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Association of IREB2 and CHRNA3 polymorphisms with airflow obstruction in severe alpha-1 antitrypsin deficiency
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Title: Association of IREB2 and CHRNA3 polymorphisms with airflow obstruction in severe alpha-1 antitrypsin deficiency
Abbreviated Title: Respiratory Research
Physical Description: Archival
Language: English
Creator: Kim, Woo Jin
Wood, Alice M.
Barker, Alan F.
Brantly, Mark L.
Campbell, Edward J.
Eden, Edward
McElvaney, Gerard
Rennard, Stephen I.
Sandhaus, Robert A.
Stocks, James M.
Stoller, James K.
Strange, Charlie
Turino, Gerard
Silverman, Edwin K.
Stockley, Robert A.
DeMeo, Dawn L.
Publisher: BioMed Central
Publication Date: 2012
 Subjects
Subjects / Keywords: CHRNA3
Chronic obstructive pulmonary disease
Genetic association analysis
Genetic modifiers
IREB2
COPD
 Notes
Abstract: Background: The development of COPD in subjects with alpha-1 antitrypsin (AAT) deficiency is likely to be influenced by modifier genes. Genome-wide association studies and integrative genomics approaches in COPD have demonstrated significant associations with SNPs in the chromosome 15q region that includes CHRNA3 (cholinergic nicotine receptor alpha3) and IREB2 (iron regulatory binding protein 2). We investigated whether SNPs in the chromosome 15q region would be modifiers for lung function and COPD in AAT deficiency. Methods: The current analysis included 378 PIZZ subjects in the AAT Genetic Modifiers Study and a replication cohort of 458 subjects from the UK AAT Deficiency National Registry. Nine SNPs in LOC123688, CHRNA3 and IREB2 were selected for genotyping. FEV1 percent of predicted and FEV1/FVC ratio were analyzed as quantitative phenotypes. Family-based association analysis was performed in the AAT Genetic Modifiers Study. In the replication set, general linear models were used for quantitative phenotypes and logistic regression models were used for the presence/absence of emphysema or COPD. Results: Three SNPs (rs2568494 in IREB2, rs8034191 in LOC123688, and rs1051730 in CHRNA3) were associated with pre-bronchodilator FEV1 percent of predicted in the AAT Genetic Modifiers Study. Two SNPs (rs2568494 and rs1051730) were associated with the post-bronchodilator FEV1 percent of predicted and pre-bronchodilator FEV1/FVC ratio; SNP-by-gender interactions were observed. In the UK National Registry dataset, rs2568494 was significantly associated with emphysema in the male subgroup; significant SNP-by-smoking interactions were observed. Conclusions: IREB2 and CHRNA3 are potential genetic modifiers of COPD phenotypes in individuals with severe AAT deficiency and may be sex-specific in their impact. Keywords: CHRNA3, Chronic obstructive pulmonary disease, Genetic association analysis, Genetic modifiers, IREB2
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Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution.
Resource Identifier: doi - 10.1186/1465-9921-13-16
System ID: AA00010467:00001

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airflow obstruction in severe alpha-1 antitrypsin deficiency
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Abstract
Background
The development of COPD in subjects with alpha-1 antitrypsin (AAT) deficiency is likely to be influenced by modifier genes. Genome-wide association studies and integrative genomics approaches in COPD have demonstrated significant associations with SNPs in the chromosome 15q region that includes CHRNA3 (cholinergic nicotine receptor alpha3) and IREB2 (iron regulatory binding protein 2).
We investigated whether SNPs in the chromosome 15q region would be modifiers for lung function and COPD in AAT deficiency.
Methods
The current analysis included 378 PIZZ subjects in the AAT Genetic Modifiers Study and a replication cohort of 458 subjects from the UK AAT Deficiency National Registry. Nine SNPs in LOC123688, CHRNA3 and IREB2 were selected for genotyping. FEV1 percent of predicted and FEV1/FVC ratio were analyzed as quantitative phenotypes. Family-based association analysis was performed in the AAT Genetic Modifiers Study. In the replication set, general linear models were used for quantitative phenotypes and logistic regression models were used for the presence/absence of emphysema or COPD.
Results
Three SNPs (rs2568494 in IREB2, rs8034191 in LOC123688, and rs1051730 in CHRNA3) were associated with pre-bronchodilator FEV1 percent of predicted in the AAT Genetic Modifiers Study. Two SNPs (rs2568494 and rs1051730) were associated with the post-bronchodilator FEV1 percent of predicted and pre-bronchodilator FEV1/FVC ratio; SNP-by-gender interactions were observed. In the UK National Registry dataset, rs2568494 was significantly associated with emphysema in the male subgroup; significant SNP-by-smoking interactions were observed.
Conclusions
IREB2 and CHRNA3 are potential genetic modifiers of COPD phenotypes in individuals with severe AAT deficiency and may be sex-specific in their impact.
http:purl.orgdcelements1.1creator
Kim, Woo Jin
Wood, Alice M
Barker, Alan F
Brantly, Mark L
Campbell, Edward J
Eden, Edward
McElvaney, Gerard
Rennard, Stephen I
Sandhaus, Robert A
Stocks, James M
Stoller, James K
Strange, Charlie
Turino, Gerard
Silverman, Edwin K
Stockley, Robert A
DeMeo, Dawn L
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BioMed Central Ltd
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Kim et al.; licensee BioMed Central Ltd.
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Respiratory Research. 2012 Feb 22;13(1):16
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dochead Research
bibl
title
p Association of it IREB2 and CHRNA3 polymorphisms with airflow obstruction in severe alpha-1 antitrypsin deficiency
aug
au id A1 snm Kimfnm Woo Jininsr iid I1 email pulmo2@kangwon.ac.kr
A2 Woodmi MAliceI2 a.m.wood@bham.ac.uk
A3 BarkerFAlanI3 barkera@ohsu.edu
A4 BrantlyLMarkI4 mark.brantly@medicine.ufl.edu
A5 CampbellJEdwardI5 ECampbell@aatdetection.com
A6 EdenEdwardI6 eeden@chpnet.org
A7 McElvaneyGerardI7 gmcelvaney@rcsi.ie
A8 RennardIStephenI8 srennard@unmc.edu
A9 SandhausARobertI9 rasandhaus@alphaone.org
A10 StocksMJamesI10 James.Stocks@uthct.edu
A11 StollerKJamesI11 STOLLEJ@ccf.org
A12 StrangeCharlieI12 strangec@musc.edu
A13 TurinoGerardI13 gmt1@columbia.edu
A14 SilvermanKEdwinI14 reeks@channing.harvard.edu
A15 StockleyARobertI15 Rob.Stockley@uhb.nhs.uk
A16 ca yes DeMeoLDawndawn.demeo@channing.harvard.edu
insg
ins Department of Internal Medicine, Kangwon National University, Chuncheon, South Korea
School of Clinical and Experimental Medicine, University of Birmingham, Birmingham, UK
Oregon Health and Science University, Portland, OR, USA
University of Florida, Gainesville, FL, USA
Intermountain Health Care, Provo, and Heredilab, Inc, Salt Lake City, UT, USA
St. Luke's/Roosevelt Hospital, New York, NY, USA
Beaumont Hospital, Dublin, Ireland
University of Nebraska, Omaha, NE, USA
National Jewish Health, Denver, CO, USA
University of Texas at Tyler, Tyler, TX, USA
Cleveland Clinic, Cleveland, OH, USA
Medical University of South Carolina, Charleston, SC, USA
St. Luke's/Roosevelt Hospital, New York, NY, USA
Channing Laboratory and the Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, MA, USA
Lung Investigation Unit, University Hospitals Birmingham, Birmingham, UK
source Respiratory Research
issn 1465-9921
pubdate 2012
volume 13
issue 1
fpage 16
url http://respiratory-research.com/content/13/1/16
xrefbib pubidlist pubid idtype doi 10.1186/1465-9921-13-16pmpid 22356581
history rec date day 11month 12year 2011acc 2222012pub 2222012
cpyrt 2012collab Kim et al; licensee BioMed Central Ltd.note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
kwdg
kwd
CHRNA3
Chronic obstructive pulmonary disease
Genetic association analysis
Genetic modifiers
IREB2
abs
sec
st
Abstract
Background
The development of COPD in subjects with alpha-1 antitrypsin (AAT) deficiency is likely to be influenced by modifier genes. Genome-wide association studies and integrative genomics approaches in COPD have demonstrated significant associations with SNPs in the chromosome 15q region that includes CHRNA3 (cholinergic nicotine receptor alpha3) and IREB2 (iron regulatory binding protein 2).
