Published ahead of print on May 8, 2003, doi:10.1164/rccm.200212-1491BC
© 2003 American Thoracic Society
Association of a Missense Mutation in the NOS3 Gene with Exhaled Nitric Oxide LevelsDepartment of Medicine, Pulmonary Division, Brigham and Women's Hospital, Boston; Channing Laboratory, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts; University Children's Hospital, Essen University Children's Hospital, Freiburg, Germany; and Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, Ohio Correspondence and requests for reprints should be addressed to Jeffrey M. Drazen, M.D., Division of Pulmonary and Critical Care Medicine, Tower 4B, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115. E-mail: jdrazen{at}nejm.org
There is evidence that genetic factors affect nitric oxide formation and that sequence variants in the nitric oxide synthase genes contribute to the observed variance of nitric oxide levels in exhaled air (fraction of expired nitric oxide, FENO) in subjects with asthma. We identified a strong association between a known functional NOS3 missense sequence variant in the endothelial nitric oxide gene (G894T) and FENO level in a cohort of subjects with asthma. Age- and sex-adjusted FENO levels were lowest in asthmatic subjects with the TT genotype (geometric mean FENO [95% CI] = 7.17 [4.48 to 11.48] ppb) and were significantly higher in those with either the GT genotype (geometric mean FENO [95% CI] = 17.11 [13.80 to 21.23] ppb) or the GG genotype (geometric mean FENO [95% CI] = 12.06 [9.91 to 14.67] ppb) (F2,59 = 5.97, p = 0.004). The G894T DNA variant explained 16.3% of the residual variance in FENO levels. Our results demonstrate that the endothelial nitric oxide synthase, a nitric oxide synthase constitutively expressed in epithelial cells, plays an important role in determining measured levels of exhaled nitric oxide, a marker of the asthmatic condition.
Key Words: asthma • endothelial nitric oxide synthase • exhaled nitric oxide • polymorphism Nitric oxide (NO) is produced endogenously by nitric oxide synthases (NOS) in the lung and can be measured in exhaled air. NO is present at higher levels in the expired air (fraction of expired nitric oxide, FENO) of subjects with asthma compared with normal subjects (1–6). Although the cellular sources responsible for the higher FENO values are not known, it is reasonable to speculate that the increased levels are the product of enhanced expression or activity of nitric oxide synthases (NOS) in various cells of the respiratory tract (7–9). Whereas NOS-1 (neuronal NOS) has been observed in nonadrenergic noncholinergic nerves (10), and NOS-2 (inducible NOS) has been detected in alveolar macrophages (11), the NOS-3 isoform (endothelial NOS) has been localized in the bronchial and pulmonary circulation (12). All three NOS isoforms (NOS-1, NOS-2, and NOS-3) are expressed by airway epithelial cells (13–17), which have been implicated as the primary source of levels of NO in expired air (FENO) (18). Even though mean FENO levels are elevated among subjects with asthma, there is substantial variance among these subjects (19–22). Elevated levels of FENO among subjects with asthma are associated with induction of NOS-2 (inducible NOS) (23). However, it is not known whether the constitutive isoforms, such as NOS-1 (neuronal NOS) and NOS-3 (endothelial NOS), also contribute to FENO levels. We reasoned that if FENO levels were significantly associated with a functional sequence variant of NOS-3, that this would provide evidence of the contribution of this NOS isoform to exhaled NO in subjects with asthma. To test this hypothesis, we sought an association between a previously described functional DNA missense variant in the NOS3 gene, G893T, and the variability of FENO levels among patients with asthma.
