Genes in the vicinity of <Emphasis Type="Italic">CFTR </Emphasis>modulate the cystic fibrosis phenotype in highly concordant or discordant F508del homozygous sib pairs

Cystic fibrosis (CF) is the most common severe autosomal recessive disease among Caucasians and is caused by lesions within the cystic fibrosis transmembrane conductance regulator ( CFTR) gene. The variability of CF disease severity suggests the effect of modifying factors. Thirty-four highly concordant and highly discordant F508del homozygous sib pairs, who have been selected out of a group of 114 pairs for extreme disease phenotypes by nutritional and pulmonary status, were typed at single nucleotide polymorphisms (SNPs) and short tandem repeat polymorphisms (STRPs) in the 24-cM CFTR-spanning region between D7S525 and D7S495. Allele frequencies differed significantly at D7S495, located within a 21-cM distance 3' of CFTR, comparing concordant mildly affected, concordant severely affected and discordant sib pairs, as judged by hypothesis-free permutation analysis by Monte Carlo simulation. A rare haplotype of two SNPs within the leptin gene promotor was found exclusively among the concordant mildly affected pairs. All concordant sib pairs shared the paternal F508del chromosome between CFTR and D7S495, whilst the cohort of discordant sib pairs inherited equal proportions of recombined and non-recombined parental chromosomes. We conclude that disease manifestation in CF is modulated by loci in the partially imprinted region 3' of CFTR that determine stature, food intake and energy homeostasis, such as the Silver-Russel-Syndrome candidate gene region and LEP.     

Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in allergic bronchopulmonary aspergillosis.

The etiology of allergic bronchopulmonary aspergillosis (ABPA) is not well understood. A clinical phenotype resembling the pulmonary disease seen in cystic fibrosis (CF) patients can occur in some individuals with ABPA. Reports of familial occurrence of ABPA and increased incidence in CF patients suggest a possible genetic basis for the disease. To test this possibility, the entire coding region of the cystic fibrosis transmembrane regulator (CFTR) gene was analyzed in 11 individuals who met strict criteria for the diagnosis of ABPA and had normal sweat electrolytes (< or = 40 mmol/liter). One patient carried two CF mutations (deltaF508/R347H), and five were found to carry one CF mutation (four deltaF508; one R117H). The frequency of the deltaF508 mutation in patients with ABPA was significantly higher than in 53 Caucasian patients with chronic bronchitis (P < .0003) and the general population (P < .003). These results suggest that CFTR plays an etiologic role in a subset of ABPA patients.     

Global proteomic approach unmasks involvement of keratins 8 and 18 in the delivery of cystic fibrosis transmembrane conductance regulator (CFTR)/deltaF508-CFTR to the plasma membrane

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF gene (cftr). Physiologically, CF is characterized by an abnormal chloride secretion in epithelia due to a dysfunction of a mutated cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a cAMP-dependent chloride channel whose most frequent mutation, deltaF508, leads to an aberrantly folded protein which causes a dysfunction of the channel. However, a growing number of reports suggest that modifier genes and environmental factors are involved in the physiology of CF. To identify proteins whose expression depends on wild-type WT-CFTR or deltaF508-CFTR, we chose a global proteomic approach based on the use of two-dimensional gel electrophoresis (2-DE) and mass spectrometry. The experiments were carried out with HeLa cells stably transfected with WT-CFTR (pTCFWT) or deltaF508-CFTR (pTCFdeltaF508). These experiments unmasked keratin 8 (K8) and 18 (K18) which were differentially expressed in pTCFWT vs. pTCFdeltaF508. An immunoblot of K18 confirmed the 2-DE results. Intracellular localization experiments of WT-CFTR, deltaF508-CFTR, K8, and K18 suggest that the expression of these proteins are linked, and that the concentrations of K8 and K18 and/or their distribution may be involved in the traffic of WT-CFTR/deltaF508-CFTR. A functional assay for CFTR revealed that specifically lowering K18 expression or changing its distribution leads to the delivery of functional deltaF508-CFTR to the plasma membrane. This work suggests a novel function of K18 in CF.     

