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1.  Determinants of airflow obstruction in severe alpha‐1‐antitrypsin deficiency 
Thorax  2007;62(9):806-813.
Severe α1‐antitrypsin (AAT) deficiency is an autosomal recessive genetic condition associated with an increased but variable risk for chronic obstructive pulmonary disease (COPD). A study was undertaken to assess the impact of chronic bronchitis, pneumonia, asthma and sex on the development of COPD in individuals with severe AAT deficiency.
The AAT Genetic Modifier Study is a multicentre family‐based cohort study designed to study the genetic and epidemiological determinants of COPD in AAT deficiency. 378 individuals (age range 33–80 years), confirmed to be homozygous for the SERPINA1 Z mutation, were included in the analyses. The primary outcomes of interest were a quantitative outcome, forced expiratory volume in 1 s (FEV1) percentage predicted, and a qualitative outcome, severe airflow obstruction (FEV1 <50% predicted).
In multivariate analysis of the overall cohort, cigarette smoking, sex, asthma, chronic bronchitis and pneumonia were risk factors for reduced FEV1 percentage predicted and severe airflow obstruction (p<0.01). Index cases had lower FEV1 values, higher smoking histories and more reports of adult asthma, pneumonia and asthma before age 16 than non‐index cases (p<0.01). Men had lower pre‐ and post‐bronchodilator FEV1 percentage predicted than women (p<0.0001); the lowest FEV1 values were observed in men reporting a history of childhood asthma (26.9%). This trend for more severe obstruction in men remained when index and non‐index groups were examined separately, with men representing the majority of non‐index individuals with airflow obstruction (71%). Chronic bronchitis (OR 3.8, CI 1.8 to 12.0) and a physician's report of asthma (OR 4.2, CI 1.4 to 13.1) were predictors of severe airflow obstruction in multivariate analysis of non‐index men but not women.
In individuals with severe AAT deficiency, sex, asthma, chronic bronchitis and pneumonia are risk factors for severe COPD, in addition to cigarette smoking. These results suggest that, in subjects severely deficient in AAT, men, individuals with symptoms of chronic bronchitis and/or a past diagnosis of asthma or pneumonia may benefit from closer monitoring and potentially earlier treatment.
PMCID: PMC2117297  PMID: 17389752
2.  Association of cigarette smoking and CRP levels with DNA methylation in α-1 antitrypsin deficiency 
Epigenetics  2012;7(7):720-728.
Alpha-1 antitrypsin (AAT) deficiency and tobacco smoking are confirmed risk factors for Chronic Obstructive Pulmonary Disease. We hypothesized that variable DNA methylation would be associated with smoking and inflammation, as reflected by the level of C-Reactive Protein (CRP) in AAT-deficient subjects. Methylation levels of 1,411 autosomal CpG sites from the Illumina GoldenGate Methylation Cancer Panel I were analyzed in 316 subjects. Associations of five smoking behaviors and CRP levels with individual CpG sites and average methylation levels were assessed using non-parametric testing, linear regression and linear mixed effect models, with and without adjustment for age and gender. Univariate linear regression analysis revealed that methylation levels of 16 CpG sites significantly associated with ever-smoking status. A CpG site in the TGFBI gene was the only site associated with ever-smoking after adjustment for age and gender. No highly significant associations existed between age at smoking initiation, pack-years smoked, duration of smoking, and time since quitting smoking as predictors of individual CpG site methylation levels. However, ever-smoking and younger age at smoking initiation associated with lower methylation level averaged across all sites. DNA methylation at CpG sites in the RUNX3, JAK3 and KRT1 genes associated with CRP levels. The most significantly associated CpG sites with gender and age mapped to the CASP6 and FZD9 genes, respectively. In summary, this study identified multiple potential candidate CpG sites associated with ever-smoking and CRP level in AAT-deficient subjects. Phenotypic variability in Mendelian diseases may be due to epigenetic factors.
PMCID: PMC3414392  PMID: 22617718
68kDa (TGFBI); C-Reactive Protein (CRP); Chronic Obstructive Pulmonary Disease (COPD); Illumina GoldenGate Methylation Cancer Panel I; alpha-1 antitrypsin (AAT) deficiency; beta-induced; methylation; smoking behaviors; transforming growth factor
3.  Variable DNA Methylation Is Associated with Chronic Obstructive Pulmonary Disease and Lung Function 
Rationale: Chronic obstructive pulmonary disease (COPD) is associated with local (lung) and systemic (blood) inflammation and manifestations. DNA methylation is an important regulator of gene transcription, and global and specific gene methylation marks may vary with cigarette smoke exposure.
Objectives: To perform a comprehensive assessment of methylation marks in DNA from subjects well phenotyped for nonneoplastic lung disease.
Methods: We conducted array-based methylation screens, using a test-replication approach, in two family-based cohorts (n = 1,085 and 369 subjects).
Measurements and Main Results: We observed 349 CpG sites significantly associated with the presence and severity of COPD in both cohorts. Seventy percent of the associated CpG sites were outside of CpG islands, with the majority of CpG sites relatively hypomethylated. Gene ontology analysis based on these 349 CpGs (330 genes) suggested the involvement of a number of genes responsible for immune and inflammatory system pathways, responses to stress and external stimuli, as well as wound healing and coagulation cascades. Interestingly, our observations include significant, replicable associations between SERPINA1 hypomethylation and COPD and lower average lung function phenotypes (combined P values: COPD, 1.5 × 10−23; FEV1/FVC, 1.5 × 10−35; FEV1, 2.2 × 10−40).
