Four adult patients with the missense mutation L206W and a mild cystic fibrosis phenotype

We report molecular and clinical analyses in four unrelated patients with cystic fibrosis (CF) with compound heterozygosity for the L206W mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR). This uncommon missense mutation (frequency less than 1% in a sample of 336 CF chromosomes from Southern France) replaces a leucine by a tryptophan residue in the middle of the third transmembrane domain of CFTR. On the basis of the clinical features presented by the four patients, we postulate that the L206W might be associated with pancreatic sufficiency and residual transmembrane transport of chloride in lung.     

CFTR: domains, structure, and function

Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF) (Collins, 1992). Over 500 naturally occurring mutations have been identified in CF gene which are located in all of the domains of the protein (Kerem et al., 1990; Mercier et al., 1993; Ghanem et al., 1994; Fanen et al., 1992; Ferec et al., 1992; Cutting et al., 1990). Early studies by several investigators characterized CFTR as a chloride channel (Anderson et al.; 1991b,c; Bear et al., 1991). The complex secondary structure of the protein suggested that CFTR might possess other functions in addition to being a chloride channel. Studies have established that the CFTR functions not only as a chloride channel but is indeed a regulator of sodium channels (Stutts et al., 1995), outwardly rectifying chloride channels (ORCC) (Gray et al., 1989; Garber et al., 1992; Egan et al., 1992; Hwang et al., 1989; Schwiebert et al., 1995) and also the transport of ATP (Schwiebert et al., 1995; Reisin et al., 1994). This mini-review deals with the studies which elucidate the functions of the various domains of CFTR, namely the transmembrane domains, TMD1 and TMD2, the two cytoplasmic nucleotide binding domains, NBD1 and NBD2, and the regulatory, R, domain.     

Effect of cystic fibrosis-associated mutations in the fourth intracellular loop of cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) contains multiple membrane spanning sequences that form a Cl- channel pore and cytosolic domains that control the opening and closing of the channel. The fourth intracellular loop (ICL4), which connects the tenth and eleventh transmembrane spans, has a primary sequence that is highly conserved across species, is the site of a preserved sequence motif in the ABC transporter family, and contains a relatively large number of missense mutations associated with cystic fibrosis (CF). To investigate the role of ICL4 in CFTR function and to learn how CF mutations in this region disrupt function, we studied several CF-associated ICL4 mutants. We found that most ICL4 mutants disrupted the biosynthetic processing of CFTR, although not as severely as the most common DeltaF508 mutation. The mutations had no discernible effect on the channel's pore properties; but some altered gating behavior, the response to increasing concentrations of ATP, and stimulation in response to pyrophosphate. These effects on activity were similar to those observed with mutations in the nucleotide-binding domains, suggesting that ICL4 might help couple activity of the nucleotide-binding domains to gating of the Cl- channel pore. The data also explain how these mutations cause a loss of CFTR function and suggest that some patients with mutations in ICL4 may have a milder clinical phenotype because they retain partial activity of CFTR at the cell membrane.     

Flat adenoma: are western colonoscopists careful enough?

There has been recent interest in the risk of various cancers in cystic fibrosis (CF) patients and carriers of cystic fibrosis transmembrane conductance regulator (CFTR) mutations. It has been proposed that a CFTR mutation may protect against breast cancer, based on evidence that elevated extracellular adenosine triphosphate (ATP) is known to inhibit breast cancer cell line growth and that CFTR pumps ATP out of epithelial cells. A CFTR mutation would therefore result in higher concentrations of serum ATP. A CFTR knockout mouse model had high serum concentrations of ATP and showed reduced breast tumour implantibility and decreased breast cancer growth rates. We have evaluated the relationship between the deltaF508 CFTR mutation and the risk of breast cancer before the age of 40. The deltaF508 CFTR mutation carrier rate in 272 cases (2.2%) was no different from the carrier rate observed in 171 controls (1.8%). If there was a protective effect resulting from the postulated elevation in serum ATP levels, tumours arising in deltaF508 CFTR carriers would have been expected to be generally less aggressive. When the histological features of the breast cancers with a deltaF508 CFTR mutation were reviewed and graded using a combined architectural and cytological grading system, all were found to be grade III, poorly differentiated tumours, contrary to the predictions. A combination of our data with other large population-based samples of cases and controls is required to resolve this issue.     

Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel, but its relationship to the primary clinical manifestation of CF, chronic Pseudomonas aeruginosa pulmonary infection, is unclear. We report that CFTR is a cellular receptor for binding, endocytosing, and clearing P. aeruginosa from the normal lung. Murine cells expressing recombinant human wild-type CFTR ingested 30-100 times as many P. aeruginosa as cells lacking CFTR or expressing mutant DeltaF508 CFTR protein. Purified CFTR inhibited ingestion of P. aeruginosa by human airway epithelial cells. The first extracellular domain of CFTR specifically bound to P. aeruginosa and a synthetic peptide of this region inhibited P. aeruginosa internalization in vivo, leading to increased bacterial lung burdens. CFTR clears P. aeruginosa from the lung, indicating a direct connection between mutations in CFTR and the clinical consequences of CF.     

Purified cystic fibrosis transmembrane conductance regulator (CFTR) does not function as an ATP channel

The gene mutated in cystic fibrosis codes for the cystic fibrosis transmembrane conductance regulator (CFTR). Previously, we provided definitive evidence that CFTR functions as a phosphorylation-regulated chloride channel in our planar lipid bilayer studies of the purified, reconstituted protein. Recent patch-clamp studies have lead to the suggestion that CFTR may also be capable of conducting ATP or inducing this function in neighboring channels. In the present study, we assessed the ATP channel activity of purified CFTR and found that the purified protein does not function as an ATP channel in planar bilayer studies of single channel activity nor in ATP flux measurements in proteoliposomes. Hence, CFTR does not possess intrinsic ATP channel activity and its putative role in cellular ATP transport may be indirect.     

An approach for treating the hepatobiliary disease of cystic fibrosis by somatic gene transfer

Cystic fibrosis (CF) is an inherited disease of epithelial cell ion transport that is associated with pathology in multiple organ systems, including lung, pancreas, and liver. As treatment of the pulmonary manifestations of CF has improved, management of CF liver disease has become increasingly important in adult patients. This report describes an approach for treating CF liver disease by somatic gene transfer. In situ hybridization and immunocytochemistry analysis of rat liver sections indicated that the endogenous CFTR (cystic fibrosis transmembrane conductance regulator) gene is primarily expressed in the intrahepatic biliary epithelial cells. To specifically target recombinant genes to the biliary epithelium in vivo, recombinant adenoviruses expressing lacZ or human CFTR were infused retrograde into the biliary tract through the common bile duct. Conditions were established for achieving recombinant gene expression in virtually all cells of the intrahepatic bile ducts in vivo. Expression persisted in the smaller bile ducts for the duration of the experiment, which was 21 days. These studies suggest that it may be feasible to prevent CF liver disease by genetically reconstituting CFTR expression in the biliary tract, using an approach that is clinically feasible.     

Mucociliary clearance in cystic fibrosis knockout mice infected with Pseudomonas aeruginosa

In this study, we examined whether mucociliary clearance differed between cystic fibrosis (CF) knockout mice and wildtype controls. Additionally, we investigated whether infection with Pseudomonas aeruginosa, a common pathogen in the CF lung, affected this important host defence mechanism. Ciliary beat frequency (fcb) and particle transport (PT) were recorded using an in vitro lung explant preparation. Measurements were made from uninfected cystic fibrosis transmembrane conductance regulator (CFTR) knockout (-/-) mice and littermate controls (+/+) and compared to measurements from infected animals. While there were no differences detectable in fcb between CFTR -/- mice and their +/+ controls either in the presence or absence of P. aeruginosa, PT rates were different between these groups; interestingly, PT rates appeared dependent on both CFTR and infection status, with uninfected CFTR +/+ animals demonstrating higher rates of PT than their -/- littermates, while CFTR +/+ P. aeruginosa-infected mice demonstrated lower PT than knockout mice. These data demonstrate differences in mucociliary clearance between cystic fibrosis transmembrane conductance regulator knockout mice and controls, and further that Pseudomonas aeruginosa infection affects mucociliary clearance in the peripheral airways of mice. Additionally, the observed differences in particle transport suggest that cystic fibrosis transmembrane conductance regulator knockout mice demonstrate different mucociliary responses to infection.     