Activating cystic fibrosis transmembrane conductance regulator channels with pore blocker analogs

Cystic fibrosis (CF) is caused by mutations that disrupt the surface localization and/or gating of the CF transmembrane conductance regulator (CFTR) chloride channel. The most common CF mutant is deltaF508-CFTR, which inefficiently traffics to the surfaces of most cells. The deltaF508 mutation may also disrupt the opening of CFTR channels once they reach the cell surface, but the extent of this gating defect is unclear. Here, we describe potent activators of wild-type and deltaF508-CFTR channels that are structurally related to 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), a negatively charged pore blocker that we show to have mixed agonistic activity (channel activation plus voltage-dependent pore block). These CFTR agonists include 1) an uncharged NPPB analog that stimulates channel opening at submicromolar concentrations without blocking the pore and 2) curcumin, a dietary compound recently reported to augment deltaF508-CFTR function in mice by an unknown mechanism. The uncharged NPPB analog enhanced the activities of wild-type and deltaF508-CFTR channels both in excised membrane patches and in intact epithelial monolayers. This compound increased the open probabilities of deltaF508-CFTR channels in excised membrane patches by 10-15-fold under conditions in which wild-type channels were already maximally active. Our results support the emerging view that CFTR channel activity is substantially reduced by the deltaF508 mutation and that effective CF therapies may require the use of channel openers to activate mutant CFTR channels at the cell surface.     

Functional cystic fibrosis transmembrane conductance regulator tagged with an epitope of the vesicular stomatis virus glycoprotein can be addressed to the apical domain of polarized cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated chloride channel apically localized in epithelial cells. In cystic fibrosis patients, the gene encoding this N-linked glycoprotein is mutated. About 70% of CF patients express a mutated form of CFTR, deleted at the phenylalanine residue at position 508 (deltaF508). CFTR-deltaF508 fails to exit the endoplasmic reticulum; it remains incompletely glycosylated and is rapidly degraded. To optimize CFTR detection for membrane localization studies and biochemical studies, we tagged wild-type and deltaF508 CFTR with the VSV-G epitope at their carboxy-terminal ends. We have generated pig kidney epithelial cell clones (LLCPK1) expressing VSV-G-tagged human wild-type and deltaF508-CFTR. In CFTR-expressing cells, the transfected protein is maturated and transported to the apical membrane where it is concentrated. The cells exhibit a strong anion channel activity after stimulation by cAMP, as demonstrated by a halide sensitive fluorescent dye assay (6-methoxy-N-ethylquinominium, SPQ), and whole-cell patch-clamp approach. This activity of CFTR-VSV-G is indistinguishable from the wild-type CFTR. In contrast, in cells expressing tagged deltaF508-CFTR or in non-transfected cells, no anion channel activity could be detected after stimulation by cAMP. In deltaF508-CFTR-VSV-G-expressing cells, the mutated CFTR remained in the incompletely glycosylated form and was localized in the endoplasmic reticulum. These cell lines reproduce the cellular fate of wild-type and mutated CFTR-deltaF508. To our knowledge, they are the first differentiated epithelial cell lines stably expressing tagged CFTR and CFTR-deltaF508 in which cellular processing and functional activity of these two proteins are reproduced. Thus the addition of the VSV-G epitope does not impair the localization and function of CFTR, and these cell lines can be used to examine CFTR function in vitro.     