Conclusions: Genetic and epigenetic pathways may both contribute to COPD. Many of the top associations between COPD and DNA methylation occur in biologically plausible pathways. This large-scale analysis suggests that DNA methylation may be a biomarker of COPD and may highlight new pathways of COPD pathogenesis.
PMCID: PMC3297093  PMID: 22161163
chronic obstructive pulmonary disease; epigenetics; DNA methylation; smoking
4.  Early-Onset Chronic Obstructive Pulmonary Disease Is Associated with Female Sex, Maternal Factors, and African American Race in the COPDGene Study 
Rationale: The characterization of young adults who develop late-onset diseases may augment the detection of novel genes and promote new pathogenic insights.
Methods: We analyzed data from 2,500 individuals of African and European ancestry in the COPDGene Study. Subjects with severe, early-onset chronic obstructive pulmonary disease (COPD) (n = 70, age < 55 yr, FEV1 < 50% predicted) were compared with older subjects with COPD (n = 306, age > 64 yr, FEV1 < 50% predicted).
Measurements and Main Results: Subjects with severe, early-onset COPD were predominantly females (66%), P = 0.0004. Proportionally, early-onset COPD was seen in 42% (25 of 59) of African Americans versus 14% (45 of 317) of non-Hispanic whites, P < 0.0001. Other risk factors included current smoking (56 vs. 17%, P < 0.0001) and self-report of asthma (39 vs. 25%, P = 0.008). Maternal smoking (70 vs. 44%, P = 0.0001) and maternal COPD (23 vs. 12%, P = 0.03) were reported more commonly in subjects with early-onset COPD. Multivariable regression analysis found association with African American race, odds ratio (OR), 7.5 (95% confidence interval [CI], 2.3–24; P = 0.0007); maternal COPD, OR, 4.7 (95% CI, 1.3–17; P = 0.02); female sex, OR, 3.1 (95% CI, 1.1–8.7; P = 0.03); and each pack-year of smoking, OR, 0.98 (95% CI, 0.96–1.0; P = 0.03).
Conclusions: These observations support the hypothesis that severe, early-onset COPD is prevalent in females and is influenced by maternal factors. Future genetic studies should evaluate (1) gene-by-sex interactions to address sex-specific genetic contributions and (2) gene-by-race interactions.
PMCID: PMC3175544  PMID: 21562134
chronic obstructive pulmonary disease; female; African Americans
5.  Genome-Wide Association Analysis of Body Mass in Chronic Obstructive Pulmonary Disease 
Cachexia, whether assessed by body mass index (BMI) or fat-free mass index (FFMI), affects a significant proportion of patients with chronic obstructive pulmonary disease (COPD), and is an independent risk factor for increased mortality, increased emphysema, and more severe airflow obstruction. The variable development of cachexia among patients with COPD suggests a role for genetic susceptibility. The objective of the present study was to determine genetic susceptibility loci involved in the development of low BMI and FFMI in subjects with COPD. A genome-wide association study (GWAS) of BMI was conducted in three independent cohorts of European descent with Global Initiative for Chronic Obstructive Lung Disease stage II or higher COPD: Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-Points (ECLIPSE; n = 1,734); Norway-Bergen cohort (n = 851); and a subset of subjects from the National Emphysema Treatment Trial (NETT; n = 365). A genome-wide association of FFMI was conducted in two of the cohorts (ECLIPSE and Norway). In the combined analyses, a significant association was found between rs8050136, located in the first intron of the fat mass and obesity–associated (FTO) gene, and BMI (P = 4.97 × 10−7) and FFMI (P = 1.19 × 10−7). We replicated the association in a fourth, independent cohort consisting of 502 subjects with COPD from COPDGene (P = 6 × 10−3). Within the largest contributing cohort of our analysis, lung function, as assessed by forced expiratory volume at 1 second, varied significantly by FTO genotype. Our analysis suggests a potential role for the FTO locus in the determination of anthropomorphic measures associated with COPD.
PMCID: PMC3266061  PMID: 21037115
chronic obstructive pulmonary disease genetics; chronic obstructive pulmonary disease epidemiology; chronic obstructive pulmonary disease metabolism; genome-wide association study
7.  IL10 Polymorphisms Are Associated with Airflow Obstruction in Severe α1-Antitrypsin Deficiency 
Severe α1-antitrypsin (AAT) deficiency is a proven genetic risk factor for chronic obstructive pulmonary disease (COPD), especially in individuals who smoke. There is marked variability in the development of lung disease in individuals homozygous (PI ZZ) for this autosomal recessive condition, suggesting that modifier genes could be important. We hypothesized that genetic determinants of obstructive lung disease may be modifiers of airflow obstruction in individuals with severe AAT deficiency. To identify modifier genes, we performed family-based association analyses for 10 genes previously associated with asthma and/or COPD, including IL10, TNF, GSTP1, NOS1, NOS3, SERPINA3, SERPINE2, SFTPB, TGFB1, and EPHX1. All analyses were performed in a cohort of 378 PI ZZ individuals from 167 families. Quantitative spirometric phenotypes included forced expiratory volume in one second (FEV1) and the ratio of FEV1/forced vital capacity (FVC). A qualitative phenotype of moderate-to-severe COPD was defined for individuals with FEV1 ⩽ 50 percent predicted. Six of 11 single-nucleotide polymorphisms (SNPs) in IL10 (P = 0.0005–0.05) and 3 of 5 SNPs in TNF (P = 0.01–0.05) were associated with FEV1 and/or FEV1/FVC. IL10 SNPs also demonstrated association with the qualitative COPD phenotype. When phenotypes of individuals with a physician's diagnosis of asthma were excluded, IL10 SNPs remained significantly associated, suggesting that the association with airflow obstruction was independent of an association with asthma. Haplotype analysis of IL10 SNPs suggested the strongest association with IL10 promoter SNPs. IL10 is likely an important modifier gene for the development of COPD in individuals with severe AAT deficiency.
