INTRODUCTION

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The messenger molecule nitric oxide (NO) is formed by a group of enzymes called NO synthases (NOS). These enzymes catalyze the five-electron oxidation of L-arginine to form L-citrulline and NO. There are three known isoforms of NOS: neuronal NOS, or NOS1; inducible NOS (iNOS) or NOS2; and endothelial NOS, or NOS3. Human airways have been shown to express all three variants, but little is known about the relative contribution of these various NOS isoforms to the amount of NO recovered in expired air. In inflammatory lung conditions such as bronchiectasis or asthma, exhaled levels of airway NO (FENO) are known to be increased relative to the levels found in normal subjects, presumably because of a cytokine-mediated upregulation of NOS2 (1, 2). However, despite the inflammatory nature of cystic fibrosis (CF) lung disease, FENO values in CF patients have been reported to be either normal or low (3).

The importance of NOS1 as an isoform involved in NO homeostasis was demonstrated in mice lacking a functional nos1 gene. These mice had significantly lower FENO levels than did wild-type mice and were hyporesponsive to methacholine (MCh) challenge (6, 7). The concept that NOS1 contributes to FENO in human airways is also supported by a recent investigation in which variability of expired NO concentrations in individuals with stable asthma was related to an intronic AAT repeat polymorphism in the NOS1 gene (8). In the current study, we sought an association between this NOS1 gene polymorphism and FENO levels in patients with CF. We found that higher repeat numbers (i.e., both alleles  12 repeats) at this genetic locus were significantly associated with low FENO values; in addition, colonization with Pseudomonas aeruginosa and Aspergillus fumigatus was significantly more common in individuals with high AAT repeat numbers than in those with low repeat numbers (i.e., at least one allele < 12 repeats).

	METHODS

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Subjects

Seventy-five white patients (33 females and 42 males) with CF were included in the study. The diagnosis of CF was confirmed by repeated sweat tests with chloride concentrations > 60 mmol/L and by cystic fibrosis transmembrane regulator (CFTR) gene mutation analysis in all patients. Patients were 15 ± 0.8 (mean ± SEM) yr of age, had a mean FEV₁ of 68.4 ± 2.9%, and had a mean FVC of 78.0 ± 2.4% of predicted normal values, respectively, as assessed by spirometry. Forty- three of the patients with CF were homozygous for the F508 mutation, the most common CFTR gene mutation in white persons with CF; 15 other patients were compound heterozygous for the F508 mutation; and 17 patients had other CFTR mutations. The basic clinical characteristics of the total CF population, consisting of patients homozygous for the F508 mutation and all others (non-F508/F508), are presented in Table 1.

All subjects were studied when they were clinically stable; patients had to be without a pulmonary exacerbation of their CF, and without a need for intravenous antibiotics or systemic or inhaled steroids for at least 2 wk before their study visit. Pulmonary function tests and FENO measurements were performed during a routine visit to our outpatient department. Sputum was collected for microbiologic analysis. Written informed consent, including consent for genetic screening, was obtained from each patient or the patient's parents. The study was approved by the institutional review board of the University of Essen.

Measurement of FENO

FENO was recorded online as previously described (4, 9) and in accordance with American Thoracic Society guidelines (10). The seated subjects performed a slow VC maneuver through a mouthpiece, after inspiration to TLC. The mouthpiece was connected to a flow resistor (2-mm diameter at its open end) and a Fleisch 2 pneumotachograph (Fleisch, Lausanne, Switzerland), to achieve a flow rate of 0.1 to 0.2 L/s, and, via side ports, to a pressure transducer (Validyne, Northridge, CA), a carbon dioxide analyzer (Capnograph IV; Gould Medical BV, Bilthoven, The Netherlands), and a chemiluminescence NO analyzer (NOA 280; Sievers, Boulder, CO). NO was collected at a constant sample gas flow of 1.1 ml/s. The minimal detectable NO concentration was 1 ppb. The NO analyzer was calibrated before each study with a 195-ppb NO calibration gas (Linde AG, Unterschleissheim, Germany). Carbon dioxide levels, NO concentration, air flow, and mouth pressure were displayed on a four-channel chart recorder (Multi-Pen Recorder; Hugo Sachs Elektronic, March-Hugstetten, Germany) during each maneuver. NO plateau concentration were determined from published criteria. Each maneuver was performed three times with a short rest in between. The average FENO for each subject was reported.