Subjects and Genotyping Seventy-three white subjects with mild to moderate asthma were recruited from the database of the Partners Asthma Center at the Brigham and Women's Hospital (Boston, MA). For these subjects, data were available on FENO levels, FEV1, asthmatic medication history, and allergic status. The age of the subjects ranged from 19 to 54 years and the FEV1 ranged from 77 to 85% predicted. All except four subjects, who did not give any information about sensitization or who rejected the skin prick test, were atopic. Subjects were considered asthmatic if they had a clinical history of asthma (as defined by the American Thoracic Society [24]) for at least 1 year and had a history within the year preceding the study of at least one of the following: 12% reversibility of FEV1 in response to a bronchodilator, 25% decrease in FEV1 in response to a cold air challenge, or a methacholine challenge with a provocative concentration that decreased FEV1 by 20% (PC20) of less than 8 mg/ml. Atopic status was evaluated by questionnaire and skin prick test. All phenotype assessments were done without knowledge of the patient's genotype. Subjects were excluded if they had used inhaled or systemic corticosteroids within 30 days of the study or if they had had an upper respiratory tract infection within the preceding 30 days, had smoked tobacco products within the preceding 6 months or had a greater than 10-pack-year smoking history, were pregnant, had a history of other documented pulmonary disease (such as chronic obstructive pulmonary disease, cystic fibrosis, or bronchiectasis), or had a history of other documented medical problems. Upon presentation to the Partners Asthma Center, each subject gave written informed consent for spirometry, NO measurement, and genetic screening (approved by the institutional review board at Brigham and Women's Hospital) and completed a patient information sheet. Measurement of FENO was performed according to the guidelines for off-line measurements (25). While seated and wearing a noseclip, qualified subjects performed normal tidal breathing for 30 seconds from a source of air containing a low concentration of NO gas. Gas exhaled by each subject was collected in three separate Mylar bags at 2-minute intervals under conditions of controlled flow and airway opening pressure (375 ml/second, 10 mm Hg). Three measurements per subjects were performed between 9 and 12 A.M. NO concentration in exhaled gas was determined by chemiluminescence (NOA 280; Sievers Instruments, Boulder, CO), and median NO values were recorded. The mean coefficient of variation for the three measures in each individual was 13.8%. White control subjects (without asthma, atopy, elevated IgE values, or significant other medical conditions) were recruited as normal subjects for genetic studies and compared with patients with asthma. FENO measurements were not available for these subjects. The region of interest of NOS3 was amplified from purified genomic DNA by polymerase chain reaction (PCR) with appropriate primers (sense primer, 5'-AAG GCA GGA GAC AGT GGA TG-3'; antisense primer, 5'-TCC CTT TGG TGC TAC GT-3'). The G893T polymorphism was typed by a restriction fragment length polymorphism-PCR analysis strategy. The amplified PCR product was digested with Sau3A (New England BioLabs, Beverly, MA) for 16 hours and separated on a 2.5% agarose gel for visualization. The genotyping method was confirmed by direct sequence analysis.
Statistical Analysis FENO was skewed with a long right-hand tail at higher values, and was thus loge transformed before analysis. Sex was analyzed as a binary variable; all other variables were analyzed as continuous. Bivariate analysis used analysis of variance to compare mean FENO across the possible genotypes. Generalized linear models (linear regression) (26) were used to investigate the multivariate relationships between the G894T variant and FENO level. Age, sex, and FEV1 level were also investigated as potential explanatory covariates in the multivariate analyses. Checks of goodness of fit (27) included an investigation of the need for interaction of polynomial terms, analyses of Pearson residuals, and examination of the effect of observations with high-regression leverage. In addition to the analyses of genotype as a categorical variable, evidence of a linear (genetically additive) effect on phenotype across the three genotypes of each polymorphism was sought by entering the genotypes as continuous covariates in the multivariate models. Minitab for Windows, version 12.1 (Minitab, College Station, TX), and S-Plus, version 4.5 (Mathsoft, Cambridge, MA), were used to manage and analyze data. Statistical significance was defined at the standard 5% level.
The demographic data are summarized in Table 1 . The frequency of the G allele was 0.67 and 0.73 in the asthmatic and control population, respectively. The genotype frequencies were consistent with Hardy–Weinberg equilibrium. Although neither age nor sex was formally significant in our multivariate analyses, we adjusted for these covariates to demonstrate that our associations remained unchanged and to allow for any subtle confounding.