PMCID: PMC2176135  PMID: 17690329
chronic obstructive pulmonary disease; genetic modifiers; interleukin 10; family-based association analysis
8.  Genetic Determinants of Emphysema Distribution in the National Emphysema Treatment Trial 
Rationale: Computed tomography (CT) scanning of the lung may reduce phenotypic heterogeneity in defining subjects with chronic obstructive pulmonary disease (COPD), and allow identification of genetic determinants of emphysema severity and distribution.
Objectives: We sought to identify genes associated with CT scan distribution of emphysema in individuals without α1-antitrypsin deficiency but with severe COPD.
Methods: We evaluated baseline CT densitometry phenotypes in 282 individuals with emphysema enrolled in the Genetics Ancillary Study of the National Emphysema Treatment Trial, and used regression models to identify genetic variants associated with emphysema distribution.
Measurements and Main Results: Emphysema distribution was assessed by two methods—assessment by radiologists and by computerized density mask quantitation, using a threshold of −950 Hounsfield units. A total of 77 polymorphisms in 20 candidate genes were analyzed for association with distribution of emphysema. GSTP1, EPHX1, and MMP1 polymorphisms were associated with the densitometric, apical-predominant distribution of emphysema (p value range = 0.001–0.050). When an apical-predominant phenotype was defined by the radiologist scoring method, GSTP1 and EPHX1 single-nucleotide polymorphisms were found to be significantly associated. In a case–control analysis of COPD susceptibility limited to cases with densitometric upper-lobe–predominant cases, the EPHX1 His139Arg single-nucleotide polymorphism was associated with COPD (p = 0.005).
Conclusions: Apical and basal emphysematous destruction appears to be influenced by different genes. Polymorphisms in the xenobiotic enzymes, GSTP1 and EPHX1, are associated with apical-predominant emphysema. Altered detoxification of cigarette smoke metabolites may contribute to emphysema distribution, and these findings may lead to further insight into genetic determinants of emphysema.
PMCID: PMC2049064  PMID: 17363767
COPD; genetics; association analysis; computed tomography; emphysema
9.  Concordance of Genotypes in Pre– and Post–Lung Transplantation DNA Samples 
Genetic epidemiology studies of end-stage lung disease are potentially hindered by low numbers of participants due to early death of patients from the underlying disease, or due to exclusion from studies after patients have had lung transplants, because of concern about bias of genotype data due to chimerism. The number of participants enrolled in genetic studies of end-stage lung disease could be increased by including those individuals who have undergone lung transplant. We hypothesized that individuals who have had lung transplants can be included in genetic epidemiology studies that use single nucleotide polymorphism and short tandem repeat marker data, without confounding due to chimerism. Ten probands with severe, early-onset chronic obstructive pulmonary disease were included in this analysis. Pre– and post–lung transplant DNA samples were used in the investigation of concordance of genotype results for 12 short tandem repeat markers and 23 single nucleotide polymorphisms. Concordance was observed for all genotypes before and after lung transplant. We conclude that the risk of biasing genetic epidemiology studies due to donor lung–related DNA microchimerism is low, and that the inclusion of post–lung transplantation participants will allow for larger genetic epidemiology studies of individuals with end-stage lung disease.
PMCID: PMC2715347  PMID: 15994430
genetic epidemiology; lung; chimerism; transplantation
10.  Cluster Analysis in the COPDGene Study Identifies Subtypes of Smokers with Distinct Patterns of Airway Disease and Emphysema 
Thorax  2014;69(5):416-423.
There is notable heterogeneity in the clinical presentation of patients with COPD. To characterize this heterogeneity, we sought to identify subgroups of smokers by applying cluster analysis to data from the COPDGene Study.
We applied a clustering method, k-means, to data from 10,192 smokers in the COPDGene Study. After splitting the sample into a training and validation set, we evaluated three sets of input features across a range of k (user-specified number of clusters). Stable solutions were tested for association with four COPD-related measures and five genetic variants previously associated with COPD at genome-wide significance. The results were confirmed in the validation set.
