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Association of Forced Vital Capacity with the Developmental Gene NCOR2

  • Cosetta Minelli ,

    cosetta.minelli1@imperial.ac.uk

    Affiliation Respiratory Epidemiology, Occupational Medicine and Public Health, National Heart and Lung Institute, Imperial College, London, United Kingdom

  • Charlotte H. Dean,

    Affiliations Leukocyte Biology, National Heart and Lung Institute, Imperial College London, London, United Kingdom, Mammalian Genetics Unit, MRC Harwell, Oxon, United Kingdom

  • Matthew Hind,

    Affiliation Respiratory Department, Royal Brompton and Harefield NHS Foundation Trust, London, United Kingdom

  • Alexessander Couto Alves,

    Affiliation Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London, London, United Kingdom

  • André F. S. Amaral,

    Affiliations Respiratory Epidemiology, Occupational Medicine and Public Health, National Heart and Lung Institute, Imperial College, London, United Kingdom, MRC-PHE Centre for Environment & Health, London, United Kingdom

  • Valerie Siroux,

    Affiliations Univ. Grenoble Alpes, IAB, Team of Environmental Epidemiology applied to Reproduction and Respiratory Health, F-38000, Grenoble, France, INSERM, IAB, Team of Environmental Epidemiology applied to Reproduction and Respiratory Health, F-38000, Grenoble, France, CHU de Grenoble, IAB, Team of Environmental Epidemiology applied to Reproduction and Respiratory Health, F-38000, Grenoble, France

  • Ville Huikari,

    Affiliation Biocenter Oulu, University of Oulu, Oulu, Finland

  • María Soler Artigas,

    Affiliation Genetic Epidemiology Group, Department of Health Sciences, University of Leicester, Leicester, United Kingdom

  • David M. Evans,

    Affiliations University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Australia, MRC Integrative Epidemiology Unit, University of Bristol, Bristol, United Kingdom

  • Daan W. Loth,

    Affiliation Department of Epidemiology, Erasmus Medical Center, Rotterdam, The Netherlands

  • Yohan Bossé,

    Affiliation Institut universitaire de cardiologie et de pneumologie de Québec, Department of Molecular Medicine, Laval University, Québec, Canada

  • Dirkje S. Postma,

    Affiliation Department of Pulmonary Diseases, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

  • Don Sin,

    Affiliation The University of British Columbia Center for Heart Lung Innovation, St-Paul’s Hospital, Vancouver, Canada

  • John Thompson,

    Affiliation Department of Health Sciences, University of Leicester, Leicester, United Kingdom

  • Florence Demenais,

    Affiliations INSERM, UMRS-946, Genetic Variation of Human Diseases Unit, Paris, France, Univ. Paris Diderot, Sorbonne Paris Cité, Institut Universitaire d’Hématologie, F-75007, Paris, France

  • John Henderson,

    Affiliation School of Social and Community Medicine, University of Bristol, Bristol, United Kingdom

  • SpiroMeta consortium ,

    Membership of the SpiroMeta consortium and CHARGE consortium is listed in the Acknowledgments.

    Affiliation SpiroMeta consortium, Genetic Epidemiology Group, Department of Health Sciences, University of Leicester, Leicester, United Kingdom

  • CHARGE consortium ,

    Membership of the SpiroMeta consortium and CHARGE consortium is listed in the Acknowledgments.

    Affiliation CHARGE consortium, Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, United States of America

  • Emmanuelle Bouzigon,

    Affiliations INSERM, UMRS-946, Genetic Variation of Human Diseases Unit, Paris, France, Univ. Paris Diderot, Sorbonne Paris Cité, Institut Universitaire d’Hématologie, F-75007, Paris, France

  • Deborah Jarvis,

    Affiliations Respiratory Epidemiology, Occupational Medicine and Public Health, National Heart and Lung Institute, Imperial College, London, United Kingdom, MRC-PHE Centre for Environment & Health, London, United Kingdom

  • Marjo-Riitta Järvelin,

    Affiliations Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London, London, United Kingdom, MRC-PHE Centre for Environment & Health, London, United Kingdom, Biocenter Oulu, University of Oulu, Oulu, Finland, Center for Life Course Epidemiology, Faculty of Medicine, P.O. Box 5000, FI-90014 University of Oulu, Oulu, Finland, Unit of Primary Care, Oulu University Hospital, Kajaanintie 50, P.O. Box 20, FI-90220, Oulu, 90029 OYS, Finland

  •  [ ... ],
  • Peter Burney

    Affiliations Respiratory Epidemiology, Occupational Medicine and Public Health, National Heart and Lung Institute, Imperial College, London, United Kingdom, MRC-PHE Centre for Environment & Health, London, United Kingdom

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Association of Forced Vital Capacity with the Developmental Gene NCOR2

  • Cosetta Minelli, 
  • Charlotte H. Dean, 
  • Matthew Hind, 
  • Alexessander Couto Alves, 
  • André F. S. Amaral, 
  • Valerie Siroux, 
  • Ville Huikari, 
  • María Soler Artigas, 
  • David M. Evans, 
  • Daan W. Loth
PLOS
x

Abstract

Background

Forced Vital Capacity (FVC) is an important predictor of all-cause mortality in the absence of chronic respiratory conditions. Epidemiological evidence highlights the role of early life factors on adult FVC, pointing to environmental exposures and genes affecting lung development as risk factors for low FVC later in life. Although highly heritable, a small number of genes have been found associated with FVC, and we aimed at identifying further genetic variants by focusing on lung development genes.

Methods

Per-allele effects of 24,728 SNPs in 403 genes involved in lung development were tested in 7,749 adults from three studies (NFBC1966, ECRHS, EGEA). The most significant SNP for the top 25 genes was followed-up in 46,103 adults (CHARGE and SpiroMeta consortia) and 5,062 children (ALSPAC). Associations were considered replicated if the replication p-value survived Bonferroni correction (p<0.002; 0.05/25), with a nominal p-value considered as suggestive evidence. For SNPs with evidence of replication, effects on the expression levels of nearby genes in lung tissue were tested in 1,111 lung samples (Lung eQTL consortium), with further functional investigation performed using public epigenomic profiling data (ENCODE).

