysis with Differential Inclusion of Smoking-related Covariates
Phenotype: Postbronchodilator FEF25–75%
Phenotype: Postbronchodilator FEF25–75%/FVC Increased evidence for linkage was observed for the smokers only analysis of FEF25–75%/FVC on chromosome 2 (LOD of 3.74 at 224 cM), compared with linkage evidence for chromosome 2 with the inclusion of pack-years of cigarettes in the model (LOD of 2.6 at 216 cM; Table 3) or without smoking covariates in the model (LOD 2.75 at 215 cM; Table 3). Modest evidence for linkage was observed on chromosome 16 for the smokers-only linkage analysis (LOD of 2.04 at 86 cM), with a trend for linkage with the smoking stratified analysis for chromosome 12 (LOD of 1.84 at 36 cM). The inclusion of additional chromosome 2 markers increased the evidence for linkage for the baseline model without pack-years (LOD of 2.52 at 214 cM); for the smokers-only model, there remained strong evidence for linkage (LOD of 4.12 at 221 cM; Figure 1C). The analysis with additional chromosome 12 markers resulted in an increased LOD score for the model with smoking covariates (LOD increase from 0.71 to 2.17; Figure 1D) and for the smokers-only model (LOD increase from 1.84 to 3.46; Figure 1D).
Phenotype: Postbronchodilator FEV1
Phenotype: Postbronchodilator FEV1/FVC Evidence for linkage was observed on chromosome 2 for postbronchodilator FEV1/FVC in the smoking covariate model (as previously reported) (2) with an increase in LOD score for the smokers-only model (LOD of 4.60 at 225 cM; Table 4). Evidence for linkage also was observed on chromosomes 1 and 17, which did not increase with smoking stratification. On chromosome 20, there was an increasing trend in LOD scores from models without smoking covariates to the model stratified by smoking (LOD 2.21 at 20 cM; Table 4). The analysis of chromosome 2 with additional markers still demonstrated strong evidence for linkage with models that considered smoking covariates in all subjects and in the smokers only analysis (LOD 4.30 at 230 cM and LOD 4.13 at 229 cM, respectively; Figure 1C). On chromosome 12, evidence for linkage increased with the additional markers for all three models, with an increase in LOD score for models with pack-years of smoking as a covariate (LOD of 2.51 at 37 cM; Figure 1D) and the model with smokers only (LOD 3.04 at 37 cM; Figure 1D).
Simulated Results and Inclusion of Multivariate t Distribution Further analysis of our models with the additional STR markers on chromosomes 2 and 12 was undertaken using the multivariate "t" distribution; all findings remained robust with this approach. Regarding the maximum LOD scores for each phenotype for the smokers-only analyses using the multivariate t distribution, for FEF25–75% and chromosome 12, the maximum LOD score was slightly attenuated to 4.64; there was no attenuation of the LOD score on chromosome 2 for FEF25–75%/FVC; for FEV1 and chromosome 12, the LOD score was slightly attenuated to 3.22, and for FEV1/FVC and chromosome 2, the LOD score was slightly attenuated to 3.89.
COPD is a complex human disease that is likely influenced by multiple genes, environmental factors, and gene-by-environment interactions. Unlike many complex diseases, the major environmental risk factor for COPD (cigarette smoking) is recognized and readily quantifiable. The Boston Early-Onset COPD Study represents a unique collection of individuals with severe, early-onset COPD in the absence of  1-antitrypsin deficiency (a proven genetic cause of COPD). Previously reported genome scan linkage analyses of quantitative and qualitative phenotypes in this population have been performed for FEV1 and FEV1/FVC, using both prebronchodilator and postbronchodilator spirometry measures (1, 2, 8); to date, there have been no genome scan linkage results reported for FEF25–75% and FEF25–75%/FVC. Here we present linkage results for FEF25–75% and FEF25–75%/FVC and linkage results for FEV1, FEV1/FVC, FEF25–75%, and FEF25–75%/FVC incorporating additional STR markers on chromosomes 2 and 12. We observed strong evidence for linkage with chromosomes 2 and 12 for the spirometric flow-related phenotypes of FEF25–75% and FEF25–75%/FVC, suggesting that these measures are important to consider in linkage and association studies in families at risk for COPD. We also examined linkage for all four spirometric phenotypes (FEV1, FEV1/FVC, FEF25–75%, and FEF25–75%/FVC) using modeling of cigarette smoking to attempt to differentiate main effects and gene-by-environment influences of cigarette smoking. By stratifying our analysis by cigarette smoking, in addition to observing robust LOD scores on chromosomes 2 and 12 (despite the potential loss of power associated with eliminating individuals who are nonsmokers), we have now identified chromosomes 16, 20, and 22 as new, previously unreported regions that may be important in COPD through gene-by-smoking interactions. For all of the models considered, the inclusion of additional STR markers on chromosomes 2 and 12 and variable inclusion of smoking in the models did not change appreciably the 1.5-LOD support interval around the linkage peaks, although the evidence for linkage did remain stable or increase with the flanking markers. DeMeo and colleagues (3) demonstrated significant familial aggregation of FEF25–75% and FEF25–75%/FVC in the Boston Early-Onset COPD Study. The current linkage analysis of flow