METHODS

Patients

White adult patients with CF and healthy volunteers without recent respiratory tract infections, corticosteroid medication, or tobacco use participated in the study. The diagnosis of CF was based on medical records, repeated sweat chloride tests, and the identification of CFTR gene mutations. Patients with one or two CFTR mutations of classes 4 or 5 were classified in the “mild” CFTR genotype group whereas patients with two mutations of classes 1, 2 or 3 were considered as having “severe” CFTR genotype.¹

Annual spirometric data (collected from the 5 years before study entry) were used to calculate annual rate of decline (based on a mean of five measurements) of forced expiratory volume in 1 second (FEV₁) in each patient (simple linear regression, accepted R² value >0.30).

The study was approved by the local ethical committee and written informed consent was obtained from all subjects.

Exhaled NO measurements

Exhaled NO was measured using a chemiluminescence analyser (NOA™ 280, Sievers, Boulder, CO, USA), sampled in triplicate at a controlled outflow of 100 ml/s, and the mean values recorded according to international guidelines.⁵

NOS1 genotype determination

Genomic DNA extracted from blood mononuclear cells was used for polymerase chain reaction (PCR) amplification (95°C for 1 minute, 40 cycles of 95°C, 10 s; 67°C, 30 s; 68°C, 12 s; final extension at 68°C for 2 minutes) with 5-CCTGCGTGGCTACTACATTC-3 (forward) and 5-TGGGTGTGGGGAGGGAGAC-3 (reverse) primers. After purification (Qiaquick PCR purification kit, Qiagen Inc), PCR products were sequenced to determine the number of GT repeats in the polymorphic region. PCR products from homozygous subjects were separated by electrophoresis in 3% agarose gels and used as standards to determine the number of repeats in the PCR products obtained from subsequent individuals.

Statistical analysis

Data were expressed as mean (SD) or percentage with 95% confidence intervals (CI). To assess the combined effect of both alleles on outcome we categorised the alleles above and below the median of GT repeats (27) and the genotypes into three classes (0, 1 or 2 alleles containing more than 27 GT repeats). Mean NO levels and FEV₁ were compared between groups by one way or two way analyses of variance. The Kolmogorov-Smirnov goodness of fit test was used to ascertain that the data were normally distributed before the analysis. Distributions were compared using χ² analysis; p values of <0.05 were considered statistically significant.

RESULTS

Fifty nine volunteers (39 men) of mean (SD) age 37.4 (2.3) years and 59 adult patients with CF were studied (table 1). Nineteen NOS1 alleles were identified, the number of GT repeats in the 5′-UTR region ranging from 18 to 36. NOS1 genotype distribution was similar in both groups with 24%, 54% and 22% of controls and 27%, 41% and 32% of patients displaying 0, 1, and 2 alleles, respectively, with more than 27 repeats (χ² = 2.401, NS).

Exhaled NO values were (plausibly) normally distributed (Z = 1.284, NS). Global comparison by one way ANOVA showed a significant association between NOS1 genotype classes and exhaled NO (F = 5.078, p = 0.008; fig 1). Two way ANOVA showed that the effect of the number of GT repeats on exhaled NO was independent of the group (controls or patients, p = 0.003). Interaction between group and genotype did not reach statistical significance (p = 0.08).

Annual decline in FEV₁ was (plausibly) normally distributed among patients (Z = 1.211, NS). The retrospective analysis of lung function during the 5 years preceding the study showed different trends according to NOS1 genotype class (p = 0.025) which could not be explained by other clinical parameters (table 1). The annual percentage loss of FEV₁ was 3.3% (95% CI 1.1 to 5.4), 3.2% (95% CI 2.4 to 4.0), and 0.8% (−0.5 to 2.1) for patients with 0, 1, and 2 alleles with >27 repeats, respectively.

DISCUSSION

Over a 5 year period patients with CF displaying more than 27 GT repeats in the 5′-UTR of each NOS1 allele had a lower annual FEV₁ loss than patients with 27 repeats or less in at least one allele.

Previous studies have indicated that the NOS1 locus affects lung disease in patients with CF,⁶ although the pathophysiological basis for this association remains elusive. Expression of NOS1 is dynamically regulated by changes in gene transcription occurring via alternative splicing within the 5′-UTR.⁴ Because the GT polymorphism investigated here occurs immediately upstream of the transcription start site of NOS1, it might affect the binding affinity of transcription factors⁷ which target DNA binding sites located in the vicinity of the repetitive sequence. The number of GT repeats of NOS1 correlated with exhaled NO concentrations, as reported in previous studies of NOS1 polymorphism affecting intronic regions.^6,8 Correlation, however, might be partially blunted by factors affecting exhaled NO measurements in CF—for example, altered NO diffusion across the thickened airway mucosal barrier, increased NO consumption by denitrifying bacteria, and combination of NO with reactive oxygen species.⁹

Although CF is a monogenic disease, previous studies have suggested that additional genes could modulate its clinical outcome.¹ Variations in exhaled NO concentrations, associated with an intronic repeat polymorphism of NOS1, were found to modulate chronic colonisation of the airway with Pseudomonas aeruginosa and Aspergillus fumigatus.⁶ This association was not found in our study, probably because of the high prevalence of chronic bronchial colonisation in this cohort of adult patients. In addition, except for a decline in lung function, no clinical differences were seen between the three NOS1 genotype groups (table 1). Other parameters which are not readily accessible to clinical investigation such as transepithelial ion transport, bronchomotor tone, or pulmonary inflammation¹⁰ can vary as a function of NO production, thus affecting the mechanisms controlling lung function decline in CF patients.

Although our results are preliminary and limited by the size of the investigated population, they show an association between the NOS1 gene locus and progression of lung disease in patients with CF which is independent of the CFTR genotype. These findings suggest that NOS1 variants leading to reduced NO production might be important for understanding the phenotypic disparities of patients with the same CFTR mutations. Further investigations are needed to establish the biological consequences of this repeat polymorphism on NOS1 function.