Low frequency of the deltaAF508 mutation of the CFTR gene in a highly admixed population in Bahia, Brazil

Cystic fibrosis (CF) is the most common autosomal recessive disease in the European (Caucasian) population, with an incidence of 1:2000 to 1:8000. The deltaF508 mutation (66%) is predominant among more than 1300 different mutations of the CFTR gene. The population of the state of Bahia, in northeastern Brazil, is highly admixed (mainly African and Portuguese descendants), and so far, no study has been carried out to assess the molecular basis of CF in this population. We determined the deltaF508 mutation frequency in 503 individuals from the general population of Salvador, the capital of the state of Bahia, and in 144 CF patients from several cities in Bahia. In the general population samples we found 4 individuals heterozygous for the deltaF508 mutation (allele frequency of 0.4%). This frequency was lower than that found in the state of Rio de Janeiro, in southeastern Brazil, and similar to that reported for the state of Paraná, in the far south. In the CF patients we found 9 heterozygous individuals and 8 homozygous individuals (allele frequency of 8.68%) for the deltaF508 mutation. This frequency is considerably lower than the average frequency of CF in the world population and in the Brazilian CF population of European ancestry (47%). These data could be explained by the intense admixture among the population in Bahia, and they suggest a heterogeneous molecular basis for CF in this area of Brazil.     

Effect of genotype on selected clinical features of Polish cystic fibrosis adults

Cystic fibrosis (CF), the most common autosomal recessive disorder of Caucasians, is caused by the mutations in the gene encoding CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) protein. Until now, approximately 1000 mutations of the CFTR gene have been described. The genotype-phenotype relationships in CF are still not completely understood. This study was undertaken in an attempt to characterise the distribution of CFTR mutations and their effect on selected clinical parameters in a group of Polish CF adults. A total number of 38 adult CF patients (mean age 21.6 +/- 6.8); 18 females & 20 males were enrolled in the study. The CFTR gene identification was conducted with the use of PCR & InnoLipa-CF set. The assessed clinical parameters included: age at diagnosis, age, lung function test, X-ray scored in Brasfield score, weight & height. We found that: (1) the genotypes of the studied population were unevenly distributed (65.8%- genotype deltaF508/M), (2) a high percentage of 3849+10kbC-->T was noted, (3) patients homozygous for the deltaF508 mutation were diagnosed significantly earlier and had a lower body mass index, (4) no differences were observed in the patients' length of life or the progression of lung disease. Conclusions: 1. In comparison to other populations, Polish adult CF patients display a relatively higher frequency of mild mutations. 2. Late diagnosis of CF in the studied group may be partially caused by a high percentage of CFTR mutations connected with the mild course of the disease that are difficult to identify. 3. Cystic fibrosis should be more commonly taken into consideration in the differential diagnosis in adult patients with milder symptoms.     

Cystic fibrosis: S158N (605G --> A) is a rare genetic variant found in coupling with deltaF508

A single nucleotide change at codon 158 in exon 4 of the CFTR gene ABCC7 was detected in an asymptomatic individual who carried deltaF508 and had a family history of cystic fibrosis (CF). Further study, using linkage, revealed that S158N was coupled with deltaF508, both having been inherited from the same parent. The clinical implications of double mutations in the same allele are discussed.     

Characterization of wild-type and deltaF508 cystic fibrosis transmembrane regulator in human respiratory epithelia

Previous studies in native tissues have produced conflicting data on the localization and metabolic fate of WT and deltaF508 cystic fibrosis transmembrane regulator (CFTR) in the lung. Combining immunocytochemical and biochemical studies utilizing new high-affinity CFTR mAbs with ion transport assays, we examined both 1) the cell type and region specific expression of CFTR in normal airways and 2) the metabolic fate of deltaF508 CFTR and associated ERM proteins in the cystic fibrosis lung. Studies of lungs from a large number of normal subjects revealed that WT CFTR protein localized to the apical membrane of ciliated cells within the superficial epithelium and gland ducts. In contrast, other cell types in the superficial, gland acinar, and alveolar epithelia expressed little WT CFTR protein. No deltaF508 CFTR mature protein or function could be detected in airway specimens freshly excised from a large number of deltaF508 homozygous subjects, despite an intact ERM complex. In sum, our data demonstrate that WT CFTR is predominantly expressed in ciliated cells, and deltaF508 CFTR pathogenesis in native tissues, like heterologous cells, reflects loss of normal protein processing.     