parameters includes both prebronchodilator and postbronchodilator values, but postbronchodilator values may be preferable to consider in linkage studies because of the reduced variability of spirometric measures after relaxation of airway smooth muscles with bronchodilator medications (2). The initial analyses of prebronchodilator and postbronchodilator FEF25–75% and FEF25–75%/FVC suggested the presence of a susceptibility locus on chromosome 2, with the highest LOD score for FEF25–75%/FVC (LOD 2.60 at 216 cM). Abnormalities of FEF25–75% have been suggested to represent evidence for small airway disease (13), but others maintain that this phenotype does not provide evidence beyond FEV1/FVC for capturing the presence of small airway alterations (14–16). The observation of increasing LOD scores with analyses of smokers only indicates that a gene-by-smoking interaction may be more important in these regions for these phenotypes. The measure of FEF25–75%/FVC has been considered a surrogate measure for dysanaptic lung growth, the physiologically normal but nonisotropic lung growth between airways and lung parenchyma (17, 18). Although dysanapsis may be a characteristic of the healthy individual, Green and colleagues also hypothesized that it may represent a susceptibility state for the development of COPD (17). Through segregation analysis, Chen and colleagues recently demonstrated that measures suggestive of dysanaptic lung growth may be under major genetic control (19). Further research is required to determine whether genes within the linkage peaks for FEF25–75%, FEF25–75%/FVC, and FEV1/FVC are important for lung growth and development and/or early inflammatory effects of smoking on airflow obstruction. To investigate the effect of cigarette smoking on our linkage results, we performed genome-wide linkage analysis on FEF25–75% and FEF25–75%/FVC, as well as FEV1 and FEV1/FVC, using three different approaches to model tobacco smoking. We hypothesized that models that demonstrate the highest LOD scores after stratification by smoking (the major risk environment for the development of COPD) most strongly suggest a gene-by-smoking interactions with our phenotypes of interest. Models that include pack-years of cigarette smoking as a covariate and result in the highest LOD score may suggest a main effect of smoking but potentially no gene-by-environment interaction; those models with the highest LOD scores on exclusion of cigarette smoking covariates suggest no smoking effect. A similar approach to stratifying individuals on the basis of the risk exposure has been used in an investigation of asthma and environmental tobacco smoke exposure (20). Colilla and colleagues suggest that stratification by tobacco smoke exposure may lead to less heterogeneity of the phenotypes of interest, with a resultant increase in power to detect susceptibility loci (20). The utility of stratification for revealing novel loci has been corroborated by other authors using simulated data (21–23). Stratification may be a useful analytic strategy to use when there are known environmental risk factors and likely multiple susceptibility loci contributing to disease. Analyses of various models in our study have resulted in LOD scores that have met standard criteria for suggestive (LOD score 1.9 or greater) and significant (LOD score 3.3 or greater) linkage (24). These criteria were not designed for the complex, multistep analytic approach that we use here. However, focusing on models in which LOD scores increase with smoking stratification and in which maximal LOD scores are greater than 2, we have identified chromosomes 2, 12, 16, 20, and 22 as potentially harboring loci that may influence phenotypic manifestations of COPD susceptibility via interactions with tobacco smoke. In a general population sample, Joost and colleagues have identified suggestive linkage for FEV1 on chromosomes 6q. We did not find evidence for linkage in that region to any spirometric phenotypes. However, both the results from Joost and colleagues and our analysis found nominal evidence for linkage (LOD > 1) on chromosomes 4 and 19 (25). In the National Heart, Lung, and Blood Institute Family Heart Study, FEV1/FVC was linked to chromosome 4, but this linkage signal was approximately 30 cM from our modest linkage to FEF25–75% and FEV1 on 4p (26). Both the study by Wilk and colleagues and our study demonstrated some evidence for linkage of 1p to FEV1/FVC. Support for evidence of linkage for FEV1/FVC to 2q in the general population was demonstrated by Malhotra and colleagues in a region similar to the region of our linkage signal (27). As such, some of the linkage signals observed in our current analysis may be relevant to genetic determinants of lung function in general. The ratio of FEF25–75%/FVC has been observed to be associated with airways hyperresponsiveness, a key feature of asthma. Some evidence for linkage with asthma and asthma-related phenotypes that overlap with regions identified by our analyses has been observed for chromosomes 2q and 12p (28–33). These results suggest that some of the intermediate phenotypes that are shared disease characteristics in asthma and COPD may be due to shared genetic features of development or interactions with environmental exposures. This study is limited by potential generalizability of these linkage results from families with severe, early-onset