Cystic fibrosis F508del patients have apically localized CFTR in a reduced number of airway cells

Present state of knowledge, mostly based on heterologous expression studies, indicates that the cystic fibrosis transmembrane conductance regulator (CFTR) protein bearing the F508del mutation is misprocessed and mislocalized in the cytoplasm, unable to reach the cell surface. Recently, however, it was described that protein levels and localization are similar between F508del and wild-type CFTR in airway and intestinal tissues, but not in the sweat glands. In this study, we used immunocytochemistry with three different anti-CFTR antibodies to investigate endogenous CFTR expression and localization in nasal epithelial cells from F508del homozygous patients, F508del carriers, and non-CF individuals. On average, 300 cells were observed per individual. No significant differences were observed for cell type distributions among CF, carrier, and non-CF samples; epithelial cells made up approximately 80% to 95% of all cells present. CFTR was detected mostly in the apical region (AR) of the tall columnar epithelial (TCE) cells, ciliated or nonciliated. By confocal microscopy analysis, we show that the CFTR apical region-staining does not overlap with either anti-calnexin (endoplasmic reticulum), anti-p58 (Golgi), or anti-tubulin (cilia) stainings. The median from results with three antibodies indicate that the apical localization of CFTR happens in 22% of TCE cells from F508del homozygous patients with CF (n = 12), in 42% of cells from F508del carriers (n = 20), and in 56% of cells from healthy individuals (n = 12). Statistical analysis indicates that differences are significant among all groups studied and for the three antibodies (p < 0.05). These results confirm the presence of CFTR in the apical region of airway cells from F508del homozygous patients; however, they also reveal that the number of cells in which this occurs is significantly lower than in F508del carriers and much lower than in healthy individuals. These findings may have an impact on the design of novel pharmacological strategies aimed at circumventing the CF defect caused by the F508del mutation.     

A cystic fibrosis allele encoding missense mutations in both nucleotide binding folds of the cystic fibrosis transmembrane conductance regulator.

German cystic fibrosis (CF) chromosomes were screened for molecular lesions in exon 20 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by chemical cleavage of mismatch. An 3884G-to-A transition was detected in two patients which leads to an exchange of a serine by an asparagine in the Walker motif A of the second nucleotide binding fold. The affected serine residue is evolutionarily strongly conserved among the pro- and eukaryotic members of the protein superfamily of traffic ATPases. The two S1251N alleles were linked to the benign missense mutation F508C which is located in another conserved region of CFTR, the center region of the first nucleotide binding fold. Both patients with the complex allele F508C-S1251N are carrying delta F508 on the other CF chromosome and are suffering from severe pulmonary and gastrointestinal CF disease. Although F508C has been classified as a neutral sequence variation because of its discovery in healthy delta F508 gene carriers, it may nevertheless influence CFTR dysfunction caused by the S1251N mutation.     

Prediction of cellular immune responses against CFTR in patients with cystic fibrosis after gene therapy

Different classes of mutations (class I-VI) of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene are responsible for lung/pancreatic disease. The most common mutation, DeltaF508, is characterized by expression of precursor forms of CFTR but no functional CFTR. Since only 5-10% of normal CFTR function is required to correct the electrophysiologic defect across the airway epithelium, gene therapy holds promise for treatment of patients with CF lung disease. However, efficient delivery and transgene expression are not the only parameters that may influence the success of gene therapy. Host-specific immune responses generated against the therapeutic CFTR protein may pose a problem, especially when the coding sequence between the normal CFTR and mutated CFTR differ. This phenomenon is more pertinent to class I mutations in which large fragments of the protein are not expressed. However, T cells directed against epitopes that span sequences containing class II-V mutations are also possible. We used MHC-binding prediction programs to predict the probability of cellular immune responses that may be generated against CFTR in DeltaF508 homozygote patients. Results obtained from running the prediction algorithms yielded a few high-scoring MHC-Class I binders within the specific sequences, suggesting that there is a possibility of the host to mount a cellular immune response against CFTR, even when the difference between therapeutic and host CFTR is a single amino acid (F) at position 508.     