Terminal glycosylation in cystic fibrosis

Cystic fibrosis (CF) is a common genetic disease for which the gene was identified within the last decade. Pulmonary disease predominates in this ultimately fatal disease and current therapy only slows the progression. CF transmembrane regulator (CFTR), the gene product, is an integral membrane glycoprotein that normally functions as a chloride channel in epithelial cells. The most common mutation, deltaF508, results in mislocalization and altered glycosylation of CFTR. Altered fucosylation and sialylation are hallmarks of both membrane and secreted glycoproteins in CF and the focus here is on these investigations. Oligosaccharides from CF membrane glycoproteins have the Lewis x, selectin ligand in terminal positions. In addition, two major bacterial pathogens in CF, Pseudomonas aeruginosa and Haemophilus influenzae, have binding proteins, which recognize fucose in alpha1,3 linkage and asialoglycoconjugates. We speculate that the altered terminal glycosylation of airway epithelial glycoproteins in CF contributes to the chronic infection and robust inflammatory response in the CF lung. Understanding the effects of mutant CFTR on glycosylation may provide further insight into the regulation of glycoconjugate processing as well as therapy for CF.     

Different activation mechanisms of cystic fibrosis transmembrane conductance regulator expressed in Xenopus laevis oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP sensitive Cl- channel that is defective in cystic fibrosis (CF). The most frequent mutation, namely deltaF508-CFTR, accounts for 66% of CF. Here we show that cAMP-activation of CFTR occurs via at least two distinct pathways: activation of CFTR molecules already present in the plasma membrane and protein kinase A (PKA)-mediated vesicular transport of new CFTR molecules to the plasma membrane and functional insertion into the membrane. We investigated the mechanisms that are responsible for these activation pathways using the Xenopus laevis oocytes expression system. We expressed CFTR and recorded continuously membrane current (Im), conductance (Gm) and capacitance (Cm), which is a direct measure of membrane surface area. Expression of CFTR alone did not change the plasma membrane surface area. However, activation of CFTR with cAMP increased Im, Gm and Cm while deltaF508-CFTR-expressing oocytes showed no response on cAMP. Inhibition of protein kinase A or buffering intracellular Ca2+ abolished the cAMP-induced increase in Cm while increases of Im and Gm were still present. ATP or the xanthine derivative 8-cyclopentyl-1,3-dipropylxanthine (CPX) did not further activate CFTR. Insertion of pre-formed CFTR into the plasma membrane could be prevented by compounds that interfere with intracellular transport mechanisms such as primaquine, brefeldin A, nocodazole. From these data we conclude that cAMP activates CFTR by at least two distinct pathways: activation of CFTR already present in the plasma membrane and exocytotic delivery of new CFTR molecules to the oocyte membrane and functional insertion into it.     

Apical CFTR Expression in Human Nasal Epithelium Correlates with Lung Disease in Cystic Fibrosis

Introduction

Although most individuals with cystic fibrosis (CF) develop progressive obstructive lung disease, disease severity is highly variable, even for individuals with similar CFTR mutations. Measurements of chloride transport as expression of CFTR function in nasal epithelial cells correlate with pulmonary function and suggest that F508del-CFTR is expressed at the apical membrane. However, an association between quantitative apical CFTR expression in nasal epithelium and CF disease severity is still missing.

Methods and Materials

Nasal epithelial cells from healthy individuals and individuals with CF between 12–18 years were obtained by nasal brushing. Apical CFTR expression was measured by confocal microscopy using CFTR mAb 596. Expression was compared between both groups and expression in CF nasal epithelial cells was associated with standardized pulmonary function (FEV₁%).

Results

The proportion of cells expressing apical CFTR in columnar epithelium is lower in CF compared to non-CF. The apical CFTR expression level was significantly correlated with FEV₁% in F508del homozygous subjects (r = 0.63, p = 0.012).

Conclusion

CFTR expression in nasal epithelial cells is lower in subjects with CF compared to healthy subjects. The proportion of cells expressing F508del-CFTR at the apical membrane is variable between subjects and is positively correlated with FEV₁% in F508del-CFTR homozygous subjects.     

