Published ahead of print on January 16, 2003, doi:10.1164/rccm.200211-1342OC
© 2003 American Thoracic Society
Effects of Sex and of Gene Variants in Constitutive Nitric Oxide Synthases on Exhaled Nitric OxideDepartment of Pediatrics, University of Essen, Essen, Germany; and Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts Correspondence and requests for reprints should be addressed to Hartmut Grasemann, M.D., Children's Hospital, University of Essen, Hufeland Strasse 55, D-45122 Essen, Germany. E-mail: hartmutg{at}hotmail.com
Genetic factors may contribute to the variability of exhaled nitric oxide in healthy individuals. We studied exhaled nitric oxide and genetic variants in both neuronal and endothelial nitric oxide synthases in 105 healthy nonsmoking and smoking subjects. Genomic DNA was screened for a repeat polymorphism in intron 20 of the neuronal nitric oxide synthase gene and for the 894G/T mutation of the endothelial nitric oxide synthase gene. Exhaled nitric oxide was significantly higher in males than females among both nonsmokers (p < 0.0001) and smokers (p = 0.003). No association was found between exhaled nitric oxide and the endothelial nitric oxide synthase gene variant. However, healthy nonsmoking females with greater numbers of repeats (i.e., both alleles with 12 or more repeats) in neuronal nitric oxide synthase had significantly lower nitric oxide levels than did females with fewer numbers of repeats (i.e., at least one allele with fewer than 12 repeats) (13.6 ± 1.6 versus 19.4 ± 1.6 ppb, p = 0.02). No association was found between exhaled nitric oxide and neuronal nitric oxide synthase genotype in males. These data suggest that variants in the neuronal nitric oxide synthase gene contribute to the variability of airway nitric oxide concentrations in healthy females.
Key Words: nitric oxide • polymorphism • sex factors The messenger molecule nitric oxide (NO), which can be measured in exhaled air, is enzymatically produced by NO synthases (NOS). The inducible isoform of NOS (NOS2), is transcriptionally regulated in response to cytokines and can be distinguished from constitutive NOS, which exist in two forms: neuronal NOS (NOS1) and endothelial NOS (NOS3). Different cell types within the lung have been shown to express NOS, including alveolar macrophages, airway smooth muscle, and airway epithelial cells (1, 2). Presently, the relative contribution of the different NOS isoforms to airway NO is unknown. The fraction of exhaled NO (FENO) is increased in asthma (3), and was shown to correlate with airway inflammation. For instance, increased FENO declines in response to antiinflammatory drugs such as corticosteroids or leukotriene receptor antagonists in patients with asthma (4, 5). FENO can therefore be used as a noninvasive marker of asthma disease activity. Although it has been suggested that increased FENO in asthma reflects overexpression of inducible NO synthase (NOS2) in airway epithelial cells (6), there is evidence that the variability of FENO in asthma is significantly related to natural variants in the gene encoding NOS1. It was demonstrated that in patients with stable asthma small numbers of an intronic AATn repeat in NOS1 were associated with high FENO, whereas larger repeat size of that polymorphism was associated with lower FENO (7). Similarly, we could demonstrate that in patients with cystic fibrosis (CF), a disease characterized by chronic airway inflammation and infection, but decreased expression of NOS2 in airway epithelium, both FENO and nasal NO were also associated with the size of the same genetic marker in NOS1 (8, 9). However, it remained unclear from these studies whether the association between FENO and NOS1 genotypes is limited to inflammatory airway conditions or may also be found in healthy individuals. The other constitutive NOS isoform, NOS3, has been shown to be constitutively expressed in pulmonary blood vessels, airway epithelial cells, and airway neutrophils (10–12), and there is increasing evidence that NOS3 contributes to NO-related physiology and pathophysiology in the respiratory tract (12–14). The NOS3 gene contains a G/T polymorphism in exon 7 that results in a sequence variant at position 298 of the NOS3 protein (15). The 894T allele was demonstrated to be associated with higher FENO and a decreased rate of airway colonization with Pseudomonas aeruginosa in female but not male patients with CF (16). Of further interest, the expression of NOS3 was shown to be reduced in the lung tissue of smokers (17), who are known to have decreased levels of FENO (3). Furthermore, in a different study, the smoking-related reduction of NOS3 protein activity in placenta tissue was dependent on NOS3 genotype (18). In the current study, we therefore analyzed the relationship between sex, variants in the genes encoding NOS1 and NOS3, and FENO in both nonsmoking and smoking healthy subjects. Some of the results of these studies have been previously reported in the form of an abstract (19).