We investigated whether SNPs in the chromosome 15q region would be modifiers for lung function and COPD in AAT deficiency.
Methods
The current analysis included 378 PIZZ subjects in the AAT Genetic Modifiers Study and a replication cohort of 458 subjects from the UK AAT Deficiency National Registry. Nine SNPs in LOC123688, CHRNA3 and IREB2 were selected for genotyping. FEVsub 1 percent of predicted and FEV1/FVC ratio were analyzed as quantitative phenotypes. Family-based association analysis was performed in the AAT Genetic Modifiers Study. In the replication set, general linear models were used for quantitative phenotypes and logistic regression models were used for the presence/absence of emphysema or COPD.
Results
Three SNPs (rs2568494 in IREB2, rs8034191 in LOC123688, and rs1051730 in CHRNA3) were associated with pre-bronchodilator FEV1 percent of predicted in the AAT Genetic Modifiers Study. Two SNPs (rs2568494 and rs1051730) were associated with the post-bronchodilator FEV1 percent of predicted and pre-bronchodilator FEV1/FVC ratio; SNP-by-gender interactions were observed. In the UK National Registry dataset, rs2568494 was significantly associated with emphysema in the male subgroup; significant SNP-by-smoking interactions were observed.
Conclusions
IREB2 and CHRNA3 are potential genetic modifiers of COPD phenotypes in individuals with severe AAT deficiency and may be sex-specific in their impact.
bdy
Background
Chronic obstructive pulmonary disease (COPD) is a complex disease characterized by persistent airflow limitation. COPD risk likely results from the cumulative effect of environmental factors (especially cigarette smoking), genetic factors, and gene-by-environment interactions abbrgrp
abbr bid B1 1
. Alpha-1 antitrypsin (AAT) deficiency, typically caused by homozygosity for the Z allele at the AAT gene (SERPINA1), is a proven genetic cause of COPD. However, the development of COPD and emphysema in subjects with AAT deficiency is highly variable and is likely influenced by modifier genes and environmental factors
B2 2
B3 3
B4 4
.
Spirometric measurements of pulmonary function are widely used phenotypes in evaluating AAT deficient subjects and monitoring lung function decline
B5 5
; CT scan assessments for emphysema have been used as additional intermediate phenotypes of COPD to overcome some of the heterogeneity inherent in spirometric classifications alone. Familial aggregation studies of pulmonary function have suggested additional modifier genes in AAT deficiency subjects
B6 6
B7 7
. A few potential AAT candidate modifier genes, including NOS3
B8 8
, GSTP1
B9 9
, TNF
B10 10
, and IL10
B11 11
, have been reported so far, but these results have not been consistently replicated.
Genome-wide association (GWA) studies have revolutionized the identification of susceptibility genes for complex diseases. Three recent GWA studies showed that SNPs in a region of chromosome 15q25 were significantly associated with lung cancer; several nicotinic acetylcholine receptor genes, including CHRNA3 and LOC123688, are located in this region
B12 12
B13 13
B14 14
. A genome-wide association (GWA) study in COPD also showed significant associations between COPD susceptibility and SNPs in this region
B15 15
. This region was also associated with airflow obstruction and emphysema
B16 16
B17 17
. Interestingly, in addition to nicotinic acetylcholine receptor genes, this region also includes IREB2 (iron regulatory binding protein 2). IREB2 was identified as a potential COPD susceptibility gene using an integrative genomics approach with gene expression analysis of lung tissue samples followed by genetic association analysis
B18 18
. We hypothesized that SNPs in this chromosome 15q region may be modifiers of intermediate phenotypes of COPD in subjects with severe AAT deficiency.
Methods
Study subjects
The current analysis included 378 subjects with severe AAT deficiency (protease inhibitor [PI] ZZ) from 167 families in the AAT Genetic Modifiers Study. Ascertainment of eligible sibling pairs was based on homozygosity for the Z allele at the SERPINA1 locus as previously described
B19 19
. Pre- and post-bronchodilator study spirometry testing was performed according to American Thoracic Society (ATS) standards as described previously
19
. Percent predicted values for FEV1 were calculated using equations of Crapo and colleagues for Caucasian subjects
B20 20
. The FEV1/FVC ratio was analyzed using unadjusted values. Pack-years of cigarette smoking were calculated by multiplying the number of years smoked by the average number of daily cigarettes smoked, divided by 20. All participants provided written informed consent, and the study protocol was approved by individual Institutional Review Boards at each of the participating clinical centers (Partners IRB, 2001P001237). 458 unrelated Caucasian subjects from the UK AATD National Registry were also genotyped. Approval for the study was given by the local ethics committee. All patients had a serum alpha-1 antitrypsin (AAT) level of < 11 μM and PI ZZ genotype confirmed by allele-specific PCR (Heredilab, Salt Lake City, Utah, USA). None of the UK subjects had ever received AAT augmentation therapy. A full clinical assessment including smoke exposure, presence of chronic bronchitis (defined as a productive cough for at least 3 months in at least 2 consecutive years
B21 21
), lung function testing and high resolution CT scanning of the chest was undertaken, as described previously
B22 22
. The presence of emphysema was determined by the appearance of the scan and density mask analysis of slices at the level of the aortic arch (representing the upper zone) and the inferior pulmonary vein (representing the lower zone) using a threshold of -910 Hounsfield Units (HU). This HU threshold has been validated against physiological measures in AATD
22
. Patients whose voxel index exceeded values seen in normal subjects in either zone
B23 23
were classified as having emphysema.
Genotyping
Two SNPs (rs8034191 and rs1051730) in chromosome 15 were selected from the previous GWA in COPD
15
. Additionally, 7 SNPs in IREB2 were selected using pairwise linkage disequilibrium (LD)-tagging in Tagger with a minimum minor allele frequency of 0.05 and rsup 2-threshold of 0.8
B24 24
. SNPs were genotyped using Sequenom (San Diego, CA) assays in the AAT Genetic Modifiers Study. All family data were evaluated for familial inconsistencies using the PEDCHECK program
B25 25
.
In UK AATD National Registry study, genotyping was carried out using TaqMan® technologies (Applied Biosystems, UK) on an ABI7900 HT for 3 SNPs associated in the test dataset (rs2568494, IREB2; rs8034191, LOC123688; rs1051730, CHRNA3). All genotyping assays were pre-validated by the suppliers, and all plates included appropriate negative controls.
Statistical analysis
Hardy-Weinberg equilibrium was assessed using goodness of fit tests. Pre- and post-bronchodilator FEV1 percent of predicted and pre- and post-bronchodilator FEV1/FVC ratio were analyzed as quantitative phenotypes. Family-based association analysis was performed using PBAT software version 3.6
B26 26
assuming additive genetic models, adjusting for pack-years and pack-years2 of cigarette smoking, under the null hypothesis of no linkage and no association in the AAT Genetic Modifiers Study. In addition to the overall model, we evaluated gender-stratified models and models that included a SNP-by-smoking or a SNP-by-gender interaction term. Haplotype analysis was performed using 8, 4, 3, 2 SNP adjacent sliding windows in PBAT.
