- Research article
- Open Access
- Open Peer Review
Nitric oxide synthase polymorphisms, gene expression and lung function in chronic obstructive pulmonary disease
© Aminuddin et al.; licensee BioMed Central Ltd. 2013
- Received: 31 July 2013
- Accepted: 31 October 2013
- Published: 6 November 2013
Due to the pleiotropic effects of nitric oxide (NO) within the lungs, it is likely that NO is a significant factor in the pathogenesis of chronic obstructive pulmonary disease (COPD). The aim of this study was to test for association between single nucleotide polymorphisms (SNPs) in three NO synthase (NOS) genes and lung function, as well as to examine gene expression and protein levels in relation to the genetic variation.
One SNP in each NOS gene (neuronal NOS (NOS1), inducible NOS (NOS2), and endothelial NOS (NOS3)) was genotyped in the Lung Health Study (LHS) and correlated with lung function. One SNP (rs1800779) was also analyzed for association with COPD and lung function in four COPD case–control populations. Lung tissue expression of NOS3 mRNA and protein was tested in individuals of known genotype for rs1800779. Immunohistochemistry of lung tissue was used to localize NOS3 expression.
For the NOS3 rs1800779 SNP, the baseline forced expiratory volume in one second in the LHS was significantly higher in the combined AG + GG genotypic groups compared with the AA genotypic group. Gene expression and protein levels in lung tissue were significantly lower in subjects with the AG + GG genotypes than in AA subjects. NOS3 protein was expressed in the airway epithelium and subjects with the AA genotype demonstrated higher NOS3 expression compared with AG and GG individuals. However, we were not able to replicate the associations with COPD or lung function in the other COPD study groups.
Variants in the NOS genes were not associated with lung function or COPD status. However, the G allele of rs1800779 resulted in a decrease of NOS3 gene expression and protein levels and this has implications for the numerous disease states that have been associated with this polymorphism.
- Chronic obstructive pulmonary disease
- Nitric oxide synthase
- Gene expression
Nitric oxide (NO) is a molecule that is involved in many physiological and pathological pathways and can have either beneficial or detrimental effects. Many studies have shown that NO has protective effects on human airways such as muscle relaxation, attenuation of airway hyper-responsiveness to bronchoconstrictor stimuli, and the killing of invading microorganisms . In contrast, adverse effects of NO have also been observed, such as vasodilation of the bronchial circulation, increased airway secretions and the promotion of pro-inflammatory pathways, as well as necrosis and apoptosis .
Endogenous NO is primarily synthesized by enzymes known as NO synthases (NOS) which catalyze the cellular production of NO from arginine. There are three known NOS isoforms: neuronal NOS (NOS1), inducible NOS (NOS2), and endothelial NOS (NOS3). In humans, NOS1 can be found in neurons and endothelial cells in the lung , while NOS3 is found in bronchiolar epithelial cells and the endothelium [3, 4]. NOS2 is expressed in the human airway epithelium , lung endothelium , and alveolar macrophages .
Due to its pleiotropic effects, it is likely that NO is a significant factor in the pathogenesis of lung diseases such as chronic obstructive pulmonary disease (COPD), which is characterized by airflow limitation that is not fully reversible. There is evidence to suggest that NOS genes are associated with COPD. Recently, the mRNA and protein expression of NOS1 and NOS2 were observed to be increased in the peripheral lung tissue of smokers with COPD compared with nonsmoker controls, whereas the opposite effect was detected for NOS3 expression . Another study reported that the numbers of NOS2+ and NOS3+ cells were increased in the bronchial submucosa of smokers with COPD compared with nonsmoker controls . Furthermore, deficiency of NOS2 has been shown to be protective against cigarette smoke-induced emphysema in a mouse model .
To further determine the effects of NOS in COPD, it is important to determine whether single nucleotide polymorphisms (SNPs) in NOS genes are associated with phenotypes related to the disease. It has been widely acknowledged that genetic factors account for some of the variability of lung function among smokers [9, 10], suggesting an interaction between genetic and environmental influences on disease progression. The aim of this study was to determine whether NOS gene variants were associated with phenotypes related to COPD. We examined the rate of decline of lung function and baseline lung function in smokers with mild to moderate airflow obstruction from the Lung Health Study (LHS) in relation to polymorphisms in three NOS genes. The LHS was a randomized trial of an anti-smoking intervention and bronchodilator treatment in volunteer smokers . We selected polymorphisms in NOS genes that had previously been associated with gene function or COPD-related traits [12–14]. We sought to determine whether these polymorphisms were associated with lung function decline and baseline level in COPD patients in the LHS as well as with COPD and lung function in four replication case–control sets.
The investigation of the LHS and lung tissue samples was approved by the University of British Columbia/Providence Health Care Research Ethics Board and all subjects provided written informed consent. We attempted to replicate the associations in subjects from the following previously recruited populations: Norway COPD Cohort (GenKOLS) , National Emphysema Treatment Trial (NETT) [16, 17], Normative Aging Study (NAS) , Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE)  and COPDGene . These studies were approved by the relevant institutional review boards and all subjects provided written informed consent. For the NAS, anonymized data were used, as approved by the institutional review boards of Partners Healthcare System and the Boston VA.
