ONLINE DATA SUPPLEMENT Genomewide Linkage of Forced Mid-Expiratory Flow in Chronic Obstructive Pulmonary Disease Dawn L. DeMeo, M.D., M.P.H.,Juan C. Celedón, M.D., Dr.P.H., Christoph Lange, John J. Reilly, M.D., Harold A. Chapman M.D.,Jody S. Sylvia, Frank E. Speizer, M.D., Scott T. Weiss, M.D., M.S.,Edwin K. Silverman, M.D., Ph.D.
Probands and Relative Enrollment The enrollment procedures for the Boston Early-Onset COPD Study have been described previously(1, 2). Briefly, probands were identified as individuals with a physician s diagnosis of COPD and FEV 1 < 40% of predicted, age < 53 years and no evidence of severe alpha 1-antitrypsin deficiency. All available first- and older second- degree relatives of the probands were invited to participate. Seventy-two pedigrees with mean size of 8 individuals (range 2-15) were included in the initial genome-scan linkage analysis of FEV 1 and FEV 1 /FVC (3). The current analysis represents a subset of the original 585 participants from 72 pedigrees. For post bronchodilator analysis of FEV 1 and FEV 1 /FVC (n=562), 23 individuals were excluded from the original data, as they were lacking post-bronchodilator values. For linkage analysis of FEF 25-75 and FEF 25-75 /FVC (n=556 for pre and post-bronchodilator analyses), 6 additional individuals were excluded due to the measurement of these values with a different spirometer. All participants provided written informed consent and completed a protocol that included a questionnaire, spirometry and a blood specimen from which DNA was extracted. Questionnaire Each participant completed a modified version of the 1978 American Thoracic Society- Division of Lung Diseases Epidemiology Questionnaire(4). 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. Non-smokers were classified as individuals answering no to the question have you ever smoked cigarettes?.
Pulmonary Function Tests Spirometry testing was performed in these pedigrees using a Survey Tach Spirometer (Warren E. Collins, Braintree, MA) as previously described(1) and in accordance with guidelines of the American Thoracic Society (5). Participants were asked to refrain, when possible, from the use of inhaled bronchodilator medications for at least four hours prior to spirometric evaluation to obtain adequate assessments of pre- and post-bronchodilator measures for FEV 1, FEV 1 /FVC, FEF 25-75 and FEF 25-75 /FVC. Post-bronchodilator spirometry was assessed approximately 15 minutes after the administration of albuterol. Absolute volume measurements with adjustments for relevant covariates were analyzed for this series of linkage analyses. Genotyping DNA was extracted at our laboratory using Puregene kits (Gentra Systems). Genome scan genotyping of short tandem repeat (STR) markers was performed by the Mammalian Genotyping Service of the National Heart, Lung and Blood Institute (Marshfield, WI). 377 autosomal STR markers separated by an average distance of 9.1 cm were included in the initial analysis, and marker locations were identified on Version 10 of the Marshfield Map (http://research.marshfield.org/genetics/map_markers/maps). On chromosome 2, nine additional STR markers were genotyped at Brigham and Women s Hospital; these additional markers were located between 215 and 233 cm. For these additional markers map locations were determined on the basis of the Marshfield Map with corroboration from the UCSC Human Genome Project Working Draft. On chromosome 12, 12 additional STR markers were genotyped between 18 and 49 cm on chromosome 12p(6). Primers (fluorescent labeled and unlabeled) were obtained from
Research Genetics and Applied Biosystems (ABI, Foster City, CA). Polymerase chain reactions were performed with Taq Gold Polymerase and products were analyzed on an ABI 3100 machine. GENESCAN and GENOTYPER (version 3.7) software programs were used to assist with genotype determinations; two reviewers manually verified all calls. As previously described, the RELCHECK program was used to assess for Mendelian inconsistencies in these pedigrees using the genome-scan marker data(3). Twenty-two subjects were excluded based on these results. Individual marker inconsistencies in the remaining 585 individuals were resolved using the PEDCHECK program(7). The SOLAR program was used to estimate marker allele frequencies through maximum likelihood methods(8). Linkage Analysis The phenotypes for the unstratified