SERCA pump inhibitors do not correct biosynthetic arrest of deltaF508 CFTR in cystic fibrosis

Deletion of phenylalanine 508 (deltaF508) accounts for nearly 70% of all mutations that occur in the cystic fibrosis transmembrane conductance regulator (CFTR). The deltaF508 mutation is a class II processing mutation that results in very little or no mature CFTR protein reaching the apical membrane and thus no cAMP-mediated Cl- conductance. Therapeutic strategies have been developed to enhance processing of the defective deltaF508 CFTR molecule so that a functional cAMP-regulated Cl- channel targets to the apical membrane. Sarcoplasmic/endoplasmic reticulum calcium (SERCA) inhibitors, curcumin and thapsigargin, have been reported to effectively correct the CF ion transport defects observed in the deltaF508 CF mice. We investigated the effect of these compounds in human airway epithelial cells to determine if they could induce deltaF508 CFTR maturation, and Cl- secretion. We also used Baby Hamster Kidney cells, heterologously expressing deltaF508 CFTR, to determine if SERCA inhibitors could interfere with the interaction between calnexin and CFTR and thereby correct the deltaF508 CFTR misfolding defect. Finally, at the whole animal level, we tested the ability of curcumin and thapsigargin to (1) induce Cl- secretion and reduce hyperabsorption of Na+ in the nasal epithelia of the deltaF508 mouse in vivo, and (2) induce Cl- secretion in intestine (jejunum and distal colon) and the gallbladder of the deltaF508 CF mouse. We conclude that curcumin and thapsigargin failed to induce maturation of deltaF508 CFTR, or induce Cl- secretion, as measured by biochemical and electrophysiologic techniques in a variety of model systems ranging from cultured cells to in vivo studies.     

Asialo GM1 is a receptor for Pseudomonas aeruginosa adherence to regenerating respiratory epithelial cells.

We investigated the implication of asialo GM1 as an epithelial receptor in the increased Pseudomonas aeruginosa affinity for regenerating respiratory epithelial cells from cystic fibrosis (CF) and non-CF patients. Human respiratory epithelial cells were obtained from nasal polyps of non-CF subjects and of CF patients homozygous for the delta F 508 transmembrane conductance regulator protein (CFTR) mutation and cultured according to the explant-outgrowth model. At the periphery of the outgrowth, regenerating respiratory epithelial cells spreading over the collagen I matrix with lamellipodia were observed, characteristic of respiratory epithelial wound repair after injury. P aeruginosa adherence to regenerating respiratory epithelial cells was found to be significantly greater in the delta F 508 homozygous CF group than in the non-CF group (P < 0.001). In vitro competitive binding inhibition assays performed with rabbit polyclonal antibody against asialo GM1 demonstrated that blocking asialo GM1 reduces P. aeruginosa adherence to regenerating respiratory epithelial cells in delta F 508 homozygous cultures (P < 0.001) as well as in non-CF cultures (P < 0.001). Blocking of asialo GM1 was significantly more efficient in CF patients than in non-CF subjects (P < 0.05). Distribution of asialo GM1 as determined by preembedding labelling and immunoelectron microscopy clearly demonstrated the specific apical membrane expression of asialo GM1 by regenerating respiratory epithelial cells, whereas other cell phenotypes did not apically express asialo GM1. These results demonstrate that (i) asialo GM1 is an apical membrane receptor for P. aeruginosa expressed at the surface of CF and non-CF regenerating respiratory epithelial cells and (ii) asialo GM1 is specifically recovered in regenerating respiratory epithelium. These results suggest that in CF, epithelial repair represents the major event which exposes asialo GM1 for P. aeruginosa adherence.     

Identification of SNPs in the cystic fibrosis interactome influencing pulmonary progression in cystic fibrosis

There is growing evidence that the great phenotypic variability in patients with cystic fibrosis (CF) not only depends on the genotype, but apart from a combination of environmental and stochastic factors predominantly also on modifier gene effects. It has been proposed that genes interacting with CF transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) are potential modifiers. Therefore, we assessed the impact of single-nucleotide polymorphisms (SNPs) of several of these interacters on CF disease outcome. SNPs that potentially alter gene function were genotyped in 95 well-characterized p.Phe508del homozygous CF patients. Linear mixed-effect model analysis was used to assess the relationship between sequence variants and the repeated measurements of lung function parameters. In total, we genotyped 72 SNPs in 10 genes. Twenty-five SNPs were used for statistical analysis, where we found strong associations for one SNP in PPP2R4 with the lung clearance index (P≤0.01), the specific effective airway resistance (P≤0.005) and the forced expiratory volume in 1 s (P≤0.005). In addition, we identified one SNP in SNAP23 to be significantly associated with three lung function parameters as well as one SNP in PPP2R1A and three in KRT19 to show a significant influence on one lung function parameter each. Our findings indicate that direct interacters with CFTR, such as SNAP23, PPP2R4 and PPP2R1A, may modify the residual function of p.Phe508del-CFTR while variants in KRT19 may modulate the amount of p.Phe508del-CFTR at the apical membrane and consequently modify CF disease.     