Characteristics of the 4132 Lung Health Study participants (2611 male, 1521 female)
Age (mean ± SD), years
48.5 ± 6.7
Smoking history (mean ± SD), pack-yearsa
40.4 ± 18.4
Baseline FEV1 post-bronchodilator (mean ± SD), % predictedb
78.55 ± 9.10
FEV1 post-bronchodilator rate of decline (mean ± SD), % predicted / yearc
-0.97 ± 1.78
Characteristics of the case–control replication study groups
Number of participants
Age (mean ± SD), years
64.77 ± 8.12
60.20 ± 8.66
63.63 ± 7.10
57.48 ± 9.44
67.47 ± 5.78
69.8 ± 7.49
65.53 ± 10.03
55.62 ± 9.71
Smoking history (mean ± SD), pack-yearsa
54.76 ± 26.69
38.87 ± 21.07
50.29 ± 27.42
32.11 ± 24.84
66.43 ± 30.68
40.66 ± 27.85
31.98 ± 18.46
19.66 ± 13.58
FEV1 (mean ± SD), % predictedb
48.73 ± 18.41
97.98 ± 11.32
47.63 ± 15.62
107.83 ± 13.56
28.12 ± 7.38
99.97 ± 13.20
50.63 ± 17.44
94.91 ± 9.24
Sex (% male)
Gene variants and genotyping
Description of the polymorphisms studied
C = > T
Promoter for exon 1c
Reduced gene expression 
G = > A
Decreased DNA-protein complex 
A = > G
Lower FEV1 % predicted in COPD patients 
Quantitative polymerase chain reaction (PCR)
RNA was extracted from lung tissue samples using the RNeasy Mini Kit (Qiagen) and cDNA was synthesized using SuperScript®III Reverse Transcriptase (Life Technologies, Grand Island, NY, USA). The cDNA samples were used to determine the gene expression of NOS3. The reference gene used was GNB2L1 as this was previously shown to be stably expressed in lung tissue . Gene expression assays for NOS3 (Hs01574659_m1) and GNB2L1 (Hs00272002_m1) were purchased from Applied Biosystems. Gene expression was calculated using cycle threshold (CT) values for NOS3 and GNB2L1, as previously described .
Protein expression levels
Human lung tissue fragments (30 mg) were homogenized in protein extraction buffer with protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Protein lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes, and probed with anti NOS3 rabbit polyclonal antibody NOS3 (C-20): sc-654 at a concentration of 200 μg/mL (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-β-tubulin monoclonal antibody clone AA2 at a concentration of 1 mg/mL (Upstate Co., Lake Placid, NY, USA). Detection was performed with IR700 and IR800 anti-mouse and anti-rabbit antibodies (Cell Signaling Technology, Danvers, MA, USA). The density of the bands was analyzed with the Odyssey Infrared Imaging System (LI-COR Biotechnology, Lincoln, NE, USA) using two infrared channels independently. The results were expressed as NOS3 / β-tubulin density ratios.
Sections were de-paraffinized, rehydrated, and antigen retrieved by autoclaving (15 min, 120°C, 30 psi) for 20 min in citrate target retrieval solution (Dako, Mississauga, ON, Canada). Endogenous peroxidase was quenched with 3% H2O2 and non-specific interactions blocked for 20 min with 10% goat serum. Antibody directed against human NOS3 (NOS3 (C-20): sc-654, Santa Cruz Biotechnology, Inc.) at 200 μg/mL was added overnight at 4°C in 5% goat serum. Sections were then incubated with biotinylated goat anti-mouse (1:100, Vector Labs Burlingame, CA, USA) for 60 min followed by a 10 min treatment with Streptavidin-HRP (Dako). The NOS3 antigen was visualized using the brown chromogen 3, 3-diaminobenzidine (Dako) and counterstained with Harris Hematoxylin Solution (Sigma-Aldrich). Finally, sections were then dehydrated and mounted with Cytoseal 60 (Richard-Allan Scientific, Kalamazoo, MI, USA). Antibody dilutions and all washes were in TRIS-buffered saline solution.
The JMP 5.1 statistical software package (SAS Institute Inc., Cary, NC, USA) was used for analysis of the relationship between the genetic variants and the measures of lung function in the LHS. Agreement of the genotype distributions with Hardy-Weinberg equilibrium was assessed using a X2 goodness-of-fit analysis. The two outcomes used in the LHS were baseline and rate of decline of post-bronchodilator FEV1, expressed as a percent of predicted value. Statistical analyses were performed by multiple linear regression. For the COPDGene, ECLIPSE, NETT-NAS, and GenKOLS, after removal of principal component outliers, genotype imputation within each study was performed using MaCH and CEU samples from HapMap2 and the 1000 Genomes Project as a reference population. Association analysis of SNPs with case–control status was performed in each cohort using logistic regression, adjusting for age, pack-years of cigarette smoking, and genetic ancestry using PLINK 1.07. Results were combined among the four cohorts using fixed effect meta-analyses using METAL and R 2.12 (manuscript submitted). Differences in gene expression and protein levels between the three genotypes were assessed using two-tailed t-tests.