We identified four clusters that can be characterized as 1) relatively resistant smokers (i.e. no/mild obstruction and minimal emphysema despite heavy smoking), 2) mild upper zone emphysema predominant, 3) airway disease predominant, and 4) severe emphysema. All clusters are strongly associated with COPD-related clinical characteristics, including exacerbations and dyspnea (p<0.001). We found strong genetic associations between the mild upper zone emphysema group and rs1980057 near HHIP, and between the severe emphysema group and rs8034191 in the chromosome 15q region (p<0.001). All significant associations were replicated at p<0.05 in the validation sample (12/12 associations with clinical measures and 2/2 genetic associations).
Cluster analysis identifies four subgroups of smokers that show robust associations with clinical characteristics of COPD and known COPD-associated genetic variants.
PMCID: PMC4004338  PMID: 24563194
11.  Menthol cigarette smoking in the COPDGene cohort: Relationship with COPD, comorbidities and CT metrics 
Respirology (Carlton, Vic.)  2014;20(1):108-114.
Background and objective
Menthol cigarettes contain higher levels of menthol to produce a characteristic mint flavour and cooling sensation. Compared with non-menthol cigarettes, little information exists on the effects of menthol cigarette smoking on clinical and radiological characteristics of chronic obstructive pulmonary disease (COPD). The main objective of the present study was to examine associations between menthol cigarette use and the risk of COPD and its characteristics, such as exacerbation, comorbidities and computed tomography (CT) abnormalities.
We analysed the data from 5699 current smokers in the COPDGene cohort to evaluate whether lung function, comorbidities, exacerbations and CT parameters were different between menthol and non-menthol cigarette smokers.
There were 3758 (65.9%) who reported use of menthol cigarettes. Multivariable regression analysis revealed that younger age, female gender and African-American ethnicity were significantly associated with smoking of menthol cigarettes. No significant associations were found between menthol cigarette use and COPD, major CT findings or comorbidities, such as cardiovascular disease, congestive heart failure, peripheral vascular disease, cerebrovascular disease, hypertension, diabetes, gastro-oesophageal reflux and osteoporosis; however, menthol cigarette smokers were more likely to experience a severe exacerbation of COPD during longitudinal follow-up (odds ratio 1.29; 95% confidence interval: 1.01–1.54) compared with the non-menthol cigarette smokers.
These results confirm that menthol cigarettes are not safer than traditional cigarettes and suggest that menthol cigarette smokers may have more frequent severe exacerbations than non-menthol cigarette smokers.
PMCID: PMC4415502  PMID: 25328036
chronic obstructive pulmonary disease; computed tomography; exacerbation; menthol; smoking
12.  Risk loci for chronic obstructive pulmonary disease: a genome-wide association study and meta-analysis 
The Lancet. Respiratory medicine  2014;2(3):214-225.
The genetic risk factors for susceptibility to chronic obstructive pulmonary disease (COPD) are still largely unknown. Additional genetic variants are likely to be identified by genome-wide association studies in larger cohorts or specific subgroups.
Genome-wide association analysis in COPDGene (non-Hispanic whites and African-Americans) was combined with existing data from the ECLIPSE, NETT/NAS, and GenKOLS (Norway) studies. Analyses were performed both using all moderate-to-severe cases and the subset of severe cases. Top loci not previously described as genome-wide significant were genotyped in the ICGN study, and results combined in a joint meta-analysis.
Analysis of a total of 6,633 moderate-to-severe cases and 5,704 controls confirmed association at three known loci: CHRNA3/CHRNA5/IREB2, FAM13A, and HHIP (10−12 < P < 10−14), and also showed significant evidence of association at a novel locus near RIN3 (overall P, including ICGN = 5•4×10−9). In the severe COPD analysis (n=3,497), the effects at two of three previously described loci were significantly stronger; we also identified two additional loci previously reported to affect gene expression of MMP12 and TGFB2 (overall P = 2•6x10−9 and 8•3×10−9). RIN3 and TGFB2 expression levels were reduced in a set of Lung Tissue Research Consortium COPD lung tissue samples compared with controls.
In a genome-wide study of COPD, we confirmed associations at three known loci and found additional genome-wide significant associations with moderate-to-severe COPD near RIN3 and with severe COPD near MMP12 and TGFB2. Genetic variants, apart from alpha-1 antitrypsin deficiency, increase the risk of COPD. Our analysis of severe COPD suggests additional genetic variants may be identified by focusing on this subgroup.
National Heart, Lung, and Blood Institute; the COPD Foundation through contributions from AstraZeneca, Boehringer Ingelheim, Novartis, and Sepracor; GlaxoSmithKline; Centers for Medicare and Medicaid Services; Agency for Healthcare Research and Quality; US Department of Veterans Affairs.
PMCID: PMC4176924  PMID: 24621683
13.  Racial Differences in CT Phenotypes in COPD 
COPD  2013;10(1):20-27.
Whether African Americans (AA) are more susceptible to COPD than non-Hispanic Whites (NHW) and whether racial differences in disease phenotype exist is controversial. The objective is to determine racial differences in the extent of emphysema and airway remodeling in COPD.
First, 2,500 subjects enrolled in the COPDGene study were used to evaluate racial differences in quantitative CT (QCT) parameters of % emphysema, air trapping and airway wall thickness. Independent variables studied included race, age, gender, education, BMI, pack-years, smoking status, age at smoking initiation, asthma, previous work in dusty job, CT scanner and center of recruitment.
