ABCMdb

PMID: 22866751

Baekvad-Hansen M, Nordestgaard BG, Dahl M
Heterozygosity for E292V in ABCA3, lung function and COPD in 64,000 individuals.
Respir Res. 2012 Aug 6;13(1):67., [PubMed]

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No. Mutations Sentence Comment

0 ABCA3 p.Glu292Val RESEARCH Open Access Heterozygosity for E292V in ABCA3, lung function and COPD in 64,000 individuals Marie B&#e6;kvad-Hansen1,4 , B&#f8;rge G Nordestgaard1,2,4 and Morten Dahl1,3,4* Abstract Background: Mutations in ATP-binding-cassette-member A3 (ABCA3) are related to severe chronic lung disease in neonates and children, but frequency of chronic lung disease due to ABCA3 mutations in the general population is unknown. Login to comment 2 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Ala320Thr ABCA3 p.Ala1086Asp ABCA3 p.Arg1474Trp ABCA3 p.Pro766Ser ABCA3 p.His86Tyr ABCA3 p.Ser1262Gly Heterozygosity for E292V in ABCA3, lung function and COPD in 64,000 individuals Respiratory Research 2012, 13:67 doi:10.1186/1465-9921-13-67 Marie Bækvad-Hansen (baekvad@gmail.com}) Børge G Nordestgaard (brno@heh.regionh.dk}) Morten Dahl (dahlos2003@yahoo.dk}) ISSN 1465-9921 Article type Research Submission date 23 March 2012 Acceptance date 27 July 2012 Publication date 6 August 2012 Article URL http://respiratory-research.com/content/13/1/67 This peer-reviewed article was published immediately upon acceptance. Login to comment 5 ABCA3 p.Glu292Val Results: In the Copenhagen City Heart Study individuals heterozygous for E292V had 5% reduced FEV1 % predicted compared with noncarriers (t-test: p = 0.008), and an increased odds ratio for COPD of 1.9 (95% CI: 1.1-3.1). Login to comment 6 ABCA3 p.Ala1086Asp In contrast, the A1086D mutation was associated with increased FEV1 % predicted (p = 0.03). Login to comment 7 ABCA3 p.Glu292Val Heterozygosity for E292V in ABCA3, lung function and COPD in 64,000 individuals Marie Bækvad-Hansen1,4 Email: baekvad@gmail.com Børge G Nordestgaard1,2,4 Email: brno@heh.regionh.dk Morten Dahl1,3,4,* Email: morten.dahl@rh.regionh.dk 1 Department of Clinical Biochemistry, Herlev Hospital, Copenhagen University Hospital, Copenhagen, Denmark 2 Copenhagen City Heart Study, Bispebjerg Hospital, Copenhagen University Hospital, Copenhagen, Denmark 3 Department of Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, Copenhagen DK-2100, Denmark 4 Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark * Corresponding author. Login to comment 8 ABCA3 p.Glu292Val In the larger Copenhagen General Population Study, and in the two studies combined, E292V heterozygotes did not have reduced lung function or increased risk of COPD (p = 0.11-0.98), while this was the case for the positive controls, surfactant protein-B 121ins2 heterozygotes and b1;1-antitrypsin ZZ homozygotes. Login to comment 9 ABCA3 p.Glu292Val Conclusion: Our results indicate that partially reduced ABCA3 activity due to E292V is not a major risk factor for reduced lung function and COPD in the general population. Login to comment 10 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Ala320Thr ABCA3 p.Ala1086Asp ABCA3 p.Arg1474Trp ABCA3 p.Pro766Ser ABCA3 p.His86Tyr ABCA3 p.Ser1262Gly Methods We screened 760 individuals with extreme pulmonary phenotypes and identified three novel (H86Y, A320T, A1086D) and four previously described mutations (E292V, P766S, S1262G, R1474W) in the ABCA3 gene. Login to comment 13 ABCA3 p.Glu292Val Results In the Copenhagen City Heart Study individuals heterozygous for E292V had 5 % reduced FEV1 % predicted compared with noncarriers (t-test: p = 0.008), and an increased odds ratio for COPD of 1.9 (95 % CI: 1.1-3.1). Login