Understanding how cystic fibrosis mutations cause a loss of Cl- channel function

Defective epithelial Cl- secretion is the hallmark of the lethal genetic disease cystic fibrosis (CF). This abnormality is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a regulated Cl- channel. Since the identification of the single gene encoding CFTR, several hundred disease-causing mutations, associated with a wide variety of clinical phenotypes, have been reported. To understand the relationship between genotype and clinical phenotype, researchers have investigated how mutations in CFTR disrupt its function. Here, we review the recent progress in understanding how CF-associated mutations in CFTR produce defective Cl- channels, and discuss the implications of this knowledge for the development of therapy for CF.     

Role of secondary prevention in congestive heart failure due to coronary artery disease

BACKGROUND: It is unknown whether genetic factors predispose patients to idiopathic pancreatitis. In patients with cystic fibrosis, mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene typically cause pulmonary and pancreatic insufficiency while rarely causing pancreatitis. We examined whether idiopathic pancreatitis is associated with CFTR mutations in persons who do not have lung disease of cystic fibrosis. METHODS: We studied 27 patients (mean age at diagnosis, 36 years), 22 of whom were female, who had been referred for an evaluation of idiopathic pancreatitis. DNA was tested for 17 CFTR mutations and for the 5T allele in intron 8 of the CFTR gene. The 5T allele reduces the level of functional CFTR and is associated with an inherited form of infertility in males. Patients with two abnormal CFTR alleles were further evaluated for unrecognized cystic fibrosis-related lung disease, and both base-line and CFTR-mediated ion transport were measured in the nasal mucosa. RESULTS: Ten patients with idiopathic chronic pancreatitis (37 percent) had at least one abnormal CFTR allele. Eight CFTR mutations were detected (prevalence ratio, 11:1; 95 percent confidence interval, 5 to 23; P<0.001). In three patients both alleles were affected (prevalence ratio, 80:1; 95 percent confidence interval, 17 to 379; P<0.001). These three patients did not have lung disease typical of cystic fibrosis on the basis of sweat testing, spirometry, or base-line nasal potential-difference measurements. Nonetheless, each had abnormal nasal cyclic AMP-mediated chloride transport. CONCLUSION: In a group of patients referred for evaluation of idiopathic pancreatitis, there was a strong association between mutations in the CFTR gene and pancreatitis. The abnormal CFTR genotypes in these patients with pancreatitis resemble those associated with male infertility.     

Infection with hepatitis G virus and its strain variant, the GB agent (GBV-C), among blood donors in Japan

Much of the morbidity and mortality seen in cystic fibrosis (CF) is related to chronic infection of the respiratory tract with Pseudomonas aeruginosa. Some studies have attributed the strong relationship between CF and Pseudomonas colonization to the presence of increased numbers of specific cell-surface receptors, although other work suggests that this relates to the presence of mucus. Several groups are now assessing the use of gene transfer as a novel form of treatment for CF. We have examined whether P. aeruginosa binding to freshly obtained CF respiratory epithelial cells is increased, and have studied the effects of transfer of the CF transmembrane conductance regulator (CFTR) gene on this attachment. Binding of P. aeruginosa to noncultured nasal epithelial cells from both CF patients (n = 31) and healthy controls (n = 15) was studied with scanning electron microscopy. Binding was also assessed for CF cells following transfection with CFTR/liposome complexes. Epifluorescence microscopy was used to assess the effects of gene transfer on chloride fluxes. Adherence of P. aeruginosa directly to the cell surface of CF airway epithelium was significantly (P < 0.001) increased over that in non-CF controls. Liposome-mediated CFTR gene transfer resulted in a significant (P < 0.01) reduction in the numbers of bacteria bound to ciliated epithelial cells. Fluorescence microscopy confirmed correction of the basic chloride defect. Thus, in CF, the absence of normal CFTR results in increased binding of P. aeruginosa to respiratory epithelial cells. This abnormality can be corrected in vitro by restoration of CFTR function. This has important implications both for the pathogenesis of CF and for the future application and assessment of gene therapy for this disease.     

