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Centre de Ressources et de Compeetences en Mucoviscidose, Laboratoire d'Explorations Fonctionnelles Reenales, Service de Biochimie A, Service d'ORL
Centre d'Investigation Clinique, Hpital Necker-Enfants Malades
INSERM U 467, Universitee Paris 5
Service d'Histologie-Biologie Tumorale, Hpital Tenon, Paris
Service de Biochimie-Geeneetique, Hpital Henri Mondor
INSERM U 468, Creeteil, France
ABSTRACT
Studying subjects heterozygous for mutations of the cystic fibrosis (CF) gene may help clarify the impact on disease onset of CF transmembrane conductance regulator protein (CFTR-)eCdependent chloride secretion. CFTR-mediated chloride transport was evaluated in 52 heterozygous subjects, 32 healthy control subjects, and 77 patients with CF with class I or II mutations. We measured the change in nasal potential difference in response to chloride-free isoproterenol solution for each subject and used a video-imaging fluorescent dye assay to assess the percentage of nasal ciliated cells with cAMP-dependent anion conductance. Our findings did not confirm the standard assumption that heterozygosity implies 50% of normal CFTR function. Half the heterozygous subjects had CFTR-mediated chloride transport levels below 50% of the normal range, and one-third had levels similar to those of the patients with CF. This reduced CFTR function was not associated with an elevated prevalence of CF-like symptoms in heterozygous subjects but was highly related to respiratory status in the patients with CF. These data suggest that CFTR-dependent chloride conductance does not directly modulate disease severity but may be part of a more global defect in patients with CF involving other CFTR functions or currently unknown modulatory factors.
Key Words: cystic fibrosis transmembrane conductance regulator heterozygosity nasal potential difference
Cystic fibrosis (CF) is caused by mutations in the gene encoding for the CF transmembrane conductance regulator (CFTR) protein, which acts as a chloride channel after activation by cyclic AMP (cAMP) (1). Classic CF is characterized by chronic bacterial infection in the airways, fat maldigestion caused by pancreatic exocrine insufficiency, and elevated chloride concentrations in sweat (2). More than 1,300 mutations have been described. Premature termination signals (class I mutations) and abnormal protein folding (class II mutations, including F508, the most common CFTR mutation) are characterized by the absence of CFTR protein at the apical membrane, whereas class III, IV, and V mutations produce proteins that are inserted in the apical membrane but either do not function normally (class III and IV) or result in significantly decreased production of normal CFTR (class V) (3). However, although pancreatic insufficiency is associated with class I, II, and III mutations, there is no correlation between genotype and severity of pulmonary disease (4).
In a previous study, we found residual cAMP-dependent chloride conductance in the nasal ciliated cells (NCC) of F508 homozygous patients with mild lung disease (5). This finding led us to wonder whether clinical heterogeneity in patients with CF might be linked to heterogeneity in CFTR function. Studying heterozygous subjects for CFTR mutations may help clarify the relation between CFTR function and onset of CF symptoms because, in this population, the mutation's clinical expression is not obscured by the secondary consequences of the disease (6). Furthermore, heterozygous subjects with CF are known to be overrepresented among patients with isolated features of the classic CF phenotype, such as asthma (7), disseminated bronchiectasis (8), allergic bronchopulmonary aspergillosis (9), chronic sinusitis (10), chronic pancreatitis (11), congenital bilateral absence of the vas deferens (12), and elevated sweat chloride concentrations (13).
CFTR functioning in heterozygous subjects is generally assumed to be half of the normal level, which is believed to be sufficient to remain disease-free (6, 14). Two studies of heterozygous subjects with isolated CF symptoms, however, reported low CFTR-dependent chloride levels (15, 16). They suggested that a single mutation might be sufficient to reduce CFTR function to the range found in patients with CF and that this may, in turn, be related to the expression, albeit limited, of a CF phenotype (17).
This study investigated CFTR-mediated chloride transport in nasal epithelium with nasal potential difference (NPD) measurements and a halide-sensitive fluorescent dye assay to determine whether the level of this chloride secretion is related to clinical expression of CF in a population of heterozygotes and patients with CF with class I and class II CFTR mutations.
