Frequency of ΔF508 and haplotype association in Austrian cystic fibrosis families

Summary The frequency of the major mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene was analyzed for 113 Austrian cystic fibrosis (CF) patients. An overall frequency of 55% for F508 was found with values of 72% and 13% for patients with pancreatic insufficiency (CF-PI) and those with pancreatic sufficiency (CF-PS), respectively. Furthermore, the distribution of the alleles of the closely linked DNA markers XV2c/KM19/MP6d-9 in our families is described.     

Lung involvement,the ΔF508 mutation and DNA haplotype analysis in cystic fibrosis

Summary Molecular studies of cystic fibrosis (CF) have allowed the genetic analysis of patients by means of DNA markers and the direct analysis of the CF gene. Some limited observations are available on the correlation between phenotype and genotype. Here, we report a study on the correlation of DNA haplotypes identified by KM-19 and XV-2c, the presence of the F508 mutation and lung involvement in 82 unrelated CF patients. Pulmonary involvement was defined by Chrispin's chest X-ray score, pulmonary function, sputum microbiology, serum immunoglobulin (SIg) levels and Shwachman's clinical score. Patients homozygous for haplotype B showed worse X-ray and clinical scores, more frequent sputum colonization byPseudomonas aeruginosa andStaphylococcus aureus, lower spirometric values and raised concentrations of SIg G, A and M, compared with patients with other haplotypes. When lung involvement parameters were examined in patients homozygous, heterozygous or null for the F508 mutation, no difference was found among the three groups. Our data indicate a significant occurrence of severe pulmonary involvement in patients homozygous for the B haplotype; this is not influenced by the F508 mutation. We suggest that simple DNA haplotypes may provide data of both diagnostic and prognostic value, without the need for extensive and expensive molecular analyses.     

Allosteric modulation balances thermodynamic stability and restores function of ΔF508 CFTR

Most cystic fibrosis is caused by a deletion of a single residue (F508) in CFTR (cystic fibrosis transmembrane conductance regulator) that disrupts the folding and biosynthetic maturation of the ion channel protein. Progress towards understanding the underlying mechanisms and overcoming the defect remains incomplete. Here, we show that the thermal instability of human ΔF508 CFTR channel activity evident in both cell-attached membrane patches and planar phospholipid bilayers is not observed in corresponding mutant CFTRs of several non-mammalian species. These more stable orthologs are distinguished from their mammalian counterparts by the substitution of proline residues at several key dynamic locations in first N-terminal nucleotide-binding domain (NBD1), including the structurally diverse region, the γ-phosphate switch loop, and the regulatory insertion. Molecular dynamics analyses revealed that addition of the prolines could reduce flexibility at these locations and increase the temperatures of unfolding transitions of ΔF508 NBD1 to that of the wild type. Introduction of these prolines experimentally into full-length human ΔF508 CFTR together with the already recognized I539T suppressor mutation, also in the structurally diverse region, restored channel function and thermodynamic stability as well as its trafficking to and lifetime at the cell surface. Thus, while cellular manipulations that circumvent its culling by quality control systems leave ΔF508 CFTR dysfunctional at physiological temperature, restoration of the delicate balance between the dynamic protein's inherent stability and channel activity returns a near-normal state.     

The haplotype distribution of the ΔF508 mutation in cystic fibrosis families in Scotland

Summary The gene defective in cystic fibrosis (CF) has recently been isolated and the major mutation identified. The haplotype distribution of this mutation (ΔF508) has been determined for 215 CF chromosomes in the Scottish population. ΔF508 represents 73% of all CF mutations in this group. There remains considerable linkage disequilibrium between XV2c and KM19 and other mutations in the CF gene.     

The frequency of the CF ΔF508 deletion in CF chromosomes of different ethnic origin

Summary The identification of the molecular defect in a significant proportion of cystic fibrosis families (in our series up to 60%) allows direct DNA diagnosis.     

Polymorphic DNA haplotypes and ΔF508 deletion in 212 Italian CF families

Summary Haplotype data based on the DNA markers closely linked to the cystic fibrosis (CF) gene have been used to correlate the presence of the 3 by specific deletion (ΔF508) in 424 CF chromosomes from 212 Italian CF families. The distribution and the frequency of the F508 deletion on CF chromosomes in our sample suggests the presence of at least a second mutation in the same ancestral haplotype.     