In the UK AATD National Registry study, data were analyzed using SPSS (version 16, Chicago: SPSS Inc). Quantitative genetic association analysis was carried out for FEV1 and FEV1/FVC using general linear models, adjusting for age, gender and smoke exposure (as pack-years and pack-years2). Logistic regression models were used for the presence of emphysema or COPD (defined as post bronchodilator FEV1/FVC < 0.7) accounting for covariates as before. Additive models were assumed for all SNPs. Gender stratification and SNP-by-gender and SNP-by-smoking interaction analyses were also carried out, as in the test dataset.
Results
Demographic characteristics
The mean age of subjects was 52.2 years, and mean post-bronchodilator FEV1 was 65.9% predicted in the AAT Genetic Modifiers Study and 50.3 years and 53.8% predicted in the UK AATD National Registry, respectively (Table tblr tid T1 1). Male subjects were 46% in AAT Genetic Modifiers Study and 59% in UK AATD National Registry, respectively. Three hundred and sixty-six subjects (79.8%) had emphysema in UK AATD National Registry.
tbl Table 1caption Baseline characteristics for PI ZZ individuals in the AAT Genetic Modifiers Study and the UK AATD National Registrytblbdy cols 4
r
c left
b Characteristics
AAT Genetic Modifiers Study
(n = 378)
UK national registry for AATD
(n = 458)
p value
cspan
hr
Age, years
52.2 ± 9.7
50.3 ± 10.4
0.01
Male sex (%)
173 (46%)
252 (55%)
< 0.0001
FEV1% predicted
65.9 ± 33.5
53.8 ± 32.2
0.0001
FEV1/FVC
0.551 ± 0.207
0.445 ± 0.194
< 0.0001
Pack-years for ever smokers
18.2 ± 14.5
15.9 ± 14.7
< 0.0001
tblfn
Data are presented as means (± S.D.) in the AAT Genetic Modifiers Study and the UK national registry for AATD, unless otherwise noted
Association analysis
There were no deviations from Hardy Weinberg equilibrium for any of the genotyped SNPs.
In the AAT Genetic Modifiers Study, three SNPs (rs2568494 in IREB2, rs8034191 in LOC123688, and rs1051730 in CHRNA3) were associated with pre-bronchodilator FEV1 percent of predicted (p < 0.05 Table T2 2 Figure figr fid F1 1). Two SNPs (rs2568494 and rs1051730) were associated with post-bronchodilator FEV1 percent of predicted and pre-bronchodilator FEV1/FVC ratio. One SNP (rs1051730) was associated with post-bronchodilator FEV1/FVC ratio (Table 2). Linkage disequilibrium (assessed with r2) between rs8034191 and the 1051730 was 0.9 (Figure F2 2). There was significant association only with a 2 SNP haplotype including rs8034191 and rs1051730 with pre- bronchodilator FEV1/FVC ratio (global test statistic; p = 0.05) using PBAT.
Table 2Genetic association results between SNPs in chromosome 15 and lung function in the AAT Genetic Modifiers Study8
center
P-Values for Different Phenotypes
Gene
SNP
MAF
pre-FEV1% predicted
post-FEV1% predicted
pre-
FEV1/FVC
post-
FEV1/FVC
IREB2
rs2568494
intron
0.30
0.02*
0.03*
0.05*
0.06
rs2656069
intron
0.22
0.48
0.29
0.59
0.46
rs1964678
intron
0.43
0.75
0.91
0.58
0.83
rs12593229
intron
0.43
0.82
0.79
0.64
0.93
rs10851906
intron
0.23
0.29
0.17
0.41
0.31
rs965604
intron
0.43
0.76
0.99
0.54
0.65
rs13180
exon
0.43
0.69
0.96
0.53
0.79
LOC123688
rs8034191
intron
0.29
0.04*
0.07
0.09
0.14
CHRNA3
rs1051730
exon
0.31
0.02*
0.03*
0.03*
0.05*
MAF = minor allele frequency
Each model was analyzed assuming an additive mode of inheritance adjusting for pack-years and pack-years2
* p ≤ 0.05
fig Figure 1FEV1 by CHRNA3 genotype (rs1051730) in the AAT Genetic Modifiers Study cohorttext
FEV1 by CHRNA3 genotype (rs1051730) in the AAT Genetic Modifiers Study cohort. Mean values (+ SEM) for a percent of predicted FEV1 are shown (p value = 0.02).
graphic file 1465-9921-13-16-1 hint_layout single
Figure 2Linkage disequilibrium (LD) among SNPs analyzed in chromosome 15. LD values are presented as r2
Linkage disequilibrium (LD) among SNPs analyzed in chromosome 15. LD values are presented as r2.
1465-9921-13-16-2
Interactions with cigarette smoking
There was no association between any of the genotyped SNPs and pack-years of smoking as the outcome in the AAT Genetic Modifiers Study. Inclusion of a SNP-by-pack-years interaction term for the lung function phenotypes showed significant interaction of rs1051730 with pack-years of smoking for the pre- bronchodilator FEV1/FVC ratio (main effect; p = 0.02, interaction effect; p = 0.04). There was no significant association with lung function phenotypes when the study population was stratified into groups of ever-smokers (n = 233) and never-smokers (n = 145) although this stratified analysis reduced the number of informative families considerably.
Genotype-by-gender interactions and gender stratification
SNP-by-gender interaction analysis showed significant interaction of rs2568494, rs8034191, and rs1051730 with gender in models for the post-bronchodilator FEV1/FVC ratio (main effect; p = 0.04, 0.04, 0.02, interaction effect; p = 0.02, 0.008, 0.004, respectively). Additionally, rs1051730 showed significant interaction in models for pre and post-bronchodilator FEV1 and pre-bronchodilator FEV1/FVC ratio (main effect; p = 0.04, 0.04, 0.08, interaction effect; p = 0.04, 0.02, 0.04, respectively).
In the stratified analysis, for the male subgroup, the p values were similar to the whole cohort results, with rs8034191 showing significant association with pre- and post-bronchodilator FEV1 percent of predicted and post-bronchodilator FEV1/FVC ratio (Table T3 3). However, in the female subgroup, there was no significant association with lung function phenotypes.
Table 3Genetic association results between SNPs in chromosome 15 and lung function of male subgroup in the AAT Genetic Modifiers Study cohort7
P-Values for Different Phenotypes
Gene
SNP
pre-FEV1% predicted
post-FEV1% predicted
pre-
FEV1/FVC
post-
FEV1/FVC
IREB2
rs2568494
intron
0.03*
0.04*
0.07
0.03*
rs2656069
intron
0.97
0.87
0.64
0.65
rs1964678
intron
0.99
0.96
0.85
0.78
rs12593229
intron
0.95
0.93
0.87
0.76
rs10851906
intron
0.70
0.61
0.84
0.80
rs965604
intron
0.84
0.90
0.75
0.70
rs13180
exon
0.85
0.91
0.76
0.71
LOC123688
rs8034191
intron
0.04*
0.04*
0.12
0.04*
CHRNA3
rs1051730
exon
0.02*
0.02*
0.07
0.02*
Each model was analyzed assuming an additive mode of inheritance adjusting for pack-years and pack-years2
* p ≤ 0.05
Replication analysis
In the initial analyses in the whole UK dataset, no significant associations with quantitative phenotypes including FEV1 and FEV1/FVC and qualitative presence of emphysema and COPD were observed (all p > 0.05). Gender interaction was apparent for rs2568494 with both COPD and emphysema (main effect p = 0.10, interaction p = 0.04 and 0.06, 0.03 respectively). No other statistically significant gender interactions were observed. In the sex-stratified models, evidence of association for SNPs in IREB2 and LOC123688 was observed. A trend was observed for association of rs8034191 and rs2568494 with COPD in the male subgroup, the risk alleles being C and A respectively (both p = 0.09). The SNP rs2568494 in IREB2 was significantly associated with emphysema in the male subgroup, the A allele conferring an odds ratio and 95% confidence interval (OR and 95% CI) of disease of 2.67 (1.10-6.51, p = 0.03). No association was seen with rs1051730 with emphysema.