Cystic fibrosis transmembrane conductance regulator does not affect neutrophil migration across cystic fibrosis airway epithelial monolayers

Recent studies have shown that airway inflammation dominated by neutrophils, ie, polymorphonuclear cells (PMN) was observed in infants and children with cystic fibrosis (CF) even in the absence of detectable infection. To assess whether there is a CF-related anomaly of PMN migration across airway epithelial cells, we developed an in vitro model of chemotactic migration across tight and polarized CF(15) cells, a CF human nasal epithelial cell line, seeded on porous filters. To compare PMN migration across a pair of CF and control monolayers in the physiological direction, inverted CF(15) cells were infected with increasing concentrations of recombinant adenoviruses containing either the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, the DeltaF508 CFTR cDNA, or the beta-galactosidase gene. The number of PMN migrating in response to N-formyl-Met-Leu-Phe across inverted CF(15) monolayers expressing beta-galactosidase was similar to that seen across CF(15) monolayers rescued with CFTR, whatever the proportion of cells expressing the transgene. Moreover, PMN migration across monolayers expressing various amounts of mutated CFTR was not different from that observed across matched counterparts expressing normal CFTR. Finally, PMN migration in response to adherent or Pseudomonas aeruginosa was equivalent across CF and corrected monolayers. The possibility that mutated CFTR may exert indirect effects on PMN recruitment, via an abnormal production of the chemotactic cytokine interleukin-8, was also explored. Apical and basolateral production of interleukin-8 by polarized CF cells expressing mutated CFTR was not different from that observed with rescued cells, either in baseline or stimulated conditions. CF(15) cells displayed a CF phenotype that could be corrected by CFTR-containing adenoviruses, because two known CF defects, Cl(-) secretion and increased P. aeruginosa adherence, were normalized after infection with those viruses. Thus, we conclude that the presence of a mutated CFTR does not per se lead to an exaggerated inflammatory response of CF surface epithelial cells in the absence or presence of a bacterial infection.     

Salmeterol restores secretory functions in cystic fibrosis airway submucosal gland serous cells

The activity of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) can be mediated by surface G protein-coupled receptors such as the beta(2)-adrenergic receptor. In this study, we explored the effect of a long-acting beta(2)-adrenergic agonist, salmeterol, on the CFTR-dependent secretory capacity of a human CF tracheal gland serous cell line (CF-KM4), homozygous for the delF508 mutation. We showed that, compared with the untreated CF serous cells, a 24-hour pre-incubation period with 200 nM salmeterol induced an 83% increase in delF508-CFTR-mediated chloride efflux. The restoration of the bioelectric properties is associated with increased apical surface pool of delF508-CFTR. Salmeterol induced a decrease in ion concentration and an increase in the level of hydration of the mucus packaged inside the CF secretory granules. The effects of salmeterol are not associated with a persistent production of cAMP. Western blotting on isolated secretory granules demonstrated immunoreactivity for CFTR and lysozyme. In parallel, we measured by atomic force microscopy an increased size of secretory granules isolated from CF serous cells compared with non-CF serous cells (MM39 cell line) and showed that salmeterol was able to restore a CF cell granule size similar to that of non-CF cells. To demonstrate that the salmeterol effect was a CFTR-dependent mechanism, we showed that the incubation of salmeterol-treated CF serous cells with CFTR-inh172 suppressed the restoration of normal secretory functions. The capacity of salmeterol to restore the secretory capacity of glandular serous cells suggests that it could also improve the airway mucociliary clearance in patients with CF.     

DNA analysis and gene diagnosis of cystic fibrosis]