Results

NCOR2-rs12708369 showed strong replication in children (p = 0.0002), with replication unavailable in adults due to low imputation quality. This intronic variant is in a strong transcriptional enhancer element in lung fibroblasts, but its eQTL effects could not be tested due to low imputation quality in the eQTL dataset. SERPINE2-rs6754561 replicated at nominal level in both adults (p = 0.036) and children (p = 0.045), while WNT16-rs2707469 replicated at nominal level only in adults (p = 0.026). The eQTL analyses showed association of WNT16-rs2707469 with expression levels of the nearby gene CPED1. We found no statistically significant eQTL effects for SERPINE2-rs6754561.

Conclusions

We have identified a new gene, NCOR2, in the retinoic acid signalling pathway pointing to a role of vitamin A metabolism in the regulation of FVC. Our findings also support SERPINE2, a COPD gene with weak previous evidence of association with FVC, and suggest WNT16 as a further promising candidate.

Introduction

Forced vital capacity (FVC), a spirometric measure routinely used in clinical practice to approximate vital capacity, is increasingly recognised as an important parameter beyond its diagnostic and prognostic role in restrictive lung diseases. Unlike the ratio of forced expiratory volume in 1 second (FEV1) to FVC, an indicator of airway obstruction, FVC is a strong predictor of all-cause mortality in asymptomatic adults without chronic respiratory conditions[1]. Although the origins of a low FVC in the general population are poorly understood, there is a strong link to poverty[2], and in particular to low socio-economic status in early life[3]. Endemic vitamin A deficiency is associated with low FVC, and maternal supplementation with vitamin A before, during and after pregnancy, improves FVC in offspring[4]. Low FVC has also been associated with early exposure to particulate air pollution[5]. The deviation of an individual’s FVC values (and lung function in general) from the population mean has been shown to remain stable over time, with future values being predicted by early measurements (“tracking”)[6], which means that early life and genetic effects that manifest in childhood will influence the individual’s whole FVC life trajectory. Taken together, this evidence highlights the role of early life factors on adult FVC, which points to environmental exposures and genes affecting the development of the lung. Severe defects in lung development lead to neonatal death, but milder structural or functional defects could affect lung function and increase susceptibility to lung diseases that become clinically detectable during childhood or later life, including asthma and COPD[7]. This is supported by experimental work on in-vitro and animal models of lung function and disease[8].

Knowledge of the genetics of FVC is still limited. Biological candidates for FVC, mainly related to host defense, inflammatory pathway, pulmonary surfactant and oxidative stress, have been evaluated in candidate-gene association studies, but replication has been difficult. New candidates for FVC have been provided by genome-wide association (GWA) studies, the largest being a recent meta-analysis from the joint CHARGE and SpiroMeta consortia on 52,253 individuals, with replication of the top associations in 24,840 individuals[9]. It identified eight loci, of which six new (EFEMP1, BMP6, MIR129-2-HSD17B12, PRDM11, WWOX, KCNJ2), and two previously associated with FEV1 and FEV1/FVC (GSTCD and PTCH1). The eight loci explain 1.8% of FVC variation, and yet FVC heritability (proportion of FVC variation attributable to genetic factors) is estimated around 40–60% by familial aggregation and twin studies[10, 11] and, more recently, genome-wide data[12].

Available GWA datasets represent an invaluable resource to test hypotheses about the role of genetic pathways involved in specific pathophysiological mechanisms. We hypothesised that focusing on genes lying in pathways related to lung development could help identify new candidates for FVC and further our understanding of the underlying biological mechanisms.

Materials and Methods

We evaluated the effect on FVC of 403 genes (24,728 SNPs) related to lung development in two stages. In Stage 1, all SNPs were tested for association with FVC in a meta-analysis of three European adult studies (ECRHS[13], NFBC1966[14], EGEA[15]). For replication in adults (CHARGE and SpiroMeta consortia)[9] and children (ALSPAC[16]) in Stage 2, we selected the best signal for the top 25 genes, defined as the SNP with the lowest meta-analysis p-value which satisfied the following criteria: minor allele frequency >0.05 and imputation quality (imputation R2) >0.7 in all three studies; low between-study heterogeneity defined as I2<30%, with I2 representing the percentage of total variation in effect estimates across studies due to heterogeneity rather than chance.

The rationale for limiting our replication analysis to the best signal for the top 25 genes was to maximise the probability of successful replication in children, where the sample size was only 5,062. With this sample size, testing for replication of 25 SNPs gives a power of about 80% to detect a variant explaining 0.3% of FVC residual variance, at a Bonferroni corrected p-value threshold of 0.002 (0.05/25). This assuming that genetic effects in children may be slightly stronger than in adults, where the variance explained by the eight loci previously identified[9] was 1.8%, an average of 0.23% per SNP.

Selection of candidate genes and SNPs

Two experts in lung development, a basic scientist (C.H.D.) and a clinician scientist (M.H.), compiled a list of genes involved in lung development, first independently and then through agreement. The selection of genes was based on their knowledge of the topic, mainly using genetic evidence from animal models[8, 17, 18]. This initial list was extended to include additional genes suggested by: 1) pathways information obtained from KEGG[19]–relevant genes lying in the same pathways as those in the initial list; 2) information from published literature identified using HuGE Navigator[20]–genes considered as associated with lung development in previous genetic association studies. When in doubt about which genes to select from large gene families, those with higher gene expression in foetal lung were chosen, with information retrieved from the Human U133A/GNF1H Gene Atlas database using BioGPS[21].

The final list included 403 genes (S1 Table). According to NCBI gene definition, we retrieved SNPs within 2 kb upstream and 500 bp downstream of each gene, using the R package NCBI2R (http://cran.r-project.org/web/packages/NCBI2R). We identified 24,728 SNPs for which imputed data (based on HapMap release 22) were available for all three studies in Stage 1 (S1 Table).

Study populations

Stage 1.

Below and in Table 1 we briefly describe the three studies, with details on spirometry and genotyping methods summarised in S2 and S3 Tables.

thumbnail
Table 1. Characteristics of studies in Stage 1.

N = number of subjects included in the analyses.

https://doi.org/10.1371/journal.pone.0147388.t001

The Northern Finland Birth Cohort 1966 (NFBC1966) is a birth-cohort study in the provinces of Oulu and Lapland that recruited pregnant women with an expected date of delivery in 1966. A total of 12,231 children were recruited and followed-up in adulthood[14], with 6,033 participating in the clinical follow-up at 31 years. Of these, 5,218 individuals with GWA and spirometry data were included in this study.

