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Highly informative intragenic microsatellite markers within the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene allow the analysis of associations between specific mutations and haplotypes. We have analysed 440 Spanish CF families carrying 22 different CF mutations and have established haplotypes in 1,036 chromosomes for microsatellites IVS8CA, IVS17BTA and IVS17BCA. No new alleles were detected at the three CFTR microsatellites, in more than 3,000 meiosis analysed (estimated mutation rate of less than 3.3 x 10(-4)). The evolution of 16 haplotypes associated with the most common CF mutation, delta F508, and the low mutation rate at these microsatellite loci suggest that delta F508 originated within the 23-31-13 haplotype at least 53,000 years ago, very early in the history of the European population. The number of haplotype changes seen for two other common mutations, G542X (haplotype 23-33-13) and N1303K (23-31-13), suggests that they originated at least 35,000 years ago. Microsatellite allele variability associated with delta F508, G542X and N1303K demonstrates that slippage and mispairing is the main mechanism generating microsatellite alleles. In spite of the haplotype variability detected for these 3 common mutations, the association between haplotype and mutations is very strong. Mutations 1609delCA, 3667del4, delta I507 and G551D are all associated with haplotype 16-7-17, which has a frequency of 14.5% in normal chromosomes. 5 haplotypes bearing specific CF mutations were not found in normal chromosomes. Haplotype 16-46-13 is strongly associated with CF mutations E92K and 3601-111G-->C. About 23% of CF chromosomes with unknown mutations show significant linkage disequilibrium for microsatellite haplotypes.(ABSTRACT TRUNCATED AT 250 WORDS)
Hum Mol Genet 1993 Jul
PMID:Microsatellite haplotypes for cystic fibrosis: mutation frameworks and evolutionary tracers. 768 96

Cystic fibrosis (CF) is characterized by a dramatic neutrophil recruitment and repeated Pseudomonas infections in the lungs. To evaluate cytokine releasibility by airway epithelial cells in the context of CF, we studied primary nasal epithelial cells isolated from the upper airways and continuous epithelial cell lines from normal and CF subjects. Relatively low levels of interleukin (IL)-8, IL-6, and granulocyte/macrophage colony-stimulating factor (GM-CSF) were produced spontaneously by primary epithelial cells (< 50 pg/10(6) cells) and higher levels of colony-stimulating factor-1 (CSF-1) (1 to 2 ng/10(6) cells). Cells were stimulated with substances that are likely to be present in the inflamed lungs of CF patients-namely, the proinflammatory monokines IL-1 and tumor necrosis factor-alpha (TNF alpha) as well as neutrophil elastase and bacterial products from Pseudomonas (mucoid exopolysaccharide [MEP] and rhamnolipids). Both IL-1 and TNF alpha induced a dose-dependent release of IL-6 (5 to 10 ng/10(6) cells) and GM-CSF (2 to 3 ng/10(6) cells) by primary epithelial cells from eight normal volunteers. The TNF alpha/IL-1-stimulated GM-CSF release was blocked by the addition of 1 microM dexamethasone, whereas basal CSF-1 release was unaffected. Neutrophil elastase was a potent inducer of IL-8 and GM-CSF both in primary epithelial cells and in cell lines. Dexamethasone (1 microM) did not inhibit elastase-induced IL-8 release in either normal or CF epithelial cells. Rhamnolipids and MEP were found to stimulate the copious release of IL-8, GM-CSF, and IL-6 from epithelial cells, in a steroid-sensitive fashion.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1993 Oct
PMID:Release of interleukin-8, interleukin-6, and colony-stimulating factors by upper airway epithelial cells: implications for cystic fibrosis. 769 Nov 10

In order to characterize the non-delta F508 mutations that account for 36% of cystic fibrosis (CF) chromosomes in Southern France in a sample of 137 patients, we have systematically screened the entire coding region and adjacent sequences of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by the single strand conformation polymorphism (SSCP) technique followed by direct sequencing of the mutant DNAs. We identified 13 novel mutations (9 reported in this paper) and 4 novel rare nucleotide sequence variations. Forty different mutations including delta F508, located in 15 exons, account for only 91.2% of mutants in a population originating from Southern France, in contrast with a recent report on the Celtic population of Brittany demonstrating that 90% of mutations can be detected with only three mutations. We present a very large spectrum of different CF mutations identified in a small geographical area.
Hum Mol Genet 1993 Aug
PMID:Analysis of the 27 exons and flanking regions of the cystic fibrosis gene: 40 different mutations account for 91.2% of the mutant alleles in southern France. 769 44