Allelic discrimination in the lung health study
Genotype frequencies of NOS polymorphisms among participants in the LHS cohort and their associations with lung function
Mean ± SE Baseline FEV1(% predicted)
Mean ± SE Rate of Decline in FEV1(% predicted)
78.39 ± 0.17
-0.97 ± 0.04
78.84 ± 0.37
-1.02 ± 0.07
79.33 ± 1.19
-1.27 ± 0.26
78.64 ± 0.17
-0.96 ± 0.03
78.28 ± 0.32
-1.03 ± 0.07
77.82 ± 1.00
-1.12 ± 0.20
77.97 ± 0.24
-1.05 ± 0.05
78.81 ± 0.23
-0.92 ± 0.05
79.31 ± 0.39
-1.02 ± 0.07
Genetic association of NOS3 rs1800779 SNP with COPD affection status in each case–control study
COPDGene β coefficient (SE) p-value*
ECLIPSE β coefficient (SE) p-value*
NETT/NAS β coefficient (SE) p-value*
GenKOLS β coefficient (SE) p-value*
Meta-analysis β coefficient (SE) p-value*
0.03 (0.15) 0.83
0.01 (0.19) 0.95
0.36 (0.19) 0.06
0.03 (0.13) 0.81
0.08 (0.08) 0.30
0.15 (0.22) 0.49
0.13 (0.28) 0.6310
0.10 (0.27) 0.70
-0.05 (0.19) 0.79
0.07 (0.12) 0.57
Genetic association of NOS3 rs1800779 SNP with FEV 1 % predicted in each case–control study
COPDGene β coefficient (SE) p-value*
ECLIPSE β coefficient (SE) p-value*
NETT/NAS β coefficient (SE) p-value*
GenKOLS β coefficient (SE) p-value*
Meta-analysis β coefficient (SE) p-value*
0.75 (1.84) 0.68
-1.43 (0.85) 0.09
-1.46 (0.84) 0.08
1.50 (1.30) 0.25
0.80 (0.52) 0.12
2.08 (2.60) 0.42
-0.52 (1.23) 0.67
-1.42 (1.17) 0.23
-2.03 (2.04) 0.32
-0.88 (0.75) 0.24
1.69 (1.10) 0.12
3.53 (2.35) 0.13
-0.49 (1.37) 0.72
-0.98 (0.68) 0.15
-0.09 (0.52) 0.86
-0.73 (1.64) 0.66
0.36 (3.47) 0.92
-1.62 (1.81) 0.37
-0.12 (1.05) 0.91
-0.51 (0.78) 0.51
NOS3mRNA expression in lung tissue
NOS3 protein levels in lung tissue
In this study we investigated three polymorphisms in the NOS genes in relation to cross-sectional lung function and rate of decline of lung function in COPD patients. Although we observed a significant association with rs1800779 in NOS3 with baseline lung function in the derivation cohort we were unable to replicate the association in additional patient groups. Nevertheless, we were able to demonstrate that the rs1800779 SNP has a functional effect on the expression of the NOS3 gene and this has implications for the numerous disease states that have been associated with this polymorphism [24–27].
The NOS gene variants that we investigated were limited to those that had strong a priori evidence for involvement in regulation of gene expression or in a trait related to COPD. We utilized this approach to maximize power by limiting the number of comparisons that were made. If a tag SNP approach had been used, a total of 137 polymorphisms would have had to be genotyped (using a minor allele cut off of 1 %, an r2 cut off for linkage disequilibrium of 0.8 in the European population, and a region 10 kb up- and downstream of each gene). Thus, the correction for multiple comparisons would be substantially more severe and the power of the study greatly reduced. The rationale for the SNP selection is described below.
The NOS1 rs41279104 polymorphism that was selected for this study was previously shown to be associated with reduced gene expression . The minor (T) allele was associated with a 30 % reduction in expression in a reporter gene assay . The minor (A) allele of the NOS2 rs8078340 polymorphism was associated with considerably decreased affinity for nuclear protein(s)  suggesting that it has functional significance. We prioritized these polymorphisms for investigation in this study, as we reasoned that these functional effects could be relevant to a variety of traits, including COPD. The rs1800779 polymorphism in NOS3 was associated with COPD status and lower FEV1 % predicted in COPD patients . This is the only published report of a NOS polymorphism associated with our disease of interest in the Caucasian population.
The rs1800779 polymorphism has been previously associated with a number of additional phenotypes but only one of these traits, cytokine responses in children at risk for asthma, was related to respiratory disease . rs1800779 is in strong LD (r2 > 0.8) with ten other polymorphisms but none of these have been associated with phenotypes related to lung function or pulmonary disease.
Other NOS polymorphisms have been investigated with respect to phenotypes related to COPD. Arif et al.  studied four NOS3 polymorphisms in north Indian COPD patients and controls: -786 T/C (rs3918161), -922A/G (rs1800779), 894G/T (rs1799983), and the 4B/4A variable number of tandem repeats (VNTR). rs3918161, rs1800779 and the VNTR were associated with COPD. However, data from the HapMap project (http://hapmap.ncbi.nlm.nih.gov/) show that rs3918161 is not polymorphic in individuals of European descent. It was not feasible to genotype the VNTR with the large sample sizes in this study and therefore rs1800779 was the only relevant polymorphism in our study populations.
Ahsan et al. investigated NOS3 rs1799983 in 27 COPD patients and 66 controls but there was no significant difference (p = 0.18) in genotype frequency between the groups . An earlier study that examined the NOS3 VNTR did not find an association with COPD although there was an association with pulmonary hypertension in the patients . Novoradovsky and colleagues genotyped six NOS3 SNPs in patients with α1-antitrypsin deficiency and found that rs1799983 and rs1549758 were increased in severely affected cases compared with healthy controls . However, these associations were not confirmed by a subsequent study of α1-antitrypsin deficient patients  and none of our patients were α1-antitrypsin deficient. NOS2 polymorphisms have been investigated in the context of lung function growth and childhood asthma . These investigators included 24 SNPs in their analysis - seven of which were in the NOS2 promoter. The haplotype block including the promoter SNPs was associated with incident asthma and impaired lung function growth during adolescence. However, as the associations were seen in children and were with asthma, the relevance to COPD, a disease affecting an older demographic with a different pathology, is not clear. Several studies have examined the relationship between NOS gene variants and the fraction of exhaled NO in asthmatics and/or healthy subjects but the results have not been consistent [33–40] and therefore we did not consider them in this study.
The NOS1 and NOS2 polymorphisms that we investigated were both associated with functional effects on their respective gene expression [12, 13]. Therefore, we hypothesized that these variants could be important factors in COPD, and that an association of these variants with FEV1 would be observed in smokers. However, we found no significant associations between the NOS1 and NOS2 polymorphisms and lung function in COPD patients.