METHODS
Subjects
All patients with CF had class I or class II mutations on both alleles. The heterozygous subjects, also carrying a class I or II mutation, were either parents or siblings of patients with CF with known mutations. Control subjects had no family history of CF and were asymptomatic. Before inclusion, they were screened and found negative for the 30 CFTR mutations most prevalent in the French population. The study was approved by the Necker-Enfants Malades Ethics Committee, and written, informed consent was obtained from each adult and parent.
Procedures
We recorded the medical history and performed a chest film and pulmonary function tests for each subject and assessed the Shwachman clinical score for patients with CF (18).
Sweat chloride concentrations were determined by conductivity with the Sweat-Chek conductivity analyzer (J2L Elitech; Wescor, Labarthe Inard, France) (19). Because sweat conductivity methods yield sodium chloride values approximately 15 mEq/L higher than pad-absorption iontophoresis methods, chloride concentrations above 70 mEq/L were defined as abnormal and those between 50 and 70 mEq/L as borderline.
We used the procedure of Knowles and collaborators (20) to determine NPD. Baseline NPD was measured after perfusion of nasal epithelium with saline solution. NPD changes were recorded after perfusion with the following solutions: 100 e amiloride in saline solution (amiloride), chloride-free solution with 100 e amiloride (chloride-free), and 100 e amiloride plus 10 e isoproterenol in chloride-free solution (isoproterenol). The sum of chloride-free and isoproterenol (chloride-freeeCisoproterenol) served as an index of transepithelial CFTR-dependent chloride transport because it reflects the cAMP activation of nasal mucosa chloride permeability.
We used a halide-sensitive video-imaging assay with fluorescent dye 6-methoxy-N-3'-sulfopropylquinolinium (SPQ) to measure chloride transport at the cellular level, as described by Verkman (21). After loading the cells with 10 mM SPQ (Molecular Probes Europe, Leiden, The Netherlands), the cells were continuously perfused with the isotonic NaI solution, followed by the NaNO3 solution, to which cAMP agonists were finally added (5). Details on this method are available in the online supplement. The maximum change in fluorescence rate after addition of cAMP agonists was defined as cAMP-dependent anion conductance (FcAMP/t) and the CFTR-mediated cAMP-stimulated anion conductive pathway was considered present when FcAMP/t was greater than 0.1 (Figure 1). At least five NCC were studied for each experiment, and the SPQ assay endpoint was the percentage of cells with cAMP response (%cAMP) in a given experiment. Details on this method are available in the online supplement.
CFTR transcript levels were analyzed in nasal cells by reverse transcriptaseeCpolymerase chain reaction as previously reported (5). CFTR mRNA content was expressed as the ratio of the densitometric value of CFTR amplification product to that of 2-microglobulin amplification product.
The variable IVS8 (TGmTn) sequence at 1342eC12 was investigated in heterozygous individuals to ascertain if the IVS8eC5T variant might be related to a defect in CFTR functioning. This test was performed either by direct sequencing or by denaturing gradient gel electrophoresis of a fragment containing exon 9 and its surrounding sequences (22). Each DGGE pattern obtained could be allocated to a specific combination of TGmTn.
Statistical Analysis
Data are presented as mean (SEM) for n subjects. Groups were compared with an unpaired t test or with the Mann-Whitney U test if there were fewer than 30 subjects per group. Because CFTR functioning in heterozygous subjects is generally assumed to be 50% of normal, the results of these assays were compared with 50% of the corresponding mean value for control subjects, a value representing a 50% reduction of normal CFTR function. Correlation coefficients were calculated with simple regression analysis or, if there were fewer than 30 values, with Spearman's rank correlation test. A p value less than or equal to 0.05 was considered statistically significant.