Decreased expression of the cystic fibrosis transmembrane conductance regulator protein in remodeled airway epithelium from lung transplanted patients

The absence or mislocalization of cystic fibrosis transmembrane conductance regulator (CFTR) is regarded as being specific for cystic fibrosis (CF). In principle, the supply of a non-CF lung transplant to a CF patient should bring up normal CFTR expression in the lower airways. Immunolocalization of CFTR and of epithelial differentiation markers (ie, cytokeratins 13, 14, and 18, and desmoplakins 1 and 2) was carried out on 21 mucosal biopsies from the upper lobe of grafts in non-CF (n = 12) and CF patients (n = 9) retrieved between days 23 and 1,608 after lung transplantation. Biopsy specimens from seven non-CF and four CF patients presented either a pseudostratified respiratory epithelium or slight basal cell hyperplasia. CFTR was distributed at the apical membrane of the ciliated cells. In remodeled epithelia with basal cell hyperplasia or squamous metaplasia, CFTR was either weakly expressed in the cytoplasm of the superficial epithelial cells or was undetectable. The extent of epithelium remodeling was significantly correlated with an impairment of lung function. The results suggest that posttransplant airway epithelium dedifferentiation of the graft leads to the loss of properly targeted CFTR irrespective of the underlying disease of the recipient.     

Regulation of CFTR chloride channels by syntaxin and Munc18 isoforms

The cystic fibrosis gene encodes a cyclic AMP-gated chloride channel (CFTR) that mediates electrolyte transport across the luminal surfaces of a variety of epithelial cells. The molecular mechanisms that modulate CFTR activity in epithelial tissues are poorly understood. Here we show that CFTR is regulated by an epithelially expressed syntaxin (syntaxin 1A), a membrane protein that also modulates neurosecretion and calcium-channel gating in brain. Syntaxin 1A physically interacts with CFTR chloride channels and regulates CFTR-mediated currents both in Xenopus oocytes and in epithelial cells that normally express these proteins. The physical and functional interactions between syntaxin 1A and CFTR are blocked by a syntaxin-binding protein of the Munc18 protein family (also called n-Secl). Our results indicate that CFTR function in epithelial cells is regulated by an interplay between syntaxin and Munc18 isoforms.     

Mutations of the cystic fibrosis gene in patients with chronic pancreatitis

BACKGROUND: The pancreatic lesions of cystic fibrosis develop in utero and closely resemble those of chronic pancreatitis. Therefore, we hypothesized that mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene may be more common than expected among patients with chronic pancreatitis. METHODS: We studied 134 consecutive patients with chronic pancreatitis (alcohol-related disease in 71, hyperparathyroidism in 2, hypertriglyceridemia in 1, and idiopathic disease in 60). We examined DNA for 22 mutations of the CFTR gene that together account for 95 percent of all mutations in patients with cystic fibrosis in the northwest of England. We also determined the length of the noncoding sequence of thymidines in intron 8, since the shorter the sequence, the lower the proportion of normal CFTR messenger RNA. RESULTS: The 94 male and 40 female patients ranged in age from 16 to 86 years. None had a mutation on both copies of the CFTR gene. Eighteen patients (13.4 percent), including 12 without alcoholism, had a CFTR mutation on one chromosome, as compared with a frequency of 5.3 percent among 600 local unrelated partners of persons with a family history of cystic fibrosis (P<0.001). A total of 10.4 percent of the patients had the 5T allele in intron 8 (14 of 134), which is twice the expected frequency (P=0.008). Four patients were heterozygous for both a CFTR mutation and the 5T allele. Patients with a CFTR mutation were younger than those with no mutations (P=0.03). None had the combination of sinopulmonary disease, high sweat electrolyte concentrations, and low nasal potential-difference values that are diagnostic of cystic fibrosis. CONCLUSIONS: Mutations of the CFTR gene and the 5T genotype are associated with chronic pancreatitis.     