The rs1800779 polymorphism in the promoter of NOS3 was associated with lung function in the LHS participants. This SNP was not in Hardy-Weinberg equilibrium; however this could be due to several factors including random chance, genotyping assay failure, population stratification or because the SNP has a true genetic effect. In this study, it is unlikely a genotyping assay failure occurred since the positive controls were in 100% concordance with genotypes from the HapMap database (n = 73). The HapMap genotypes were generated with different technologies than the one used in this study and therefore the concordance is strongly indicative that the genotypes are accurate. Furthermore, approximately 13% of the subjects were re-genotyped and were in 100% concordance with the initial genotype results (n = 573). In addition, the LHS subjects were selected based on the presence of mild/moderate COPD and therefore the testing for Hardy-Weinberg equilibrium in this cohort may not be appropriate.
The results of the study in the LHS cohort demonstrated that the rs1800779 G allele was associated with a higher baseline FEV1. Although we found an association in the LHS we did not find any association of this SNP with COPD in four case–control populations. This may indicate that the association in the LHS is a false positive result, even though a limited number of polymorphisms were tested. Alternatively, the lack of replication may be a reflection of the different recruitment strategies and hence demographic factors in the cohorts involved e.g. the LHS participants were younger and had less severe airflow obstruction than the subjects in the other cohorts. The data presented in this study are contradictory to results in a previous paper which reported that the G allele of the SNP is associated with reduced lung function in COPD . A reason for this discrepancy could be differences in the study populations. The cohort used in the previous study was composed of Indian subjects, whereas the cohort in this study only included Caucasian subjects.
A follow-up experiment was performed to determine the effect of the rs1800779 SNP on NOS3 gene expression in lung tissue. It was observed that subjects who had the AG + GG genotypes had lower NOS3 gene and protein expression compared with the AA genotype. In addition, immunohistochemical analysis demonstrated higher NOS3 expression in the airway epithelium of COPD patients with the AA genotype. Taken together, these results strongly suggest that the G allele is associated with decreased NOS3 expression.
The function of NO as a deleterious pro-inflammatory or protective anti-inflammatory agent has yet to be fully understood. However, there is evidence that NO is implicated in the pathogenesis of lung diseases such as COPD. NO is a radical molecule that can rapidly react with superoxides, yielding a cytotoxic molecule known as peroxynitrite. This compound has been shown to be an important factor contributing to tissue damage in chronic inflammation, as well as impairing key cellular functions . In particular, nitrosative stress mediated by peroxynitrite is evident in patients with COPD, suggesting the toxic compound is a key contributor to the pathogenesis of the disease . Therefore, it can be speculated that once exposed to an environmental pollutant, such as cigarette smoke, airway cells and tissues experience oxidative and nitrosative stress due to production of endogenous NO.
Variants in the NOS genes were not associated with lung function or COPD status. However, the G allele of rs1800779 resulted in a decrease of NOS3 gene expression and protein levels and this has implications for the numerous disease states that have been associated with this polymorphism. The rs1800779 polymorphism is also an excellent candidate for traits that are influenced by NO levels.
This work was supported by grants from the Canadian Institutes of Health Research and National Institutes of Health Grant 5R01HL064068-04. LA is the recipient of a UBC Four Year Doctoral Fellowship and an AllerGen NCE Inc. Canadian Allergy and Immune Diseases Training Award. AJS is the recipient of a Canada Research Chair in genetics and a Michael Smith Foundation for Health Research Senior Scholar Award.
The Lung Health Study was supported by contract N01-HR-46002 from the Division of Lung Diseases of the National Heart, Lung, and Blood Institute.
The COPDGene® project is supported by the COPD Foundation through contributions made to an Industry Advisory Board comprised of AstraZeneca, Boehringer Ingelheim, Novartis, Pfizer, and Sunovion. The COPDGene® project is also supported by the National Heart, Lung, and Blood Institute contracts R01HL089856 and R01HL089897. The members of the COPDGene® study group include: Ann Arbor VA: Jeffrey Curtis, MD (PI), Ella Kazerooni, MD (RAD). Baylor College of Medicine, Houston, TX: Nicola Hanania, MD, MS (PI), Philip Alapat, MD, Venkata Bandi, MD, Kalpalatha Guntupalli, MD, Elizabeth Guy, MD, Antara Mallampalli, MD, Charles Trinh, MD (RAD), Mustafa Atik, MD. Brigham and Women’s Hospital, Boston, MA: Dawn DeMeo, MD, MPH (Co-PI), Craig Hersh, MD, MPH (Co-PI), George Washko, MD, Francine Jacobson, MD, MPH (RAD). Columbia University, New York, NY: R. Graham Barr, MD, DrPH (PI), Byron Thomashow, MD, John Austin, MD (RAD). Duke University Medical Center, Durham, NC: Neil MacIntyre, Jr., MD (PI), Lacey Washington, MD (RAD), H Page McAdams, MD (RAD). Fallon Clinic, Worcester, MA: Richard Rosiello, MD (PI), Timothy Bresnahan, MD (RAD). Health Partners Research Foundation, Minneapolis, MN: Charlene McEvoy, MD, MPH (PI), Joseph Tashjian, MD (RAD). Johns Hopkins University, Baltimore, MD: Robert Wise, MD (PI), Nadia Hansel, MD, MPH, Robert Brown, MD (RAD), Gregory Diette, MD. Los Angeles Biomedical Research Institute at Harbor UCLA Medical Center, Los Angeles, CA: Richard Casaburi, MD (PI), Janos Porszasz, MD, PhD, Hans Fischer, MD, PhD (RAD), Matt Budoff, MD. Michael E. DeBakey VAMC, Houston, TX: Amir Sharafkhaneh, MD (PI), Charles Trinh, MD (RAD), Hirani Kamal, MD, Roham Darvishi, MD. Minneapolis VA: Dennis Niewoehner, MD (PI), Tadashi Allen, MD (RAD), Quentin Anderson, MD (RAD), Kathryn Rice, MD. Morehouse School of Medicine, Atlanta, GA: Marilyn Foreman, MD, MS (PI), Gloria Westney, MD, MS, Eugene Berkowitz, MD, PhD (RAD). National Jewish Health, Denver, CO: Russell Bowler, MD, PhD (PI), Adam Friedlander, MD, David Lynch, MB (RAD), Joyce Schroeder, MD (RAD), John Newell, Jr., MD (RAD). Temple University, Philadelphia, PA: Gerard Criner, MD (PI), Victor Kim, MD, Nathaniel Marchetti, DO, Aditi Satti, MD, A. James Mamary, MD, Robert Steiner, MD (RAD), Chandra Dass, MD (RAD). University of Alabama, Birmingham, AL: William Bailey, MD (PI), Mark Dransfield, MD (Co-PI), Hrudaya Nath, MD (RAD). University of California, San Diego, CA: Joe Ramsdell, MD (PI), Paul Friedman, MD (RAD) University of Iowa, Iowa City, IA: Geoffrey McLennan, MD, PhD (PI), Edwin JR van Beek, MD, PhD (RAD), Brad Thompson, MD (RAD), Dwight Look, MD. University of Michigan, Ann Arbor, MI: Fernando Martinez, MD (PI), MeiLan Han, MD, Ella Kazerooni, MD (RAD). University of Minnesota, Minneapolis, MN: Christine Wendt, MD (PI), Tadashi Allen, MD (RAD). University of Pittsburgh, Pittsburgh, PA: Frank Sciurba, MD (PI), Joel Weissfeld, MD, MPH, Carl Fuhrman, MD (RAD), Jessica Bon, MD. University of Texas Health Science Center at San Antonio, San Antonio, TX: Antonio Anzueto, MD (PI), Sandra Adams, MD, Carlos Orozco, MD, Mario Ruiz, MD (RAD). Administrative Core: James Crapo, MD (PI), Edwin Silverman, MD, PhD (PI), Barry Make, MD, Elizabeth Regan, MD, Sarah Moyle, MS, Douglas Stinson. Genetic Analysis Core: Terri Beaty, PhD, Barbara Klanderman, PhD, Nan Laird, PhD, Christoph Lange, PhD, Michael Cho, MD, Stephanie Santorico, PhD, John Hokanson, MPH, PhD, Dawn DeMeo, MD, MPH, Nadia Hansel, MD, MPH, Craig Hersh, MD, MPH, Jacqueline Hetmanski, MS, Tanda Murray. Imaging Core: David Lynch, MB, Joyce Schroeder, MD, John Newell, Jr., MD, John Reilly, MD, Harvey Coxson, PhD, Philip Judy, PhD, Eric Hoffman, PhD, George Washko, MD, Raul San Jose Estepar, PhD, James Ross, MSc, Rebecca Leek, Jordan Zach, Alex Kluiber, Jered Sieren, Heather Baumhauer, Verity McArthur, Dzimitry Kazlouski, Andrew Allen, Tanya Mann, Anastasia Rodionova. PFT QA Core, LDS Hospital, Salt Lake City, UT: Robert Jensen, PhD. Biological Repository, Johns Hopkins University, Baltimore, MD: Homayoon Farzadegan, PhD, Stacey Meyerer, Shivam Chandan, Samantha Bragan. Data Coordinating Center and Biostatistics, National Jewish Health, Denver, CO: James Murphy, PhD, Douglas Everett, PhD, Carla Wilson, MS, Ruthie Knowles, Amber Powell, Joe Piccoli, Maura Robinson, Margaret Forbes, Martina Wamboldt. Epidemiology Core, University of Colorado School of Public Health, Denver, CO: John Hokanson, MPH, PhD, Marci Sontag, PhD, Jennifer Black-Shinn, MPH, Gregory Kinney, MPH.
The Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE) study (clinicaltrials.govidentifier NCT00292552; GSK code SCO104960) is funded by GlaxoSmithKline. Principal investigators and centers participating in the ECLIPSE study (NCT00292552): Bulgaria: Y. Ivanov (Pleven), and K. Kostov (Sofia); Canada: J. Bourbeau (Montreal, QC), M. Fitzgerald (Vancouver, BC), P. Hernandez (Halifax, NS), K. Killian (Hamilton, ON), R. Levy (Vancouver, BC), F. Maltais (Montreal, QC), and D. O’Donnell (Kingston, ON); Czech Republic: J. Krepelka (Prague); Denmark: J. Vestbo (Hvidovre); the Netherlands: E. Wouters (Horn and Maastricht); New Zealand: D. Quinn (Wellington); Norway: P. Bakke (Bergen); Slovenia: M. Kosnik (Golnik); Spain: A. Agustı’ (Palma de Mallorca), and J. Sauleda (Palma de Mallorca); Ukraine: Y. Feschenko (Kiev), V. Gavrisyuk (Kiev), N. Monogarova (Donetsk), and L. Yashina (Kiev); UK: P. Calverley (Liverpool), D. Lomas (Cambridge), W. MacNee (Edinburgh), D. Singh (Manchester), and J. Wedzicha (London); and USA: A. Anzueto (San Antonio, TX), S. Braman (Providence, RI), R. Casaburi (Torrance CA), B. Celli (Boston, MA), G. Giessel (Richmond, VA), M. Gotfried (Phoenix, AZ), G. Greenwald (Rancho Mirage, CA), N. Hanania (Houston, TX), D. Mahler (Lebanon, NH), B. Make (Denver, CO), S. Rennard (Omaha, NE), C. Rochester (New Haven, CT), P. Scanlon (Rochester, MN), D. Schuller (Omaha, NE), F. Sciurba (Pittsburgh, PA), A. Sharafkhaneh (Houston, TX), T. Siler (St Charles, MO), E. Silverman (Boston, MA), A. Wanner (Miami, FL), R. Wise (Baltimore, MD), and R. ZuWallack (Hartford, CT). Steering committee: H. Coxson (Vancouver, Canada); L. Edwards (GlaxoSmithKline, Research Triangle Park, NC, USA); K. Knobil (cochair; GlaxoSmithKline, Research Triangle Park, NC, USA); D. Lomas (Cambridge, UK); W. MacNee (Edinburgh, UK); E. Silverman (Boston, MA, USA); R. Tal-Singer (GlaxoSmithKline, King of Prussia, PA, USA); J. Vestbo (co-chair; Hvidovre, Denmark); and J. Yates (GlaxoSmithKline, Research Triangle Park, NC, USA). Scientific committee: A. Agustı’ (Barcelona, Spain); P. Calverley (Liverpool, UK); B. Celli (Boston, MA, USA); C. Crim (GlaxoSmithKline, Research Triangle Park, NC, USA); B. Miller (GlaxoSmithKline, King of Prussia, PA, USA); W. MacNee (chair; Edinburgh, UK); S. Rennard (Omaha, NE, USA); R. Tal-Singer (GlaxoSmithKline, King of Prussia, PA, USA); E. Wouters (Horn, Maastricht, the Netherlands); and J. Yates (GlaxoSmithKline, Research Triangle Park, NC, USA).