Cystic fibrosis (CF) is the most common autosomal recessive disorder in the Caucasian population, affecting approximately 1 in 2,000 newborns but the actual estimate varies with the geographic location. The incidence of CF in non-Caucasian populations is low. Intensive efforts using genetic linkage information ultimately led to the cloning of the CF gene prior to the identification of the gene product or its function. The gene encodes what is believed to be a transmembrane protein, which has been named the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR contains two nucleotide-binding folds (NBF) which show homology to numerous transport proteins with the greatest homology to the P-glycoproteins that are encoded by the multiple drug-resistance loci. A three- base deletion resulting in the loss of phenylalanine residue (delta F508) in the tenth exon of the CFTR gene is the mutation occurring on the majority of CF chromosomes. The overall frequency of delta F508 in the present mutant CF gene pool is about 70%, but the study populations are not equally represented: there is marked variation in the population of delta F508 among different geographic populations. Recently, numerous additional, less common mutations have been found. Some mutations occur on 2-5% of the CF chromosomes. Many of these are rare 'private' mutations, occurring in individual families of all racial and ethnic backgrounds. By contrast over 80% of Western European CF mutations have been identified. The highly heterogeneous nature of the remaining CF mutations provides important insights into the structure and function of the protein, but further improvements are needed in DNA-based genetic screening for CF carrier status.     

Cystic fibrosis patients with mutation 1949del84 in exon 13 of the CFTR gene have a similar clinical severity as delta F508 homozygotes.

The majority of the identified cystic fibrosis (CF) mutations are very uncommon in the total patient population, making the correlation between the clinical presentation and the molecular alterations difficult. The largest deletion that has been described so far in CF is of 84 bp in exon 13, which corresponds to the regulatory (R) domain of the CF transmembrane conductance regulator (CFTR) protein. We have analysed 340 Spanish CF patients for this deletion, named 1949del84, and found three further compound heterozygous patients for mutations 1949del84 and delta F508, and one for 1949del84 and an unknown mutation. Evaluation of the clinical data in these patients suggests that this in-frame deletion, when associated with delta F508, has a similar disease severity to that of delta F508 homozygous patients.     

Mucin glycosylation and sulphation in airway epithelial cells is not influenced by cystic fibrosis transmembrane conductance regulator expression

Abnormalities in mucus properties and clearance make a major contribution to the pathology of cystic fibrosis (CF). Our aim was to test the hypothesis that the defects in CF mucus are a direct result of mutations in the CF transmembrane conductance regulator (CFTR) protein. We evaluated a single mucin molecule MUC1F/5ACTR that carries tandem repeat sequence from MUC5AC, a major secreted airway mucin, in a MUC1 mucin vector. To establish whether the presence of mutant or normal CFTR directly influences the O-glycosylation and sulphation of mucins in airway epithelial cells, we used the CFT1-LC3 (DeltaF508 CFTR mutant) and CFT1-LCFSN (wild-type CFTR corrected) human airway epithelial cell lines. MUC1F/5ACTR mucin was immunoprecipitated, centricon purified, and O-glycosylation was evaluated by Matrix-assisted laser desorption ionization and electrospray tandem mass spectrometry to determine the composition of different carbohydrate structures. Mass spectrometry data showed the same O-glycans in both CFTR mutant and wild-type CFTR corrected cells. Metabolic labeling assays were performed to evaluate gross glycosylation and sulphation of the mucins and showed no significant difference in mucin synthesized in six independent clones of these cell lines. Our results show that the absence of functional CFTR protein causes neither an abnormality in mucin O-glycosylation nor an increase in mucin sulphation.     

Pseudomonas aeruginosa-induced apoptosis is defective in respiratory epithelial cells expressing mutant cystic fibrosis transmembrane conductance regulator

Chronic lung infection with Pseudomonas aeruginosa constitutes the most severe manifestation of cystic fibrosis, a scenario that results from defects in early clearance of the microbe. Early clearance involves epithelial cell ingestion of bacteria, rapid activation of nuclear factor-kappa B and cellular desquamation within minutes of P. aeruginosa infection, processes that are deficient in cells with mutant alleles of Cftr. Analyzing the effect of Cftr genotype on the apoptotic response of airway epithelial cells to P. aeruginosa, we found that human bronchial epithelial cells expressing Delta F508 cystic fibrosis transmembrane conductance regulator (CFTR) underwent significantly delayed apoptosis compared with cells expressing wild-type (WT) CFTR. Mice with a WT Cftr allele had apoptotic cells in their lungs after P. aeruginosa infections, whereas mice homozygous for the Delta F508 or G551D Cftr alleles showed little apoptosis in response to acute infection. Pseudomonal infection induced expression of CD95 and CD95 ligand, a response that was also delayed in cells homozygous for mutant Cftr alleles. Thus, WT CFTR expression promotes a rapid expression of CD95/CD95 ligand and apoptotic response to P. aeruginosa infection. Prompt apoptosis of infected epithelial cells may be critical for clearance of P. aeruginosa, and CFTR-associated defects in apoptosis may contribute to the pathogenesis of the lung disease in cystic fibrosis.     