Cystic fibrosis transmembrane conductance regulator-associated ATP release is controlled by a chloride sensor

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis, and has also been closely associated with ATP permeability in cells. Using a Xenopus oocyte cRNA expression system, we have evaluated the molecular mechanisms that control CFTR-modulated ATP release. CFTR-modulated ATP release was dependent on both cAMP activation and a gradient change in the extracellular chloride concentration. Activation of ATP release occurred within a narrow concentration range of external Cl- that was similar to that reported in airway surface fluid. Mutagenesis of CFTR demonstrated that Cl- conductance and ATP release regulatory properties could be dissociated to different regions of the CFTR protein. Despite the lack of a need for Cl- conductance through CFTR to modulate ATP release, alterations in channel pore residues R347 and R334 caused changes in the relative ability of different halides to activate ATP efflux (wtCFTR, Cl > Br; R347P, Cl > Br; R347E, Br > Cl; R334W, Cl = Br). We hypothesize that residues R347 and R334 may contribute a Cl- binding site within the CFTR channel pore that is necessary for activation of ATP efflux in response to increases of extracellular Cl-. In summary, these findings suggest a novel chloride sensor mechanism by which CFTR is capable of responding to changes in the extracellular chloride concentration by modulating the activity of an unidentified ATP efflux pathway. This pathway may play an important role in maintaining fluid and electrolyte balance in the airway through purinergic regulation of epithelial cells. Insight into these molecular mechanisms enhances our understanding of pathogenesis in the cystic fibrosis lung.     

Effect of deletion mutations on the function of CFTR chloride channels

Mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a unique R domain; CFTR functions as a Cl- channel regulated by phosphorylation and by nucleoside triphosphates. To study the domains of CFTR involved in Cl- channel function, we expressed mutants lacking various domains and assayed cAMP-stimulated Cl- channel activity using the halide-sensitive fluorophore, 6-methoxy-N-(3'-sulfopropyl)-quinolinium. We previously reported that deletion of part of the R domain (residues 708-835) produced Cl- channels that were constitutively open. Here we show that more extensive deletions within the R domain failed to generate functional CFTR Cl- channels; the portion of protein that could be deleted without destroying function corresponds to sequences that are not conserved in related proteins. In contrast, when we deleted the two NBDs (either alone, together, or in combination with the R domain), we did not observe functional Cl- channels. CFTR has a unique carboxyl terminus that is conserved across species. However, truncation of the carboxyl terminus (up to, but not including, NBD2) produced a regulated anion permeability similar to that of wild-type CFTR, suggesting that this region is not essential for channel function. Expression of two CF-associated nonsense mutants (G542X and W1316X) also failed to generate functional CFTR Cl- channels. These results help define structure:function relationships for CFTR and identify the domains that are required for Cl- channel function.     

Primary hyperoxaluria

Cystic fibrosis (CF) is the most common lethal autosomal recessive disorder among Caucasians and is caused by abnormalities in the cystic fibrosis transmembrane conductance regulator gene (CFTR). CFTR gene encodes a chloride channel that regulates secretion in many exocrine tissues especially pancreatic and pulmonary tissues. The clinical presentation of cystic fibrosis is highly variable with isolated CAVD (congenital absence of vas deferens) and/or typical pancreatic and pulmonary manifestations. Over 500 mutations in the CFTR gene have been described and vary among different geographic locations. The severity of clinical manifestations and specially the pulmonary disease is poorly correlated with genotype. It is interesting to collect clinical and genetical data by analysing a larger cohort of CF patients. These results are likely to improve our understanding of the physiopathology of CF and the genetic counselling; particular biochemical defect could lead to more specific treatments in the future. From our 110 patients selected in Champagne-Ardenne country, we analysed the entire coding sequence of CFTR gene and detected 95% of CF mutations and in fact, 89.5% if we include the CAVD patients; 59.4% of CF mutations were detected for these patients. Three new mutations have been here reported. We found numerous CF mutations with a large distribution throughout the gene. Nevertheless, three exons are mainly involved: 10, 11 and 21. Relationships between the genotype and phenotype are difficult to assess.     