The European Community Respiratory Health Survey (ECRHS) is an international cohort study designed to identify risk factors for asthma[13] that started in 1992–1994, with follow-up performed twice in the following 20 years. Included in this study are 1,662 subjects from the first survey (ECRHS I, age 20–48) with GWA and spirometry data available, recruited from 16 centres that used random sampling frameworks.

The Epidemiological study on the Genetics and Environment of Asthma (EGEA), which combines a case-control and a family-based study of asthma, was conducted in 1991–1995 (EGEA1), with follow-up after 12 years (EGEA2, 2003–2007)[15]. The study included 388 nuclear families, ascertained by one or two asthmatic adult or paediatric probands, and 415 population-based controls, totalling 2,120 subjects. This analysis only includes 869 non-asthmatic adults, using spirometry data from EGEA1 for subjects ≥18 year old at baseline and EGEA2 for those <18 in EGEA1.

Stage 2.

The joint CHARGE (Cohorts for Heart and Aging Research in Genomic Epidemiology) and SpiroMeta consortia performed a GWA investigation of FVC in 52,253 individuals of European ancestry from 26 studies[9], which included ECRHS and NFBC1966. Included here are 46,103 individuals from 24 studies, after subtracting the contribution of ECRHS and NFBC1966. New effect estimates and standard errors were derived by taking a weighted difference between the original fixed-effect meta-analysis estimate and the pooled estimate of ECRHS and NFBC1966.

The Avon Longitudinal Study of Parents and their Children (ALSPAC) is a birth cohort study consisting initially of 14,541 women and their children recruited in the county of Avon, UK, in the early 1990s[16]. Included in this study are 5,062 white European children (50.3% male) of 8–9 years of age with GWA and spirometry data. Their mean height was 132.6 cm (standard deviation, SD: 5.8) and mean FVC 1,931 ml (SD: 319).

Statistical analyses

Stage 1.

Study-specific estimates for the three studies were obtained assuming an additive mode of inheritance. In ECRHS, linear regression analyses of the effects of the SNPs on FVC (in ml) were adjusted for age, age2, height, sex, centre, and first four ancestry principal components to control for residual population stratification. In NFBC1966, all subjects were 31 year olds and linear regression analyses were only adjusted for height, sex and first two principal components. In the family-based EGEA, the regression analyses were performed using linear mixed models to account for family structure, adjusting for age, age2, height, sex and first two principal components.

Inverse-variance weighted meta-analysis of the three studies using a fixed effect model was performed on a total of 7,749 individuals.

The association analyses for NFBC1966 were carried out using SNPTEST[22], while the analyses for ECRHS and EGEA and the meta-analysis were performed using R, version 3.0.1 (www.R-project.org).

Stage 2.

Individual cohorts within CHARGE and SpiroMeta performed GWA analyses for FVC (ml) using linear regression adjusted for age, age2, height and sex (plus height2 and weight for CHARGE), as well as centre and/or principal components if appropriate[9].

In ALSPAC, linear regression analyses on FVC (ml) were performed adjusting for age, age2, height and sex. Principal components were not included since no evidence of population stratification was found in the study.

Replication of a SNP was defined based on evidence from Stage 2 only, rather than on combined evidence from Stage 1 and Stage 2, since this protects against the winner’s curse, an upwards bias typical of the screening stage[23]. We considered a SNP replicated if the effect estimate was in the same direction as in Stage 1 and the one-side p-value survived Bonferroni correction for multiple testing (p<0.002) in either adults or children. We considered replication evidence as suggestive if the p-value was significant only at nominal level.

Lung eQTL data

For SNPs with evidence of replication, we investigated their effects on the expression of nearby genes (genes within 100 kb up and downstream from the SNP) in lung samples from the Lung QTL consortium. This includes data on 1,111 individuals undergoing lung surgery, recruited at Laval University (n = 409), University of British Columbia (n = 339) and University of Groningen (n = 363)[24].

Gene expression and genotyping profiles were obtained using a custom Affymetrix array (GEO platform GPL10379) and the Illumina Human1M-Duo BeadChip array, respectively. Expression values were extracted using the Robust Multichip Average method[25] implemented in the Affymetrix Power Tools software. Expression values were analysed with a robust regression model adjusted for age, sex and smoking status, using the R statistical package MASS (rlm function).

Genetic associations were performed in PLINK 1.9. A fixed-effect meta-analysis was used to pool the results across the three sites.

Results

Stage 1 study-specific and meta-analysis results are reported in Table 2 for the best SNP of the top 25 genes, and in S1 Table for all 24,728 SNPs. Replication could only be performed for 24 SNPs, since no data were available for EYA1 rs12549242 or any proxy (defined as a SNP with linkage disequilibrium, LD, R2>0.8) in CHARGE and SpiroMeta and ALSPAC. In Stage 2, one gene showed strong replication in children, NCOR2, with replication unavailable for adults due to low imputation quality; other two genes showed suggestive evidence of replication, one in both adults and children, SERPINE2, and the other in adults but not in children, WNT16 (Table 3). The regional association plots for their lead SNP are presented in S1 Fig.

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Table 2. Results for the best SNP of the top 25 genes in Stage 1: NFBC 1966, ECRHS, EGEA, and meta-analysis.

Chr: chromosome; EA: effect allele; EAF: effect allele frequency, calculated as weighted average across the three studies; β (standard error, SE): estimate of the per-allele effect on FVC (ml); I2: magnitude of the between-study heterogeneity of effect estimates

https://doi.org/10.1371/journal.pone.0147388.t002

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Table 3. Replication findings for the best SNP of the top 25 genes.

Chr: chromosome; EA: effect allele; EAF: effect allele frequency; β (standard error, SE): per-allele effect on FVC (ml); Repl P: one-side replication p-value, calculated and reported only for estimates in the same direction as the original ones; I2: between-study heterogeneity; Imp R2 = imputation quality R2 (for CHARGE and SpiroMeta: average imputation R2 across studies)

https://doi.org/10.1371/journal.pone.0147388.t003

NCOR2-rs12708369 replicated in ALSPAC children with an effect of 26.9 ml/allele (95% confidence interval: 12.0 to 41.8) and a p-value well below Bonferroni correction (p = 0.0002). The estimate was very similar to that of Stage 1 (26.1; 7.5 to 44.7), suggesting a relatively stronger effect in children given their lower FVC, although the confidence intervals are wide and conclusions as to a difference in effect sizes cannot be deduced. In line with this, the proportion of FVC residual variance explained by this SNP was much higher in children than in adults from Stage 1, 0.65% vs. 0.11%. Replication of NCOR2-rs12708369 could not be performed in adults because of low imputation quality (imputation R2 = 0.4) and no proxy available. Using publicly available epigenomic profiling data (ChIP-seq) from ENCODE[26] via the UCSC Genome Browser (http://genome.cse.ucsc.edu), we found that the intronic variant NCOR2-rs12708369 is in a region with regulatory function in lung tissue. The SNP is located within a DNase I hypersensitivity site, in a strong enhancer element with histone mark H3K27ac indicating active chromatin in lung fibroblasts. Unfortunately neither NCOR2-rs12708369 nor any proxy could be tested in the lung eQTL analysis due to failed imputation quality control.