Cystic fibrosis (CF) is caused by mutations in the gene encoding a chloride channel called the CF transmembrane conductance regulator (CFTR). A single mutation in this gene, deletion of three nucleotides that leads to the absence of phenylalanine 508 (i.e., delta F508), is found on 70% of all CF chromosomes. To explore the molecular mechanism(s) responsible for defective chloride transport in patients with CF, we have studied the processing, localization, and function of wild type (W.T.), delta F508 and G551D CFTR (a G-->D missense mutation at position 551) in retrovirus transduced L cells. Cell transduced with W.T. CFTR expressed a 170 kd CFTR protein that was endoglycosidase H (Endo H) resistant, localized to the plasma membrane, and generated a cAMP-mediated anion conductance (GCl) when stimulated with standard concentrations of forskolin (5 microM), cpt cAMP (400 microM) and IBMX (100 microM). The G551D CFTR was indistinguishable from W.T. CFTR with respect to post-translational processing and localization, but it did not produce a cAMP-activated GCl in response to the standard stimulation cocktail. However, raising the IBMX concentration to 4 mM produced GCl in G551D expressing cells. Cells transduced with delta F508 CFTR expressed an Endo H sensitive CFTR protein (approximately 140 kd) that was found in a cytosolic, perinuclear location. These cells did not respond to the standard cocktail, but approximately 20% of cells increased GCl when the cocktail contained 4 mM IBMX.(ABSTRACT TRUNCATED AT 250 WORDS)
Hum Mol Genet 1993 Aug
PMID:Molecular basis of defective anion transport in L cells expressing recombinant forms of CFTR. 769 45

The past decade of research in cystic fibrosis has produced a wealth of information about the underlying defect responsible for the disease. The initial finding that the physiological disturbance in CF is one of abnormal electrolyte transport across epithelial tissues led to the elucidation of a pathway in which epithelial chloride transport is normally elicited in response to beta-adrenergic stimuli and involves the second messenger cAMP to activate protein kinase A. While that pathway was being described, work on the genetic front was concurrently providing information about the genomic location of the gene causing CF, which ultimately led to the identification and cloning of the gene encoding the cystic fibrosis transmembrane conductance regulator. The cloned CFTR gene provided a powerful reagent to use in the next generation of cell physiology experiments, in which it was determined that CFTR is not only the substrate of PKA phosphorylation, a step previously determined to be in the activation pathway of the chloride channel, but is in fact a cAMP-dependent chloride conducting channel itself. Further analysis of the gene has shown that although there is a single mutation that accounts for most of CF, there are well over 200 other lesions within the gene that can cause disease as well. Identification of these mutations has provided information into the normal function of CFTR by studying these variants in heterologous expression systems. As a result, the molecular mechanism of CFTR function is beginning to unfold, as well as the mechanism by which particular mutations impair that function. From a clinical perspective, the research brings optimism from two directions. First, understanding how disease-causing mutations impair function may culminate in pharmacologic approaches that can restore function to some of these mutants. Second, treating the disease at the level of the gene appears to be a realistic goal: Gene transfer experiments in cultured CF cells have shown that the procedure will restore cAMP-dependent chloride conductance to the cells, laying the groundwork for somatic cell gene therapy as a feasible treatment for CF. Currently, work is rapidly progressing in developing delivery systems for this purpose. Finally, animal models that should not only aid in understanding the physiology of electrolyte transport in epithelia but should serve as indicators for tests of therapeutic approaches to treating CF are being developed, either by pharmacological means or by gene delivery protocols.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Genet Med 1993
PMID:Molecular biology of cystic fibrosis. 769 8

We have identified a novel CFTR missense mutation associated with a protein trafficking defect in mammalian cells but normal chloride channel properties in a Xenopus oocyte assay. The mutation, a cysteine for glycine substitution at residue 480 (G480C), was detected in a pancreatic insufficient, African-American, cystic fibrosis (CF) patient. G480C was found on one additional CF chromosome and on none of 220 normal chromosomes, including 160 chromosomes from normal African-American individuals. Western blot analysis and immunofluorescence studies revealed that, in 293T cells, the encoded mutant protein was not fully glycosylated and failed to reach the plasma membrane, suggesting that the G480C protein was subject to defective intracellular processing. However, in Xenopus oocytes, a system in which mutant CFTR proteins are less likely to experience an intracellular processing/trafficking deficit, expression of G480C CFTR was associated with a chloride conductance that exhibited a sensitivity to activation by forskolin and 3-isobutyl-1-methylxanthine (IBMX) that was similar to that of wild-type CFTR. This appears to be the first identification of a CFTR mutant with a single amino acid substitution in which the sole basis for disease is mislocalization of the protein.
Hum Mol Genet 1995 Feb
PMID:Missense mutation (G480C) in the CFTR gene associated with protein mislocalization but normal chloride channel activity. 775 78