With addition of a SNP-by-smoking interaction term, both rs8034191 and rs2568494 were associated with COPD in the male subgroup (main effect, p = 0.03; interaction effect, p = 0.02; and main effect, p = 0.04; interaction effect, p = 0.003, respectively). Similar associations with emphysema were seen for rs2568494 (p = 0.03 and 0.02 respectively). Positive findings in the two datasets are summarized in Table T4 4.
Table 4Positive findings of genetic association analysis in the AAT Genetic Modifiers Study and the UK AATD National Registry3
AAT Genetic Modifiers Study
UK AATD National Registry
All subjects
Association with pre- and post- FEV1% predicted and pre- and post- FEV1/FVC
none
Interaction with gender
yes
yes
Interaction with smoking
yes
only in the male subgroup
Male subgroup
association with FEV1 and FEV1/FVC
association with emphysema
association with COPD after adding smoking interaction term
Discussion
SNPs in the chromosome 15 CHRNA3/CHRNA5/LOC123688/IREB2 region have been shown to have associations with lung cancer and COPD unrelated to AAT deficiency. In our current analysis, SNPs in IREB2, LOC123688 and CHRNA3 genes were shown to be associated with lung function phenotypes in AAT deficient subjects (all PI ZZ) from the AAT Genetic Modifiers Study, and suggested a potential sex-specific effect. Replication in another cohort of AAT deficient subjects from the UK showed that a SNP in IREB2 was also associated with emphysema in men. This suggests that chromosome 15q region genes that were found by GWA studies and gene expression analysis of lung tissue samples may also be modifier genes of COPD and emphysema in AAT deficient subjects.
CHRNA3 was associated with lung cancer in three separate large GWA studies. This gene was associated with COPD by GWA and the association was replicated in two other COPD cohorts. There have also been recent reports of an association with smoking addiction
B27 27
, so it is unclear whether the lung cancer and COPD associations relate to smoking behavior, another aspect of lung biology, or both. CHRNA3 is a subunit gene of the nicotinic cholinergic receptor and expressed in autonomic ganglia and brain but is also expressed in bronchial and non-bronchial epithelial cells
B28 28
. Expression in lung cancer cells and signal transduction and apoptosis studies suggests a potential role in carcinogenesis
B29 29
. Interestingly, there are not many observations of lung cancer in patients with AAT deficiency, perhaps because of mortality associated with the development of severe COPD at an early age. Whether there is a common mechanism unrelated to smoking in the pathogenesis of lung cancer and COPD, or whether these previously reported associations relate to smoking addiction is unclear.
IREB2 is a protein of iron-responsive elements (IREs) and is regulated in response to iron and oxygen supply
B30 30
. IREB2-/- mice have aberrant iron homeostasis and accumulate iron in the intestine and the central nervous system(CNS); the CNS accumulation may lead to neurodegenerative disease
B31 31
. Excess iron can be toxic, but the mechanism of neurodegenerative disease is unclear; work is in progress to further characterize the functional pathways impacted by IREB2 in the lung. IREB2 was found to be differentially expressed according to lung function by microarray experiments, and the SNPs in IREB2 showed associations in both a COPD case-control study and family-based studies including the Boston Early-onset COPD and International COPD Genetics Network studies
18
. In a recent report, IREB2 polymorphisms were associated with COPD susceptibility in a European population
B32 32
. Interestingly, rs2568494 was significantly associated with COPD in three studies including our current study.
Previous studies of AAT deficient subjects showed that lung function was lower in men than women
B33 33
, and previous analyses in the AAT Genetic Modifiers Study also showed lower lung function in men
19
. Our current study suggests that genetic modifier effects of IREB2 and CHRNA3 may be more prominent in males--potentially contributing to some of the sex-specific features of COPD susceptibility and severity among PI ZZ subjects, although a larger sample size is needed to verify a gene-by-sex interaction.
In this study, there was no association between IREB2 and CHRNA3 genes and smoking intensity. In the AAT Genetic Modifiers Study, results showed no association when the cohort was stratified by smoking history (ever smokers versus never smokers). However, there was a marginal interaction of rs1051730 with smoking. In the UK study, there were significant smoking interactions of rs2568494 and rs8034191. Smoking markedly increases the risk of COPD and lowers the age-of-onset of COPD in AAT deficient subjects
6
19
, and despite small sample sizes, we found reasonable evidence for gene-by-smoking interactions in the chromosome 15q region.
There are several limitations in this study. Multiple statistical comparisons are a potential concern in any complex disease genetics study. Adjusting for either 3 genes or 9 SNPs tested, a p value of 0.02 is marginal. Additionally, the association with pulmonary function did not replicate in the UK population, potentially due to phenotypic heterogeneity between the two cohorts. Specifically, the UK subjects have lower mean FEV1 and potentially more emphysema, both of which could influence non-replication. Of note, the association with emphysema was investigated only in the UK population as chest CT scan data collection was not part of the AAT Genetic Modifiers Study. Considering that these SNPs (rs2568494 in IREB2, rs8034191 in LOC123688, and rs1051730 in CHRNA3) were associated with intermediate phenotypes of COPD in other populations and that we include an independent AAT deficient replication cohort, our result are likely meaningful. Additionally, this test- replication approach is even more appealing since all subjects were homozygous recessive for the AAT risk locus (PI ZZ). Also, replication of our results showed association with emphysema, a less heterogeneous pulmonary phenotype. The associated SNPs included two intronic (rs2568494, rs8034191) and one synonymous exonic (rs1051730) SNP. The exonic SNP was not associated with COPD-related phenotypes in the UK cohort. Another limitation of our current study is that rare functional variants in this chromosome 15 region may be contributing to the role of these genes in COPD; genome sequencing efforts in AAT deficient cohorts would be valuable to study rare variant associations. Functional data for associated variants are currently lacking, but many groups are pursuing functional work on this chromosome 15 region.
Conclusion
We have identified that the chromosome 15q25 region likely contains at least one potential modifier gene of COPD phenotypes in individuals with severe AAT deficiency. The association may be due to smoking behavior, but this is less likely; additionally, these associations may have sex-specific effects. Future directions will include further evaluation of the gene-by-sex interaction in larger cohort with AAT deficiency and identification of the functional variant or variants in this region.
Abbreviations
AAT: Alpha-1 antitrypsin; CHRNA3: Cholinergic nicotine receptor alpha3; GWA: Genome-wide association; IREB2: Iron regulatory binding protein 2; MAF: Minor allele frequency
Competing interests
WJK, AMW, AFB, MLB, EJC, EE, GM, RAS and GT have reported that no potential conflicts of interest. SIR was supported from GlaxoSmithKline for travel to meetings for the study. JMS received grant support from Talecris, Baxter. JKS received grant support from AstraZeneca, and honoraria from Talecris, Baxter, CSL Behring, Boehringer Ingelheim, Kamada, Grifols, and has received fees for participation in review activities from Shire and AsthmaTx. CS received consulting fees from AstraZeneca, Talecris, Baxter, Forest, Phamaceuticals, Uptake Medical, Pulmonx and payment for lectures from Talecris and AstraZeneca. EKS received grant support and consulting fees from GlaxoSmithKline for studies of COPD genetics and honoraria and consulting fees from AstraZeneca. RAS received grant support, honoraria and consulting fees and supported for travel to meetings for the study from Talecris. DLD received grant support from Doris Duke Charitable Foundation.