Genetic Analysis

Genomic DNA was extracted by standard techniques (Genomic DNA Extraction Kit; Pharmacia, Piscataway, NJ) from each subject's blood. Alleles containing the intronic AAT repeat were amplified with the polymerase chain reaction (PCR). The primers used were 5'-TGC AGG AAC TAG GCA CAA GC-3' and 5'-GAT CGA CAC ACT TGT GCA GG-3' (11). Forward and reverse primers (20 pmol) were radiolabeled with [-³²p]deoxyadenosine triphosphate ([³²P]dATP) through the use of polynucleotide kinase (Boehringer Mannheim, Mannheim, Germany). The PCR reaction mixture contained 100 ng of genomic DNA; 1 µl of the radiolabled primer set; 200 µM dATP, deoxycytosine triphosphate, deoxyguanidine triphosphate, and deoxythymidine triphosphate; and 1.5 U of Taq polymerase in a 25-µl volume. PCR conditions were 6 min at 94° C, followed by 35 cycles of 94° C for 1 min, 59° C for 1 min, and 72° C for 1 min. Chain elongation was continued after the last cycle for 5 min. Simple sequence length polymorphism (SSLP) (12), as modified by ourselves, was used for allele assignment (13). Gels were run at room temperature at 60 W and were dried and exposed to X-ray film as required. The numbers of AAT repeats constituting the different alleles were assigned by sequencing (Applied Biotechnologies) from DNA minipreps (QIAprep Miniprep; Quiagene Inc., Valencia, CA), as recently described (13).

Pulmonary Function Testing

Pulmonary function tests were performed with a bell-spirometer (Volugraph; Mijnhardt, Bunnik, The Netherlands). Results were expressed as percentages of normal reference values (14, 15).

Statistics

Results were expressed as mean ± SEM. Statistical differences in expired NO between individuals homozygous for alleles with < 12 repeats, heterozygous (one allele with less than 12 and the other allele with at least 12 repeats), or homozygous for alleles with  12 repeats at the NOS1 locus were analyzed with analysis of variance and the Kruskal-Wallis test. Intergroup comparisons were made with Wilcoxon's test or the t test; depending on the metric distribution of data. The F test was used to assess differences in variance. Differences in proportions of colonized airways were determined with Fisher's exact test. A value of p < 0.05 was considered statistically significant.

	RESULTS

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Total CF Population

In the study cohort of 75 CF patients, we identified eight distinct alleles of the intronic AAT repeat in NOS1. The alleles were numbered according to the number of repeats (range: nine to 16 repeats). Allele frequencies, as displayed in Table 2, were similar to previously reported frequencies in healthy white subjects (13). FENO for the total group was 5.7 ± 0.7 (mean ± SEM) ppb; (range: 0.4 to 38.3 ppb), which is in the same range as previously reported values for CF patients (4, 8). Since the NOS1 alleles were distributed in a bimodal pattern, we segregated the patients into two groups on the basis of their allele complement. The group with high repeat numbers consisted of patients harboring two alleles with at least 12 repeats, whereas individuals in the group with low repeat numbers had at least one allele with less than 12 repeats. Since the data for the total study population were not equally distributed, differences in FENO levels across genotypes defined by repeat size were assessed with Wilcoxon's test. NO concentrations were significantly lower in patients with high-repeat alleles (n = 20) than in subjects with at least one low-repeat allele (n = 55) in NOS1 (4.0 ± 0.8 ppb versus 6.4 ± 0.9 ppb, p = 0.027). Variance around the mean was also significantly different between the groups (F_54;19 = 3.616, p = 0.003, F test).