Integrative gene therapy typically requires dividing cells. This requirement has been perceived as an impediment for gene transfer to mature, uninjured airways where proliferation rates are very low. In diseases such as cystic fibrosis (CF) that may be candidates for integrative gene therapy, airway cell turnover is not known but may be increased as a result of chronic inflammation. To determine if cells in airway surface epithelium and submucosal glands of CF patients proliferate at an increased rate, paraffin sections of bronchial segments removed from CF patients (n = 6) at the time of lung transplantation or rapid autopsy and from non-CF patients (n = 4) undergoing lung resection or transplantation were immunostained with PC10, a monoclonal antibody to proliferating cell nuclear antigen (PCNA), a marker of proliferating cells. The PCNA index (percentage of nuclei immunostaining for PCNA) in CF bronchial surface epithelium was 17.0 +/- 4.6% (mean +/- SEM), substantially greater than in non-CF airways (less than 0.2%). Within submucosal glands, PCNA-positive cells were more prevalent in the collecting ducts of CF patients than in those of normal subjects, but only rare mucous or serous cells were PCNA positive. These studies show that airway epithelial cell proliferation rates can be very high in inflamed CF airways. This prevalence of proliferating cells suggests that CF airway epithelium and submucosal gland ducts may be amenable to gene transfer using vectors, such as retroviruses, that require cell replication for stable integrative expression. Further studies are needed to evaluate cell proliferation in CF airways with less extensive airway injury.
Am J Respir Cell Mol Biol 1995 Jun
PMID:Cell proliferation in bronchial epithelium and submucosal glands of cystic fibrosis patients. 776 25

We have cloned a cDNA from the human epithelial cell line T84 whose predicted amino acid sequence shows 93.9% identity with rat CIC-2. Mapping by somatic cell hybrids and polymerase chain reaction localizes the gene corresponding to this cDNA to chromosome 3q26-qter. The major transcription start site assessed by RNA primer extension is 100 nt upstream of the putative translation initiation codon. Analysis of the 5' flanking sequence revealed a high GC content and lack of common transcriptional elements such as TATA and CCAAT boxes. Northern blot analysis indicated wide organ distribution including tissues affected in cystic fibrosis (CF) and expression in an airway epithelial cell line derived from a CF patient. The high degree of sequence similarity and similar tissue distribution to rat CIC-2 suggests that this cDNA encodes the human CIC-2 voltage-gated chloride channel. Since this chloride channel is present in epithelial tissues it may be amenable to manipulation to circumvent the chloride secretion defect observed in CF.
Hum Mol Genet 1995 Mar
PMID:Cloning of a putative human voltage-gated chloride channel (CIC-2) cDNA widely expressed in human tissues. 779 95

There is considerable potential to ameliorate the pulmonary disease in cystic fibrosis (CF) using somatic gene therapy. Even low levels of expression of the gene in airways epithelium may be beneficial. Adenoviral vectors, DNA-liposome complexes, adeno-associated viral vectors and DNA-ligand complexes have been used effectively in vitro and have been tested in animals to varying extent. Adenoviral vectors and DNA-liposome complexes are being used to deliver the CF gene to patient airways in phase I clinical trials. Transient correction of the electrophysiological defect in human CF nasal epithelium has been achieved. Major goals are (i) to demonstrate that expression of the CF gene in airways epithelium will ameliorate lung disease in CF patients, and (ii) to achieve long-term expression of the introduced gene either through a single delivery with persistent expression or through the ability to use a delivery system repetitively with safety and efficacy.
Hum Mol Genet 1994
PMID:Somatic gene therapy for cystic fibrosis. 784 44

This paper describes low-density mucus glycoconjugates released from feline trachea by dirhamnolipid (DRL), a toxin from Pseudomonas aeruginosa. Mucus glycoconjugates in feline tracheas were radiolabeled in vivo with 3H-proline and 14C-glucose. Control mucus and that released by 200 micrograms/ml DRL were dissolved in guanidine hydrochloride buffer (GuHCl) and chromatographed on Sepharose CL-2B. Molecules eluting in the void volume (V0) of the column were isolated by isopycnic density gradient centrifugation in CsCl/GuHCl. All samples gave peaks of radiolabeled and periodic acid/Schiff (PAS)-reactive material at rho = approximately 1.50 and approximately 1.60 g/ml, but DRL-stimulated samples contained low-density material (rho < 1.32 g/ml), also PAS-reactive and radiolabeled. Control secretions incubated with DRL in vitro did not form low-density material. In Triton X-100 (1% vol/vol), a nonionic detergent, low-density material behaved as smaller molecules, running in the partially included volume (Vi) of the column of Sepharose CL-2B, but still in the V0 of Sephacryl S-300. Incubation with chondroitinase ABC, heparinase II and III, and keratanase failed to change its elution profile on S-300, evidence against glycosaminoglycans; but proteolysis with trypsin or proteinase K gave two peaks, peptide fragments near the totally included volume of the column and glycopeptides in V0. The V0 glycopeptides banded between 1.50 and 1.55 g/ml in a CsCl gradient and eluted as a single peak in the Vi of Sephacryl S-400, suggesting a distinct homogeneous glycopeptide, smaller than those from normal mucins. The main 14C-labeled sugars in this glycopeptide were fucose, glucosamine, galactosamine, and galactose, consistent with a mucin. Thus, DRL releases stable but noncovalent complexes containing one or more distinct mucinlike glycoconjugates, probably combined with lipids and peptides. We discuss their possible relevance to airway diseases, including cystic fibrosis.
Am J Respir Cell Mol Biol 1995 Mar
PMID:Mucus glycoconjugate complexes released from feline trachea by a bacterial toxin. 787 96


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