XV-2c and KM.19 haplotype analysis in Chilean patients with cystic fibrosis and unknown CFTR gene mutations

Cystic fibrosis (CF) is caused by mutations in the CFTR gene. More than 1600 mutations have been described, with frequencies that differ worldwide according to the ethnic origin of patients. A small group of mutations are recurrent on several populations. It has been shown that they each tend occur on specific chromosome 7 haplotypes, supporting the notion of a single origin for them. Less than 50% of mutations in Chilean patients have been identified to date. To indirectly assess the possible presence of a predominant founder mutation in the remaining unknown alleles, we evaluated 2 polymorphic markers, XV-2c and KM.19, tightly linked to the CFTR locus. The study was done in Chilean CF patients with unknown or deltaF508 (DeltaF508) CFTR mutations and their haplotypes were compared to affected family-based controls. DeltaF508 showed marked linkage disequilibrium with XV-2c/KM.19 haplotype B, with 90% of alleles on that haplotype. There was no difference in haplotype distribution between unknown mutations and normal controls. These results support a European origin for DeltaF508 alleles in Chilean patients, and make unlikely the presence of a predominant founder mutation in the so-far unknown alleles.     

Spectrum of CFTR mutations in Mexican cystic fibrosis patients: identification of five novel mutations (W1098C, 846delT, P750L, 4160insGGGG and 297–1G→A)

We have analyzed 97 CF unrelated Mexican families for mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Our initial screening for 12 selected CFTR mutations led to mutation detection in 56.66% of the tested chromosomes. In patients with at least one unknown mutation after preliminary screening, an extensive analysis of the CFTR gene by single stranded conformation polymorphism (SSCP) or by multiplex heteroduplex (mHET) analysis was performed. A total of 34 different mutations representing 74.58% of the CF chromosomes were identified, including five novel CFTR mutations: W1098C, P750L, 846delT, 4160insGGGG and 297-1G-->A. The level of detection of the CF mutations in Mexico is still lower than that observed in other populations with a relatively low frequency of the deltaF508 mutation, mainly from southern Europe. The CFTR gene analysis described here clearly demonstrated the high heterogeneity of our CF population, which could be explained by the complex ethnic composition of the Mexican population, in particular by the strong impact of the genetic pool from southern European countries.     

Height discordance in monozygotic females is not attributable to discordant inactivation of X-linked stature determining genes.

We tested the hypothesis that X-linked genes determining stature which are subject to skewed or non-random X-inactivation can account for discordance in height in monozygotic female twins. Height discordant female monozygotic adult twins (20 pairs) were identified from the Australian Twin Registry, employing the selection criteria of proven monozygosity and a measured height discordance of at least 5 cm. Differential X-inactivation was examined in genomic DNA extracted from peripheral lymphocytes by estimating differential methylation of alleles at the polymorphic CAG triplet repeat of the Androgen receptor gene (XAR). There were 17/20 MZ pairs heterozygous at this locus and informative for analysis. Of these, 10/17 both had random X-inactivation, 5/17 showed identical X-inactivation patterns of non random inactivation and 2/17 (12%) showed discordant X-inactivation. There was no relationship between inactivation patterns and self-report chorionicity. We conclude that non-random X-inactivation does not appear to be a major contributor to intra-pair height discordance in female MZ twins.     

Haplotype distribution of and linkage disequilibrium between four polymorphic markers near the CFTR locus in Brazilian cystic fibrosis patients