CFTR F508/F508 Patients

The 43 patients who were homozygous for the most common disease-causing CFTR mutation in white persons (F508/ F508) were analyzed as a subgroup of the total CF patient population since they formed a genetically more homogenous CF population. The F508/F508 patients did not differ significantly from all other CF patients (non-F508/F508) except for having a better mean FEV₁ and MEF_50% (Table 1). The FENO in these patients was 4.4 ± 0.6 ppb. Differences among the groups having both alleles with < 12 AAT repeats (5.7 ± 1.6 ppb), one allele with < 12 AAT repeats (5.2 ± 0.8 ppb), and both alleles with  12 AAT repeats (2.3 ± 0.4 ppb) were statistically significant (p = 0.01, Kruskal-Wallis test). On the basis of these findings, we segregated the patients into two groups, one consisting of patients with both alleles having  12 AAT repeats (n = 14) and the other with at least one allele having < 12 repeats (n = 29). Mean NO was significantly lower in the group with high repeat numbers as compared with patients having low repeat numbers (2.3 ± 0.4 ppb versus 5.3 ± 0.7 ppb, p = 0.0006) (Figure 1). The distribution of expired NO for CFTR F508/F508 patients with high- or low-repeat- number alleles in NOS1 is shown in Figure 2. As already shown for the total group, variance around the mean was also significantly different between the two groups of CFTR F508/ F508 patients (F_27;13 = 4.692, p = 0.005, F test). No correlation was found between FENO and either FEV₁ or FVC.

View larger version (12K): [in this window] [in a new window]   Figure 1.   FENO in cystic fibrosis (CF) patients homozygous for the cystic fibrosis transmembrane regulator (CFTR) F508 mutation, stratified by the size of an intronic AAT-repeat polymorphism in the NOS1 gene. FENO was measured in parts per billion. Each symbol represents one individual. Horizontal lines show the mean of the respective group. Mean FENO was significantly lower in the group with both NOS1 alleles having more than 12 AAT repeats (high repeat numbers) than in individuals carrying at least one allele with fewer than 12 repeats (low repeat numbers) (p < 0.0006, t test).

Colonization of Airways

It has been speculated that colonization of airways with denitrifying pathogens such as P. aeruginosa may contribute to decreased NO levels in CF patients (16). A comparison of airway NO concentrations in P. aeruginosa-positive (n = 34) and P. aeruginosa-negative (n = 41) CF patients in the total study cohort revealed significantly lower NO concentrations in patients positive for P. aeruginosa (3.8 ± 0.5 ppb versus 7.4 ± 1.1 ppb, p = 0.0047). Airway NO was also lower in A. fumigatus-positive (n = 18) than in A. fumigatus-negative (n = 57) patients (4.3 ± 1.6 ppb versus 6.2 ± 0.7 ppb, p = 0.0054) (Table 3). Similar differences were observed in CFTR F508/F508 patients for both P. aeruginosa (2.9 ± 0.6 ppb [n=19] versus 5.5 ± 0.8 ppb [n = 24], p=0.017) and A. fumigatus (1.7 ± 0.5 ppb [n = 9] versus 5.0 ± 0.6 ppb [n = 34], p = 0.002) (Table 3). Furthermore, the relative proportion of all CF patients colonized with P. aeruginosa was significantly greater in the group with NOS1 alleles having high AAT repeat numbers (low NO levels) than in the group having alleles with low AAT repeat numbers (high NO levels) (65% versus 38%, p = 0.0358). Similarly, among the F508/F508 patients, the group with high repeat numbers was significantly more often colonized with both P. aeruginosa (71% versus 31%, p = 0.0147) and A. fumigatus (43% versus 10%, p = 0.0221) than was the group with low NOS1 AAT repeat numbers. No differences in airway colonization was observed for the non-F508/F508 patients (Table 4).

Interestingly, among the F508/F508 patients, a low FENO was not only associated with high AAT repeat numbers in NOS1 in terms of the overall group (Table 1), but also in terms of the patients not colonized with P. aeruginosa or A. fumigatus (Table 3). This suggests that differences in FENO between the groups stratified by NOS1 genotype were not due to differences in colonization of airways alone.

	DISCUSSION

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FENO values in patients with CF are known to be lower than FENO values found in normal subjects (3, 4). A number of factors, including retention of NO in airway secretions, bacterial consumption of NO, and decreased NO formation due to a lack of NO synthase expression have previously been discussed as contributing to low airway NO levels in CF (5, 16). In the present study, we established an association between the length of a repeat polymorphism in the NOS1 gene and FENO in patients with CF. High numbers of intronic NOS1 repeats are associated with low FENO levels and are also associated with colonization of the airways of CF patients with P. aeruginosa and A. fumigatus.