Of the 1,063 subjects with GOLD Stage II-IV COPD, 200 self-reported as AA. AAs had a lower mean % emphysema (13.1 % vs. 16.1%, p = 0.005) than NHW and proportionately less emphysema in the lower lung zones. After adjustment for covariates, there was no statistical difference by race in air trapping or airway wall thickness. Measured QCT parameters were more predictive of poor functional status in NHWs compared to AAs.
AAs have less emphysema than NHWs but the same degree of airway disease. Additional factors not easily assessed by current QCT techniques may account for the poor functional status in AAs.
PMCID: PMC4321889  PMID: 23413893
Airway wall thickness; Air trapping; Chronic obstructive pulmonary disease; Emphysema; Quantitative CT; Race
14.  Characterising the association of latency with α1-antitrypsin polymerisation using a novel monoclonal antibody 
α1-Antitrypsin is primarily synthesised in the liver, circulates to the lung and protects pulmonary tissues from proteolytic damage. The Z mutant (Glu342Lys) undergoes inactivating conformational change and polymerises. Polymers are retained within the hepatocyte endoplasmic reticulum (ER) in homozygous (PiZZ) individuals, predisposing the individuals to hepatic cirrhosis and emphysema. Latency is an analogous process of inactivating, intra-molecular conformational change and may co-occur with polymerisation. However, the relationship between latency and polymerisation remained unexplored in the absence of a suitable probe. We have developed a novel monoclonal antibody specific for latent α1-antitrypsin and used it in combination with a polymer-specific antibody, to assess the association of both conformers in vitro, in disease and during augmentation therapy. In vitro kinetics analysis showed polymerisation dominated the pathway but latency could be promoted by stabilising monomeric α1-antitrypsin. Polymers were extensively produced in hepatocytes and a cell line expressing Z α1-antitrypsin but the latent protein was not detected despite manipulation of the secretory pathway. However, α1-antitrypsin augmentation therapy contains latent α1-antitrypsin, as did the plasma of 63/274 PiZZ individuals treated with augmentation therapy but 0/264 who were not receiving this medication (p < 10−14). We conclude that latent α1-antitrypsin is a by-product of the polymerisation pathway, that the intracellular folding environment is resistant to formation of the latent conformer but that augmentation therapy introduces latent α1-antitrypsin into the circulation. A suite of monoclonal antibodies and methodologies developed in this study can characterise α1-antitrypsin folding and conformational transitions, and screen methods to improve augmentation therapy.
PMCID: PMC4305080  PMID: 25462157
α1-Antitrypsin; Latency; Polymerisation; Monoclonal antibodies; Augmentation therapy
15.  A Simplified Score to Quantify Comorbidity in COPD 
PLoS ONE  2014;9(12):e114438.
Comorbidities are common in COPD, but quantifying their burden is difficult. Currently there is a COPD-specific comorbidity index to predict mortality and another to predict general quality of life. We sought to develop and validate a COPD-specific comorbidity score that reflects comorbidity burden on patient-centered outcomes.
Materials and Methods
Using the COPDGene study (GOLD II-IV COPD), we developed comorbidity scores to describe patient-centered outcomes employing three techniques: 1) simple count, 2) weighted score, and 3) weighted score based upon statistical selection procedure. We tested associations, area under the Curve (AUC) and calibration statistics to validate scores internally with outcomes of respiratory disease-specific quality of life (St. George's Respiratory Questionnaire, SGRQ), six minute walk distance (6MWD), modified Medical Research Council (mMRC) dyspnea score and exacerbation risk, ultimately choosing one score for external validation in SPIROMICS.
Associations between comorbidities and all outcomes were comparable across the three scores. All scores added predictive ability to models including age, gender, race, current smoking status, pack-years smoked and FEV1 (p<0.001 for all comparisons). Area under the curve (AUC) was similar between all three scores across outcomes: SGRQ (range 0·7624–0·7676), MMRC (0·7590–0·7644), 6MWD (0·7531–0·7560) and exacerbation risk (0·6831–0·6919). Because of similar performance, the comorbidity count was used for external validation. In the SPIROMICS cohort, the comorbidity count performed well to predict SGRQ (AUC 0·7891), MMRC (AUC 0·7611), 6MWD (AUC 0·7086), and exacerbation risk (AUC 0·7341).
Quantifying comorbidity provides a more thorough understanding of the risk for patient-centered outcomes in COPD. A comorbidity count performs well to quantify comorbidity in a diverse population with COPD.
PMCID: PMC4267736  PMID: 25514500
16.  Sexually-dimorphic targeting of functionally-related genes in COPD 
BMC Systems Biology  2014;8:118.
There is growing evidence that many diseases develop, progress, and respond to therapy differently in men and women. This variability may manifest as a result of sex-specific structures in gene regulatory networks that influence how those networks operate. However, there are few methods to identify and characterize differences in network structure, slowing progress in understanding mechanisms driving sexual dimorphism.
Here we apply an integrative network inference method, PANDA (Passing Attributes between Networks for Data Assimilation), to model sex-specific networks in blood and sputum samples from subjects with Chronic Obstructive Pulmonary Disease (COPD). We used a jack-knifing approach to build an ensemble of likely networks for each sex. By adapting statistical methods to compare these network ensembles, we were able to identify strong differential-targeting patterns associated with functionally-related sets of genes, including those involved in mitochondrial function and energy metabolism. Network analysis also identified several potential sex- and disease-specific transcriptional regulators of these pathways.