to comment 14 ABCA3 p.Ala1086Asp In contrast, the A1086D mutation was associated with increased FEV1 % predicted (p = 0.03). Login to comment 16 ABCA3 p.Glu292Val In the larger Copenhagen General Population Study, and in the two studies combined, E292V heterozygotes did not have reduced lung function or increased risk of COPD (p = 0.11-0.98), while this was the case for the positive controls, surfactant protein-B 121ins2 heterozygotes and α1-antitrypsin ZZ homozygotes. Login to comment 17 ABCA3 p.Glu292Val Conclusion Our results indicate that partially reduced ABCA3 activity due to E292V is not a major risk factor for reduced lung function and COPD in the general population. Login to comment 18 ABCA3 p.Glu292Val ABCA3 p.Glu292Val This is an important finding as 1.3 % in the Danish population has partially reduced ABCA3 function due to E292V. Login to comment 20 ABCA3 p.Glu292Val The E292V mutation * Correspondence: morten.dahl@rh.regionh.dk 1 Department of Clinical Biochemistry, Herlev Hospital, Copenhagen University Hospital, Copenhagen, Denmark 3 Department of Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, Copenhagen DK-2100, Denmark Full list of author information is available at the end of the article (c) 2012 B&#e6;kvad-Hansen et al. Login to comment 25 ABCA3 p.Glu292Val Given this, ABCA3 variants, and in particular E292V, could play an important role on development of common pulmonary disorders in the general population. Login to comment 27 ABCA3 p.Glu292Val A relatively common mutation in ABCA3, E292V, is associated with partially impaired ABCA3 function [6] and with milder chronic lung disease in childhood [3,6]. Login to comment 29 ABCA3 p.Glu292Val The E292V mutation has been found in about 0.3-0.4 % of individuals in a heterogeneous US population [7] and may affect both heterozygous [8,9] and compound heterozygous carriers [8,10]. Login to comment 31 ABCA3 p.Glu292Val Given this, ABCA3 variants, and in particular E292V, could play an important role on development of common pulmonary disorders in the general population. Login to comment 58 ABCA3 p.Ala320Thr ABCA3 p.Ala1086Asp It was not possible to design TaqMan genotyping assays for two of the mutations (A320T and A1086D), and genotyping for these variants was instead performed using the LightScanner (Additional file 3). Login to comment 64 ABCA3 p.Ala320Thr ABCA3 p.Ala1086Asp It was not possible to design TaqMan genotyping assays for two of the mutations (A320T and A1086D), and genotyping for these variants was instead performed using the LightScanner (Additional file 3). Login to comment 71 ABCA3 p.Glu292Val ABCA3 p.Ala320Thr ABCA3 p.Ala1086Asp ABCA3 p.Arg1474Trp ABCA3 p.Pro766Ser ABCA3 p.His86Tyr ABCA3 p.Ser1262Gly H86Y, A320T and A1086D were novel variants, whereas E292V, P766S, S1262G and R1474W have been described previously. Login to comment 72 ABCA3 p.Arg1474Trp ABCA3 p.Pro766Ser Two individuals heterozygous for P766S and one individual heterozygous for R1474W suffered from interstitial lung disease (Additional file 4). Login to comment 76 ABCA3 p.Glu292Val SIFT and Polyphen both predicted E292V to be damaging, but none of the other six mutations. Login to comment 77 ABCA3 p.Glu292Val ABCA3 p.Ala320Thr ABCA3 p.Ala1086Asp ABCA3 p.Arg1474Trp ABCA3 p.Pro766Ser ABCA3 p.His86Tyr ABCA3 p.Ser1262Gly H86Y, A320T and A1086D were novel variants, whereas E292V, P766S, S1262G and R1474W have been described previously. Login to comment 78 ABCA3 p.Arg1474Trp ABCA3 p.Pro766Ser Two individuals heterozygous for P766S and one individual heterozygous for R1474W suffered from interstitial lung disease (Additional file 4). Login to comment 81 