Regulatory Insertion Removal Restores Maturation, Stability and Function of ΔF508 CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel is a large multidomain membrane protein that matures inefficiently during biosynthesis. Its assembly is further perturbed by the deletion of F508 from the first nucleotide-binding domain (NBD1) responsible for most cystic fibrosis. The mutant polypeptide is recognized by cellular quality control systems and is proteolyzed. CFTR NBD1 contains a 32-residue segment termed the regulatory insertion (RI) not present in other ATP-binding cassette transporters. We report here that RI deletion enabled F508 CFTR to mature and traffic to the cell surface where it mediated regulated anion efflux and exhibited robust single chloride channel activity. Long-term pulse-chase experiments showed that the mature ΔRI/ΔF508 had a T_1/2 of ∼ 14 h in cells, similar to the wild type. RI deletion restored ATP occlusion by NBD1 of ΔF508 CFTR and had a strong thermostabilizing influence on the channel with gating up to at least 40 °C. None of these effects of RI removal were achieved by deletion of only portions of RI. Discrete molecular dynamics simulations of NBD1 indicated that RI might indirectly influence the interaction of NBD1 with the rest of the protein by attenuating the coupling of the F508-containing loop with the F1-like ATP-binding core subdomain so that RI removal overcame the perturbations caused by F508 deletion. Restriction of RI to a particular conformational state may ameliorate the impact of the disease-causing mutation.     

The incidence of ΔF508 CF mutation,and associated haplotypes,in a sample of English CF families

Summary Data are presented for ΔF508 screening and KM19/XV2c haplotype analysis of 195 cystic fibrosis (CF) chromosomes from the British Caucasian population. We report the frequency of ΔF508 in this group to be 80% and find pronounced disequilibrium between the deletion and the KM 2, XV 1 haplotype. Haplotype analysis of 71 normal chromosomes is also presented. We report one individual who had meconium ileus and who does not have the ΔF508 mutation on either chromosome.     

Regulation of endogenous ENaC functional expression by CFTR and ΔF508-CFTR in airway epithelial cells

The functional expression of the epithelial sodium channel (ENaC) appears elevated in cystic fibrosis (CF) airway epithelia, but the mechanism by which this occurs is not clear. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) alters the trafficking of endogenously expressed human ENaC in the CFBE41o? model of CF bronchial epithelia. Functional expression of ENaC, as defined by amiloride-inhibited short-circuit current (I(sc)) in Ussing chambers, was absent under control conditions but present in CFBE41o? parental and ΔF508-CFTR-overexpressing cells after treatment with 1 μM dexamethasone (Dex) for 24 h. The effect of Dex was mimicked by incubation with the glucocorticoid hydrocortisone but not with the mineralocorticoid aldosterone. Application of trypsin to the apical surface to activate uncleaved, "near-silent" ENaC caused an additional increase in amiloride-sensitive I(sc) in the Dex-treated cells and was without effect in the control cells, suggesting that Dex increased ENaC cell surface expression. In contrast, Dex treatment did not stimulate amiloride-sensitive I(sc) in CFBE41o? cells that stably express wild-type (wt) CFTR. CFBE41o? wt cells also had reduced expression of α- and γ-ENaC compared with parental and ΔF508-CFTR-overexpressing cells. Furthermore, application of trypsin to the apical surface of Dex-treated CFBE41o? wt cells did not stimulate amiloride-sensitive I(sc), suggesting that ENaC remained absent from the surface of these cells even after Dex treatment. We also tested the effect of trafficking-corrected ΔF508-CFTR on ENaC functional expression. Incubation with 1 mM 4-phenylbutyrate synergistically increased Dex-induced ENaC functional expression in ΔF508-CFTR-overexpressing cells. These data support the hypothesis that wt CFTR can regulate the whole cell, functional, and surface expression of endogenous ENaC in airway epithelial cells and that absence of this regulation may foster ENaC hyperactivity in CF airway epithelia.     

Benzbromarone stabilizes ΔF508 CFTR at the cell surface

Deletion of Phe508 from the first nucleotide-binding domain of the CFTR chloride channel causes cystic fibrosis because it inhibits protein folding. Indirect approaches such as incubation at low temperatures can partially rescue ΔF508 CFTR, but the protein is unstable at the cell surface. Here, we show that direct binding of benzbromarone to the transmembrane domains promoted maturation and stabilized ΔF508 CFTR because its half-life at the cell surface was ~10-fold longer than that for low-temperature rescue. Therefore, a search for small molecules that can rescue and stabilize ΔF508 CFTR could lead to the development of an effective therapy for cystic fibrosis.     

CFTR: folding, misfolding and correcting the ΔF508 conformational defect

Cystic fibrosis (CF), the most common lethal genetic disease in the Caucasian population, is caused by loss-of-function mutations of the CF transmembrane conductance regulator (CFTR), a cyclic AMP-regulated plasma membrane chloride channel. The most common mutation, deletion of phenylalanine 508 (ΔF508), impairs CFTR folding and, consequently, its biosynthetic and endocytic processing as well as chloride channel function. Pharmacological treatments may target the ΔF508 CFTR structural defect directly by binding to the mutant protein and/or indirectly by altering cellular protein homeostasis (proteostasis) to promote ΔF508 CFTR plasma membrane targeting and stability. This review discusses recent basic research aimed at elucidating the structural and trafficking defects of ΔF508 CFTR, a prerequisite for the rational design of CF therapy to correct the loss-of-function phenotype.     