Authors' contributions
All authors contributed to the study design, data collection and analysis, and writing of the manuscript. WJK, AMW, DLD contributed to data analysis. All authors read and approved the final manuscript.
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ack
Acknowledgements
This work was supported by US National Institutes of Health [Grant R01 HL68926, R01 HL075478, R01 HL084323, P01 HL083069 (EKS) and HL089438]. The UK AATD Registry was supported by an unrestricted grant by Talecris as part of the ADAPT program. Additionally, DLD is supported by a Clinician Scientist Development Award from the Doris Duke Foundation.
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RESEARCH OpenAccessAssociationof IREB2 and CHRNA3 polymorphisms withairflowobstructioninseverealpha-1 antitrypsindeficiencyWooJinKim1,AliceMWood2,AlanFBarker3,MarkLBrantly4,EdwardJCampbell5,EdwardEden6, GerardMcElvaney7,StephenIRennard8,RobertASandhaus9,JamesMStocks10,JamesKStoller11, CharlieStrange12,GerardTurino13,EdwinKSilverman14,RobertAStockley15andDawnLDeMeo14*AbstractBackground: ThedevelopmentofCOPDinsubjectswithalpha-1antitrypsin(AAT)deficiencyislikelytobe influencedbymodifiergenes.Genome-wideassociationstudiesandintegrativegenomicsapproachesinCOPD havedemonstratedsignificantassociationswithSNPsinthechromosome15qregionthatincludes CHRNA3 (cholinergicnicotinereceptoralpha3)and IREB2 (ironregulatorybindingprotein2). WeinvestigatedwhetherSNPsinthechromosome15qregionwouldbemodifiersforlungfunctionandCOPDin AATdeficiency. Methods: Thecurrentanalysisincluded378PIZZsubjectsintheAATGeneticModifiersStudyandareplication cohortof458subjectsfromtheUKAATDeficiencyNationalRegistry.NineSNPsin LOC123688,CHRNA3 and IREB2 wereselectedforgenotyping.FEV1percentofpredictedandFEV1/FVCratiowereanalyzedasquantitative phenotypes.Family-basedassociationanalysiswasperformedintheAATGeneticModifiersStudy.Inthereplication set,generallinearmodelswereusedforquantitativephenotypesandlogisticregressionmodelswereusedforthe presence/absenceofemphysemaorCOPD. Results: ThreeSNPs(rs2568494in IREB2 ,rs8034191in LOC123688 ,andrs1051730in CHRNA3)wereassociatedwith pre-bronchodilatorFEV1percentofpredictedintheAATGeneticModifiersStudy.TwoSNPs(rs2568494and rs1051730)wereassociatedwiththepost-bronchodilatorFEV1percentofpredictedandpre-bronchodilatorFEV1/FVC ratio;SNP-by-genderinteractionswereobserved.IntheUKNationalRegistrydataset,rs2568494wassignificantly associatedwithemphysemainthemalesubgroup;significantSNP-by-smokinginteractionswereobserved. Conclusions: IREB2 and CHRNA3 arepotentialgeneticmodifiersofCOPDphenotypesinindividualswithsevere AATdeficiencyandmaybesex-specificintheirimpact. Keywords: CHRNA3 ,Chronicobstructivepulmonarydisease,Geneticassociationanalysis,Geneticmodifiers, IREB2BackgroundChronicobstructivepulmonarydisease(COPD)isa complexdiseasecharacterizedbypersistentairflowlimitation.COPDrisklikelyresultsfromthecumulative effectofenvironmentalfactors(especiallycigarette smoking),geneticfactors,andgene-by-environment interactions[1].Alpha-1antitrypsin(AAT)deficiency, typicallycausedbyhomozygosityfortheZalleleatthe AATgene( SERPINA1) ,isaprovengeneticcauseof COPD.However,thedevelopmentofCOPDand emphysemainsubjectswithAATdeficiencyishighly variableandislikelyinfluencedbymodifiergenesand environmentalfactors[2-4]. Spirometricmeasurementsofpulmonaryfunctionare widelyusedphenotypesinevaluatingAATdeficient subjectsandmonitoringlungfunctiondecline[5];CT scanassessmentsforemphysemahavebeenusedas *Correspondence:dawn.demeo@channing.harvard.edu14ChanningLaboratoryandtheDivisionofPulmonaryandCriticalCare Medicine,BrighamandWomen sHospital,andHarvardMedicalSchool, Boston,MA,USA FulllistofauthorinformationisavailableattheendofthearticleKim etal RespiratoryResearch 2012, 13 :16 http://respiratory-research.com/content/13/1/16 2012Kimetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

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additionalintermediatephenotypesofCOPDtoovercomesomeoftheheterogeneityinherentinspirometric classificationsalone.Familialaggregationstudiesofpulmonaryfunctionhavesuggestedadditionalmodifier genesinAATdeficiencysubjects[6,7].Afewpotential AATcandidatemodifiergenes,including NOS3 [8], GSTP1 [9], TNF [10],and IL10 [11],havebeenreported sofar,buttheseresultshavenotbeenconsistently replicated. Genome-wideassociation(GWA)studieshaverevolutionizedtheidentificationofsusceptibilitygenesforcomplexdiseases.ThreerecentGWAstudiesshowedthat SNPsinaregionofchromosome15q25weresignificantlyassociatedwithlungcancer;severalnicotinicacetylcholinereceptorgenes,including CHRNA3 and LOC123688,arelocatedinthisregion[12-14].Agenome-wideassociation(GWA)studyinCOPDalso showedsignificantassociationsbetweenCOPDsusceptibilityandSNPsinthisregion[15].Thisregionwasalso associatedwithairflowobstructionandemphysema [16,17].Interestingly,inaddi tiontonicotinicacetylcholinereceptorgenes,thisregionalsoincludes IREB2 (iron regulatorybindingprotein2). IREB2 wasidentifiedasa potentialCOPDsusceptibilitygeneusinganintegrative genomicsapproachwithgeneexpressionanalysisoflung tissuesamplesfollowedbygeneticassociationanalysis [18].WehypothesizedthatSNPsinthischromosome 15qregionmaybemodifiersofintermediatephenotypes ofCOPDinsubjectswithsevereAATdeficiency.MethodsStudysubjectsThecurrentanalysisincluded378subjectswithsevere AATdeficiency(proteaseinhibitor[PI]ZZ)from167 familiesintheAATGeneticModifiersStudy.Ascertainmentofeligiblesiblingpairswasbasedonhomozygosity fortheZalleleatthe SERPINA1 locusaspreviously described[19].Pre-andpost-bronchodilatorstudyspirometrytestingwasperformedaccordingtoAmerican ThoracicSociety(ATS)standardsasdescribedpreviously [19].PercentpredictedvaluesforFEV1werecalculated usingequationsofCrapoand colleaguesforCaucasian subjects[20].TheFEV1/FVCratiowasanalyzedusing unadjustedvalues.Pack-year sofcigarettesmokingwere calculatedbymultiplyingthenumberofyearssmokedby theaveragenumberofdailycigarettessmoked,divided by20.Allparticipantsprovidedwritteninformedconsent,andthestudyprotocol wasapprovedbyindividual InstitutionalReviewBoardsateachoftheparticipating clinicalcenters(PartnersIRB,2001P001237).458unrelatedCaucasiansubjectsfromtheUKAATDNational Registrywerealsogenotyped.Approvalforthestudywas