The National Emphysema Treatment Trial (NETT) was supported by the National Heart, Lung, and Blood Institute contracts N01HR76101, N01HR76102, N01HR76103, N01HR76104, N01HR76105, N01HR76106, N01HR76107, N01HR76108, N01HR76109, N01HR76110, N01HR76111, N01HR76112, N01HR76113, N01HR76114, N01HR76115, N01HR76116, N01HR76118, and N01HR76119. The NETT was also supported by the Centers for Medicare and Medicaid Services and the Agency for Healthcare Research and Quality. Co-investigators in the NETT Genetics Ancillary Study also include J. Benditt, G. Criner, M. DeCamp, P. Diaz, M. Ginsburg, L. Kaiser, M. Katz, M. Krasna, N. MacIntyre, R. McKenna, F. Martinez, Z. Mosenifar, J. Reilly, A. Ries, P. Scanlon, F. Sciurba, and J. Utz.
The Normative Aging Study (NAS) is supported by the Cooperative Studies Program/ Epidemiology Research and Information Center (ERIC) of the US Department of Veterans Affairs and is a component of the Massachusetts Veterans Epidemiology Research and Information Center (MAVERIC), Boston, Massachusetts.
The Norway GenKOLS study (Genetics of Chronic Obstructive Lung Disease, GSK code RES11080) is funded by GlaxoSmithKline.
- Ricciardolo FL, Sterk PJ, Gaston B, Folkerts G: Nitric oxide in health and disease of the respiratory system. Physiol Rev. 2004, 84: 731-765. 10.1152/physrev.00034.2003.View ArticlePubMedGoogle Scholar
- Kobzik L, Bredt DS, Lowenstein CJ, Drazen J, Gaston B, Sugarbaker D, Stamler JS: Nitric oxide synthase in human and rat lung: immunocytochemical and histochemical localization. Am J Respir Cell Mol Biol. 1993, 9: 371-377. 10.1165/ajrcmb/9.4.371.View ArticlePubMedGoogle Scholar
- Shaul PW, North AJ, Wu LC, Wells LB, Brannon TS, Lau KS, Michel T, Margraf LR, Star RA: Endothelial nitric oxide synthase is expressed in cultured human bronchiolar epithelium. J Clin Invest. 1994, 94: 2231-2236. 10.1172/JCI117585.View ArticlePubMedPubMed CentralGoogle Scholar
- Giaid A, Saleh D: Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med. 1995, 333: 214-221. 10.1056/NEJM199507273330403.View ArticlePubMedGoogle Scholar
- Guo FH, De Raeve HR, Rice TW, Stuehr DJ, Thunnissen FB, Erzurum SC: Continuous nitric oxide synthesis by inducible nitric oxide synthase in normal human airway epithelium in vivo. Proc Natl Acad Sci USA. 1995, 92: 7809-7813. 10.1073/pnas.92.17.7809.View ArticlePubMedPubMed CentralGoogle Scholar
- Brindicci C, Kharitonov SA, Ito M, Elliott MW, Hogg JC, Barnes PJ, Ito K: Nitric oxide synthase isoenzyme expression and activity in peripheral lung tissue of patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2010, 181: 21-30. 10.1164/rccm.200904-0493OC.View ArticlePubMedGoogle Scholar
- Ricciardolo FL, Caramori G, Ito K, Capelli A, Brun P, Abatangelo G, Papi A, Chung KF, Adcock I, Barnes PJ, et al: Nitrosative stress in the bronchial mucosa of severe chronic obstructive pulmonary disease. J Allergy Clin Immunol. 2005, 116: 1028-1035. 10.1016/j.jaci.2005.06.034.View ArticlePubMedGoogle Scholar
- Seimetz M, Parajuli N, Pichl A, Veit F, Kwapiszewska G, Weisel FC, Milger K, Egemnazarov B, Turowska A, Fuchs B, et al: Inducible NOS inhibition reverses tobacco-smoke-induced emphysema and pulmonary hypertension in mice. Cell. 2011, 147: 293-305. 10.1016/j.cell.2011.08.035.View ArticlePubMedGoogle Scholar
- Berndt A, Leme AS, Shapiro SD: Emerging genetics of COPD. EMBO Mol Med. 2012, 4: 1144-1155. 10.1002/emmm.201100627.View ArticlePubMedPubMed CentralGoogle Scholar
- Foreman MG, Campos M, Celedon JC: Genes and chronic obstructive pulmonary disease. Med Clin North Am. 2012, 96: 699-711. 10.1016/j.mcna.2012.02.006.View ArticlePubMedPubMed CentralGoogle Scholar
- Connett JE, Kusek JW, Bailey WC, O’ Hara P, Wu M: Design of the Lung Health Study: a randomized clinical trial of early intervention for chronic obstructive pulmonary disease. Control Clin Trials. 1993, 14: 3S-19S. 10.1016/0197-2456(93)90021-5.View ArticlePubMedGoogle Scholar
- Saur D, Vanderwinden JM, Seidler B, Schmid RM, De Laet MH, Allescher HD: Single-nucleotide promoter polymorphism alters transcription of neuronal nitric oxide synthase exon 1c in infantile hypertrophic pyloric stenosis. Proc Natl Acad Sci USA. 2004, 101: 1662-1667. 10.1073/pnas.0305473101.View ArticlePubMedPubMed CentralGoogle Scholar