Subjects A total of 150 randomly selected adults (18 to 61 years of age) were studied. Individuals with a history of asthma or atopy as self-reported, or with a medication for any other medical condition, were excluded. The remaining 105 individuals were assigned to one of two groups: (1) current or recent smokers, and (2) healthy nonsmoking subjects. Demographic data of the study population are displayed in Table 1 . At the time of study subjects had to be free of upper or lower airway infections.
NO Measurement FENO was measured in all subjects with a chemiluminescence analyzer (NOA 280; Sievers, Boulder, CO). Single-breath on-line measurements for the assessment of lower airway NO were performed at a constant expiratory flow of 3 and 6 L · minute–1 in each individual, respectively, in accordance with published American Thoracic Society standards (20). The mean value of three end-expiratory NO concentrations was calculated for each subject.
Genotyping Restriction fragment length polymorphism analysis (RFLP-PCR) was used for the 894G/T variant in the NOS3 gene, as described (16). Forward primer (5'-AAG GCA GGA GAC AGT GGA TG-3') and reverse primer (5'-TCC CTT TGG TGC TAC GT-3') were used for the following PCR conditions: 6 minutes at 94°C, followed by 35 cycles of 94°C for 1 minute, 59°C for 1 minute, and 72°C for 1 minute. Chain elongation was continued for 5 minutes after the last cycle. The amplified PCR product was digested with Sau3AI (New England BioLabs, Beverly, MA) for 16 hours and separated on a 2.5% agarose gel for visualization.
Statistical Analysis
FENO in healthy nonsmoking individuals was significantly (p = 0.0002) higher than in smokers (Table 1). A significant effect of sex on FENO, with higher NO values in males than females, was observed in both healthy individuals (35.5 ± 2.9 versus 18.6 ± 1.4 ppb, p < 0.0001) and smokers (19.8 ± 2.1 versus 11.4 ± 1.6 ppb, p = 0.003) (Table 1). Allele frequencies (G, 0.67; T, 0.33) and distribution of NOS3 genotypes (GG, 0.45; GT, 0.44; TT, 0.11) in our population were in Hardy–Weinberg equilibrium. There were no differences in NOS3 genotype distribution between smokers and healthy nonsmokers. No association was found between the 894G/T mutation in NOS3 and FENO in any group. The reduction of FENO in smokers was independent of NOS3 genotype (Table 2) .
The NOS1 alleles showed the characteristic bimodal distribution pattern of this particular repeat polymorphism, as described (7, 8), with the 10 AAT repeats allele (48.3%), and the 13 and 14 AAT repeats alleles (21.8 and 11.8%, respectively) being the most common alleles. On the basis of statistical analysis performed in our two articles on the relation of NOS1 gene polymorphisms and lower airway FENO (7, 8), we segregated the populations into two groups: one consisting of individuals harboring two NOS1 alleles with at least 12 repeats (large repeat numbers), and the other consisting of individuals harboring at least one NOS1 allele with fewer than 12 repeats (small repeat numbers). In this analysis FENO was associated with the size of the NOS1 AATn repeat polymorphism in healthy women. FENO was significantly lower in females with large repeat numbers (n = 7) compared with females harboring small repeat numbers (n = 42) in NOS1 (13.6 ± 1.6 versus 19.4 ± 1.6 ppb, p = 0.02), when using an expiratory flow rate of 3 L/minute for the measurement of FENO (Figure 1) . The association between FENO and NOS1 genotype in females was even more pronounced when using an expiratory flow of 6 L/minute (6.7 ± 0.9 versus 10.4 ± 0.8 ppb, p = 0.007). In contrast, no association could be observed between FENO and NOS1 in healthy males (32.5 ± 3.7 versus 38.1 ± 6.6 ppb, p = 0.47).