Network analysis yielded insight into potential mechanisms driving sexual dimorphism in COPD that were not evident from gene expression analysis alone. We believe our ensemble approach to network analysis provides a principled way to capture sex-specific regulatory relationships and could be applied to identify differences in gene regulatory patterns in a wide variety of diseases and contexts.
Electronic supplementary material
The online version of this article (doi:10.1186/s12918-014-0118-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4269917  PMID: 25431000
Network modeling; Gene regulation; Regulatory networks; Sexual-dimorphism; Chronic Obstructive Lung Disease
17.  Non-emphysematous chronic obstructive pulmonary disease is associated with diabetes mellitus 
BMC Pulmonary Medicine  2014;14:164.
Chronic obstructive pulmonary disease (COPD) has been classically divided into blue bloaters and pink puffers. The utility of these clinical subtypes is unclear. However, the broader distinction between airway-predominant and emphysema-predominant COPD may be clinically relevant. The objective was to define clinical features of emphysema-predominant and non-emphysematous COPD patients.
Current and former smokers from the Genetic Epidemiology of COPD Study (COPDGene) had chest computed tomography (CT) scans with quantitative image analysis. Emphysema-predominant COPD was defined by low attenuation area at -950 Hounsfield Units (LAA-950) ≥10%. Non-emphysematous COPD was defined by airflow obstruction with minimal to no emphysema (LAA-950 < 5%).
Out of 4197 COPD subjects, 1687 were classified as emphysema-predominant and 1817 as non-emphysematous; 693 had LAA-950 between 5–10% and were not categorized. Subjects with emphysema-predominant COPD were older (65.6 vs 60.6 years, p < 0.0001) with more severe COPD based on airflow obstruction (FEV1 44.5 vs 68.4%, p < 0.0001), greater exercise limitation (6-minute walk distance 1138 vs 1331 ft, p < 0.0001) and reduced quality of life (St. George’s Respiratory Questionnaire score 43 vs 31, p < 0.0001). Self-reported diabetes was more frequent in non-emphysematous COPD (OR 2.13, p < 0.001), which was also confirmed using a strict definition of diabetes based on medication use. The association between diabetes and non-emphysematous COPD was replicated in the ECLIPSE study.
Non-emphysematous COPD, defined by airflow obstruction with a paucity of emphysema on chest CT scan, is associated with an increased risk of diabetes. COPD patients without emphysema may warrant closer monitoring for diabetes, hypertension, and hyperlipidemia and vice versa.
Trial registration identifiers: COPDGene NCT00608764, ECLIPSE NCT00292552.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2466-14-164) contains supplementary material, which is available to authorized users.
PMCID: PMC4216374  PMID: 25341556
Airway disease; CT scan; Diabetes mellitus; Emphysema; Spirometry
18.  Epidemiology, genetics, and subtyping of preserved ratio impaired spirometry (PRISm) in COPDGene 
Respiratory Research  2014;15(1):89.
Preserved Ratio Impaired Spirometry (PRISm), defined as a reduced FEV1 in the setting of a preserved FEV1/FVC ratio, is highly prevalent and is associated with increased respiratory symptoms, systemic inflammation, and mortality. Studies investigating quantitative chest tomographic features, genetic associations, and subtypes in PRISm subjects have not been reported.
Data from current and former smokers enrolled in COPDGene (n = 10,192), an observational, cross-sectional study which recruited subjects aged 45–80 with ≥10 pack years of smoking, were analyzed. To identify epidemiological and radiographic predictors of PRISm, we performed univariate and multivariate analyses comparing PRISm subjects both to control subjects with normal spirometry and to subjects with COPD. To investigate common genetic predictors of PRISm, we performed a genome-wide association study (GWAS). To explore potential subgroups within PRISm, we performed unsupervised k-means clustering.
The prevalence of PRISm in COPDGene is 12.3%. Increased dyspnea, reduced 6-minute walk distance, increased percent emphysema and decreased total lung capacity, as well as increased segmental bronchial wall area percentage were significant predictors (p-value <0.05) of PRISm status when compared to control subjects in multivariate models. Although no common genetic variants were identified on GWAS testing, a significant association with Klinefelter’s syndrome (47XXY) was observed (p-value < 0.001). Subgroups identified through k-means clustering include a putative “COPD-subtype”, “Restrictive-subtype”, and a highly symptomatic “Metabolic-subtype”.
PRISm subjects are clinically and genetically heterogeneous. Future investigations into the pathophysiological mechanisms behind and potential treatment options for subgroups within PRISm are warranted.
Trial registration Identifier: NCT000608764.
Electronic supplementary material
The online version of this article (doi:10.1186/s12931-014-0089-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4256936  PMID: 25096860
Spirometry; Restriction; Lung diseases; Smoking
19.  Systemic Steroid Exposure Is Associated with Differential Methylation in Chronic Obstructive Pulmonary Disease 
Rationale: Systemic glucocorticoids are used therapeutically to treat a variety of medical conditions. Epigenetic processes such as DNA methylation may reflect exposure to glucocorticoids and may be involved in mediating the responses and side effects associated with these medications.