ABCA3 p.Glu292Val Individuals heterozygous for E292V had 5% reduced FEV1 % predicted (t-test:p = 0.008), 3% reduced FVC % predicted (p = 0.04) and 0.02 reduced FEV1/FVC (p = 0.03), compared with wildtypes (Figure 1). Login to comment 82 ABCA3 p.Glu292Val ABCA3 p.Glu292Val SIFT and Polyphen both predicted E292V to be damaging, but none of the other six mutations. Login to comment 84 ABCA3 p.Ala1086Asp In contrast, A1086D heterozygotes had increased FEV1 % predicted (p = 0.03) and FVC % predicted (p = 0.008) compared with wildtypes, whereas the FEV1/FVC ratio showed no statistical significant difference (p = 0.51). Login to comment 87 ABCA3 p.Glu292Val Individuals heterozygous for E292V had 5 % reduced FEV1%predicted (t-test:p = 0.008), 3 % reduced FVC%predicted (p = 0.04) and 0.02 reduced FEV1/FVC (p = 0.03), compared with wildtypes (figure 1). Login to comment 88 ABCA3 p.Glu292Val ABCA3 p.Ala320Thr ABCA3 p.Arg1474Trp ABCA3 p.Pro766Ser ABCA3 p.His86Tyr ABCA3 p.Ser1262Gly The observed reductions in pulmonary function were almost similar in absolute numbers to those observed for surfactant protein-B 121ins2, a mutation which in the homozygous state associates with a phenotype somewhat similar to that for E292V compound heterozygosity. Login to comment 90 ABCA3 p.Pro766Ser ABCA3 p.Ser1262Gly This individual was compound heterozygous for P766S and S1262G and was not identified among the extreme phenotypes group. Login to comment 92 ABCA3 p.Ala1086Asp P-values are by Student`s t-test. Lung function according to surfactant protein-B 121ins2 heterozygosity and α1-antitrysin ZZ homozygosity serve as positive controls. Numbers of individuals with surfactant protein-B 121ins2 heterozygosity and α1-antitrypsin ZZ homozygosity differ slightly from the number of individuals with ABCA3 genotypes due to different number of study subjects available for analysis within the study period In contrast, A1086D heterozygotes had increased FEV1%predicted (p = 0.03) and FVC%predicted (p = 0.008) compared with wildtypes, whereas the FEV1/FVC ratio showed no statistical significant difference (p = 0.51). Login to comment 93 ABCA3 p.Glu292Val COPD by ABCA3 genotype in the Copenhagen City Heart Study Individuals heterozygous for E292V had a multivariate adjusted odds ratio for COPD of 1.9 (95% CI 1.1-3.1) compared with wildtypes in the Copenhagen City Heart Study (Figure 2). Login to comment 95 ABCA3 p.Arg1474Trp ABCA3 p.Pro766Ser ABCA3 p.His86Tyr For the H86Y, P766S and R1474W mutations, risk of COPD did not deviate significantly from 1.0. Login to comment 96 ABCA3 p.Ala320Thr ABCA3 p.Arg1474Trp ABCA3 p.Pro766Ser ABCA3 p.His86Tyr ABCA3 p.Ser1262Gly Individuals heterozygous for H86Y, A320T, P766S, S1262G, or R1474W did not differ from wildtypes in FEV1%predicted, FVC%predicted or FEV1/FVC (p≥0.19). Login to comment 97 ABCA3 p.Ala320Thr ABCA3 p.Ala1086Asp ABCA3 p.Ser1262Gly Due to lack of events among A320T, A1086D and S1262G heterozygotes, we were not able to calculate risk of COPD for these variants. Login to comment 98 ABCA3 p.Pro766Ser ABCA3 p.Ser1262Gly This individual was compound heterozygous for P766S and S1262G and was not identified among the extreme phenotypes group. Login to comment 99 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Glu292Val Pulmonary function and COPD by ABCA3 E292V in the Copenhagen General Population Study To further validate the findings for E292V, we genotyped the Copenhagen General Population Study (n = 54,395) for the E292V variant. Login to comment 101 ABCA3 p.Glu292Val COPD by ABCA3 genotype in the Copenhagen City Heart Study Individuals heterozygous for E292V had a multivariate adjusted