ARTICLE Received 5 Feb 2015 | Accepted 17 Sep 2015 | Published 4 Dec 2015 DOI: 10.1038/ncomms9658 OPEN Sixteen new lung function signals identified through 1000 Genomes Project reference panel imputation Marı´a Soler Artigas et al.# Lung function measures are used in the diagnosis of chronic obstructive pulmonary disease. In 38,199 European ancestry individuals, we studied genome-wide association of forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC) and FEV1/FVC with 1000 Genomes Project (phase 1)-imputed genotypes and followed up top associations in 54,550 Europeans. We identify 14 novel loci (Po5 Â 10 À 8) in or near ENSA, RNU5F-1, KCNS3, AK097794, ASTN2, LHX3, CCDC91, TBX3, TRIP11, RIN3, TEKT5, LTBP4, MN1 and AP1S2, and two novel signals at known loci NPNT and GPR126, providing a basis for new understanding of the genetic determinants of these traits and pulmonary diseases in which they are altered. Correspondence and requests for materials should be addressed to I.P.H. (email: [email protected]) or to M.D.T. (email: [email protected]). #A full list of authors and their affiliations appears at the end of the paper. NATURE COMMUNICATIONS | 6:8658 | DOI: 10.1038/ncomms9658 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9658 ung function, as measured by spirometry, predicts morbidity STAGE 1 (genome-wide association studies) and mortality1,2. Altered lung function is a key criterion for n=38,199 Lthe diagnosis of chronic obstructive pulmonary disease B58C (n=5,934) (COPD), a leading cause of death worldwide3,4. The ratio of BHS1&2 (n=4,355) CROATIA-Korcula (n=826) STAGE 2 (follow-up studies) forced expiratory volume in 1 s (FEV1) over forced vital capacity (FVC) defines patients with airflow obstruction, while FEV is CROATIA-Split (n=493) n=54,550 1 CROATIA-Vis (n=925) used to assess the severity of the obstruction. Reduced FVC values 5 EPIC population based (n=2,339) ECRHS (n=1,747) are seen in restrictive lung diseases such as pulmonary fibrosis . GS:SFHS (n=8,093) PIVUS (n=837) While environmental risk factors, such as tobacco smoking or air H2000 (n=821) TwinsUK (n=3,023) 6,7 pollution, play a significant role in determining lung function , KORA F4 (n=1,474) UK BiLEVE (n=48,943) genetic factors are also important contributors, with estimates of KORA S3 (n=1,147) heritability ranging between 39 and 54% (refs 8,9). LBC1936 (n=991) NFBC1966 (n=4,563) Genome-wide association studies (GWAS) of around 2.5 NSPHS (n=871) million common (minor allele frequency (MAF)45%) single- ORCADES (n=1,802) nucleotide polymorphisms (SNPs) in Europeans have identified SAPALDIA (n=1,378) 32 loci associated with lung function at genome-wide significance SHIP (n=1,768) level (Po5 Â 10 À 8)10–14. However, as for other complex YFS (n=419) traits15,16, these loci only explain a limited proportion of the Figure 1 | Study design for autosomal chromosome analyses. The heritability11,13. Among explanations for the ‘missing heritability’ are a large number of, as yet, undetected common variants discovery stage (stage 1) included 17 studies and 38,199 individuals. Fifty- five variants were followed up in stage 2, which comprised four studies and with modest effect sizes, in addition to low-frequency 54,550 individuals. (1%oMAFr5%) and rare (MAFr1%) variants with larger effect sizes16,17. Of particular relevance to low-frequency variants, phase 1 of the 1000 Genomes Project18 sequenced 1,092 individuals from 14 populations, providing an imputation across X chromosome variants was 1.04 for FEV1 and 1.00 for B reference panel of 38 million SNPs and 1.4 million indels, FEV1/FVC and FVC. Quantile–quantile plots are presented in including autosomal and X chromosome variants. Supplementary Fig. 2a. Variants with effective sample sizes (N The aim of the current study, undertaken within the SpiroMeta effective, product of sample size and imputation quality summed consortium, was to improve coverage of low-frequency variants across studies) o70% were filtered out, and a total of 8,694,268 and detect novel loci associated with lung function by under- variants were included in this genome-wide study. taking imputation of GWAS data to the 1000 Genomes Project18 Forty-eight SNPs and seven indels in independent autosomal Phase-1 reference panel in 38,199 individuals of European chromosome regions (±500 kb either side of sentinel variant) ancestry. We meta-analysed GWAS results across 17 studies with stage 1 Po5 Â 10 À 6 were followed up in stage 2 using in and followed up the most significant associations with in silico silico data from four studies comprising 54,550 