Objectives: To test the hypothesis that differences in DNA methylation are associated with current systemic steroid use.
Methods: We obtained DNA methylation data at 27,578 CpG sites in 14,475 genes throughout the genome in two large, independent cohorts: the International COPD Genetics Network (ndiscovery = 1,085) and the Boston Early Onset COPD study (nreplication = 369). Sites were tested for association with current systemic steroid use using generalized linear mixed models.
Measurements and Main Results: A total of 511 sites demonstrated significant differential methylation by systemic corticosteroid use in all three of our primary models. Pyrosequencing validation confirmed robust differential methylation at CpG sites annotated to genes such as SLC22A18, LRP3, HIPK3, SCNN1A, FXYD1, IRF7, AZU1, SIT1, GPR97, ABHD16B, and RABGEF1. Functional annotation clustering demonstrated significant enrichment in intrinsic membrane components, hemostasis and coagulation, cellular ion homeostasis, leukocyte and lymphocyte activation and chemotaxis, protein transport, and responses to nutrients.
Conclusions: Our analyses suggest that systemic steroid use is associated with site-specific differential methylation throughout the genome. Differentially methylated CpG sites were found in biologically plausible and previously unsuspected pathways; these genes and pathways may be relevant in the development of novel targeted therapies.
PMCID: PMC3622442  PMID: 23065012
DNA methylation; glucocorticoids; chronic obstructive pulmonary disease
20.  Gene–environment interaction testing in family-based association studies with phenotypically ascertained samples: a causal inference approach 
Biostatistics (Oxford, England)  2011;13(3):468-481.
We propose a method for testing gene–environment (G × E) interactions on a complex trait in family-based studies in which a phenotypic ascertainment criterion has been imposed. This novel approach employs G-estimation, a semiparametric estimation technique from the causal inference literature, to avoid modeling of the association between the environmental exposure and the phenotype, to gain robustness against unmeasured confounding due to population substructure, and to acknowledge the ascertainment conditions. The proposed test allows for incomplete parental genotypes. It is compared by simulation studies to an analogous conditional likelihood–based approach and to the QBAT-I test, which also invokes the G-estimation principle but ignores ascertainment. We apply our approach to a study of chronic obstructive pulmonary disorder.
PMCID: PMC3372944  PMID: 22084302
Causal inference; COPD; Family-based association; G-estimation; Gene–environment interaction
21.  Cigarette smoking behaviors and time since quitting are associated with differential DNA methylation across the human genome 
Human Molecular Genetics  2012;21(13):3073-3082.
The impact of cigarette smoking can persist for extended periods following smoking cessation and may involve epigenetic reprogramming. Changes in DNA methylation associated with smoking may help to identify molecular pathways that contribute to the latency between exposure and disease onset. Cross-sectional cohort data from subjects in the International COPD Genetics Network (n = 1085) and the Boston Early-Onset COPD study (n = 369) were analyzed as the discovery and replication cohorts, respectively. Genome-wide methylation data on 27 578 CpG sites in 14 475 genes were obtained on DNA from peripheral blood leukocytes using the Illumina HumanMethylation27K Beadchip in both cohorts. We identified 15 sites significantly associated with current smoking, 2 sites associated with cumulative smoke exposure, and, within the subset of former smokers, 3 sites associated with time since quitting cigarettes. Two loci, factor II receptor-like 3 (F2RL3) and G-protein-coupled receptor 15 (GPR15), were significantly associated in all three analyses and were validated by pyrosequencing. These findings (i) identify a novel locus (GPR15) associated with cigarette smoking and (ii) suggest the existence of dynamic, site-specific methylation changes in response to smoking which may contribute to the extended risks associated with cigarette smoking that persist after cessation.
PMCID: PMC3373248  PMID: 22492999
22.  A genome-wide association study of COPD identifies a susceptibility locus on chromosome 19q13 
Human Molecular Genetics  2011;21(4):947-957.
The genetic risk factors for chronic obstructive pulmonary disease (COPD) are still largely unknown. To date, genome-wide association studies (GWASs) of limited size have identified several novel risk loci for COPD at CHRNA3/CHRNA5/IREB2, HHIP and FAM13A; additional loci may be identified through larger studies. We performed a GWAS using a total of 3499 cases and 1922 control subjects from four cohorts: the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE); the Normative Aging Study (NAS) and National Emphysema Treatment Trial (NETT); Bergen, Norway (GenKOLS); and the COPDGene study. Genotyping was performed on Illumina platforms with additional markers imputed using 1000 Genomes data; results were summarized using fixed-effect meta-analysis. We identified a new genome-wide significant locus on chromosome 19q13 (rs7937, OR = 0.74, P = 2.9 × 10−9). Genotyping this single nucleotide polymorphism (SNP) and another nearby SNP in linkage disequilibrium (rs2604894) in 2859 subjects from the family-based International COPD Genetics Network study (ICGN) demonstrated supportive evidence for association for COPD (P = 0.28 and 0.11 for rs7937 and rs2604894), pre-bronchodilator FEV1 (P = 0.08 and 0.04) and severe (GOLD 3&4) COPD (P = 0.09 and 0.017). This region includes RAB4B, EGLN2, MIA and CYP2A6, and has previously been identified in association with cigarette smoking behavior.