odds ratio for COPD of 1.9 (95 % CI 1.1-3.1) compared with wildtypes in the Copenhagen City Heart Study (figure 2). Login to comment 102 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Ala320Thr ABCA3 p.Ala1086Asp ABCA3 p.Arg1474Trp ABCA3 p.Pro766Ser ABCA3 p.His86Tyr ABCA3 p.Ser1262Gly Characteristics did not differ between E292V heterozygotes 60 80 100 120 60 80 100 120 0.6 0.8 Genotype N FEV1 % predicted p FVC % predicted p FEV1/FVC p H86Y Wt 9801 Het 15 0.76 0.91 0.39 E292V Wt 9706 Het 110 0.008 0.04 0.03 A320T Wt 9803 Het 13 0.19 0.27 0.74 P766S Wt 9695 Het 121 0.65 0.42 0.55 A1086D Wt 9802 Het 4 0.03 0.008 0.51 S1262G Wt 9802 Het 14 0.47 0.77 0.53 R1474W Wt 9636 Het 180 0.49 0.20 0.48 SP-B121ins2 Wt 10425 Het 21 0.67 0.68 0.10 b1;1-antitrypsin MM 8082 ZZ 6 0.01 0.55 0.0008 Figure 1 Lung function according to ATP binding cassette member 3 (ABCA3) genotype in the Copenhagen City Heart Study. Login to comment 103 ABCA3 p.Arg1474Trp ABCA3 p.Pro766Ser ABCA3 p.His86Tyr For the H86Y, P766S and R1474W mutations, risk of COPD did not deviate significantly from 1.0. Login to comment 105 ABCA3 p.Ala320Thr ABCA3 p.Ala1086Asp ABCA3 p.Ser1262Gly Due to lack of events among A320T, A1086D and S1262G heterozygotes, we were not able to calculate risk of COPD for these variants. Login to comment 108 ABCA3 p.Glu292Val We found that FEV1 % predicted, FVC % predicted, and FEV1/FVC did not differ in E292V heterozygotes vs wildtypes (pࣙ0.67), whereas FEV1 % predicted was reduced in the positive controls, surfactant protein-B 121ins2 heterozygotes and b1;1-antitrypsin ZZ homozygotes. Login to comment 109 ABCA3 p.Glu292Val In accordance with the results on pulmonary function, the multivariate adjusted odds ratio for COPD was not increased in E292V heterozygotes compared with wildtypes (odds ratio 1.10 (0.83-1.46)), while surfactant protein-B 121ins2 heterozygotes and b1;1-antitrypsin ZZ homozygotes had increased odds ratios for COPD of 2.8 (1.0-8.1) and 6.6 (1.7-26) (Additional file 9). Login to comment 110 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Glu292Val Risk of COPD according to surfactant protein-B 121ins2 heterozygosity and α1-antitrysin ZZ homozygosity serve as positive controls. Numbers of individuals with surfactant protein-B 121ins2 heterozygosity and α1-antitrypsin ZZ homozygosity differ slightly from the number of individuals with ABCA3 genotypes due to different number of study subjects available for analysis within the study period Pulmonary function and COPD by ABCA3 E292V in the Copenhagen General Population Study To further validate the findings for E292V, we genotyped the Copenhagen General Population Study (n = 54,395) for the E292V variant. Login to comment 111 ABCA3 p.Glu292Val In this analysis, we found that FEV1 % predicted, FVC % predicted, and FEV1/FVC did not differ in E292V heterozygotes vs wildtypes (pࣙ0.28), whereas FEV1 % predicted was reduced in the positive controls, surfactant protein-B 121ins2 heterozygotes and b1;1-antitrysin ZZ homozygotes (figure 3). Login to comment 112 ABCA3 p.Glu292Val ABCA3 p.Arg1474Trp ABCA3 p.Pro766Ser ABCA3 p.His86Tyr We also stratified our data for smoking status, as did our previous study of surfactant protein-B 121ins2 as a risk factor in COPD [12], but found no significant differences in lung Age adjusted Odds ratio (95% confidence interval) 0.1 1 10 Genotype Participants Events H86Y Wt 9801 1080 Het 15 2 E292V Wt 9706 1063 Het 110 19 P766S Wt 9695 1067 Het 121 15 R1474W Wt 9636 1064 Het 180 18 SP-B121ins2 Wt 10427 1209 Het 21 5 b1;1-antitrypsin MM 8082 927 ZZ 6 3 Multi variate adjusted 0.1 1 10 Figure 2 Risk of COPD according to ATP binding