Cellular heterogeneity of CFTR expression and function in the lung: implications for gene therapy of cystic fibrosis

Cystic fibrosis (CF) has become a paradigm disorder for the clinical testing of gene therapies in the treatment of inherited disease. In recent years, efforts directed at gene therapy of CF have concentrated on improving gene delivery systems to the airway. Surrogate endpoints for complementation of CFTR dysfunction in the lung have been primarily dependent on correction of chloride transport abnormalities. However, it is now clear that the pathophysiology of CF airways disease is far more complex than can be solely attributed to altered chloride permeability. For example, in addition to functioning as a chloride channel, CFTR also has been implicated in the regulation of other apical membrane conductance pathways through interactions with the amiloride sensitive epithelial sodium channel (ENaC) and the outwardly rectifying chloride channel (ORCC). Superimposed on this functional diversity of CFTR is a highly regulated pattern of CFTR expression in the lung. This heterogeneity occurs at both the level of CFTR protein expression within different cell types in the airway and the anatomical location of these cells in the lung. Potential targets for gene therapy of CF include ciliated, non-ciliated, and goblet cells in the surface airway epithelium as well as submucosal glands within the interstitium of the airways. Each of these distinct cellular compartments may have functionally distinct roles in processes which affect the pathogenesis of CF airways disease, such as fluid and electrolyte balance. However, it is presently unclear which of these cellular targets are most pathophysiologic relevant with regard to gene therapy. Elucidation of the underlying mechanisms of CFTR function in the airway will allow for the rational design of gene therapy approaches for CF lung diseases. This review will provide a summary of the field's current knowledge regarding CFTR functional diversity in the airway and the implications of such diversity for gene therapies of CF lung disease.     

Cystic fibrosis transmembrane conductance regulator: the first nucleotide binding fold targets the membrane with retention of its ATP binding function

Most cases of cystic fibrosis are caused by a single deletion mutation (deltaF508) within the first nucleotide binding fold (NBF1) of the CFTR protein (cystic fibrosis transmembrane conductance regulator). NBF1 is classically defined as amino acid residues phenylalanine 433 through serine 589, encoded by exons 10-12, and only part of exon 9, of the CFTR gene. This assignment is based on sequence homology of this region of the CFTR protein with that of other nucleotide binding proteins. Here, we report that when the complete modular unit encoded precisely by exons 9-12 is expressed in Escherichia coli as glycine 404 through serine 589, i.e., as [G404-N432]NBF1 or as deltaF508[G404-N432]NBF1, the resultant proteins target the cytoplasmic membrane. Significantly, [G404-N432]NBF1 is readily labeled from the outside of intact E. coli spheroplasts with the water soluble, membrane impermeable probe Biotin-X-NHS, sulfosuccinimidyl-6-(biotinamido)-hexanoate. Similar findings were observed with the disease causing mutant deltaF508[G404-N432]NBF1. Three different control experiments which involved (1) assays for known cytosolic E. coli enzymes, (2) immuno-gold electron microscopy with antibody having an epitope for the biotin moiety, and (3) tests for biotinylation of the cytosolic component, Enzyme 1 of the glucose phosphotransferase system, demonstrated that the spheroplasts used in this study are neither leaky nor permeable to Biotin-X-NHS. In addition, membrane-associated [G404-N432]NBF1, upon solubilization with Triton X-100, was found to bind to an ATP-agarose column and be released therefrom by elution with ATP, emphasizing retention of a native-like structure. In sharp contrast, NBF1 localizes to the cytosol when the [G404-N432]-N-terminal region is replaced with the maltose binding protein. The novel findings reported here implicate a role of the N-terminal region of NBF1 in its subcellular localization and are directly relevant to our understanding of the membrane structure, function, and trafficking of CFTR.