PMCID: PMC3298111  PMID: 22080838
23.  Integration of Mouse and Human Genome-Wide Association Data Identifies KCNIP4 as an Asthma Gene 
PLoS ONE  2013;8(2):e56179.
Asthma is a common chronic respiratory disease characterized by airway hyperresponsiveness (AHR). The genetics of asthma have been widely studied in mouse and human, and homologous genomic regions have been associated with mouse AHR and human asthma-related phenotypes. Our goal was to identify asthma-related genes by integrating AHR associations in mouse with human genome-wide association study (GWAS) data. We used Efficient Mixed Model Association (EMMA) analysis to conduct a GWAS of baseline AHR measures from males and females of 31 mouse strains. Genes near or containing SNPs with EMMA p-values <0.001 were selected for further study in human GWAS. The results of the previously reported EVE consortium asthma GWAS meta-analysis consisting of 12,958 diverse North American subjects from 9 study centers were used to select a subset of homologous genes with evidence of association with asthma in humans. Following validation attempts in three human asthma GWAS (i.e., Sepracor/LOCCS/LODO/Illumina, GABRIEL, DAG) and two human AHR GWAS (i.e., SHARP, DAG), the Kv channel interacting protein 4 (KCNIP4) gene was identified as nominally associated with both asthma and AHR at a gene- and SNP-level. In EVE, the smallest KCNIP4 association was at rs6833065 (P-value 2.9e-04), while the strongest associations for Sepracor/LOCCS/LODO/Illumina, GABRIEL, DAG were 1.5e-03, 1.0e-03, 3.1e-03 at rs7664617, rs4697177, rs4696975, respectively. At a SNP level, the strongest association across all asthma GWAS was at rs4697177 (P-value 1.1e-04). The smallest P-values for association with AHR were 2.3e-03 at rs11947661 in SHARP and 2.1e-03 at rs402802 in DAG. Functional studies are required to validate the potential involvement of KCNIP4 in modulating asthma susceptibility and/or AHR. Our results suggest that a useful approach to identify genes associated with human asthma is to leverage mouse AHR association data.
PMCID: PMC3572953  PMID: 23457522
24.  Combining Disease Models to Test for Gene-Environment Interaction in Nuclear Families 
Biometrics  2011;67(4):1260-1270.
It is useful to have robust gene-environment interaction tests that can utilize a variety of family structures in an efficient way. This paper focuses on tests for gene-environment interaction in the presence of main genetic and environmental effects. The objective is to develop powerful tests that can combine trio data with parental genotypes and discordant sibships when parents genotypes are missing. We first make a modest improvement on a method for discordant sibs (discordant on phenotype), but the approach does not allow one to use families when all offspring are affected, e.g. trios. We then make a modest improvement on a Mendelian transmission-based approach that is inefficient when discordant sibs are available, but can be applied to any nuclear family. Finally, we propose a hybrid approach that utilizes the most efficient method for a specific family type, then combines over families. We utilize this hybrid approach to analyze a chronic obstructive pulmonary disorder dataset to test for gene-environment interaction in the Serpine2 gene with smoking. The methods are freely available in the R package fbati.
PMCID: PMC3120904  PMID: 21401569
Gene-Environment Interaction; Family-Based Association Tests; Candidate Gene Analysis; Binary Trait; COPD; Serpine2
25.  Alu and LINE-1 methylation and lung function in the normative ageing study 
BMJ Open  2012;2(5):e001231.
To investigate the association between methylation of transposable elements Alu and long-interspersed nuclear elements (LINE-1) and lung function.
Cohort study.
Outpatient Veterans Administration facilities in greater Boston, Massachusetts, USA.
Individuals from the Veterans Administration Normative Aging Study, a longitudinal study of aging in men, evaluated between 1999 and 2007. The majority (97%) were white.
Primary and secondary outcome measures
Primary predictor was methylation, assessed using PCR-pyrosequencing after bisulphite treatment. Primary outcome was lung function as assessed by spirometry, performed according to American Thoracic Society/European Respiratory Society guidelines at the same visit as the blood draws.
In multivariable models adjusted for age, height, body mass index (BMI), pack-years of smoking, current smoking and race, Alu hypomethylation was associated with lower forced expiratory volume in 1 s (FEV1) (β=28 ml per 1% change in Alu methylation, p=0.017) and showed a trend towards association with a lower forced vital capacity (FVC) (β=27 ml, p=0.06) and lower FEV1/FVC (β=0.3%, p=0.058). In multivariable models adjusted for age, height, BMI, pack-years of smoking, current smoking, per cent lymphocytes, race and baseline lung function, LINE-1 hypomethylation was associated with more rapid decline of FEV1 (β=6.9 ml/year per 1% change in LINE-1 methylation, p=0.005) and of FVC (β=9.6 ml/year, p=0.002).
In multiple regression analysis, Alu hypomethylation was associated with lower lung function, and LINE-1 hypomethylation was associated with more rapid lung function decline in a cohort of older and primarily white men from North America. Future studies should aim to replicate these findings and determine if Alu or LINE-1 hypomethylation may be due to specific and modifiable environmental exposures.
PMCID: PMC3488751  PMID: 23075571
Epidemiology; Genetics

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