cassette member 3 (ABCA3) genotype in the Copenhagen City Heart Study. Login to comment 113 ABCA3 p.Glu292Val Characteristics did not differ between E292V heterozygotes and wildtypes in any of the two study cohorts. Login to comment 116 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Glu292Val Table 1 Characteristics of participants in the Copenhagen City Heart Study and the Copenhagen General Population Study according to E292V genotype Copenhagen City Heart Study Copenhagen General Population Study Wildtypes E292V heterozygotes p-value Wildtypes E292V heterozygotes p-value N 9,706 110 53,685 710 Women, % 5,374 (55) 60 (55) 0.86 29,756 (55) 394 (55) 0.97 Age, yrs 58 (44- 69) 57 (45-67) 0.57 60(50-70) 59(49-69) 0.20 Eversmokers, % 76 77 0.72 60 59 0.40 Packyears of tobacco smoked 25(11-40) 25(13-40) 0.72 17(6-31) 15(6-30) 0.44 Values represent number, median (interquartile range), or percent. Login to comment 118 ABCA3 p.Glu292Val function for E292V. Login to comment 119 ABCA3 p.Glu292Val We found that FEV1%predicted, FVC%predicted, and FEV1/FVC did not differ in E292V heterozygotes vs wildtypes (p≥0.67), whereas FEV1%predicted was reduced in the positive controls, surfactant protein-B 121ins2 heterozygotes and α1-antitrypsin ZZ homozygotes. Login to comment 120 ABCA3 p.Glu292Val ABCA3 p.Glu292Val In accordance with the results on pulmonary function, the multivariate adjusted odds ratio for COPD was not increased in E292V heterozygotes compared with wildtypes (odds ratio 1.10 (0.83-1.46)), while surfactant protein-B 121ins2 heterozygotes and α1-antitrypsin ZZ homozygotes had increased odds ratios for COPD of 2.8 (1.0-8.1) and 6.6 (1.7-26) (Additional file 9) Pulmonary function and COPD by ABCA3 E292V in the Copenhagen City Heart Study and Copenhagen General Population Study combined Finally, to maximize our statistical power, we combined the Copenhagen City Heart Study and Copenhagen General Population Study. Login to comment 121 ABCA3 p.Glu292Val In this analysis, we found that FEV1%predicted, FVC%predicted, and FEV1/FVC did not differ in E292V heterozygotes vs wildtypes (p≥0.28), whereas FEV1%predicted was reduced in the positive controls, surfactant protein-B 121ins2 heterozygotes and α1-antitrysin ZZ homozygotes (figure 3). Login to comment 122 ABCA3 p.Glu292Val ABCA3 p.Glu292Val We also stratified our data for smoking status, as did our previous study of surfactant protein-B 121ins2 as a risk factor in COPD [12], but found no significant differences in lung function for E292V. Login to comment 125 ABCA3 p.Glu292Val We had 80% power to exclude odds ratios for COPD for E292V heterozygotes of 1.3, for surfactant protein-B 121ins2 heterozygotes of 2.1, and for b1;1-antitrypsin ZZ homozygotes of 4.0. Login to comment 126 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Ala320Thr ABCA3 p.Ala1086Asp ABCA3 p.Arg1474Trp ABCA3 p.Pro766Ser ABCA3 p.His86Tyr ABCA3 p.Ser1262Gly Figure 3 Lung function according to ATP binding cassette member 3 (ABCA3) E292V, surfactant protein-B 121ins2, and α1-antitrypsin ZZ genotypes in the Copenhagen City Heart Study and Copenhagen General Population Study combined, stratified for smoking status. Login to comment 128 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Glu292Val P-values are by Student`s t-test. Lung function according to surfactant protein-B 121ins2 heterozygosity and α1-antitrysin ZZ homozygosity serve as positive controls. Numbers of individuals with surfactant protein-B 121ins2 heterozygosity and α1-antitrypsin ZZ homozygosity differ slightly from the number of individuals with an ABCA3 E292V genotype due to different number of study subjects