disease to individuals with COPD that presents at later ages. As well, the Boston Early-Onset COPD Study cohort is predominantly white; the generalizability of these findings to other families and other races is potentially limited. Reliable phenotype specification is crucial for robust linkage results. Measurement-related variability in spirometric phenotypes (especially FEF25–75% and FEF25–75%/FVC) must be considered as a potential source of error, but the same technique and spirometric equipment were used for all participants, which may increase the accuracy and reproducibility of these measures in our study. Also, the use of postbronchodilator measurements for linkage analysis has been suggested to decrease the heterogeneity of spirometric measures (2). Importantly, variance component linkage analysis can be influenced by kurtosis of the phenotype distributions, with kurtosis being a main determinant of the effect of non-normality on the likelihood ratio test statistic (34). However, none of our results demonstrated kurtosis greater than 2; simulations were highly significant, and results remained robust when analyzed using the "t" distribution. We have completed point-wise (not genome-wide) simulation estimates for our most significant linkage analysis results. Our simulated p values differ by an order of magnitude compared with what would be expected, suggesting that our highest LOD score (5.03 for FEF25–75% on chromosome 12) may be slightly inflated. In addition, we did not formally correct for multiple comparisons given the number of analyses that we performed for each phenotype, but simulations suggest that our most significant linkage results for FEF25–75% were not markedly inflated because of performing subsetted analyses. We have not corrected for the number of phenotypes that we analyzed because of the high correlation between spirometric measures. In light of these issues, instead of focusing on the exact value of LOD scores, we have used these observations to generate hypotheses regarding gene-by-smoking interactions relevant to future fine mapping in these chromosomal regions. Stratification based on an environmental exposure is one approach to assesses for genotype-by-environment interaction (35). In the Boston Early-Onset COPD Study families, a direct comparison of linkage signals in smokers and nonsmokers could not be fairly performed, as a low number of probands and relatives were nonsmokers. Instead, we compared the linkage results in the entire set of family members to the linkage results in smokers only. The identification of several regions in which linkage signals increased in smokers only, despite including a smaller number of subjects in the linkage analysis, suggests that the smokers were generating the evidence for linkage in those regions. These stratification results are suggestive of a genotype-by-smoking interaction, but formal statistical testing of interaction has not been performed. Susceptibility to develop COPD is mediated by genes, tobacco exposure, as well as the interaction between genes and smoking. Replication of our linkage results will be important to corroborate these observations. The use of intermediate phenotypes and analytic methods such as stratification by risk factors to identify loci relevant for gene-by-environment interactions may help direct investigation toward novel genetic determinants of COPD, with the ultimate goal of gaining new insight into COPD pathophysiology and disease treatment.
Supported by grants from the National Institutes of Health (HL61575, HL71393, HL75478, and HL67204 [H.A.C.]), by a Career Investigator Award from the American Lung Association (E.K.S.), and by National Institutes of Health grant K08 HL72918 (D.L.D.). This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Conflict of Interest Statement: D.L.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.C.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; C.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.J.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; H.A.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.S.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; F.E.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.T.W. received a grant for $900,065 for the Asthma Policy Modeling Study and from AstraZeneca from 1997–2003 and was co-investigator on a grant from Millenium Pharmaceuticals to pursue asthma genetics in 1996–2001 and received a grant from Pfizer to examine diabetes mellitus and its relationship to lung function between 2000 and 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 that began in 2003 and received no funds for his involvement in this project and has been a consultant for Variagenics on human subjects issue 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 has received a grant from Glaxo-Wellcome for $500,000 for genomics equipment from 2000–2003 and was a consultant for Roche Pharmaceuticals in 2000 and received no financial renumeration for this consultancy. E.K.S. has received a grant of approximately $250,000 per year from GlaxoSmithKline to study COPD genetics between 2001 and 2004 and also received a payment of $2,400 from GlaxoSmithKline in 2003 for consultation in respiratory diseases. Received in original form April 20, 2004; accepted in final form August 28, 2004
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