RESULTS
Subjects' Clinical Characteristics and Genotypes
The study included 52 heterozygous subjects, 32 control subjects, and 77 patients with CF (Table 1). Patients had the following mutations: F508/F508 (n = 46), class I/class II (n = 22), or class I on both alleles (n = 9). Most of the mutations observed in heterozygous subjects were class II: F508 (n = 40) and N1303K (n = 2) mutations. Ten had class I mutations: 3659delC, 1078delT, 3791delC, 1717eC1G>A, 2183AA>G, S466X, W1282X, R553X, or G542X (n = 2). Five of the 52 heterozygous subjects had possible CF-related symptoms: three had chronic sinusitis and two had asthma. All heterozygous and control subjects had normal chest films and pulmonary function tests. Forty-four patients had chronic Pseudomonas aeruginosa colonization.
Sweat Test
Sweat chloride concentrations were measured in all heterozygous and control subjects and a sample of 20 patients with CF (Table 1). The mean level for the heterozygote population was significantly higher than for the control group: 51 (range, 27eC109) mEq/L compared with 41 (range, 30eC55) mEq/L (p = 0.01). Twenty-seven heterozygous subjects had borderline sweat chloride concentrations, ranging from 50 to 70 mEq/L, but they had no clinical symptoms. One heterozygous subject had a sweat chloride concentration of 109 mEq/L and moderate asthma. An extensive gene search did not reveal a second CFTR mutation.
Functional Assays
Measurement of NPD.
Neither the baseline nor the amiloride NPD values of heterozygous and control subjects differed significantly, but patients' baseline NPD was significantly more negative and they had significantly greater depolarization after amiloride (Table 1, lines 6 and 7; Figures 2A and 2B). After chloride-free superfusion and after isoproterenol, NPD response for the three groups overlapped significantly (Figures 2C and 2D). Nevertheless, change in the voltage response was significantly lower among heterozygotes than among control subjects and was minimal or absent in the patient group (Table 1, lines 8, 9, and 10; Figures 2C, 2D, and 2E). Among the heterozygous subjects, the five symptomatic subjects did not differ from the others in their NPD results.
The chloride-freeeCisoproterenol response in heterozygous subjects was compared with 50% of its mean absolute value in the control group (i.e., |eC7.5| mV; from Table 1, line 10), a value that reflects 50% of the normal CFTR-regulated cAMP-dependent chloride secretion. After perfusion of isoproterenol in chloride-free solution, repolarization of less than |eC7.5| mV was observed for 17 heterozygous subjects (46%; Figure 2E). In this group, the mean chloride-freeeCisoproterenol was eC4.3(0.4) mV versus eC12.5(1.2) mV for the patients with repolarization greater than |eC7.5| mV. These subjects did not differ from the other heterozygous subjects for the prevalence of isolated CF-like symptoms, the results of respiratory function tests, or sweat chloride concentrations. This pattern did not change when the analysis was restricted to F508 heterozygotes.
In the patient group, we focused on the isoproterenol response because the low chloride response may vary with natural hormonal influences. The magnitude of this response was compared with 50% of its mean absolute value in the control group (i.e., |eC2.5| mV; from Table 1, line 9). Six patients with CF (16%) responded to isoproterenol with a voltage change of |eC2.5| mV or greater, thereby demonstrating the persistence of more than 50% of the CFTR-dependent chloride permeability. They had better respiratory function tests and Shwachman scores than the patients with isoproterenol values of less than |eC2.5| mV (Table 2).
SPQ Fluorescent Dye Video-imaging Assay.
The heterozygous subjects and the patients had overlapping %cAMP values (Figure 3). For the control subjects, the lowest proportion of NCC demonstrating a cAMP-dependent conductance was 50%, whereas heterozygous subjects and patients with CF had responses scattered between 0 and 100% (Table 1, line 11; Figure 3). Results did not differ between the symptomatic and nonsymptomatic heterozygous subjects. This pattern of results did not change when the analysis was restricted to the F508 carriers and the F508 homozygous patients. Nine of the 17 heterozygous subjects investigated with the SPQ assay (52%) had cAMP-mediated response in less than 50% of their NCC. Eight of the 17 heterozygous subjects had %cAMP levels less than or equal to the mean patient response (i.e., 38%), and four had no cells showing a cAMP-dependent conductive pathway (Figure 3). These findings reveal that CFTR function was reduced by more than 50% in half of the subjects investigated and that one-third had levels similar to those of the patients with CF. The heterozygous subjects with %cAMP of less than 50% did not differ from those with %cAMP of 50% or greater for CF-related symptoms, sweat chloride concentrations, or respiratory function tests.