available for analysis within the study period Individuals heterozygous for E292V had multivariate adjusted odds ratios for COPD of 1.2 (0.96-1.57) among all subjects, of 1.1 (0.6-2.1) among nonsmokers, and of 1.2 (0.95-1.63) among smokers, respectively, compared with wildtypes (figure 4). Login to comment 129 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Glu292Val To validate the findings for 60 80 100 Genotype N FEV1 % predicted P FVC% predicted P FEV1/FVC P All E292V Wt 63391 Het 820 0.28 0.40 0.60 SPB121ins2 Wt 46409 Het 83 0.02 0.07 0.11 b1;1 antitrypsin MM 40066 ZZ 19 0.0008 0.1 9 0.0009 Never smoker E292V Wt 23738 Het 319 0.81 0.99 0.53 SPB121ins2 Wt 16248 Het 36 0.60 0.50 0.70 b1;1 antitrypsin MM 14022 ZZ 9 0.43 0.82 0.67 Smoker E292V Wt 39653 Het 501 0.11 0.26 0.19 SPB121ins2 Wt 30175 Het 47 0.0008 0.01 0.00007 b1;1 antitrypsin MM 26044 ZZ 10 0.0001 0.10 0.0001 60 80 100 0.6 0.8 Figure 3 Lung function according to ATP binding cassette member 3 (ABCA3) E292V, surfactant protein-B 121ins2, and b1;1-antitrypsin ZZ genotypes in the Copenhagen City Heart Study and Copenhagen General Population Study combined, stratified for smoking status. Login to comment 130 ABCA3 p.Glu292Val Values are mean and standard error. P-values are by Student`s t-test. Lung function according to surfactant protein-B 121ins2 heterozygosity and b1;1-antitrysin ZZ homozygosity serve as positive controls. Numbers of individuals with surfactant protein-B 121ins2 heterozygosity and b1;1-antitrypsin ZZ homozygosity differ slightly from the number of individuals with an ABCA3 E292V genotype due to different number of study subjects available for analysis within the study period. Login to comment 131 ABCA3 p.Glu292Val ABCA3 p.Glu292Val We had 80 % power to exclude odds ratios for COPD for E292V heterozygotes of 1.3, for surfactant protein-B 121ins2 heterozygotes of 2.1, and for α1-antitrypsin ZZ homozygotes of 4.0. Login to comment 132 ABCA3 p.Glu292Val ABCA3 p.Glu292Val Figure 4 Risk of COPD according to ATP binding cassette member 3 (ABCA3) E292V, surfactant protein-B 121ins2, and α1-antitrypsin ZZ genotypes in the Copenhagen City Heart Study and Copenhagen General Population Study combined, stratified for smoking status. Login to comment 133 ABCA3 p.Glu292Val Supporting this overall negative result, the estimates for the positive controls, surfactant protein-B 121ins2 heterozygotes and b1;1-antitrypsin ZZ homozygotes, were relatively stable throughout the studies and there was no evidence of selection bias against E292V in any of the two cohorts. Login to comment 134 ABCA3 p.Glu292Val The phenotype in E292V carriers was previously reported to range from minimal changes on lung biopsy and no symptoms to severe fatal lung disease [8,9]. Login to comment 135 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Ala320Thr ABCA3 p.Ala1086Asp ABCA3 p.Arg1474Trp ABCA3 p.Pro766Ser ABCA3 p.His86Tyr ABCA3 p.Ser1262Gly Numbers of individuals with surfactant protein-B 121ins2 heterozygosity and α1-antitrypsin ZZ homozygosity differ slightly from the number of individuals with an ABCA3 E292V genotype due to different number of study subjects available for analysis within the study period Discussion To test whether individuals heterozygous for ABCA3 variants have reduced lung function and increased risk of COPD in the general population, we screened 760 individuals with extreme lung phenotypes for genetic variations in the ABCA3 gene and identified three novel (H86Y, A320T, A1086D) and four previously described variations (E292V, P766S, S1262G, R1474W). Login to comment 136 ABCA3 p.Glu292Val Tobacco smoking [23] and unidentified mutations