Abstract
Forced vital capacity (FVC), a spirometric measure of pulmonary function, reflects lung volume and is used to diagnose and monitor lung diseases. We performed genome-wide association study meta-analysis of FVC in 52,253 individuals from 26 studies and followed up the top associations in 32,917 additional individuals of European ancestry. We found six new regions associated at genome-wide significance (P < 5 × 10−8) with FVC in or near EFEMP1, BMP6, MIR129-2–HSD17B12, PRDM11, WWOX and KCNJ2. Two loci previously associated with spirometric measures (GSTCD and PTCH1) were related to FVC. Newly implicated regions were followed up in samples from African-American, Korean, Chinese and Hispanic individuals. We detected transcripts for all six newly implicated genes in human lung tissue. The new loci may inform mechanisms involved in lung development and the pathogenesis of restrictive lung disease.
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Acknowledgements
For all studies, information on funding and acknowledgments can be found in the Supplementary Note.
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Authors and Affiliations
Contributions
Project conception, design and management. Stage 1 GWAS. Ages: G.E., M.G., V.G., T.B.H., L.J.L. ARIC: S.J.L., N.F., D.C., D.B.H., B.R.J., A.C.M., K.E.N. B58C T1DGC: D.P.S. B58C WTCCC: D.P.S. CARDIA: A.S., L.J.S. CHS: S.A.G., S.R.H., B.M.P. CROATIA-Korcula: O.P., A.F.W. CROATIA-Vis: I.R. ECRHS: D.J., E.O., I.P., M. Wjst EPIC: R.A.S., N.J.W., J.H.Z. FHS: J.B.W., G.T.O., J.D. FTC: J.K., K.H.P., T.R., A. Viljanen Health ABC: P.A.C., T.B.H., S.B.K., Y.L. Health 2000: M.H., M.K. HCS: J.R.A., R.J.S. KORA S3: C.G., J. Heinrich MESA-Lung: R.G.B. NFBC1966: M.-R.J., A.P. NSPHS: U.G. ORCADES: H.C., J.F.W., S.H.W. RS I, II and III: A.H., B.H. Stricker, G.G.B. SHIP: S.G., B.K., H.V. Twins UK-I: C.J.H., T.D.S. Stage 2 follow-up studies. BHS 1 and 2: A.L.J., A.B.M., J.B. CROATIA-Split: N.D.H., C.H. Generation Scotland: D.J.P., B.H. Smith. KORA F4: H.S. LBC1936: I.J.D., J.M.S. LifeLines: H.M.B., D.S.P., J.M.V., C.W. LLFS: A.N., B.T., M. Wojczynski, R.L.M. PIVUS: E.I., L. Lind Twins UK-II and III: C.J.H., T.D.S. Multi-ancestry follow-up studies. CARe: R.G.B., K.M.B., D.J.L., R.K., L.J.S., J.B.W., N.H., M.F.P., K.M.B., S. Redline, E.G.B., G.T.O., L.R.L., W.B.W. KARE3 and Healthy Twin Study: J.S., W.J.K., Y.-M.O. Gene expression analyses. RT-PCR: K.R.B., G.G.B. eQTL analysis: J.B.J.v.M., A.G.U. Fetal lung expression analysis: I.P.H., I. Sayers, E.M.
Phenotype collection and data management. Stage 1 GWAS. Ages: T.A. ARIC: D.C., N.F., A.C.M., K.E.N. B58C T1DGC: A.R.R., D.P.S. B58C WTCCC: A.R.R., D.P.S. CARDIA: L.J.S., O.D.W. CHS: S.A.G., S.R.H., B.M.P., T.L. CROATIA-Korcula: O.P., L.Z. CROATIA-Vis: S.C., I.K. ECRHS: D.L.J., E.O., I.P., M. Wjst EPIC: J.H.Z. FHS: J.B.W., G.T.O., J.D. FTC: J.K., K.H.P., T.R., A. Viljanen Health ABC: P.A.C., W.T. Health 2000: M.H., M.K. HCS: J.R.A. KORA S3: J. Heinrich MESA-Lung: R.G.B. NFBC1966: M.-R.J., J.P., A.P. NSPHS: Å.J., S.E., U.G. ORCADES: S.H.W., J.F.W. RS I, II and III: G.G.B., L. Lahousse, D.W.L., B.H. Stricker SHIP: S.G., B.K., H.V. Twins UK-I: P.G.H., A. Viñuela. Stage 2 follow-up studies. BHS 1 and 2: A.L.J., A.B.M., J.B. CROATIA-Split: C.H., T.Z. Generation Scotland: D.J.P., B.H. Smith. KORA F4: R.H., S.K., H.S. LBC1936: I.J.D., J.M.S. LifeLines: D.S.P., J.M.V. LLFS: A.N., B.T., R.L.M. PIVUS: E.I., L. Lind Twins UK-II and III: P.G.H., A. Viñuela Multi-ancestry follow-up studies. CARe: R.G.B., T.D.P., K.M.B., D.J.L., R.K., L.J.S., J.B.W., N.H., M.F.P., K.M.B., S. Ripatti, E.G.B., G.T.O., L.R.L., W.B.W. KARE3 and Healthy Twin Study: J.S., W.J.K., Y.-M.O. Gene expression analyses. RT-PCR: K.R.B., G.G.B., F.M.V., P.S.H. eQTL analysis: J.B.J.v.M., M.J.P. Fetal lung expression analysis: I.P.H., I. Sayers, E.M.