Cystic fibrosis mutations in French Canadians: three CFTR mutations are relatively frequent in a Quebec population with an elevated incidence of cystic fibrosis.

The French-Canadian population in the Saguenay-Lac St. Jean region of northeastern Quebec has an elevated frequency of cystic fibrosis (CF). The average incidence of cystic fibrosis was 1 in 891 births and the prevalence of CF carriers was estimated to be 1 in 15. We tested for 10 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in 133 French-Canadian CF families from Quebec. Ninety-one families were from the Saguenay-Lac St. Jean region and 42 families were referred from other regions of Quebec. We detected the CFTR mutation in 93 and 92% of the CF chromosomes in the Saguenay-Lac St. Jean and the major-urban Quebec families, respectively. The two groups of French-Canadian families were significantly different for the proportions of CFTR mutations. The three most common mutations in the Saguenay-Lac St. Jean families were delta F508 (58%), 621 + 1G----T (23%), and A455E (8%); and in the major-urban Quebec families were delta F508 (71%), 711 + 1G----T (9%), and 621 + 1G----T (5%). These results provide evidence for the role of founder effect in the elevated incidence of cystic fibrosis in the Saguenay-Lac St. Jean population.     

Cystic fibrosis genotype and bacterial infection: a possible connection

Patients with cystic fibrosis (CF) have repeated bacterial infection of the airways, which can lead to chronic infection. There is evidence that disease severity is determined by the genetic mutations present. This study aims to establish if CF genotype is related to the frequency and types of airway bacterial infection. Adult patients attending the regional CF unit are followed for two years and assigned to one of three groups depending on whether they are chronically infected with Burkholderia cepacia complex (BCC) organisms, Pseudomonas aeruginosa, or neither of these organisms. Genotype analysis is performed on all patients to determine which of the cystic fibrosis transmembrane regulator (CFTR) gene mutations are present. The numbers and types of organism with the CFTR mutations isolated from sputum are identified. Data are available on 59 patients: 15 colonised with BCC, 24 colonised with P. aeruginosa, and 20 not colonised with either organism. Twenty patients were homozygous for deltaF508, 25 were heterozygous, and the deltaF508 mutation was not present in the remaining 14 patients. Patients homozygous or heterozygous for the deltaF508 mutation had an increased likelihood of colonisation with BCC or P. aeruginosa, an increased number of positive sputum cultures and a higher frequency of multiple infecting organisms. Cystic fibrosis mutational analysis identified seven patients who had the R117H mutation. These patients were less likely to be colonised with BCC or P. aeruginosa. In conclusion, patients homozygous or heterozygous for the deltaF508 deletion are more likely to suffer airway colonisation with BCC or P. aeruginosa, with increased numbers of positive sputum cultures and infecting organisms. Those with the R117H mutation are less likely to be colonised by Gram-negative organisms.     

Regulation of gap junctional communication by a pro-inflammatory cytokine in cystic fibrosis transmembrane conductance regulator-expressing but not cystic fibrosis airway cells

Airway inflammation is orchestrated by cell-cell interactions involving soluble mediators and cell adhesion molecules. Alterations in the coordination of the multicellular process of inflammation may play a major role in the chronic lung disease state of cystic fibrosis (CF). The aim of this study was to determine whether direct cell-cell interactions via gap junctional communication is affected during the inflammatory response of the airway epithelium. We have examined the strength of intercellular communication and the activation of nuclear factor-kappaB (NF-kappaB) in normal (non-CF) and CF human airway cell lines stimulated with tumor necrosis factor-alpha (TNF-alpha). TNF-alpha induced maximal translocation of NF-kappaB into the nucleus of non-CF as well as CF airway cells within 20 minutes. In non-CF cells, TNF-alpha progressively decreased the extent of intercellular communication. In contrast, gap junctional communication between CF cells exposed to TNF-alpha remained unaltered. CF results from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Interestingly, transfer of wild-type CFTR into CF cells by adenovirus-mediated infection was associated with the recovery of TNF-alpha-induced uncoupling. These results suggest that expression of functional CFTR is necessary for regulation of gap junctional communication by TNF-alpha. Gap junction channels close during the inflammatory response, therefore limiting the intercellular diffusion of signaling molecules, and thereby the recruitment of neighboring cells. Defects in this mechanism may contribute to the excessive inflammatory response of CF airway epithelium.     