To contribute to a better understanding of the origin and distribution of CFTR mutations in the Brazilian population, we have investigated the linkage between four polymorphic markers (XV2c, KM19, GATT, and TUB9) within or near the CFTR locus. The distribution of alleles for each polymorphism for both parental and cystic fibrosis (CF) chromosomes from Rio de Janeiro CF families were ascertained using a maximum-likelihood method. This same method was applied to study the distribution of the haplotypes defined by these markers. There was no significant association between the XV2c and KM19 loci on the parental and CF chromosomes. On the other hand, a strong association between GATT and TUB9 loci was observed on both CF and parental chromosomes, and striking linkage disequilibrium between the GATT-TUB9 pair and deltaF508 was observed (chi2 = 26.48, p < 0.0001). Remarkable linkage disequilibrium between the GATT-TUB9 marker pair and non-deltaF508 was also found (chi2 = 17.05, p < 0.0001). Our finding of a linkage disequilibrium between GATT-TUB9 and the CFTR locus could suggest that gene flow between different ethnic groups, mainly sub-Saharan and Mediterranean populations, with Brazilian populations could have resulted in some CF mutations originating on chromosomes that carried the GATT-TUB9 marker haplotype 7-2 (OR = 1.34 < 2.83 < 6.00; p = 0.0066).     

Targeting of carbonic anhydrase IV to plasma membranes is altered in cultured human pancreatic duct cells expressing a mutated (deltaF508) CFTR

Human pancreatic duct cells secrete HCO3- ions mediated by a Cl-/HCO3- exchanger and a HCO3- channel that may be a carbonic anhydrase IV (CA IV) in a channel-like conformation. This secretion is regulated by CFTR (Cystic Fibrosis Transmembrane conductance Regulator). In CF cells homozygous for the deltaF508 mutation, the defect in targeting of CFTR to plasma membranes leads to a disruption in the secretion of Cl- and HCO3 ions along with a defective targeting of other proteins. In this study, we analyzed the targeting of membrane CA IV in the human pancreatic duct cell line CFPAC-1, which expresses a deltaF508 CFTR, and in the same cells transfected with the wild-type CFTR (CFPAC-PLJ-CFTR6) or with the vector alone (CFPAC-PLJ6). The experiments were conducted on cells in the stationary phase the polarized state of which was checked by the distribution of occludin and actin. We show that both cell lines express a 35-kDa CA IV at comparable levels. Analysis of fractions of plasma membranes purified on a Percoll gradient evidenced lower levels of CA IV (8-fold) in the CFPAC-1 than in the CFPAC-PLJ-CFTR6 cells. Quantitative analyses showed that 6- to 10-fold fewer cells in the CFPAC-1 cell line exhibited membrane CA IV-immunoreactivity than in the CFPAC-PLJ-CFTR6 cell line. Taken together, these results suggest that the targeting of CA IV to apical plasma membranes is impaired in CFPAC-1 cells. CA IV/gamma-adaptin double labeling demonstrated the presence of CA IV in the trans-Golgi network (TGN) of numerous CFPAC-1 cells, indicating that trafficking was disrupted on the exit face of the TGN. The retargeting of CA IV observed in CFPAC-PLJ-CFTR6 cells points to a relationship between the traffic of CFTR and CA IV. On the basis of these observations, we propose that the absence of CA IV in apical plasma membranes due to the impairment in targeting in cells expressing a deltaAF508 CFTR largely contributes to the disruption in HCO3- secretion in CF epithelia.     

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.     

Cystic fibrosis gene variability in two southern Brazilian Amerindian populations: analysis of the deltaF508 mutation and the KM19 and XV2C haplotypes

The frequencies of the deltaF508 deletion, the most common cystic fibrosis mutation in Europeans and European-derived populations, and the XV2C and KM19 restriction fragment length polymorphisms that are tightly linked to the CFTR locus vary among populations. To determine the distribution of these extragenic markers and of the deltaF508 mutation, we analyzed 326 chromosomes of individuals from two South American Indian populations, the Guarani and the Kaingang. The allele and haplotype frequencies differed greatly between the two populations as well as among Amerindians and normal European Brazilians and European Brazilian cystic fibrosis patients. The absence of the deltaF508 mutation and the B haplotype are in agreement with the hypothesis that the deltaF508 mutation occurred after the divergence of these two populations. This finding is useful for populations containing a large Amerindian component and helps us to understand the origins of the deltaF508 deletion, the most common cystic fibrosis mutation in Europeans and European-derived populations, as well as the different incidences of cystic fibrosis in continental groups.