FENO and polymorphisms in the NOS genes SNP- and haplotype-based association analyses

Background Polymorphisms in nitric oxide synthasegenes (NOS1, NOS2, and NOS3) have been suggestedto have a major impact on fraction of exhaled nitric oxide(FENO), a biomarker of airway inflammation. However,the genetic contribution of NOS polymorphisms to FENOis not fully understood. The aim of this study was toinvestigate comprehensively the association betweensingle nucleotide polymorphisms (SNPs) in all three NOSgenes and FENO in an adult population, and to assesswhether such associations are modified by asthma oratopy.Method In 1737 adults from a Swedish generalpopulation sample, FENO was measured and geneticvariation in the NOS genes was assessed using 49 SNPs.The genetic effect of NOS polymorphisms on FENO,asthma, and atopy was estimated using multipleregression methods.Results In a multi-SNP model based on stepwiseregression analysis, two SNPs in NOS2 and one in NOS3showed independent associations with levels of FENO.For NOS2 SNP rs9901734, subjects had 5.3% (95% CI1.0% to 9.7%) higher levels of FENO per G allele, and forrs3729508, subjects with CC or CT genotypes had 9.4%(95% CI 3.1% to 15.2%) higher levels compared with TT.For NOS3 SNP rs7830, subjects with GT or TT had 5.6%(95% CI 0.4% to 11.1%) higher levels than GG; thegenetic effect of this SNP was stronger in asthmatics(21.9%, 95% CI 4.6% to 42.0%).Conclusion These results suggest that NOS2 is themajor NOS gene determining variability in exhaled nitricoxide in the healthy adult population, while NOS3 mayplay a more important role in asthmatic adults. INTRODUCTIONThe fraction of exhaled nitric oxide (FENO) isa non-invasive biomarker of airway inflammationand a useful clinical tool for diagnosing and moni-toring asthma. 2 It is easy to measure and resultscan be obtained in real-time (online), making it anattractive method in the clinical management ofasthma. We have recently reported that increasedFENO levels predict new onset of respiratorysymptoms in a follow-up cohort. One obstacle,however, to more widespread utility of FENO isthat the inter-individual variation in healthysubjects is relatively large, and there is limitedknowledge about factors explaining this variation.FENO levels are increased in subjects with asthmaand atopy and appear to be related to eosinophilicairway inflammation, such that 26% of thevariation in FENO could be explained by theconcentration of eosinophils in induced sputum ina large study of asthmatic adults. In healthy adultsubjects, the main predictors of FENO are atopy,height, and age, which together explain 11% of thevariability of FENO.In addition, genetic factors are suggested to havea major impact on FENO variability. In a Norwe-gian twin study, genetic factors explained 60% ofthe variability in FENO. This study also observedthat FENO and airway hyperresponsiveness sharea common genetic basis.Nitric oxide (NO) is synthesised by specific NOsynthase (NOS) enzymes, with three distinctisoforms: neuronal NOS (nNOS; NOS1), inducibleNOS (iNOS; NOS2), and endothelial NOS (eNOS;NOS3). 10 All three NOS isoforms are expressed inthe lung. The NOS1 and NOS3 genes are consti-tutively expressed resulting in a low basal synthesisof NO, 13 whereas expression of NOS2 seems tobe more strongly regulated by gene transcriptionfactors such as nuclear factor k B (NFKb), resultingin an increased NO production. A recent geneexpression study suggested that expression ofNOS2 in humans is significantly more detectableand highly correlated with FENO than NOS3.Few studies have examined associations betweenpolymorphisms in the NOS genes and FENO levelsand, the results of these studies have been incon-sistent. A reported association between AATrepeats in intron 20 in NOS1 and higher FENOlevels in asthmatic adults was not replicated inasthmatic children. 18 20 A recent population basedstudy found tag single nucleotide polymorphisms(SNPs) in NOS2 to be significantly associated FENOand the association was stronger in asthmatic chil-dren. For NOS3, an association of the missensevariant, G894T (rs1799983), to lower FENO levelswas reported in adult asthmatics but not in Chinesechildren with asthma. 20The aims of this study were to examinecomprehensively the role of SNPs in all three NOSgenes on the levels of FENO in a large adult cohort;and to study whether any risk variants might beassociated with asthma, atopy or lung function,and if the genetic effect on FENO is modified bythese respiratory phenotypes. METHODSStudy population and designThe present study is a part of the ADONIX (adult-onset asthma and exhaled nitric oxide) studycohort of 2200 randomly selected men and women< Additional materials arepublished online only. To viewthese files please visit thejournal online (http://jmg.bmj.com/content/49/3.toc). Department of Occupationaland Environmental Medicine,University of Gothenburg,Gothenburg, SwedenAstraZeneca R&D;, Mölndal,SwedenDepartment of Public Healthand Clinical Medicine,Occupational and EnvironmentalMedicine, University of Umea,Umea, SwedenDepartment of Medical andClinical Genetics, University ofGothenburg, Gothenburg,Sweden Correspondence toSantosh Dahgam, Occupationaland Environmental Medicine,University of Gothenburg, POBox 414, SE-405 30Gothenburg, Sweden;[email protected] Received 21 October 2011Revised 27 January 2012Accepted 29 January 2012 200J Med Genet 2012;49:200e205. doi:10.1136/jmedgenet-2011-100584Complex traits