Exhaled NO (FENO) is influenced by a number of factors including ambient NO concentration, soft palate closure, expiratory flow rate, and differences in individual airway NO formation. It is unclear, at present, whether genetic factors influence the production of NO in healthy subjects. Here we show that FENO is significantly lower in adult female than male subjects, and that FENO is associated with NOS1 genotype in healthy females. Kissoon and colleagues reported that body surface area, age, and FEF25–75 were significant predictors of FENO in healthy adult subjects (23). Interestingly, although not separately discussed in their article, FENO in that study was 190–210% higher in males than females at expiratory flows between 0.9 and 2.76 L/minute but not at lower flows (23). Similarly, using an expiratory flow of 3 L/minute, FENO was 190% higher in healthy males than females in our study. Findings in children revealed no differences in FENO between prepubertal boys and girls 6 to 11 years old but higher FENO in boys than girls 12 to 18 years of age (24). It is conceivable that these differences between the sexes could be related to body mass, as suggested by Tsang and coworkers, who found similar differences between men and women and a positive correlation of FENO with height and weight (25). Sex-related differences in FENO could also reflect differences in NOS activity between males and females. The activity of vascular NOS3 was reported to respond to circulating estrogen, which activates plasma membrane-associated estrogen receptors coupled to NOS3 (26). However, it is unclear to date whether vascular activity of NOS3 is reflected in FENO. Although estrogen has been shown to rapidly increase the activity of NOS3 in fetal pulmonary artery endothelial cells and human airway epithelial cells via nontranscriptional mechanisms (12, 26), it is also unknown whether the activity of NOS3 differs in pulmonary tissues between males and females. Although an early report suggested that FENO was positively related to estrogen levels during the menstrual cycle (27), this could not be confirmed in subsequent studies (28, 29). Activation of NOS3 by estrogens would be expected to increase FENO and would not explain the lower levels of FENO in healthy females.
The NOS3 gene contains a common variant that is located in exon 7 at position 894 and results in an amino acid change from glutamate to aspartate at position 298 of the protein. There is evidence that the 894T variant is of biological relevance because it was shown to be associated with the severity of lung disease in A polymorphism in the gene encoding NOS1 has been related to the level of FENO in humans with inflammatory airway diseases. In these studies associations were found between the size of an AATn repeat polymorphism in intron 20 of the NOS1 gene and FENO in patients with asthma, cystic fibrosis, and sickle cell anemia (7–9, 35). These intronic repeats are unlikely to affect the activity of NOS1 directly, but may reflect yet undetected DNA sequence variants in the nearby coding region of NOS1 that influence the catalytic activity of the enzyme. Nevertheless, these studies suggested that the level of FENO is genetically determined in subjects with inflammatory airway conditions. In the current study we now demonstrate that, the size of the AATn repeat in NOS1 is also associated with FENO in healthy nonsmoking women. We are aware that the interpretation of these results is limited by the relatively small number of healthy females studied. The lack of an association between FENO and NOS1 genotype in males may also be related to the small sample size or may represent differences in the contribution of NOS isoforms between sexes. Indirect evidence of a significant contribution of NOS1 to FENO in healthy male subjects, however, comes from a study of patients with Duchenne muscular dystrophy, a disease in which the underlying genetic defect results in a lack of NOS1 expression at the smooth muscle cell membrane. In this study FENO was found to be significantly reduced in boys with Duchenne muscular dystrophy (36). Studies suggest that genotypic differences in NOS1 may not only affect FENO but also modulate other phenotypic characteristics. For instance, the size of the AATn repeat in NOS1 was found to be associated with acute chest syndrome in patients with sickle cell anemia (35), and, in patients with cystic fibrosis, with the rate of colonization of the airways with P. aeruginosa (8, 9). Further studies are needed to confirm the association between NOS1 and FENO in healthy individuals and to detect further NOS1 related genotype–phenotype associations in healthy control subjects.
Supported by a grant from the Deutsche Forschungsgemeinschaft (to K.S.v.G.). Received in original form November 16, 2002; accepted in final form January 14, 2003
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ABSTRACT
METHODS
-32P]dATP with the use of polynucleotide kinase (Boehringer, Mannheim, Germany). The PCR mix contained 100 ng of genomic DNA, 1 µl of the radiolabeled primer set, dATP, dCTP, dGTP, dTTP (200 µM each), and 1.5 U of Taq polymerase in a 25-µl reaction mixture. PCR conditions were 6 minutes at 94°C, followed by 35 cycles of 94°C for 1 minute, 59°C for 1 minute, and 72°C for 1 minute. Chain elongation was continued after the last cycle for 5 minutes. Simple sequence length polymorphism (SSLP) analysis was used for allele assignment (
2 goodness-of-fit test on a contingency table of observed versus predicted genotype frequencies. A p value below 0.05 was considered statistically significant. PC-Statistik version 2.11 (TopSoft, Hannover, Germany) was used for statistical analysis. 
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Asp) is a major risk factor for coronary artery disease in the UK. Circulation 1999;100:1515–1520.