givenbythelocalethicscommittee.Allpatientshada serumalpha-1antitrypsin(AAT)levelof<11 MandPI ZZgenotypeconfirmedbyallele-specificPCR(Heredilab, SaltLakeCity,Utah,USA).NoneoftheUKsubjectshad everreceivedAATaugmentationtherapy.Afullclinical assessmentincludingsmokeexposure,presenceof chronicbronchitis(definedasaproductivecoughforat least3monthsinatleast2consecutiveyears[21]),lung functiontestingandhighresolutionCTscanningofthe chestwasundertaken,asdescribedpreviously[22].The presenceofemphysemawas determinedbytheappearanceofthescananddensitymaskanalysisofslicesatthe leveloftheaorticarch(representingtheupperzone)and theinferiorpulmonaryvein(representingthelower zone)usingathresholdof-910HounsfieldUnits(HU). ThisHUthresholdhasbeenvalidatedagainstphysiologicalmeasuresinAATD[22].Patientswhosevoxelindex exceededvaluesseeninnorm alsubjectsineitherzone [23]wereclassifiedashavingemphysema.GenotypingTwoSNPs(rs8034191andrs1051730)inchromosome 15wereselectedfromthepreviousGWAinCOPD [15].Additionally,7SNPsin IREB2 wereselectedusing pairwiselinkagedisequilibrium(LD)-tagginginTagger withaminimumminorallelefrequencyof0.05andr2thresholdof0.8[24].SNPsweregenotypedusingSequenom(SanDiego,CA)assaysintheAATGeneticModifiersStudy.Allfamilydatawereevaluatedforfamilial inconsistenciesusingthePEDCHECKprogram[25]. InUKAATDNationalRegistrystudy,genotypingwas carriedoutusingTaqMantechnologies(AppliedBiosystems,UK)onanABI7900HTfor3SNPsassociated inthetestdataset(rs2568494, IREB2 ;rs8034191, LOC123688;rs1051730, CHRNA3 ).Allgenotyping assayswerepre-validatedbythesuppliers,andallplates includedappropriatenegativecontrols.StatisticalanalysisHardy-Weinbergequilibriumwasassessedusinggoodnessoffittests.Pre-andpost-bronchodilatorFEV1percentofpredictedandpre-andpost-bronchodilator FEV1/FVCratiowereanalyzedasquantitativephenotypes.Family-basedassociationanalysiswasperformed usingPBATsoftwareversion3.6[26]assumingadditive geneticmodels,adjustingforpack-yearsandpack-years2ofcigarettesmoking,underthenullhypothesisofno linkageandnoassociationintheAATGeneticModifiersStudy.Inadditiontotheoverallmodel,weevaluatedgender-stratifiedmodelsandmodelsthatincluded aSNP-by-smokingoraSNP-by-genderinteractionterm. Haplotypeanalysiswasperformedusing8,4,3,2SNP adjacentslidingwindowsinPBAT. IntheUKAATDNationalRegistrystudy,datawere analyzedusingSPSS(version16,Chicago:SPSSInc). QuantitativegeneticassociationanalysiswascarriedoutKim etal RespiratoryResearch 2012, 13 :16 http://respiratory-research.com/content/13/1/16 Page2of7

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forFEV1andFEV1/FVCusinggenerallinearmodels, adjustingforage,genderandsmokeexposure(aspackyearsandpack-years2).Logisticregressionmodelswere usedforthepresenceofemphysemaorCOPD(defined aspostbronchodilatorFEV1/FVC<0.7)accountingfor covariatesasbefore.Additivemodelswereassumedfor allSNPs.GenderstratificationandSNP-by-genderand SNP-by-smokinginteractionanalyseswerealsocarried out,asinthetestdataset.ResultsDemographiccharacteristicsThemeanageofsubjectswas52.2years,andmeanpostbronchodilatorFEV1was65.9%predictedintheAAT GeneticModifiersStudyand50.3yearsand53.8%predictedintheUKAATDNationalRegistry,respectively (Table1).Malesubjectswere46%inAATGeneticModifiersStudyand59%inUKAATDNationalRegistry, respectively.Threehundredandsixty-sixsubjects (79.8%)hademphysemainUKAATDNationalRegistry.AssociationanalysisTherewerenodeviationsfromHardyWeinbergequilibriumforanyofthegenotypedSNPs. IntheAATGeneticModifiersStudy,threeSNPs (rs2568494in IREB2 ,rs8034191in LOC123688 ,and rs1051730in CHRNA3 )wereassociatedwithpre-bronchodilatorFEV1percentofpredicted( p <0.05Table2Figure1).TwoSNPs(rs2568494andrs1051730)were associatedwithpost-bronchodilatorFEV1percentofpredictedandpre-bronchodilatorFEV1/FVCratio.OneSNP (rs1051730)wasassociatedwithpost-bronchodilator FEV1/FVCratio(Table2).Linkagedisequilibrium (assessedwithr2)betweenrs8034191andthe1051730 was0.9(Figure2).Therewassignificantassociationonly witha2SNPhaplotypeincludingrs8034191and rs1051730withpre-bronchodilatorFEV1/FVCratio(globalteststatistic; p =0.05)usingPBAT.InteractionswithcigarettesmokingTherewasnoassociationbetweenanyofthegenotyped SNPsandpack-yearsofsmokingastheoutcomeinthe AATGeneticModifiersStudy.InclusionofaSNP-bypack-yearsinteractiontermforthelungfunctionphenotypesshowedsignificantin teractionofrs1051730with pack-yearsofsmokingforthepre-bronchodilator FEV1/FVCratio(maineffect; p =0.02,interactioneffect; p =0.04).Therewasnosignificantassociationwithlung functionphenotypeswhenthestudypopulationwas stratifiedintogroupsofever-smokers( n =233)and never-smokers( n =145)althoughthisstratifiedanalysis reducedthenumberofinformativefamilies considerably.Genotype-by-genderinteractionsandgender stratificationSNP-by-genderinteractionanalysisshowedsignificant interactionofrs2568494,rs8034191,andrs1051730with genderinmodelsforthepost-bronchodilatorFEV1/FVC ratio(maineffect; p =0.04,0.04,0.02,interactioneffect; p =0.02,0.008,0.004,respectively).Additionally, rs1051730showedsignificantinteractioninmodelsfor preandpost-bronchodilatorFEV1andpre-bronchodilatorFEV1/FVCratio(maineffect; p =0.04,0.04,0.08, interactioneffect; p =0.04,0.02,0.04,respectively). Inthestratifiedanalysis,forthemalesubgroup,the p valuesweresimilartothewholecohortresults,with rs8034191showingsignificantassociationwithpre-and post-bronchodilatorFEV1percentofpredictedand post-bronchodilatorFEV1/FVCratio(Table3).However, inthefemalesubgroup,therewasnosignificantassociationwithlungfunctionphenotypes.ReplicationanalysisIntheinitialanalysesinthewholeUKdataset,nosignificantassociationswithquantitativephenotypesincluding FEV1andFEV1/FVCandqualitativepresenceofemphysemaandCOPDwereobserved(all p >0.05).Gender interactionwasapparentforrs2568494withboth COPDandemphysema(maineffect p =0.10,interaction p =0.04and0.06,0.03respectively).Nootherstatisticallysignificantgenderinteractionswereobserved. Inthesex-stratifiedmodels,evidenceofassociationfor SNPsin IREB2 and LOC123688 wasobserved.Atrend Table1BaselinecharacteristicsforPIZZindividualsintheAATGeneticModifiersStudyandtheUKAATDNational RegistryCharacteristics AATGeneticModifiersStudy ( n =378) UKnationalregistryforAATD ( n =458) p value Age,years 52.29.7 50.310.4 0.01 Malesex(%) 173(46%) 252(55%) <0.0001 FEV1%predicted 65.933.5 53.832.2 0.0001 FEV1/FVC 0.5510.207 0.4450.194 <0.0001 Pack-yearsforeversmokers 18.214.5 15.914.7 <0.0001Dataarepresentedasmeans(S.D.)intheAATGeneticModifiersStudyandtheUKnationalregistryforAATD,unlessotherwisenotedKim etal RespiratoryResearch 2012, 13 :16 http://respiratory-research.com/content/13/1/16 Page3of7