individuals (Fig. 1; data in up to 54,550 Europeans. We identify 14 new loci Supplementary Table 2). One SNP on the X chromosome also associated with lung function at genome-wide significance level, met these criteria and was followed up in a subset of three studies and novel distinct signals at two previously reported loci. These comprising 52,359 individuals (Supplementary Fig. 1; include two low-frequency variant association signals, which Supplementary Table 2). Characteristics of follow-up (stage 2) seem to be explained by non-synonymous SNPs. The results of cohort participants, genotyping and imputation are shown in these analyses implicate both previously considered and novel Supplementary Table 1. Stage-2 studies adjusted the traits for age, mechanisms influencing lung function. age2, sex, height and principal components to account for population structure and ever-smoking status, and also undertook association testing on the inverse normally transformed residuals Results assuming additive genetic effects. Stage-2 estimates were We undertook a meta-analysis of 17 GWAS imputed using the combined across studies, and then with stage-1 estimates, using 1000 Genomes Project18 Phase-1 reference panel in a study of inverse variance-weighted fixed-effects meta-analysis. Thirteen 38,199 individuals of European ancestry in stage 1 (Fig. 1), of SNPs and three indels, each representing new signals of which 19,532 were individuals not included in the discovery stage association, met a genome-wide significance threshold corrected of previous meta-analyses of lung GWAS10–13. Characteristics of for multiple testing (Po5 Â 10 À 8) after combining stage-1 and cohort participants, genotyping and imputation are shown in stage-2 results (Table 1; Fig. 2), of which 10 SNPs and three indels Supplementary Table 1. Each study adjusted FEV1, FEV1/FVC achieved independent replication meeting a Bonferroni-corrected and FVC, for age, age2, sex, height and principal components for threshold for 56 tests (Po8.93 Â 10 À 4) in stage 2 alone. population structure, separately for never and ever smokers. Fourteen studies additionally undertook analyses for X chromosome variants (33,009 individuals, Supplementary Fig. 1 Sixteen novel association signals for FEV1,FEV1/FVC and FVC. and Methods). Inverse normally transformed residuals were then Of the 16 novel signals reaching genome-wide significance, two used for association testing within each smoking stratum, represent distinct new signals for FEV1/FVC in previously reported assuming an additive genetic effect. Within each study, we loci10,12 (stage-1 P value conditioned on previously reported combined smoking strata association summary statistics using variant o5 Â 10 À 6). Among the remaining 14, five new loci were inverse variance-weighted fixed-effects meta-analysis, and applied identified for FEV1, six new loci for FEV1/FVC and three new loci genomic control19 to account for residual population structure for FVC (Table 1). The sentinel variants at the 16 loci were in or not accounted for by principal components. We subsequently near the following genes: MCL1-ENSA (1q21.3), LYPLAL1- combined study-specific estimates across studies using inverse RNU5F-1 (1q41), KCNS3-NT5C1B (2p24.2), AK097794 (3q25.32), variance weighing, and applied genomic control19 after fixed- NPNT (4q24), GPR126-LOC153910 (6q24.1), ASTN2 (9q33.1), effects meta-analysis. The genomic inflation factor across LHX3 (9q33.1), PTHLH-CCDC91 (12p11.22), TBX3 (12q24.21), autosomal variants was 1.03 for each of the three traits, and TRIP11 (14q32.12), RIN3 (14q32.12), EMP2-TEKT5 (16p13.13), 2 NATURE COMMUNICATIONS | 6:8658 | DOI: 10.1038/ncomms9658 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9658 ARTICLE Table 1 | Variants associated with FEV1, FEV1/FVC or FVC. rs number Gene (function) eQTL Coded Measure Stage 1 Stage 2 Meta-analysis (chr:position) gene allele/ other allele Coded N Beta P Coded N Beta (s.e.) P Beta (s.e.) P allele (s.e.) allele freq. freq. À 7 À 3 À 9 rs6681426 MCL1 ARNT G/A FEV1 0.36 37,944 0.042 1.07 Â 10 0.35 5,4301 0.021 1.13 Â 10 0.029 4.35 Â 10 (chr1:150586971) (dist. ¼ 34,757), (0.008) (0.006) (0.005) ENSA (dist. ¼ 7,628) À 6 À 5 À 10 rs201204531 LYPLAL1 — A/ATG FEV1/FVC 0.41 34,866 À 0.038 1.98 Â 10 0.38 53,760 À 0.027 1.77 Â 10 À 0.031 2.68 Â 10 (chr1:219963090) (dist. ¼ 576,883), (0.008) (0.006) (0.005) RNU5F-1 (dist. ¼ 83,529) À 8 À 9 À 15 rs61067109 KCNS3 — G/A FEV1/FVC 0.77 37,416 À 0.050 3.09 Â 10 0.77 54,341 À 0.042 6.56 Â 10 À 0.045 1.40 Â 10 (chr2:18292452) (dist. ¼ 178,227), (0.009) (0.007) (0.006) NT5C1B (dist.