%cAMP greater than 50% was observed in 31 patients (40%). Their clinical status was less severe than that of the other patients in terms of respiratory status, number of bronchial exacerbations requiring antibiotics, and Shwachman score (Table 2).
%cAMP was significantly correlated with isoproterenol (r = 0.46, p = 0.01) but not with amiloride (r = 0.32, p = 0.08) or chloride-free (r = 0.15, p = 0.43; Spearman correlation test; results based on the entire study population; Figure 4).
Intron 8Tn Sequence Analysis
Forty heterozygous subjects underwent a functional exploration either by NPD test or the SPQ assay. Nineteen heterozygous subjects agreed to provide a blood sample to investigate the IVS8 (TGmTn) genotyping pattern. None carried the 5T allele. Nine had either chloride-freeeCisoproterenol or %cAMP below 50% of the control value. Of the 21 heterozygous subjects who could not be tested, we would expect only one to carry the 5T allele, considering its 5% frequency in the general population (12). However, in this group, 15 subjects had CFTR function reduced below the 50% level according to either the NPD test or the SPQ assay. These data suggest that the presence of 5T is not related to the function of CFTR.
CFTR Expression
CFTR mRNA levels did not differ between heterozygous subjects and patients with CF (Table 1). The transcript levels were not correlated with either isoproterenol or %cAMP.
DISCUSSION
Few studies have investigated CFTR function in carriers of CFTR mutations. Wilson and colleagues (23) reported greater sodium transport inhibition among them, whereas Knowles and colleagues (24) found PD values similar to those of a control group. Both studies involved small heterozygote samples. This is the first large-scale study to investigate CFTR function in individuals heterozygous for a CFTR mutation compared with two control groups, one healthy and one composed of patients with CF. We found significantly lower cAMP-mediated conductance in the nasal epithelium of heterozygous subjects with class I or II mutations. Our study of a large sample shows that CFTR-mediated chloride transport appears to be reduced by more than 50% in about half of the heterozygous subjects investigated and reaches levels similar to those in one-third of patients with CF. This pattern of results was similar when the analysis was restricted to the F508 mutation. These results are consistent with another study of carriers of CFTR mutations and obligate heterozygous subjects, which also found significant alterations in CFTR ion channel function (25).
Investigation of CFTR-dependent CleC Conductance in Nasal Epithelium
The cAMP-activated CleC conductance in the nasal epithelium was explored with measurements from two functional tests: chloride-freeeCisoproterenol for NPD and %cAMP for the SPQ video-imaging assay. It can be assumed that these tests reflect the cAMP-stimulated CleC transport mediated by CFTR because our experimental protocols exclude the other cAMP-dependent CleC-conductive pathways present in the nasal epithelium (i.e., the Ca2+ and the volume-mediated conductance pathways) (26). Specifically, we excluded the Ca2+-induced response to isoproterenol reflected by a rapid rise in NPD that fades to baseline over 2 to 3 minutes. We considered only the steady-state value obtained after 3 to 5 minutes of chloride perfusion, which represents the cAMP-CFTReCmediated CleC conductance (24). For SPQ assay, the cells were left to settle for 30 minutes, long enough to deactivate the cAMP-stimulated volume-dependent anion conductance.
The American Pediatric Society consensus statement on CF has validated the use of the NPD to identify CF in patients with borderline sweat tests and only one identified mutation (27). The principal disadvantage of NPD, however, is that nonspecific epithelial trauma, such as rhinitis or allergy, can damage the epithelial barrier and blunt all bioelectrical parameters. The SPQ video-imaging assay avoids this pitfall because it evaluates the chloride conductive pathway at the single-cell level, whereas NPD reflects all of the isoproterenol-activated conductive pathways across the entire epithelium (24). It must, nonetheless, be stressed that the SPQ assay requires high technology available only in specialized laboratories. The strong correlation between isoproterenol and %cAMP observed in our study suggests that the SPQ video-imaging test may be a useful tool for studying the CleC channel CFTR function. This assay is currently used in CF gene therapy trials and clinical research assessment (5, 28eC30).