- Burgner D, Usen S, Rockett K, Jallow M, Ackerman H, Cervino A, Pinder M, Kwiatkowski DP: Nucleotide and haplotypic diversity of the NOS2A promoter region and its relationship to cerebral malaria. Hum Genet. 2003, 112: 379-386.PubMedGoogle Scholar
- Arif E, Ahsan A, Vibhuti A, Rajput C, Deepak D, Athar M, Singh B, Pasha MA: Endothelial nitric oxide synthase gene variants contribute to oxidative stress in COPD. Biochem Biophys Res Commun. 2007, 361: 182-188. 10.1016/j.bbrc.2007.07.008.View ArticlePubMedGoogle Scholar
- Pillai SG, Ge D, Zhu G, Kong X, Shianna KV, Need AC, Feng S, Hersh CP, Bakke P, Gulsvik A, et al: A genome-wide association study in chronic obstructive pulmonary disease (COPD): identification of two major susceptibility loci. PLoS Genet. 2009, 5: e1000421-10.1371/journal.pgen.1000421.View ArticlePubMedPubMed CentralGoogle Scholar
- Fishman A, Martinez F, Naunheim K, Piantadosi S, Wise R, Ries A, Weinmann G, Wood DE: National Emphysema Treatment Trial Research Group: A randomized trial comparing lung-volume-reduction surgery with medical therapy for severe emphysema. N Engl J Med. 2003, 348: 2059-2073.View ArticlePubMedGoogle Scholar
- Fishman A, Martinez F, Naunheim K, Piantadosi S, Wise R, Ries A, Weinmann G, Wood DE, National Emphysema Treatment Trial Research G: A randomized trial comparing lung-volume-reduction surgery with medical therapy for severe emphysema. N Engl J Med. 2003, 348: 2059-2073.View ArticlePubMedGoogle Scholar
- Bell B, Rose C, Damon H: The Normative Aging Study: an interdisciplinary and longitudinal study of health and aging. Aging Hum Dev. 1972, 3: 5-17. 10.2190/GGVP-XLB5-PC3N-EF0G.View ArticleGoogle Scholar
- Vestbo J, Anderson W, Coxson HO, Crim C, Dawber F, Edwards L, Hagan G, Knobil K, Lomas DA, MacNee W, et al: Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points (ECLIPSE). Eur Respir J. 2008, 31: 869-873. 10.1183/09031936.00111707.View ArticlePubMedGoogle Scholar
- Regan EA, Hokanson JE, Murphy JR, Make B, Lynch DA, Beaty TH, Curran-Everett D, Silverman EK, Crapo JD: Genetic epidemiology of COPD (COPDGene) study design. COPD. 2010, 7: 32-43. 10.3109/15412550903499522.View ArticlePubMedPubMed CentralGoogle Scholar
- Cho MH, Boutaoui N, Klanderman BJ, Sylvia JS, Ziniti JP, Hersh CP, DeMeo DL, Hunninghake GM, Litonjua AA, Sparrow D, et al: Variants in FAM13A are associated with chronic obstructive pulmonary disease. Nat Genet. 2010, 42: 200-202. 10.1038/ng.535.View ArticlePubMedPubMed CentralGoogle Scholar
- He JQ, Sandford AJ, Wang IM, Stepaniants S, Knight DA, Kicic A, Stick SM, Paré PD: Selection of housekeeping genes for real-time PCR in atopic human bronchial epithelial cells. Eur Respir J. 2008, 32: 755-762. 10.1183/09031936.00129107.View ArticlePubMedGoogle Scholar
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. 2001, 25: 402-408. 10.1006/meth.2001.1262.View ArticlePubMedGoogle Scholar
- Hoffjan S, Nicolae D, Ostrovnaya I, Roberg K, Evans M, Mirel DB, Steiner L, Walker K, Shult P, Gangnon RE, et al: Gene-environment interaction effects on the development of immune responses in the 1st year of life. Am J Hum Genet. 2005, 76: 696-704. 10.1086/429418.View ArticlePubMedPubMed CentralGoogle Scholar
- Kang JH, Wiggs JL, Rosner BA, Hankinson SE, Abdrabou W, Fan BJ, Haines J, Pasquale LR: Endothelial nitric oxide synthase gene variants and primary open-angle glaucoma: interactions with sex and postmenopausal hormone use. Invest Ophthalmol Vis Sci. 2010, 51: 971-979. 10.1167/iovs.09-4266.View ArticlePubMedPubMed CentralGoogle Scholar
- Fernandez-Cadenas I, Mendioroz M, Domingues-Montanari S, Del Rio-Espinola A, Delgado P, Ruiz A, Hernandez-Guillamon M, Giralt D, Chacon P, Navarro-Sobrino M, et al: Leukoaraiosis is associated with genes regulating blood–brain barrier homeostasis in ischaemic stroke patients. Eur J Neurol. 2011, 18: 826-835. 10.1111/j.1468-1331.2010.03243.x.View ArticlePubMedGoogle Scholar
- Kuzmanic Samija R, Primorac D, Resic B, Lozic B, Krzelj V, Tomasovic M, Stoini E, Samanovic L, Benzon B, Pehlic M, et al: Association of NOS3 tag polymorphisms with hypoxic-ischemic encephalopathy. Croat Med J. 2011, 52: 396-402. 10.3325/cmj.2011.52.396.View ArticlePubMedPubMed CentralGoogle Scholar