Endocrine and exocrine pancreatic function and the ΔF508 mutation in cystic fibrosis

The relationship between the cystic fibrosis (CF) genotype and endocrine and exocrine pancreatic function was studied in 215 CF patients. In the 211 patients with the delta F508 mutation, endocrine pancreatic function (oral glucose tolerance; WHO criteria) was normal in 72.5%, impaired in 12.3%, and diabetic in 15.2% of the patients, with no difference between CF patients homozygous (N = 163, median age 15 years, range 2-40) or heterozygous (N = 48, 18 years, 3-40; age difference not significant) for the delta F508 mutation. Exocrine pancreatic sufficiency (no need for pancreatic enzyme substitution) was found in 0.6% of the patients homozygous for the delta F508 mutation and in 10.4% of the heterozygotes (p less than 0.01). Homozygous patients with pancreatic insufficiency took more pancreatic enzyme capsules (median 42 per day, range 0-192) than the heterozygotes (29 per day, 0-300; p less than 0.001). The four patients (1.9%) without the delta F508 mutation had normal glucose tolerance but exocrine pancreatic insufficiency. In conclusion, the major mutation genotype in CF (delta F508) affects the severity of the exocrine pancreatic insufficiency, whereas endocrine pancreatic function is unrelated to this genotype.     

Prevalence of cystic fibrosis mutations in the East German population.

A representative multicenter cystic fibrosis (CF) mutation analysis on about half of all known cystic fibrosis patients of the 5 East German L?nder is reported. Analyses for 17 mutations, among them Delta F508, R553X, G542X, S549R,N,I, G551D, S1255X, R347P,H, and Y122X, were performed. As expected, the delta F508 mutation in exon 10 of the CFTR gene is the major gene alteration causing CF in our patients. However, in comparison to studies from Western Germany, a significantly lower percentage of just over 60% is found in our patients, resembling data obtained from slavonic populations. The severe phenotype of cystic fibrosis is most frequently associated with homozygosity for the delta F508 mutation. No particular allele association could be found with the intermediate and mild phenotypes of this disease. The next most frequent of the investigated mutations is R553X (13.3% of non-delta F chromosomes) followed by R347P (9.2%) and G542X (4.4%).     

Frequency of delta F508 mutation in Venezuelan patients with cystic fibrosis

Cystic Fibrosis (CF) is the most common and severe autosomal recessive disease in Caucasian populations, with an incidence of 1 in 2500 live births. It is characterized by a generalized disturbance in exocrine glands and it is caused by over one thousand mutations at the cystic fibrosis conductance regulator gene (CFTR) mapped at 7q31. AF508 is the most frequent mutation worldwide and it consists in a deletion of the codon that encodes fenilalanine at the 508 protein's position. The aim of this study was to determine the frequency of the delta F508 mutation in Venezuelan patients with CF using the Polymerase Chain Reaction (PCR). We studied thirty patients of twenty eight families who were diagnosed with CF based on their clinical features and sweat chloride level > 60 mEq/l in two determinations. Detection of the mutation was performed from the amplification of a 98 pair of bases (pb) CF gene segment which contains the codon that encodes fenilalanine in the 508 position by PCR. This PCR product is absent in those who have the mutation. The delta F508 allelic frequency was 26.79%, distributed in six homozygous and seven compound heterozygote delta F508/X. The reminder mutations (no delta F508) represent 73.21%. The delta F508 frequency in our sample is less than the reported in European countries. On the other hand, a delta F508 frequency highly heterogeneous has been observed in Latin-American countries. This variation results from mixed populations with a different genetic background influenced by external migration and CF molecular alterations, which exists in the analyzed populations. In this study, the delta F508 mutation comes mainly from grandparents (79.41%) who were born in Mediterranean countries and Colombia, while the no delta F508 mutations come from grandparents who were born in Venezuela (79.27%) and Colombia (17.07%).     

Clinical features of cystic fibrosis patients with rare genotypes.

We describe the clinical features of seven cystic fibrosis patients from southern Italy who bear rare genotypes: (1) a patient homozygous for the 2183 AA-->G mutation who was affected by a very early pulmonary form of cystic fibrosis, and five patients who were compound heterozygotes either for the 2183 AA-->G mutation or for the I148T mutation, in both instances with the delta F508 mutation; and (2) a patient homozygous for the early nonsense R553X mutation who showed only a moderately severe form of cystic fibrosis. Our results confirm that environmental or genetic factors unrelated to the CF disease contribute significantly to the development of the phenotype.