Variability of CFTR Activity in Normal Nasal Epithelium
Our results show that the cAMP-dependent CleC conductance pathway can vary markedly in the NCC of control subjects without CF and can be quite low regardless of the assay. This suggests that even a low level of functional protein is sufficient to offer protection from symptoms of CF disease. These results are consistent with previous studies demonstrating that the CFTR gene is expressed at low levels (1eC2 transcripts/cell) in the epithelium of the nose, trachea, and bronchi (31) in normal, healthy individuals, and that this low level of normal CFTR is sufficient for restoration of normal airway epithelial function in gene transfer experiments (32, 33).
Residual CFTR Activity Is Related to Mild Pulmonary Disease in Patients with CF
Approximately one-third of the patients with CF showed a significant CFTR-dependent response for either the NPD test or the SPQ assay. This residual CFTR activity seems to be clinically relevant because these patients' pulmonary status is significantly better. This finding is consistent with those of previous studies that have documented mild pulmonary phenotypes in patients with residual CFTR functioning (5, 28, 34eC36).
Reduction of CFTR Function in Heterozygous Subjects and Pathophysiologic Hypotheses
The loss of CFTR function in heterozygous subjects is not associated with either increased prevalence of isolated CF-related clinical symptoms or higher sweat chloride concentrations. CFTR-mediated chloride transport at the range seen in patients with CF thus does not appear to be related per se to clinical symptoms. A growing body of evidence suggests that mechanisms other than those related to chloride secretion may underlie chronic obstructive lung disease in patients with CF (37). Possibilities include hyperactivity of the amiloride-sensitive epithelial sodium channel (38), alterations in intracellular processes, reduced host defenses, and persistent inflammatory response (39). On the other hand, our results show that residual CFTR-dependent chloride transport in patients with CF is positively correlated with better clinical condition. The explanation for the apparent contradiction between the findings for heterozygous subjects and for patients with CF may be that the cAMP-dependent chloride transport is not directly correlated with CF disease severity but rather reflects a more global defect, involving other CTFR functions or other currently unknown modulatory factors.
Molecular Basis of CFTR Function Reduction in Heterozygous Subjects
Several hypotheses may explain how heterozygosity for CF mutations is linked to CF-like functional defects. We can rule out the hypothesis of a second undetected CFTR mutation in our group of heterozygous subjects, because they are obligate heterozygotes. Modulation of the phenotype by the genotype can be excluded because we reach similar conclusions when the analysis is restricted to the homozygous F508 patients. Nor is a lower rate of CFTR transcription a plausible explanation, as we did not observe any correlation between it and the response to isoproterenol in NPD testing or to cAMP in the SPQ assay.
We analyzed the variable intron 8 (TG)mTn sequence to determine whether the 5T allele, in combination with a CF mutation, might be associated with reduced chloride secretion. Because none of the 19 heterozygotes tested carried the 5T allele and nine of them had CFTR function reduced below 50%, differential splicing of exon 9 cannot explain this reduction. A more likely explanation for this modulation of the impaired CFTR-dependent chloride transport in heterozygous subjects is that the combination of one mutation with CFTR polymorphisms or with specific alleles of modifier genes modifies a post-translational step: for example, altering biosynthesis of the CFTR protein, decreasing its stability in the plasma membrane, or directly downregulating cAMP-dependent chloride secretion (40).
In conclusion, our results provide evidence of a defect in the CFTR-regulated chloride conductive pathway in heterozygous subjects, sometimes at levels similar to those seen in patients with CF. This reduced function is correlated with better clinical condition in patients with CF but does not seem to be clinically relevant in subjects who are heterozygous. Taken together, these results point to the need for large-scale studies of CF carriers and highlight the potential importance of alternatives to reduced CFTR-dependent chloride transport as a mechanism for CF pathogenesis (41).
Acknowledgments
The authors thank all the children and parents who took part in the study as well as Delphine Bataille and Mireille Blonde for technical assistance.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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