Genotyping. Stage 1 GWAS. B58C T1DGC: W.L.M. B58C WTCCC: W.L.M. CARDIA: M.F., X.G. CHS: J.I.R., B.M.P. CROATIA-Korcula: J.E.H. CROATIA-Vis: S.C. ECRHS: M. Wjst EPIC: J.H.Z. FTC: J.K. Health ABC: Y.L., K.L. Health 2000: S. Ripatti, I. Surakka. HCS: R.J.S. KORA S3: H.G. MESA-Lung: S.S.R. NFBC1966: M.-R.J. NSPHS: Å.J., S.E., U.G. Orcades: H.C., J.F.W. RS I, II and III: F.R., A.G.U. SHIP: S.G., B.K., A.T., H.V. Twins UK-I: C.J.H., T.D.S. Stage 2 follow-up studies. BHS 1 and 2: J. Hui, J.B. CROATIA-Split: C.H., P.N., T.Z. Generation Scotland: D.J.P., B.H. Smith, H.T. LBC1936: G.D. LifeLines: C.W. LLFS: A.N., B.T. PIVUS: E.I., A.P.M. Twins UK-II and III: C.J.H., T.D.S.
Data analysis. Stage 1 GWAS. Ages: A.V.S. ARIC: N.F., D.B.H. B58C T1DGC: A.R.R., D.P.S. B58C WTCCC: A.R.R., D.P.S. CARDIA: X.G. CHS: S.A.G., G.L., S.R.H., T.L. CROATIA-Korcula: J.E.H. CROATIA-Vis: V.V. ECRHS: D.L.J., A.R. EPIC: J.H.Z. FHS: J.B.W., J.D., W.G. Health ABC: P.A.C., Y.L., K.L., W.T. Health 2000: M.K., S. Ripatti, I. Surakka. HCS: C.O., E.G.H. KORA S3: E.A. MESA-Lung: A.M., S.S.R. NFBC1966: A.C.A. NSPHS: S.E. ORCADES: P.K.J. RS I, II and III: L. Lahousse, D.W.L. SHIP: A.T. Twins UK-I: P.G.H. Stage 2 follow-up studies. KORA F4: C.F., R.H. LBC1936: L.M.L. LifeLines: K.d.J., H.M.B. LLFS: M. Wojczynski, B.T. PIVUS: T.F. Twins UK-II and III: P.G.H. Multi-ancestry follow-up studies. N.C.G. CARe: T.D.P., Q.D., L.A.L., X.-Q.W. KARE3 and Healthy Twin Study: M.K.L. Gene expression analyses. RT-PCR: K.R.B., F.M.V. eQTL analysis: M.J.P. Fetal lung expression analysis: I.P.H., I. Sayers, E.M.
Analysis group: CHARGE Consortium: D.W.L., S.A.G., S.J.L., J.D., N.F., A.V.S. CARe: T.D.P., Q.D. SpiroMeta Consortium: M.S.A., L.V.W., B.K., I.P.H., M.D.T.
Writing group: CHARGE Consortium: S.J.L., D.W.L., S.A.G., N.F., J.D., G.G.B., A.S. SpiroMeta Consortium: I.P.H., M.S.A., M.D.T., L.V.W., C.H.
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J.B.W. is employed by Pfizer, Inc. None of the other authors have declared a possible conflict of interest.
Integrated supplementary information
Supplementary Figure 1 Quantile-quantile (QQ) plot of observed genome-wide association results against expected association results for FVC.