in surfactant protein-C [24,25], ABCA3 [26], and surfactant protein-B could be risk factors of additionally impaired surfactant function in E292V heterozygotes; although the latter risk factors are probably less prevalent in the general population as compared with tobacco smoking. Login to comment 137 ABCA3 p.Glu292Val ABCA3 p.Glu292Val We found that E292V heterozygotes had reduced lung function and increased risk of COPD, whereas the novel A1086 mutation was associated with increased lung function. Login to comment 138 ABCA3 p.Glu292Val ABCA3 p.Glu292Val To validate the findings for E292V we genotyped an additional 54,395 individuals from the Copenhagen General Population Study. Login to comment 139 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Glu292Val In this larger cohort, and in the two study cohorts combined, we found that E292V heterozygotes did not have reduced lung function or increased risk of COPD. Login to comment 140 ABCA3 p.Glu292Val ABCA3 p.Glu292Val Supporting this overall negative result, the estimates for the positive controls, surfactant protein-B 121ins2 heterozygotes and α1-antitrypsin ZZ homozygotes, were relatively stable throughout the studies and there was no evidence of selection bias against E292V in any of the two cohorts. Login to comment 141 ABCA3 p.Glu292Val ABCA3 p.Ala1086Asp The phenotype in E292V carriers was previously reported to range from minimal changes on lung biopsy and no symptoms to severe fatal lung disease [8,9]. Login to comment 142 ABCA3 p.Glu292Val It is thus possible that E292V may cause lung disease in certain contexts or subgroups of individuals, e.g. when additional impairments of surfactant function are present, leaving the average carrier unaffected. Login to comment 143 ABCA3 p.Glu292Val ABCA3 p.Ala1086Asp Tobacco smoking [23] and unidentified mutations in surfactant protein-C [24,25], ABCA3 [26], and surfactant protein-B could be risk factors of additionally impaired surfactant function in E292V heterozygotes; although the latter risk factors are probably less prevalent in the general population as compared with tobacco smoking. Login to comment 144 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Ala1086Asp Because participants in the Copenhagen City Heart Study smoked more tobacco than participants from the Copenhagen General Population Study we speculate that the difference in smoking habits between the two cohorts could partly contribute to the different results observed for E292V in the two cohorts. Login to comment 145 ABCA3 p.Glu292Val ABCA3 p.His86Tyr When statistical power, however, was maximised using the Copenhagen General Population Study or the two studies combined, E292V heterozygotes did not differ from wildtypes in lung function or COPD risk. Login to comment 146 ABCA3 p.Leu101Pro ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.His86Tyr This is an important finding as 1.3 % in the Danish general population has partially reduced ABCA3 function due to E292V, and since this variant has been linked previously with severe chronic lung disease in heterozygous and compound heterozygous E292V carriers [8-10]. Login to comment 147 ABCA3 p.Glu292Val ABCA3 p.Ala320Thr Besides E292V, we identified six other variants of potential relevance to ABCA3 function. Login to comment 148 ABCA3 p.Ala1086Asp ABCA3 p.Arg1474Trp However, none of these mutations were associated with lung function or risk of COPD, except for the novel A1086D mutation. Login to comment 149 ABCA3 p.Glu292Val ABCA3 p.Glu292Val However, no association with N Events N Events N Events E292V Wt 63391 5168 23738 726 39653 4442 Het 820 75 319 10 501 65 SP-B 121ins2 