- Ahsan A, Ram R, Baig MA, Pasha MA: ACE I allele and eNOS G allele crosstalk may have a role in chronic obstructive pulmonary disease. Clin Biochem. 2004, 37: 1037-1040. 10.1016/j.clinbiochem.2004.05.026.View ArticlePubMedGoogle Scholar
- Yildiz P, Oflaz H, Cine N, Erginel-Unaltuna N, Erzengin F, Yilmaz V: Gene polymorphisms of endothelial nitric oxide synthase enzyme associated with pulmonary hypertension in patients with COPD. Respir Med. 2003, 97: 1282-1288. 10.1016/j.rmed.2003.06.001.View ArticlePubMedGoogle Scholar
- Novoradovsky A, Brantly ML, Waclawiw MA, Chaudhary PP, Ihara H, Qi L, Eissa NT, Barnes PM, Gabriele KM, Ehrmantraut ME, et al: Endothelial nitric oxide synthase as a potential susceptibility gene in the pathogenesis of emphysema in α1-antitrypsin deficiency. Am J Respir Cell Mol Biol. 1999, 20: 441-447. 10.1165/ajrcmb.20.3.3144.View ArticlePubMedGoogle Scholar
- Demeo DL, Campbell EJ, Barker AF, Brantly ML, Eden E, McElvaney NG, Rennard SI, Sandhaus RA, Stocks JM, Stoller JK, et al: IL10 polymorphisms are associated with airflow obstruction in severe alpha1-antitrypsin deficiency. Am J Respir Cell Mol Biol. 2008, 38: 114-120. 10.1165/rcmb.2007-0107OC.View ArticlePubMedGoogle Scholar
- Islam T, Breton C, Salam MT, McConnell R, Wenten M, Gauderman WJ, Conti D, Van Den Berg D, Peters JM, Gilliland FD: Role of inducible nitric oxide synthase in asthma risk and lung function growth during adolescence. Thorax. 2010, 65: 139-145. 10.1136/thx.2009.114355.View ArticlePubMedGoogle Scholar
- Wechsler ME, Grasemann H, Deykin A, Silverman EK, Yandava CN, Israel E, Wand M, Drazen JM: Exhaled nitric oxide in patients with asthma: association with NOS1 genotype. Am J Respir Crit Care Med. 2000, 162: 2043-2047. 10.1164/ajrccm.162.6.2003089.View ArticlePubMedGoogle Scholar
- Ali M, Khoo SK, Turner S, Stick S, Le Souef P, Franklin P: NOS1 polymorphism is associated with atopy but not exhaled nitric oxide levels in healthy children. Pediatr Allergy Immunol. 2003, 14: 261-265. 10.1034/j.1399-3038.2003.00065.x.View ArticlePubMedGoogle Scholar
- Storm Van's Gravesande K, Wechsler ME, Grasemann H, Silverman ES, Le L, Palmer LJ, Drazen JM: Association of a missense mutation in the NOS3 gene with exhaled nitric oxide levels. Am J Respir Crit Care Med. 2003, 168: 228-231. 10.1164/rccm.200212-1491BC.View ArticlePubMedGoogle Scholar
- Leung TF, Liu EK, Tang NL, Ko FW, Li CY, Lam CW, Wong GW: Nitric oxide synthase polymorphisms and asthma phenotypes in Chinese children. Clin Exp Allergy. 2005, 35: 1288-1294. 10.1111/j.1365-2222.2005.02342.x.View ArticlePubMedGoogle Scholar
- Leung TF, Liu EK, Li CY, Chan IH, Yung E, Lam CW, Wong GW: Lack of association between NOS2 pentanucleotide repeat polymorphism and asthma phenotypes or exhaled nitric oxide concentration. Pediatr Pulmonol. 2006, 41: 649-655. 10.1002/ppul.20428.View ArticlePubMedGoogle Scholar
- Salam MT, Bastain TM, Rappaport EB, Islam T, Berhane K, Gauderman WJ, Gilliland FD: Genetic variations in nitric oxide synthase and arginase influence exhaled nitric oxide levels in children. Allergy. 2011, 66: 412-419. 10.1111/j.1398-9995.2010.02492.x.View ArticlePubMedGoogle Scholar
- Dahgam S, Nyberg F, Modig L, Naluai AT, Olin AC: Single nucleotide polymorphisms in the NOS2 and NOS3 genes are associated with exhaled nitric oxide. J Med Genet. 2012, 49: 200-205. 10.1136/jmedgenet-2011-100584.View ArticlePubMedGoogle Scholar
- Bouzigon E, Monier F, Boussaha M, Le Moual N, Huyvaert H, Matran R, Letort S, Bousquet J, Pin I, Lathrop M, et al: Associations between nitric oxide synthase genes and exhaled NO-related phenotypes according to asthma status. PLoS ONE. 2012, 7: e36672-10.1371/journal.pone.0036672.View ArticlePubMedPubMed CentralGoogle Scholar
- Sadeghi-Hashjin G, Folkerts G, Henricks PA, Muijsers RB, Nijkamp FP: Peroxynitrite in airway diseases. Clin Exp Allergy. 1998, 28: 1464-1473. 10.1046/j.1365-2222.1998.00428.x.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/13/64/prepub
This article is published under license to BioMed Central Ltd. 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.