linkage analysis included both pre- and postbronchodilator FEF 25-75 and FEF 25-75 /FVC. Initial linkage analysis included all genotyped individuals. Multipoint linkage analysis was performed using a variance component approach as implemented in the Sequential Oligogenic Linkage Analysis Routines (SOLAR) software system, version 1.7.4(8), assuming an additive mode of inheritance. Genotypes were imputed for individuals with missing genotype information, and identity by descent matrices (IBD) were estimated for each autosomal marker. Multipoint IBD matrices were subsequently generated in SOLAR at a 1 cm resolution. IBD matrices were recalculated for chromosomes 2 and 12 with the supplemental markers. Covariates considered for inclusion in the multivariate models included age, sex, race, height, and pack-years of smoking with the higher order polynomials of age 2,
height 2 and pack-years 2. Covariates were retained in the polygenic models if P <0.05. Narrow sense heritability estimates were calculated in SOLAR using the variancecomponent linkage approach and represent the ratio of the phenotypic variance due to the additive genetic effects of a trait divided by the total trait phenotypic variance. Because pedigrees were ascertained through a single proband with severe, early-onset COPD (and thus very low spirometry) all analyses included an ascertainment correction by conditioning the pedigree likelihood on the probability of the proband s phenotype(9). 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 FEV 1, FEV 1 /FVC, FEF 25-75 and FEF 25-75 /FVC: Model 1) all subjects excluding any adjustment for smoking; Model 2) all subjects adjusting for intensity of cigarette smoking by including pack-years and pack-years 2 as covariates; and Model 3) smokers-only analysis including pack-years and pack-years 2 as covariates. For the analysis of smokers-only, the phenotypes of the non-smokers were set to missing. We performed point-wise simulations in SOLAR 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 due to due to deviations from multivariate normality, linkage analysis was repeated for chromosomes 2 and 12 using the multivariate t distribution option, as implemented in SOLAR(8).
References E1. Silverman, E. K., H. A. Chapman, J. M. Drazen, S. T. Weiss, B. Rosner, E. J. Campbell, W. J. O'Donnell, J. J. Reilly, L. Ginns, S. Mentzer, J. Wain, and F. E. Speizer. 1998. Genetic epidemiology of severe, early-onset chronic obstructive pulmonary disease. Risk to relatives for airflow obstruction and chronic bronchitis. Am J Respir Crit Care Med 157(6 Pt 1):1770-8. E2. Silverman, E. K., S. T. Weiss, J. M. Drazen, H. A. Chapman, V. Carey, E. J. Campbell, P. Denish, R. A. Silverman, J. C. Celedon, J. J. Reilly, L. C. Ginns, and F. E. Speizer. 2000. Gender-related differences in severe, early-onset chronic obstructive pulmonary disease. Am J Respir Crit Care Med 162(6):2152-8. E3. Silverman, E. K., L. J. Palmer, J. D. Mosley, M. Barth, J. M. Senter, A. Brown, J. M. Drazen, D. J. Kwiatkowski, H. A. Chapman, E. J. Campbell, M. A. Province, D. C. Rao, J. J. Reilly, L. C. Ginns, F. E. Speizer, and S. T. Weiss. 2002. Genomewide linkage analysis of quantitative spirometric phenotypes in severe early-onset chronic obstructive pulmonary disease. Am J Hum Genet 70(5):1229-39. E4. Ferris, B. G. 1978. Epidemiology Standardization Project (American Thoracic Society). Am Rev Respir Dis 118(6 Pt 2):1-120. E5. 1995. Standardization of Spirometry, 1994 Update. American Thoracic Society. Am J Respir Crit Care Med 152(3):1107-36. E6. Silverman, E. K., J. D. Mosley, L. J. Palmer, M. Barth, J. M. Senter, A. Brown, J. M. Drazen, D. J. Kwiatkowski, H. A. Chapman, E. J. Campbell, M. A. Province, D. C. Rao, J. J. Reilly, L. C. Ginns, F. E. Speizer, and S. T. Weiss. 2002. Genome-wide linkage
analysis of severe, early-onset chronic obstructive pulmonary disease: airflow obstruction and chronic bronchitis phenotypes. Hum Mol Genet 11(6):623-32. E7. O'Connell, J. R., and D. E. Weeks. 1998. PedCheck: a program for identification of genotype incompatibilities in linkage analysis. Am J Hum Genet 63(1):259-66. E8. Almasy, L., and J. Blangero. 1998. Multipoint quantitative-trait linkage analysis in general pedigrees. Am J Hum Genet 62(5):1198-211. E9. Lange, K., D. Weeks, and M. Boehnke. 1988. Programs for Pedigree Analysis: MENDEL, FISHER, and dgene. Genet Epidemiol 5(6):471-2.