Discussion
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 [
12]. The minor (T) allele was associated with a 30 % reduction in expression in a reporter gene assay [
12]. The minor (A) allele of the
NOS2 rs8078340 polymorphism was associated with considerably decreased affinity for nuclear protein(s) [
13] 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 FEV
1 % predicted in COPD patients [
14]. 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 [
24]. rs1800779 is in strong LD (r
2 > 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. [
14] 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 [
28]. 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 [
29]. Novoradovsky and colleagues genotyped six
NOS3 SNPs in patients with α
1-antitrypsin deficiency and found that rs1799983 and rs1549758 were increased in severely affected cases compared with healthy controls [
30]. However, these associations were not confirmed by a subsequent study of α
1-antitrypsin deficient patients [
31] and none of our patients were α
1-antitrypsin deficient.
NOS2 polymorphisms have been investigated in the context of lung function growth and childhood asthma [
32]. 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 FEV
1 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 FEV
1. 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 [
14]. 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 [
41]. 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 [
7]. 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.
Acknowledgements
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.