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wasobservedforassociationofrs8034191and rs2568494withCOPDinthemalesubgroup,therisk allelesbeingCandArespectively(both p =0.09).The SNPrs2568494in IREB2 wassignificantlyassociated withemphysemainthemalesubgroup,theAalleleconferringanoddsratioand95%confidenceinterval(OR and95%CI)ofdiseaseof2.67(1.10-6.51, p =0.03).No associationwasseenwithrs1051730withemphysema. WithadditionofaSNP-bysmokinginteractionterm, bothrs8034191andrs2568494wereassociatedwith COPDinthemalesubgroup(maineffect, p =0.03; interactioneffect, p =0.02;andmaineffect, p =0.04; interactioneffect, p =0.003,respectively).Similarassociationswithemphysemawereseenforrs2568494( p = 0.03and0.02respectively).P ositivefindingsinthetwo datasetsaresummarizedinTable4.DiscussionSNPsinthechromosome15 CHRNA3/CHRNA5/ LOC123688/IREB2 regionhavebeenshowntohave associationswithlungcancerandCOPDunrelatedto AATdeficiency.Inourcurrentanalysis,SNPsin IREB2, LOC123688 and CHRNA3 geneswereshowntobeassociatedwithlungfunctionphenotypesinAATdeficient subjects(allPIZZ)fromtheAATGeneticModifiers Study,andsuggestedapotentialsex-specificeffect. ReplicationinanothercohortofAATdeficientsubjects fromtheUKshowedthataSNPin IREB2 wasalso associatedwithemphysemainmen.Thissuggeststhat chromosome15qregiongenesthatwerefoundby GWAstudiesandgeneexpressionanalysisoflungtissue samplesmayalsobemodifiergenesofCOPDand emphysemainAATdeficientsubjects. Table2GeneticassociationresultsbetweenSNPsinchromosome15andlungfunctionintheAATGeneticModifiers StudyP -ValuesforDifferentPhenotypes GeneSNPMAFpre-FEV1% predicted post-FEV1% predicted preFEV1/FVC postFEV1/FVC IREB2 rs2568494intron0.300.02*0.03*0.05*0.06 rs2656069intron0.220.48 0.29 0.59 0.46 rs1964678intron0.430.75 0.91 0.58 0.83 rs12593229intron0.430.82 0.79 0.64 0.93 rs10851906intron0.230.29 0.17 0.41 0.31 rs965604intron0.430.76 0.99 0.54 0.65 rs13180 exon0.430.69 0.96 0.53 0.79 LOC123688 rs8034191intron0.290.04* 0.07 0.09 0.14 CHRNA3 rs1051730exon0.310.02* 0.03* 0.03* 0.05*MAF=minorallelefrequency Eachmodelwasanalyzedassuminganadditivemodeofinheritanceadjustingforpack-yearsandpack-years2* p 0.05 50 55 60 65 70 75 80 85 90 95 100 GG (170)GA (155)AA (33)FEV1 (%)Genotype (number of subjects) Figure1 FEV1by CHRNA3 genotype(rs1051730)intheAAT GeneticModifiersStudycohort .Meanvalues(+SEM)fora percentofpredictedFEV1areshown( p value=0.02). Figure2 Linkagedisequilibrium(LD)amongSNPsanalyzedin chromosome15.LDvaluesarepresentedasr2. Kim etal RespiratoryResearch 2012, 13 :16 http://respiratory-research.com/content/13/1/16 Page4of7

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CHRNA3 wasassociatedwithlungcancerinthree separatelargeGWAstudie s.Thisgenewasassociated withCOPDbyGWAandtheassociationwasreplicated intwootherCOPDcohorts.Therehavealsobeen recentreportsofanassociationwithsmokingaddiction [27],soitisunclearwhetherthelungcancerandCOPD associationsrelatetosmokingbehavior,anotheraspect oflungbiology,orboth. CHRNA3 isasubunitgeneof thenicotiniccholinergicreceptorandexpressedinautonomicgangliaandbrainbutisalsoexpressedinbronchialandnon-bronchialepithelialcells[28].Expression inlungcancercellsandsignaltransductionandapoptosisstudiessuggestsapotent ialroleincarcinogenesis [29].Interestingly,therearenotmanyobservationsof lungcancerinpatientswithAATdeficiency,perhaps becauseofmortalityassociatedwiththedevelopmentof severeCOPDatanearlyage.WhetherthereisacommonmechanismunrelatedtosmokinginthepathogenesisoflungcancerandCOPD,orwhetherthese previouslyreportedassociationsrelatetosmokingaddictionisunclear. IREB2isaproteinofiron-re sponsiveelements(IREs) andisregulatedinresponsetoironandoxygensupply [30].IREB2-/-micehaveaberrantironhomeostasisand accumulateironintheintestineandthecentralnervous system(CNS);theCNSaccumulationmayleadtoneurodegenerativedisease[31].Excessironcanbetoxic,but themechanismofneurodegenerativediseaseisunclear; workisinprogresstofurthercharacterizethefunctional pathwaysimpactedby IREB2 inthelung. IREB2 was foundtobedifferentiallye xpressedaccordingtolung functionbymicroarrayexperiments,andtheSNPsin IREB2 showedassociationsinbothaCOPDcase-control studyandfamily-basedstudiesincludingtheBoston Early-onsetCOPDandInternationalCOPDGenetics Networkstudies[18].Inarecentreport, IREB2 polymorphismswereassociatedwithCOPDsusceptibilityin aEuropeanpopulation[32].Interestingly,rs2568494 wassignificantlyassociatedwithCOPDinthreestudies includingourcurrentstudy. PreviousstudiesofAATdeficientsubjectsshowed thatlungfunctionwaslowerinmenthanwomen[33], andpreviousanalysesintheAATGeneticModifiers Studyalsoshowedlowerlungfunctioninmen[19].Our currentstudysuggeststhat geneticmodifiereffectsof IREB2 and CHRNA3 maybemoreprominentinmales potentiallycontributingtosomeofthesex-specificfeaturesofCOPDsusceptibilityandseverityamongPIZZ subjects,althoughalargersamplesizeisneededtoverifyagene-by-sexinteraction. Inthisstudy,therewasnoassociationbetween IREB2 and CHRNA3 genesandsmokingintensity.IntheAAT GeneticModifiersStudy,resultsshowednoassociation whenthecohortwasstratifiedbysmokinghistory(ever smokersversusneversmokers).However,therewasa marginalinteractionofrs1051730withsmoking.Inthe Table3GeneticassociationresultsbetweenSNPsinchromosome15andlungfunctionofmalesubgroupintheAAT GeneticModifiersStudycohortP -ValuesforDifferentPhenotypes GeneSNPpre-FEV1%predictedpost-FEV1%predictedpreFEV1/FVC postFEV1/FVC IREB2 rs2568494intron0.03* 0.04* 0.07 0.03* rs2656069intron0.97 0.87 0.64 0.65 rs1964678intron0.99 0.96 0.85 0.78 rs12593229intron0.95 0.93 0.87 0.76 rs10851906intron0.70 0.61 0.84 0.80 rs965604intron0.84 0.90 0.75 0.70 rs13180exon0.85 0.91 0.76 0.71 LOC123688 rs8034191intron0.04* 0.04* 0.12 0.04* CHRNA3 rs1051730exon0.02* 0.02* 0.07 0.02*Eachmodelwasanalyzedassuminganadditivemodeofinheritanceadjustingforpack-yearsandpack-years2* p 0.05 Table4Positivefindingsofgeneticassociationanalysis intheAATGeneticModifiersStudyandtheUKAATD NationalRegistryAATGeneticModifiers Study UKAATDNational Registry AllsubjectsAssociationwithpre-and post-FEV1%predictedand pre-andpost-FEV1/FVC none Interaction with gender yesyes Interaction with smoking yesonlyinthemalesubgroup Male subgroup associationwithFEV1and FEV1/FVC associationwith emphysema associationwithCOPD afteraddingsmoking interactionterm Kim etal RespiratoryResearch 2012, 13 :16 http://respiratory-research.com/content/13/1/16 Page5of7