SERPINE2-rs6754561, a variant located 133 bp downstream from the gene, replicated at nominal level in adults from the CHARGE and SpiroMeta consortia (-7.1 ml/allele; p = 0.036), where there was no heterogeneity across the 24 studies (I2 = 0%), and ALSPAC children (-12.0 ml/allele; p = 0.045). The proportion of FVC residual variance explained was only 0.01% in adults, but 0.11% in children (0.09% in adults from Stage 1). SERPINE2-rs6754561 did not show association with the expression of SERPINE2 or any nearby genes in the lung eQTL dataset.

The intronic variant WNT16-rs2707469 replicated at nominal level in adults (10.0 ml/allele; p = 0.026; I2 = 6%), but not in children (11.8 ml/allele; p = 0.105). The proportion of FVC residual variance explained was only 0.01% in adults from the CHARGE and SpiroMeta consortia (0.10% in Stage 1). This variant is in a conserved region and is located in a DNase I hypersensitivity site in lung fibroblasts. WNT16-rs2707469 was not associated with WNT16 expression but showed suggestive evidence of an effect on a nearby gene, CPED1, with the FVC-lowering allele G associated with higher CPED1 mRNA expression levels (p = 0.087; I2 = 0%; S2 Fig). We investigated this further and found that the effect on CPED1 expression was stronger (p = 0.004; I2 = 0%) for a SNP in high LD with WNT16-rs2707469 (R2 = 0.94), rs2536166 (S2 Fig).

Discussion

By testing the association of FVC with genes related to lung development, we have identified a new gene, NCOR2, in the retinoic acid signalling pathway pointing to a role of vitamin A metabolism in the regulation of FVC. Our study also provides support for SERPINE2, a gene which has previously shown weak evidence of association with FVC, and suggests WNT16 as a promising candidate requiring further investigation.

NCOR2 (nuclear receptor corepressor 2), also known as SMRT (silencing mediator of retinoid and thyroid hormone), is a potent regulator of retinoid and thyroid hormone signalling. Nuclear receptors are ligand-activated transcription factors that regulate many developmental and physiological processes. Retinoic acid is the biologically active metabolite of vitamin A (retinol) which has a well described role in organogenesis and epithelial homeostasis directing growth, patterning and differentiation of many organs including the lung[27]. NCOR2 is a transcriptional “platform” protein that acts as a repressive co-regulatory factor for multiple transcription factor pathways. Publicly available data retrieved from BioGPS[21] (Human U133A/GNF1H Gene Atlas database) show that the expression of NCOR2 in the adult lung is very high and that the gene is also expressed in foetal lung. In this study we found an association of NCOR2 (rs12708369) with FVC in adults, which strongly replicated in children. Replication in adults from the CHARGE and SpiroMeta consortia could not be performed due to low imputation quality and no data on proxies available either. The NCOR2-rs12708369 intronic variant is in a strong transcriptional enhancer element in lung fibroblasts and may therefore affect gene expression levels[28], although we were not able to test this due to the same problem of low imputation quality in the Lung eQTL dataset. The replication of NCOR2 in children and the known central developmental roles of retinoic acid and thyroid hormone signalling during alveologenesis[29] suggest that this gene may influence lung growth and ultimately FVC. Although retinoic acid has also been postulated to have a role in ongoing alveolar maintenance and regeneration[30], in our study the NCOR2-rs12708369 effect in adults could be estimated only in Stage 1 mostly based on 31-year olds, so potential effects on FVC decline would not have been detected. Interestingly, another related gene, the RARB encoding the retinoic acid receptor beta, was selected in Stage 1, although it could not be replicated possibly due to the low minor allele frequency of its selected SNP (rs11926758; MAF = 0.06). This gene has been previously associated with measures of airway obstruction in adults and children (FEV1/FVC)[31, 32], and in infants (V’maxFRC)[33]. Overall our findings point to a role of vitamin A/thyroid metabolism in the regulation of FVC, and suggest the importance of further research investigating genes in related pathways as well as gene-environment interactions with vitamin A intake.

SERPINE2 is a member of a gene family encoding serpins, highly conserved proteins that help maintain tissue integrity by controlling the activity of proteases in diverse biological processes, in particular by inhibiting serine proteases such as trypsin. SERPINE2 has a known link to airway obstruction, with strong evidence of association with COPD[34] and some evidence of association with childhood asthma[35]. Our findings support an association with a marker of lung restriction too, FVC, in both adults and children, in line with previous findings of an association with FVC in children that could not be replicated[36]. SERPINE2-rs6754561 showed no effect on the expression of SERPINE2 or nearby genes in the lung. However, although the Lung eQTL dataset represents the largest eQTL mapping study of human lung samples currently available, weak to moderate effects on gene expression may not have been detected due to insufficient statistical power. Cellular heterogeneity in lung tissue may also impair the detection of cell type-specific eQTL[37].

We also found suggestive evidence of an association of WNT16 with FVC in adults. WNT16 belongs to a family of genes encoding 19 Wnt ligands, secreted signalling proteins involved in many developmental processes. Although Wnts are critical for normal lung development[18, 38], Wnt16 has not been previously studied in relation to lung function and disease. In addition to lung development, evidence from mouse models suggests that Wnt16 plays a role in tissue repair[39] and in the response to cellular damage[40]. The WNT16-rs2707469 intronic variant is in a conserved region with regulatory function in lung fibroblasts. This variant showed no eQTL effect on WNT16 in the lung, but an effect on a nearby gene, CPED1 (cadherin-like and PC-esterase domain containing 1). CPED1 has both a cadherin-like domain, thought to have a carbohydrate binding function, and a PC-esterase domain, predicted to modify cell surface biomolecules like glycoproteins. It is possible that Wnt16, which is a glycoprotein containing carbohydrates, could bind to, and/or be modified by, CPED1.