Wt 46423 4735 16248 660 30175 4075 Het 83 15 36 2 47 13 b1;1-antitrypsin MM 40066 3588 14022 454 26044 3134 ZZ 19 6 9 1 10 5 All 0.1 1 10 Never smoker Odds ratio (95% confidence interval) 0.1 1 10 Smoker 0.1 1 10 Figure 4 Risk of COPD according to ATP binding cassette member 3 (ABCA3) E292V, surfactant protein-B 121ins2, and b1;1-antitrypsin ZZ genotypes in the Copenhagen City Heart Study and Copenhagen General Population Study combined, stratified for smoking status. Login to comment 150 ABCA3 p.Ala1086Asp However, the small number of A1086D heterozygotes (n = 4) makes this finding highly insecure. Login to comment 151 ABCA3 p.Glu292Val In addition, if correction for multiple comparisons was performed none of the results for E292V in the Copenhagen City Heart Study would be of statistical significance. Login to comment 152 ABCA3 p.Glu292Val ABCA3 p.His86Tyr The second novel mutation, H86Y, is situated in the first extracellular loop of ABCA3. Login to comment 153 ABCA3 p.Leu101Pro ABCA3 p.Arg1474Trp ABCA3 p.His86Tyr Another mutation in this loop, L101P, has been shown to affect ABCA3 protein folding leading to retention of ABCA3 in the endoplasmatic reticulum and subsequent ER stress and apoptosis [27,28], however individuals heterozygous for the H86Y mutation appeared asymptomatic in this study. Login to comment 154 ABCA3 p.Ala320Thr The third novel mutation, A320T, resides in the transmembrane helix domain of ABCA3 and did also not associate with lung function or COPD. Login to comment 155 ABCA3 p.Arg1474Trp Finally, the previously described R1474W mutation may seem particularly interesting as this mutation is situated within the conserved nucleotide binding domain which is important for binding of ATP [19]. Login to comment 156 ABCA3 p.Arg1474Trp However, no association with lung function or risk of COPD was observed for R1474W heterozygosity. Login to comment 166 ABCA3 p.Glu292Val Conclusions We found with significant statistical power that ABCA3 E292V heterozygotes do not have reduced lung function or increased risk of COPD in the general population. Login to comment 167 ABCA3 p.Glu292Val ABCA3 p.Glu292Val This is an important finding as 1.3% in the Danish general population has partially reduced ABCA3 function due to E292V and since this variant has been linked previously with severe chronic lung disease in E292V heterozygotes and compound heterozygotes. Login to comment 169 ABCA3 p.Glu292Val Conclusions We found with significant statistical power that ABCA3 E292V heterozygotes do not have reduced lung function or increased risk of COPD in the general population. Login to comment 170 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Glu292Val This is an important finding as 1.3 % in the Danish general population has partially reduced ABCA3 function due to E292V and since this variant has been linked previously with severe chronic lung disease in E292V heterozygotes and compound heterozygotes. Login to comment 171 ABCA3 p.Glu292Val Abbreviations ABCA3, ATP-binding cassette member A3; COPD, Chronic obstructive pulmonary disease; E292V, Substitution of valine for glutamic acid at aminoacid-position 292; FEV1, Forced expiratory volume in one second; FVC, Forced vital capacity. Login to comment 172 ABCA3 p.Glu292Val Abbreviations ABCA3: ATP-binding cassette member A3; COPD: Chronic obstructive pulmonary disease; E292V: Substitution of valine for glutamic acid at aminoacid-position 292; FEV1: Forced expiratory volume in one second; FVC: Forced vital capacity. Login to comment 295 ABCA3 p.Glu292Val : Heterozygosity for E292V in ABCA3, lung function and COPD in 64,000 individuals. Login to comment