Neuronal NOS is encoded by a gene (NOS1) that has been mapped to chromosomal region 12q24. NOS1 contains a number of highly polymorphic repeats, including the intronic AAT repeat that we studied (17). It is unlikely that the identified alleles directly cause changes in NOS1 mRNA transcript generation, but the alleles associated with low FENO values could reflect a yet undetected DNA sequence variant in the nearby NOS1 coding region that influences the catalytic activity of the enzyme. Our data are compatible with the hypothesis that neuronal NO production is an important contributor to airway NO, which is in accord with recent findings of an association between the same NOS1 polymorphism that we examined and expired NO levels in asthma patients (8). Evidence for a contribution of neuronally derived NO to airway NO concentrations was first provided by a study with mice. Animals lacking a functional nos1 gene were shown to have 40% less NO in their lower airways than did wild-type mice (6). Furthermore, nos1 "knockout" mice were hyporesponsive to MCh challenge at baseline. More recently, the role of NOS1 in airway physiology was confirmed in a murine model of asthma. Airway responsiveness to MCh after allergen sensitization and challenge, was decreased in mice lacking nos1, whereas mice with a targeted deletion of nos2 had airway responsiveness similar to that of wild-type mice (7).

NOS1-derived NO is thought to be the main neurotransmitter of nonadrenergic, noncholinergic (NANC) nerves, and is involved in human bronchomotor control. Belvisi and coworkers had speculated, on the basis of immunohistochemical distribution patterns and functional aspects, that abnormalities in the inducible bronchodilatatory NANC system may contribute to the development of airway obstruction in CF (18). Indeed, we and others have previously demonstrated a positive correlation of FENO and sputum levels of NO metabolites with pulmonary function in CF patients (4, 19, 20). In the present study, we did not find an association of allele assignment at the NOS1 locus studied or of FENO with either FEV₁ or FVC, but the conclusions drawn from a single evaluation of pulmonary function test data at a stable time point may not accurately reflect pulmonary function as a measure of severity of lung disease in CF patients. The interaction of neuronal airway caliber control and inflammation is complex. Genetic variations of NOS1 may not account for differences in CF airway obstruction, but since NOS1 has been shown to regulate iNOS (NOS2) expression (21), these variations may influence NO-related aspects of airway inflammation.

Although NOS1 has been shown to be constitutively expressed in human airway epithelial cells (22), antimicrobial activity is usually believed to be related to a different isoform of NOS: NOS2, or iNOS. This enzyme has been shown to be important in controlling fungal, viral, and bacterial organisms (23). Recent studies have suggested that reduced expression of NOS2 in CF airway epithelial cells may play an important role in the impaired antimicrobial activity of CF airways (24, 25). Kelley and Drumm (25) showed that the ability of wild-type mice to resist infections with P. aeruginosa was reduced after suppression of NOS2 with a specific inhibitor. Furthermore, lipopolysaccharide treatment of CF (F508/F508) mice, in contrast to wild-type mice, did not result in an increase in airway NO formation. Kelley and Drumm concluded from their experiments that decreased expression of NOS2 in CF airways may predispose to infections with P. aeruginosa (25). We speculate, on the basis of our data, that differences in the formation of NO by NOS1 could influence antimicrobial activity in CF airways. Effects could either occur directly, through NOS1 expressed in airway epithelial cells, or, as mentioned earlier, from an NOS1-NOS2 interaction.

A number of factors may contribute to the low and variable FENO levels in CF, including retention of NO in airway secretions (20) or consumption of NO by the enzyme NO-reductase, which is expressed in P. aeruginosa (16). The reported evidence for consumption of NO by P. aeruginosa would be consistent with our findings of decreased NO concentrations in airways colonized with this pathogen. However, differences in NO levels between individuals with high and low intronic AAT repeat numbers in NOS1 were also seen for patients not colonized with P. aeruginosa. These data suggest that the observed differences in P. aeruginosa and A. fumigatus positivity between the groups in our study may not have been the consequence of, but rather a factor predisposing to the colonization of airways.

In conclusion, we provide evidence that allele assignment at an intronic repeat polymorphism in the NOS1 gene is associated with exhaled NO levels in patients with CF. Patients with high AAT repeat numbers ( 12) at the relevant locus are more likely than others to have low airway NO concentrations and airway colonization with P. aeruginosa and A. fumigatus. This is the first study to show that genetic variants in NOS genes may modify characteristics of CF lung disease.