The occurrence of various non-ΔF508 CFTR gene mutations among Hungarian cystic fibrosis patients

Summary Cystic fibrosis (CF) is an autosomal recessive disease caused by different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The frequency of the major mutation (F508) in the Hungarian population is 64%. To identify other common mutations in CF families from Hungary, 30 nonF508 CF chromosomes were analyzed for selected mutations in exon 11 (G551D, R553X, G542X), intron 4 (621+1GT), intron 10 (1717–1GA), exon 20 (W1282X), and in exon 21 (N1303K) of the CFTR gene. In 6 of the 30 non-F508 CF chromosomes the following mutations were detected: R553X, G542X, 1717–1GA, W1282X, and N1303K. After analysis of the above eight mutations, 30% of CF chromosomes are as yet undefined and further analysis is planned.     

Requirements for efficient correction of ΔF508 CFTR revealed by analyses of evolved sequences

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Highlights? Statistical coupling analyses reveal positions linked to F508 ? Coupled positions reveal that the ΔF508 mutation interferes with two steps in CFTR folding ? Two defects of ΔF508 misfolding explain the efficacy ceiling observed for correctors ? Correction of both defective steps is synergistic and required to restore function     

Frequency of the ΔF508 deletion and G551D,R553X and G542X mutations in Yugoslav CF patients

Summary A study was undertaken to find the frequency of the F508 deletion and those of the G551D, R553X and G524X mutations among the mainly Slavic population of Serbia, Bosnia, Herzegovina, and Montenegro and compare the frequencies determined with those in other European populations. The F508 mutation was found to account for about 70% of CF genes in central Jugoslavia, where its frequency is significantly higher than elsewhere in Southern European populations.     

The primary folding defect and rescue of ΔF508 CFTR emerge during translation of the mutant domain

In the vast majority of cystic fibrosis (CF) patients, deletion of residue F508 from CFTR is the cause of disease. F508 resides in the first nucleotide binding domain (NBD1) and its absence leads to CFTR misfolding and degradation. We show here that the primary folding defect arises during synthesis, as soon as NBD1 is translated. Introduction of either the I539T or G550E suppressor mutation in NBD1 partially rescues ΔF508 CFTR to the cell surface, but only I539T repaired ΔF508 NBD1. We demonstrated rescue of folding and stability of NBD1 from full-length ΔF508 CFTR expressed in cells to isolated purified domain. The co-translational rescue of ΔF508 NBD1 misfolding in CFTR by I539T advocates this domain as the most important drug target for cystic fibrosis.     

Reduced PDZ Interactions of Rescued ΔF508CFTR Increases Its Cell Surface Mobility

Deletion of phenylalanine 508 (ΔF508) in the cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane chloride channel is the most common cause of cystic fibrosis (CF). Though several maneuvers can rescue endoplasmic reticulum-retained ΔF508CFTR and promote its trafficking to the plasma membrane, rescued ΔF508CFTR remains susceptible to quality control mechanisms that lead to accelerated endocytosis, ubiquitination, and lysosomal degradation. To investigate the role of scaffold protein interactions in rescued ΔF508CFTR surface instability, the plasma membrane mobility of ΔF508CFTR was measured in live cells by quantum dot single particle tracking. Following rescue by low temperature, chemical correctors, thapsigargin, or overexpression of GRASP55, ΔF508CFTR diffusion was more rapid than that of wild-type CFTR because of reduced interactions with PDZ domain-containing scaffold proteins. Knock-down of the plasma membrane quality control proteins CHIP and Hsc70 partially restored ΔF508CFTR-scaffold association. Quantitative comparisons of CFTR cell surface diffusion and endocytosis kinetics suggested an association between reduced scaffold binding and CFTR internalization. Our surface diffusion measurements in live cells indicate defective scaffold interactions of rescued ΔF508CFTR at the cell surface, which may contribute to its defective peripheral processing.     

Validation of the determination of ΔF508 mutations of the cystic fibrosis gene in over 11000 mouthwashes

Mouthwashes can be used as a DNA resource for mutation detection and, because collection and DNA isolation is simple and cheap, they could in particular, be used for large numbers of samples. To determine the failure rate (the proportion of mouth samples in which no PCR product was obtained) and the specificity of buccal epithelial cell mutation detection in large numbers of samples, we collected mouthwashes and blood samples from 11413 blood donors and tested the mouthwashes for the F508 mutation, which has an estimated frequency of 75% among cystic fibrosis chromosomes in The Netherlands. Blood samples were tested for the F508 mutations only if the mutation was identified in the mouthwash or in the case of a failure to obtain PCR products. The sensitivity of the test was determined in mouthwashes of 75 F508 carriers known from earlier family studies. These samples were offered blindly between the mouthwashes of the blood donors. Both specificity and sensitivity of the mouthwash procedure were 100%. The overall failure rate was 5.6%. This large figure was caused mainly by insufficient rinsing of the mouth in one particular blood bank. Exclusion of the results of this blood bank reduced the failure rate to 1.8%. Our results also confirm that for a large number of samples the mouthwash procedure is suitable for mutation detection and, with proper instructions, can be used in community screening.