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UKstudy,thereweresignificantsmokinginteractionsof rs2568494andrs8034191.Smokingmarkedlyincreases theriskofCOPDandlowerstheage-of-onsetofCOPD inAATdeficientsubjects[6,19],anddespitesmallsamplesizes,wefoundreasonableevidenceforgene-bysmokinginteractionsinthechromosome15qregion. Thereareseverallimitationsinthisstudy.Multiple statisticalcomparisonsareapotentialconcerninany complexdiseasegeneticsstudy.Adjustingforeither3 genesor9SNPstested,a p valueof0.02ismarginal. Additionally,theassociat ionwithpulmonaryfunction didnotreplicateintheUKpopulation,potentiallydue tophenotypicheterogeneitybetweenthetwocohorts. Specifically,theUKsubjectshavelowermeanFEV1and potentiallymoreemphysema,bothofwhichcouldinfluencenon-replication.Ofnote,theassociationwith emphysemawasinvestigatedonlyintheUKpopulation aschestCTscandatacollectionwasnotpartofthe AATGeneticModifiersStudy.Consideringthatthese SNPs(rs2568494in IREB2 ,rs8034191in LOC123688 andrs1051730in CHRNA3 )wereassociatedwithintermediatephenotypesofCOPDinotherpopulationsand thatweincludeanindependentAATdeficientreplicationcohort,ourresultarelikelymeaningful.Additionally,thistest-replicationapproachisevenmore appealingsinceallsubjectswerehomozygousrecessive fortheAATrisklocus(PIZZ).Also,replicationofour resultsshowedassociationwithemphysema,alessheterogeneouspulmonaryphenotype.TheassociatedSNPs includedtwointronic(rs2568494,rs8034191)andone synonymousexonic(rs1051730)SNP.TheexonicSNP wasnotassociatedwithCOPD-relatedphenotypesin theUKcohort.Anotherlimitationofourcurrentstudy isthatrarefunctionalvariantsinthischromosome15 regionmaybecontributingtotheroleofthesegenesin COPD;genomesequencinge ffortsinAATdeficient cohortswouldbevaluabletostudyrarevariantassociations.Functionaldataforassociatedvariantsarecurrentlylacking,butmanygroupsarepursuingfunctional workonthischromosome15region.ConclusionWehaveidentifiedthatthechromosome15q25region likelycontainsatleastonepotentialmodifiergeneof COPDphenotypesinindividualswithsevereAATdeficiency.Theassociationmaybeduetosmokingbehavior,butthisislesslikely;additionally,these associationsmayhavesex-sp ecificeffects.Futuredirectionswillincludefurtherevaluationofthegene-by-sex interactioninlargercohortwithAATdeficiencyand identificationofthefunctionalvariantorvariantsinthis region.Abbreviations AAT:Alpha-1antitrypsin;CHRNA3:Cholinergicnicotinereceptoralpha3; GWA:Genome-wideassociation;IREB2:Ironregulatorybindingprotein2; MAF:Minorallelefrequency Acknowledgements ThisworkwassupportedbyUSNationalInstitutesofHealth[GrantR01 HL68926,R01HL075478,R01HL084323,P01HL083069(EKS)andHL089438]. TheUKAATDRegistrywassupportedbyanunrestrictedgrantbyTalecrisas partoftheADAPTprogram.Additionally,DLDissupportedbyaClinician ScientistDevelopmentAwardfromtheDorisDukeFoundation. Authordetails1DepartmentofInternalMedicine,KangwonNationalUniversity,Chuncheon, SouthKorea.2SchoolofClinicalandExperimentalMedicine,Universityof Birmingham,Birmingham,UK.3OregonHealthandScienceUniversity, Portland,OR,USA.4UniversityofFlorida,Gainesville,FL,USA.5Intermountain HealthCare,Provo,andHeredilab,Inc,SaltLakeCity,UT,USA.6St.Luke s/ RooseveltHospital,NewYork,NY,USA.7BeaumontHospital,Dublin,Ireland.8UniversityofNebraska,Omaha,NE,USA.9NationalJewishHealth,Denver, CO,USA.10UniversityofTexasatTyler,Tyler,TX,USA.11ClevelandClinic, Cleveland,OH,USA.12MedicalUniversityofSouthCarolina,Charleston,SC, USA.13St.Luke s/RooseveltHospital,NewYork,NY,USA.14Channing LaboratoryandtheDivisionofPulmonaryandCriticalCareMedicine, BrighamandWomen sHospital,andHarvardMedicalSchool,Boston,MA, USA.15LungInvestigationUnit,UniversityHospitalsBirmingham, Birmingham,UK. Authors contributions Allauthorscontributedtothestudydesign,datacollectionandanalysis,and writingofthemanuscript.WJK,AMW,DLDcontributedtodataanalysis.All authorsreadandapprovedthefinalmanuscript. Competinginterests WJK,AMW,AFB,MLB,EJC,EE,GM,RASandGThavereportedthatno potentialconflictsofinterest.SIRwassupportedfromGlaxoSmithKlinefor traveltomeetingsforthestudy.JMSreceivedgrantsupportfromTalecris, Baxter.JKSreceivedgrantsupportfromAstraZeneca,andhonorariafrom Talecris,Baxter,CSLBehring,BoehringerIngelheim,Kamada,Grifols,andhas receivedfeesforparticipationinreviewactivitiesfromShireandAsthmaTx. CSreceivedconsultingfeesfromAstraZeneca,Talecris,Baxter,Forest, Phamaceuticals,UptakeMedical,Pulmonxandpaymentforlecturesfrom TalecrisandAstraZeneca.EKSreceivedgrantsupportandconsultingfees fromGlaxoSmithKlineforstudiesofCOPDgeneticsandhonorariaand consultingfeesfromAstraZeneca.RASreceivedgrantsupport,honorariaand consultingfeesandsupportedfortraveltomeetingsforthestudyfrom Talecris.DLDreceivedgrantsupportfromDorisDukeCharitableFoundation. Received:11December2011Accepted:22February2012 Published:22February2012 References1.RabeKF,HurdS,AnzuetoA,BarnesPJ,BuistSA,CalverleyP,FukuchiY, JenkinsC,Rodriguez-RoisinR,vanWeelC,ZielinskiJ: GlobalInitiativefor ChronicObstructiveLungDisease.Globalstrategyforthediagnosis, management,andpreventionofchronicobstructivepulmonarydisease: Goldexecutivesummary. AmJRespirCritCareMed 2007, 176 :532-555. 2.DeMeoDL,SilvermanEK: Alpha1-antitrypsindeficiency.2:Genetic aspectsofalpha-1-antitrypsindeficiency:Phenotypesandgenetic modifiersofemphysemarisk. 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RespiratoryResearch 2012 13 :16. Submit your next manuscript to BioMed Central and take full advantage of: Convenient online submission Thorough peer review No space constraints or color gure charges Immediate publication on acceptance Inclusion in PubMed, CAS, Scopus and Google Scholar Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Kim etal RespiratoryResearch 2012, 13 :16 http://respiratory-research.com/content/13/1/16 Page7of7