By focusing on genetic pathways related to lung development, which represent highly plausible candidates for low FVC, our study identifies a novel gene and proposes two further promising candidates which had not been identified in the previous GWA meta-analysis[9]. This shows how a comprehensive hypothesis-driven approach can complement hypothesis-free GWA analyses in identifying variants which failed to reach the strict significance level needed to protect against false positives in genome-wide investigations (typically 5x10-8). However, we did miss the association of one of the genes we tested, PTCH1, a gene which has shown association with FVC in the previous GWA meta-analysis[9] and had been identified before as associated with FEV1/FVC[32, 41]. The three SNPs previously identified in PTCH1 had non-significant p-values in our Stage 1 analysis, most likely due to their relatively low minor allele frequency (MAF between 0.08 and 0.10), which made our analysis underpowered to detect them.

In conclusion, this study identifies NCOR2 as a new gene for FVC, indicating the importance of further research into the role of vitamin A intake/supplementation and its interactions with related genes in the regulation of FVC. Our findings also suggest other biological pathways as promising candidates for future investigation. We might expect genes involved in lung development to show stronger effects in childhood, and the relatively large replication estimate of the effect of NCOR2-rs12708369 in children seems to support this. We speculate that future investigation of genes involved in lung development in larger samples of children and young adults could identify further genetic variants associated with FVC through their effect on lung growth and maximum level attained.

Supporting Information

S1 Fig. Regional association plots for NCOR2 rs12708369, SERPINE2 rs6754561 and WNT16 rs2707469.

https://doi.org/10.1371/journal.pone.0147388.s001

(DOC)

S2 Fig. Forest plots for the meta-analyses of lung gene expression levels of CPED1 associated with WNT16 variants.

https://doi.org/10.1371/journal.pone.0147388.s002

(DOC)

S1 Table. Stage 1 study-specific and meta-analysis results for all the 24,728 SNPs in the 403 genes.

https://doi.org/10.1371/journal.pone.0147388.s003

(XLSX)

S2 Table. Spirometry methods for studies in Stage 1.

https://doi.org/10.1371/journal.pone.0147388.s004

(DOC)

S3 Table. Genotyping and imputation methods for studies in Stage 1.

https://doi.org/10.1371/journal.pone.0147388.s005

(DOC)

Acknowledgments

NFBC1966 study: NFBC1966 received financial support from the Academy of Finland (project grants 104781, 120315, 129269, 1114194, 24300796, Center of Excellence in Complex Disease Genetics and SALVE), University Hospital Oulu, Biocenter, University of Oulu, Finland (75617), NHLBI grant 5R01HL087679-02 through the STAMPEED program (1RL1MH083268-01), NIH/NIMH (5R01MH63706:02), ENGAGE project and grant agreement HEALTH-F4-2007-201413, EU FP7 EurHEALTHAgeing -277849, the Medical Research Council, UK (G0500539, G0600705, G1002319, PrevMetSyn/SALVE) and the MRC, Centenary Early Career Award. The program is currently being funded by the H2020-633595 DynaHEALTH action and academy of Finland EGEA-project.

The DNA extractions, sample quality controls, biobank up-keeping and aliquotting was performed in the National Public Health Institute, Biomedicum Helsinki, Finland and supported financially by the Academy of Finland and Biocentrum Helsinki. We thank the late Professor Paula Rantakallio (launch of NFBCs), and Ms Outi Tornwall and Ms Minttu Jussila (DNA biobanking). The authors would like to acknowledge the contribution of the late Academian of Science Leena Peltonen.

ECRHS study: The authors would like to thank the participants, field workers and researchers who have participated in the ECRHS study for their time and cooperation.

This work was supported by a contract from the European Commission (018996), Fondo de Investigación Sanitaria (91/0016-060-05/E, 92/0319, 93/0393, 97/0035-01, 99/0034-01 and 99/0034-02), Hospital General de Albacete, Hospital General Ramón Jiménez, Consejería de Sanidad del Principado de Asturias, CIRIT (1997SGR 00079, 1999SGR 00241), and Servicio Andaluz de Salud, SEPAR, Public Health Service (R01 HL62633-01), RCESP (C03/09), Red RESPIRA (C03/011), Basque Health Department, Swiss National Science Foundation, Swiss Federal Office for Education and Science, Swiss National Accident Insurance Fund (SUVA), GSF-National Research Centre for Environment and Health, Deutsche Forschungsgemeinschaft (DFG) (FR 1526/1-1, MA 711/4-1), Programme Hospitalier de Recherche Clinique-DRC de Grenoble 2000 no. 2610, Ministry of Health, Direction de la Recherche Clinique, Ministere de l’Emploi et de la Solidarite, Direction Generale de la Sante, CHU de Grenoble, Comite des Maladies Respiratoires de l’Isere. UCB-Pharma (France), Aventis (France), Glaxo France. Estonian Science Foundation. AsthmaUK (formerly known as National Asthma Campaign UK).

EGEA cooperative group: Coordination: V Siroux (epidemiology, PI since 2013); F Demenais (genetics); I Pin (clinical aspects); R Nadif (biology); F Kauffmann (PI 1992–2012). Respiratory epidemiology: Inserm U 700, Paris: M Korobaeff (Egea1), F Neukirch (Egea1); Inserm U 707, Paris: I Annesi-Maesano (Egea1-2); Inserm CESP/U 1018, Villejuif: F Kauffmann, N Le Moual, R Nadif, MP Oryszczyn (Egea1-2), R Varraso; Inserm U 823, Grenoble: V Siroux. Genetics: Inserm U 393, Paris: J Feingold; Inserm U 946, Paris: E Bouzigon, F Demenais, MH Dizier; CNG, Evry: I Gut (now CNAG, Barcelona, Spain), M Lathrop (now Univ McGill, Montreal, Canada). Clinical centers: Grenoble: I Pin, C Pison; Lyon: D Ecochard (Egea1), F Gormand, Y Pacheco; Marseille: D Charpin (Egea1), D Vervloet (Egea1-2); Montpellier: J Bousquet; Paris Cochin: A Lockhart (Egea1), R Matran (now in Lille); Paris Necker: E Paty (Egea1-2), P Scheinmann (Egea1-2); Paris Trousseau: A Grimfeld (Egea1-2), J Just. Data and quality management: Inserm ex-U155 (Egea1): J Hochez; Inserm CESP/U 1018, Villejuif: N Le Moual; Inserm ex-U780: C Ravault (Egea1-2); Inserm ex-U794: N Chateigner (Egea1-2); Grenoble: J Quentin-Ferran (Egea1-2).