The quantile-quantile (QQ) plot shows –log10 (P) of observed genome-wide association results against expected association results for FVC. λGC before applying genomic control was 1.12. The QQ plot for all SNPs is shown in black. The results in red show the –log10 (P) of observed genome-wide association results for FVC after excluding SNPs within the 26 previously reported regions for FEV1 and FEV1/FVC; SNPs within 500 kb of the lead reported SNP in the SpiroMeta-CHARGE meta-analysis of FEV1 and FEV1/FVC were excluded.
Supplementary Figure 2 Regional plots.
Regional association plots of seven FVC- associated loci with P value < 5 × 10–7. The statistical significance of each SNP is shown on the –log10 (P) scale as a function of chromosome position (NCBI Build 36) in the meta-analysis of stage 1. The sentinel SNP at each locus is shown with a purple diamond with the correlations (r2) of surrounding SNPs to the sentinel indicated by color (red, r2 > 0.8; orange, r2 > 0.6; green, r2 > 0.4; light blue, r2 > 0.2; purple, unknown r2). The fine-scale recombination rate is shown in blue.
Supplementary Figure 3 Forest plots for the cohort-specific effects in stage 1.
Forest plots for the seven loci associated with FVC for stage 1. Six of the SNPs included in the figure showed a genome-wide significant association (P < 5 × 10–8) with FVC after combining both stages. The contributing effect (β, in ml) from each study is shown by a square, with confidence intervals indicated by horizontal lines. The contributing weight of each study to the meta-analysis is indicated by the size of the square, where the size per study is relative to the total contribution of the stage. The combined meta-analysis per stage is shown at the bottom of each graph.
Supplementary Figure 4 Forest plots for the cohort-specific effects in stage 2.
Forest plots for the seven loci associated with FVC for stage 2. Six of the SNPs included in the figure showed a genome-wide significant association (P < 5 × 10–8) with FVC after combining both stages. The contributing effect (β, in ml) from each study is shown by a square, with confidence intervals indicated by horizontal lines. The contributing weight of each study to the meta-analysis is indicated by the size of the square, where the size per study is relative to the total contribution of the stage. The combined meta-analysis per stage is shown at the bottom of each graph.
Supplementary Figure 5 Regional plots for loci in African Americans.
Regional plot of –log10(P value) of EFEMP1 in African American samples. The LD is based on CEU 1000G reference panel. A. rs62164511 is the most significant SNP in this locus in African American data. B. rs1430193 is the index SNP identified in European ancestry individuals. Note the low linkage disequilibrium (r2 = 0.16) between these SNPs and the evidence for allelic heterogeneity at the locus.
Supplementary Figure 6 Expression analysis of candidate genes in lung tissue and primary cells.
(a) RT-PCR profiling of gene transcripts demonstrates expression of all seven candidate genes in total lung tissue: EFEMP1 (86 bp), BMP6 (108 bp), WWOX (89 bp), TMEM163 (90 bp), KCNJ2 (160 bp), PRDM11 (77 bp) and HSD17B12 (65 bp). (b) mRNA expression profiling in human primary cells: human bronchial epithelial cells (HBECs), human airway smooth muscle (HASM) cells and peripheral blood mononuclear cells (PBMCs).
Supplementary Figure 7 Real-time RT-PCR of candidate genes in lung tissue and primary cells.
TaqMan amplification curves of EFEMP1 (a), BMP6 (b), WWOX (c), KCNJ2 (d), HSD17B12 (e), PRDM11 (f) and GAPDH (g) on total lung tissue, human bronchial epithelial cells (HBECs), human airway smooth muscle (HASM) cells and peripheral blood mononuclear cells (PMBCs). (NTC, non-template control.)
Supplementary Figure 8 eQTL association.
This graph shows the cis effects for the sentinel SNP rs4237643 on probe 3420341–HSD17B12, the only significant eQTL association we were able to identify in whole blood. eQTL analysis was performed using Illumina Whole-Genome Expression BeadChips (HumanHT-12 v4). The blue and red dots show the sex of the individual (blue, male; red, female). The black diagonal line crosses the mean gene expression level per genotype group (μ). The vertical line at the right gives the average z score and its P value.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–8, Supplementary Tables 1–12 and Supplementary Note (PDF 4342 kb)
Supplementary Data Set 1
SNPs, chromosome numbers, HapMap allele frequency for the reference allele and P value for the association with forced vital capacity in stage 1. (ZIP 50782 kb)
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Loth, D., Artigas, M., Gharib, S. et al. Genome-wide association analysis identifies six new loci associated with forced vital capacity. Nat Genet 46, 669–677 (2014). https://doi.org/10.1038/ng.3011
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DOI: https://doi.org/10.1038/ng.3011