Association of G894T Polymorphism with Asthma After genotyping a sample of white control subjects (n = 84), binary logistic regression adjusted for age and sex indicated there was no significant association between asthma risk and the G894T variant under either a dominant model (odds ratio = 1.07, 95% confidence interval [CI] = 0.51 to 2.25, p = 0.86) or a recessive model (odds ratio = 2.50, 95% CI = 0.61 to 10.25, p = 0.20).
Association of G894T Polymorphism with Pulmonary Function in Adults with Asthma
Association of G894T Polymorphism with FENO Level in Adults with Asthma
Although there is substantial variability, it is widely accepted that FENO levels are increased in individuals with asthma in the absence of treatment with inhaled or systemic corticosteroids, compared with the generally low levels observed in healthy control subjects (19, 21, 22). The underlying mechanism of this observed variability is not yet established, but might be influenced by disease severity (28, 29), atopy (30–33), or genetic factors (22). There is evidence that NOS-1 contributes to the variability of FENO levels. A variable nucleotide tandem repeat in intron 20 explains 5.1% of the variance in FENO levels in white subjects (22). However, it is not known whether the constitutive isoform, NOS-3 (endothelial NOS), contributes to FENO levels. We reasoned that we could take advantage of a known functional variant of NOS-3 to help ascertain whether NOS-3 contributes to measured exhaled NO levels. In this study we show that a known DNA sequence variant in NOS3 is associated with decreased levels of FENO. This suggests that NOS-3 may play an important role in determining the FENO level in many adults with asthma. No association was observed between the G894T missense mutation and the diagnosis of asthma. There has been evidence that the NOS3 gene plays a role in susceptibility to asthma. An association between a biallelic intronic variable nucleotide tandem repeat with either five tandem 27-bp repeats or four tandem 27-bp repeats in conjunction with an asthma diagnosis has been reported (34). Shaul and coworkers also suggested that NOS-3 is at least one of the sources of endogenous NO that may function in a paracrine fashion to modulate bronchomotor tone (15). This hypothesis is also supported by the finding that NOS3 knockout mice are more hyperresponsive to methacholine compared with wild-type mice (12). Therefore, decreased NOS-3 activity might promote bronchial hyperresponsiveness and contribute to the asthmatic phenotype. However, no association between the G894T missense mutation and FEV1 or reversibility of FEV1 after administration of inhaled albuterol was found in our study population. We observed that the T allele at this locus is associated with lower NO levels. This observation, coupled with previous studies demonstrating that the G894T missense mutation is functionally relevant (35), leads us to speculate that it may be a reason for the low FENO levels. In placental tissue, Wang and coworkers showed that the G894T variant influences NOS-3 expression and enzyme activity (35). Specifically the isoform of NOS-3 containing aspartate I allele is cleaved in the cell and thus inactivated (36). It is interesting to speculate that intracellular cleavage at position 298 of NOS-3 in individuals with asthma occurs in vivo, because airway pH is low in untreated patients with asthma (37). Therefore low airway pH in untreated individuals with asthma could result in intracellular cleavage of the NOS-3 protein. Our data demonstrate that an NOS3 missense variant contributes about one-sixth of the variance in FENO levels among patients with asthma who are not receiving inhaled steroids. No other gene or mechanism has been shown to contribute this large a fraction of the total variance in FENO, suggesting that NOS-3 may play an important role in determining the NO detected in exhaled air in many adults with asthma. Because mechanisms of control of FENO may differ among subjects, further studies are required to clarify the role of NOS-3 in the production of exhaled nitric oxide levels in healthy people. Because others have shown that the G894T sequence variant results in an allele-dependent reduction of endogenous NO production, we conclude that NOS-3 likely contributes to the elevated FENO levels found in most individuals with asthma and that NOS-3 is an effect modifier in asthma.
Supported by a grant from the United States National Institutes of Health (HL-SCOR HL 56383-05). K. Storm van's Gravesande is the recipient of a grant from the Deutsche Forschungsgemeinschaft (STO 420/1-1). Received in original form December 17, 2002; accepted in final form May 1, 2003
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ABSTRACT
METHODS
Asp variant of human endothelial nitric oxide synthase. J Biol Chem 2001;276:26674–26679.