ALSPAC study: We are extremely grateful to all the families who took part in this study, the midwives for their help in recruiting them, and the whole ALSPAC team, which includes interviewers, computer and laboratory technicians, clerical workers, research scientists, volunteers, managers, receptionists and nurses. GWAS data was generated by Sample Logistics and Genotyping Facilities at the Wellcome Trust Sanger Institute and LabCorp (Laboratory Corportation of America) using support from 23andMe. The UK Medical Research Council and the Wellcome Trust (Grant ref: 102215/2/13/2) and the University of Bristol provide core support for ALSPAC. This publication is the work of the authors and DME and AJH will serve as guarantors for the contents of this paper.

The Lung eQTL study: The authors would like to thank the staff at the Respiratory Health Network Tissue Bank of the FRQS for their valuable assistance with the lung eQTL dataset at Laval University. The lung eQTL study at Laval University was supported by the Chaire de pneumologie de la Fondation JD Bégin de l’Université Laval, the Fondation de l’Institut universitaire de cardiologie et de pneumologie de Québec, the Respiratory Health Network of the FRQS, the Canadian Institutes of Health Research (MOP—123369), and the Cancer Research Society and Read for the Cure. Y. Bossé is the recipient of a Junior 2 Research Scholar award from the Fonds de recherche Québec–Santé (FRQS). At the Groningen UMCG site Marnix Jonker is thanked for his support in selecting, handling and sending of lung tissues.

CHARGE & SpiroMeta consortia

CHARGE consortium: Infrastructure for the CHARGE Consortium is supported in part by the National Heart, Lung, and Blood Institute grant R01HL105756. Contact: Stephanie J London (london2@niehs.nih.gov)

SpiroMeta consortium: The research undertaken by MSA was part-funded funded by the National Institute for Health Research (NIHR). The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. The Universities of Leicester and Nottingham acknowledge receipt of a Collaborative Research and Development grant from the Healthcare and Bioscience iNet, a project funded by the East Midlands Development Agency (EMDA), part-financed by the European Regional Development Fund and delivered by Medilink East Midlands. Contact: Martin D Tobin (mt47@le.ac.uk)

Full list of collaborators in the CHARGE and SpiroMeta consortia:

Daan W Loth1,2, María Soler Artigas3,4, Sina A Gharib5,6, Louise V Wain3,4, Nora Franceschini7,8, Beate Koch9, Tess D Pottinger10, Albert Vernon Smith11,12, Qing Duan13, Chris Oldmeadow14,15, Mi Kyeong Lee16, David P Strachan17, Alan L James18–20, Jennifer E Huffman21, Veronique Vitart21, Adaikalavan Ramasamy22,23, Nicholas J Wareham24, Jaakko Kaprio25–27, Xin-Qun Wang28, Holly Trochet21, Mika Kähönen29, Claudia Flexeder30, Eva Albrecht31, Lorna M Lopez32,33, Kim de Jong34,35, Bharat Thyagarajan36, Alexessander Couto Alves23, Stefan Enroth37,38, Ernst Omenaas39,40, Peter K Joshi41, Tove Fall38,42, Ana Viñuela43, Lenore J Launer44, Laura R Loehr7,8, Myriam Fornage45,46, Guo Li47, Jemma B Wilk48, Wenbo Tang49, Ani Manichaikul28,50, Lies Lahousse1,51, Tamara B Harris44, Kari E North7, Alicja R Rudnicka17, Jennie Hui52, Xiangjun Gu45,46, Thomas Lumley53, Alan F Wright21, Nicholas D Hastie21, Susan Campbell21, Rajesh Kumar54, Isabelle Pin55–57, Robert A Scott24, Kirsi H Pietiläinen27,58,59, Ida Surakka27,60, Yongmei Liu61, Elizabeth G Holliday14,15, Holger Schulz30, Joachim Heinrich30,62, Gail Davies32,33,63,64, Judith M Vonk34,35, Mary Wojczynski65, Anneli Pouta66,67, Åsa Johansson37,38,68, Sarah H Wild41, Erik Ingelsson38,42,69, Fernando Rivadeneira70,71, Henry Völzke72, Pirro G Hysi43, Gudny Eiriksdottir11, Alanna C Morrison73, Jerome I Rotter74,75, Wei Gao76, Dirkje S Postma35,77, Wendy B White78, Stephen S Rich50, Albert Hofman1,71, Thor Aspelund11,12, David Couper79, Lewis J Smith54, Bruce M Psaty6,47,80,81, Kurt Lohman82, Esteban G Burchard83,84, André G Uitterlinden1,70,71, Melissa Garcia44, Bonnie R Joubert85, Wendy L McArdle86, A Bill Musk87, Nadia Hansel88, Susan R Heckbert47,80,81, Lina Zgaga89,90, Joyce B J van Meurs70,71, Pau Navarro21, Igor Rudan41, Yeon-Mok Oh91,92, Susan Redline93, Deborah L Jarvis22,94, Jing Hua Zhao24, Taina Rantanen95, George T O’Connor96,97, Samuli Ripatti27,60,98, Rodney J Scott14,15, Stefan Karrasch30,99,100, Harald Grallert101, Nathan C Gaddis102, John M Starr32,103, Cisca Wijmenga104, Ryan L Minster105, David J Lederer10,106, Juha Pekkanen107,108, Ulf Gyllensten37,38, Harry Campbell41, Andrew P Morris69, Sven Gläser9, Christopher J Hammond43, Kristin M Burkart10, John Beilby52, Stephen B Kritchevsky109, Vilmundur Gudnason11,12, Dana B Hancock85,110, O Dale Williams111, Ozren Polasek112, Tatijana Zemunik113, Ivana Kolcic112, Marcy F Petrini114, Matthias Wjst115, Woo Jin Kim116,117, David J Porteous63, Generation Scotland118, Blair H Smith119, Anne Viljanen95, Markku Heliövaara26, John R Attia14,15, Ian Sayers120, Regina Hampel121, Christian Gieger31, Ian J Deary32,33, H Marike Boezen34,35, Anne Newman122, Marjo-Riitta Järvelin23,123–126, James F Wilson41, Lars Lind127, Bruno H Stricker1,2,70,71, Alexander Teumer128, Timothy D Spector43, Erik Melén129, Marjolein J Peters70,71, Leslie A Lange13, R Graham Barr10,106, Ken R Bracke51, Fien M Verhamme51, Joohon Sung16,130, Pieter S Hiemstra131, Patricia A Cassano49,132, Akshay Sood133, Caroline Hayward21, Josée Dupuis76,97, Ian P Hall120, Guy G Brusselle1,51,134, Martin D Tobin3,4 & Stephanie J London85.

1 Department of Epidemiology, Erasmus MC, Rotterdam, the Netherlands. 2 Netherlands Health Care Inspectorate, The Hague, the Netherlands. 3 Genetic Epidemiology Group, Department of Health Sciences, University of Leicester, Leicester, UK. 4 National Institute for Health Research (NIHR) Leicester Respiratory Biomedical Research Unit, Glenfield Hospital, Leicester, UK. 5 Computational Medicine Core, Center for Lung Biology, University of Washington, Seattle, Washington, USA. 6 Department of Medicine, University of Washington, Seattle, Washington, USA. 7 Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. 8 Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. 9 Department of Internal Medicine B–Pneumology, Cardiology, Intensive Care and Infectious Diseases, University Hospital Greifswald, Greifswald, Germany. 10 Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York, USA. 11 Iceland Heart Association, Kopavogur, Iceland. 12 University of Iceland, Reykjavik, Iceland. 13 Department of Genetics, University of North Carolina, Chapel Hill, North Carolina, USA. 14 Hunter Medical Research Institute, University of Newcastle, Newcastle, New South Wales, Australia. 15 Faculty of Health, University of Newcastle, Newcastle, New South Wales, Australia. 16 Institute of Health and Environment, Seoul National University, Seoul, South Korea. 17 Division of Population Health Sciences and Education, St George’s, University of London, London, UK. 18 Department of Pulmonary Physiology and Sleep Medicine/West Australian Sleep Disorders Research Institute, Nedlands, Western Australia, Australia. 19 School of Medicine and Pharmacology, The University of Western Australia, Perth, Western Australia, Australia. 20 Busselton Population Medical Research Institute, Busselton, Western Australia, Australia. 21 Medical Research Council (MRC) Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine (IGMM), University of Edinburgh, Edinburgh, UK. 22 Respiratory Epidemiology and Public Health Group, National Heart and Lung Institute, Imperial College London, London, UK. 23 Department of Epidemiology and Biostatistics, MRC Health Protection Agency (HPA) Centre for Environment and Health, School of Public Health, Imperial College London, London, UK. 24 MRC Epidemiology Unit, Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge, UK. 25 Hjelt Institute, Department of Public Health, University of Helsinki, Helsinki, Finland. 26 National Institute for Health and Welfare (THL), Helsinki, Finland. 27 Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland. 28 Division of Biostatistics and Epidemiology, Department of Public Health Sciences, University of Virginia, Charlottesville, Virginia, USA. 29 Department of Clinical Physiology, University of Tampere and Tampere University Hospital, Tampere, Finland. 30 Institute of Epidemiology I, Helmholtz Zentrum München–German Research Center for Environmental Health, Neuherberg, Germany. 31 Institute of Genetic Epidemiology, Helmholtz Zentrum München–German Research Center for Environmental Health, Neuherberg, Germany. 32 Centre for Cognitive Ageing and Cognitive Epidemiology, University of Edinburgh, Edinburgh, UK. 33 Department of Psychology, University of Edinburgh, Edinburgh, UK. 34 Department of Epidemiology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands. 35 Groningen Research Institute for Asthma and COPD (GRIAC), University of Groningen, University Medical Center Groningen, Groningen, the Netherlands. 36 Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota, USA. 37 Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden. 38 Science for Life Laboratory, Uppsala University, Uppsala, Sweden. 39 Centre for Clinical Research, Haukeland University Hospital, Bergen, Norway. 40 Department of Clinical Sciences, University of Bergen, Bergen, Norway. 41 Centre for Population Health Sciences, Medical School, University of Edinburgh, Edinburgh, UK. 42 Molecular Epidemiology, Department of Medical Sciences, Uppsala University, Uppsala, Sweden. 43 Department of Twins Research and Genetic Epidemiology, King’s College London, London, UK. 44 Laboratory of Epidemiology, Demography and Biometry, National Institute on Aging, US National Institutes of Health, Bethesda, Maryland, USA. 45 Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, Texas, USA. 46 Human Genetics Center, University of Texas Health Science Center at Houston, Houston, Texas, USA. 47 Cardiovascular Health Research Unit, University of Washington, Seattle, Washington, USA. 48 Precision Medicine, Pfizer Global Research and Development, Cambridge, Massachusetts, USA. 49 Division of Nutritional Sciences, Cornell University, Ithaca, New York, USA. 50 Center for Public Health Genomics, University of Virginia, Charlottesville, Virginia, USA. 51 Department of Respiratory Medicine, Ghent University Hospital, Ghent, Belgium. 52 PathWest Laboratory Medicine Washington, Nedlands, Western Australia, Australia. 53 Department of Statistics, University of Auckland, Auckland, New Zealand. 54 Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA. 55 Centre Hospitalier Universitaire de Grenoble, Grenoble, France. 56 INSERM U823, Institut Albert Bonniot, Grenoble, France. 57 Université Joseph Fourier, Grenoble, France. 58 Obesity Research Unit, Research Programs Unit, Diabetes and Obesity, University of Helsinki, Helsinki, Finland. 59 Division of Internal Medicine, Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland. 60 Public Health Genomics Unit, Department of Chronic Disease Prevention, National Institute for Health and Welfare (THL), Helsinki, Finland. 61 Department of Epidemiology and Prevention, Division of Public Health Sciences, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA. 62 Comprehensive Pneumology Center Munich (CPC-M), member of the German Center for Lung Research, Munich, Germany. 63 Medical Genetics Section, Centre for Genomics and Experimental Medicine, MRC IGMM, University of Edinburgh, Edinburgh, UK. 64 MRC Institute of Genetics and Molecular Medicine, Edinburgh, UK. 65 Department of Statistical Genomics, Washington University, St. Louis, Missouri, USA. 66 National Institute for Health and Welfare, Oulu, Finland. 67 Department of Clinical Sciences/Obstetrics and Gynecology, University Hospital of Oulu, University of Oulu, Oulu, Finland. 68 Uppsala Clinical Research Center, Uppsala University, Uppsala, Sweden. 69 Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK. 70 Department of Internal Medicine, Erasmus MC, Rotterdam, the Netherlands. 71 Netherlands Genomics Initiative (NGI)-sponsored Netherlands Consortium for Healthy Aging (NCHA), Rotterdam, the Netherlands. 72 Institute for Community Medicine, University Medicine Greifswald, Greifswald, Germany. 73 School of Public Health, University of Texas Health Science Center at Houston, Houston, Texas, USA. 74 Biomedical Research Institute, Harbor–University of California, Los Angeles (UCLA) Medical Center, Torrance, California, USA. 75 Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, California, USA. 76 Department of Biostatistics, Boston University School of Public Health, Boston, Massachusetts, USA. 77 Department of Pulmonology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands. 78 Tougaloo College, Jackson, Mississippi, USA. 79 Department of Biostatistics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. 80 Department of Epidemiology, University of Washington, Seattle, Washington, USA. 81 Group Health Research Institute, Group Health Cooperative, Seattle, Washington, USA. 82 Department of Biostatistical Sciences, Division of Public Health Sciences, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA. 83 Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, California, USA. 84 Department of Medicine, University of California, San Francisco, San Francisco, California, USA. 85 Epidemiology Branch, National Institute of Environmental Health Sciences, US National Institutes of Health, US Department of Health and Human Services, Research Triangle Park, North Carolina, USA. 86 School of Social and Community Medicine, University of Bristol, Bristol, UK. 87 Department of Respiratory Medicine, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia. 88 Department of Medicine, Johns Hopkins University, Baltimore, Maryland, USA. 89 Department of Public Health and Primary Care, Trinity College Dublin, Dublin, Ireland. 90 Adrija Stampar School of Public Health, Medical School, University of Zagreb, Zagreb, Croatia. 91 Department of Pulmonary and Critical Care Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea. 92 Clinical Research Center for Chronic Obstructive Airway Diseases, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea. 93 Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA. 94 MRC-PHE Centre for Environment and Health, Imperial College London, London, UK. 95 Gerontology Research Centre, Department of Health Sciences, University of Jyväskylä, Jyväskylä, Finland. 96 Pulmonary Center, Boston University School of Medicine, Boston, Massachusetts, USA. 97 National Heart, Lung, and Blood Institute’s Framingham Heart Study, Framingham, Massachusetts, USA. 98 Genetic Epidemiology Group, Wellcome Trust Sanger Institute, Hinxton, UK. 99 Institute and Outpatient Clinic for Occupational, Social and Environmental Medicine, Ludwig Maximilians Universität, Munich, Germany. 100 Institute of General Practice, University Hospital Klinikum Rechts der Isar, Technische Universität München, Munich, Germany. 101 Research Unit of Molecular Epidemiology, Helmholtz Zentrum München–German Research Center for Environmental Health, Neuherberg, Germany. 102 Research Computing Division, Research Triangle Institute International, Research Triangle Park, North Carolina, USA. 103 Alzheimer Scotland Dementia Research Centre, University of Edinburgh, Edinburgh, UK. 104 Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands. 105 Department of Human Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. 106 Department of Epidemiology, Mailman School of Public Health, Columbia University, New York, New York, USA. 107 Department of Environmental Health, National Institute for Health and Welfare (THL), Kuopio, Finland. 108 Public Health and Clinical Nutrition, University of Eastern Finland, Kuopio, Finland. 109 Sticht Center on Aging, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA. 110 Behavioral Health Epidemiology Program, Research Triangle Institute International, Research Triangle Park, North Carolina, USA. 111 Florida International University, Miami, Florida, USA. 112 Department of Public Health, Medical School, University of Split, Split, Croatia. 113 Department of Medical Biology, Medical School, University of Split, Split, Croatia. 114 Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of Mississippi Medical Center, Jackson, Mississippi, USA. 115 Comprehensive Pneumology Center (CPC), Helmholtz Zentrum München (HMGU), Munich, Germany. 116 Department of Internal Medicine, Kangwon National University Hospital, School of Medicine, Kangwon National University, Chuncheon, South Korea. 117 Environmental Health Center, Kangwon National University Hospital, School of Medicine, Kangwon National University, Chuncheon, South Korea. 118 A collaboration between the University Medical Schools and National Health Service (NHS) in Aberdeen, Dundee, Edinburgh and Glasgow, UK. 119 Medical Research Institute, University of Dundee, Dundee, UK. 120 Division of Therapeutics and Molecular Medicine, University of Nottingham, Nottingham, UK. 121 Institute of Epidemiology II, Helmholtz Zentrum München–German Research Center for Environmental Health, Neuherberg, Germany. 122 Department of Epidemiology, Center for Aging and Population Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. 123 Institute of Health Sciences, University of Oulu, Oulu, Finland. 124 Biocenter Oulu, University of Oulu, Oulu, Finland. 125 Unit of Primary Care, Oulu University Hospital, Oulu, Finland. 126 Department of Children and Young People and Families, National Institute for Health and Welfare, Oulu, Finland. 127 Department of Medical Sciences, Uppsala University, Uppsala, Sweden. 128 Department for Genetics and Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany. 129 Institute of Environmental Medicine, Karolinska Institutet and Sachs’ Children’s Hospital, Stockholm, Sweden. 130 Complex Disease and Genetic Epidemiology Branch, Department of Epidemiology, Seoul National University School of Public Health, Seoul, South Korea. 131 Department of Pulmonology, Leiden University Medical Center, Leiden, the Netherlands. 132 Department of Public Health, Division of Biostatistics and Epidemiology, Weill Cornell Medical College, New York, New York, USA. 133 University of New Mexico Health Sciences Center School of Medicine, Albuquerque, New Mexico, USA. 134 Department of Respiratory Medicine, Erasmus MC, Rotterdam, the Netherlands.

Author Contributions

Conceived and designed the experiments: CM PB CHD MH. Analyzed the data: CM ACA AFSA VH EB MSA DME YB. Wrote the paper: CM CHD MH PB AFSA JT YB. Participated in the acquisition of the data: MRJ DJ EB MSA JH YB ACA VH VS FD DWL DME DSP DS. Revised the manuscript: CM CHD MH ACA AFSA VS VH MSA DME DWL YB DSP DS JT FD JH EB DJ MRJ PB.

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