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Characterization of Defects in Ion Transport and Tissue Development in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-Knockout Rats  [PDF]
Katherine L. Tuggle, Susan E. Birket, Xiaoxia Cui, Jeong Hong, Joe Warren, Lara Reid, Andre Chambers, Diana Ji, Kevin Gamber, Kengyeh K. Chu, Guillermo Tearney, Li Ping Tang, James A. Fortenberry, Ming Du, Joan M. Cadillac, David M. Bedwell, Steven M. Rowe, Eric J. Sorscher, Michelle V. Fanucchi
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0091253
Abstract: Animal models for cystic fibrosis (CF) have contributed significantly to our understanding of disease pathogenesis. Here we describe development and characterization of the first cystic fibrosis rat, in which the cystic fibrosis transmembrane conductance regulator gene (CFTR) was knocked out using a pair of zinc finger endonucleases (ZFN). The disrupted Cftr gene carries a 16 base pair deletion in exon 3, resulting in loss of CFTR protein expression. Breeding of heterozygous (CFTR+/?) rats resulted in Mendelian distribution of wild-type, heterozygous, and homozygous (CFTR?/?) pups. Nasal potential difference and transepithelial short circuit current measurements established a robust CF bioelectric phenotype, similar in many respects to that seen in CF patients. Young CFTR?/? rats exhibited histological abnormalities in the ileum and increased intracellular mucus in the proximal nasal septa. By six weeks of age, CFTR?/? males lacked the vas deferens bilaterally. Airway surface liquid and periciliary liquid depth were reduced, and submucosal gland size was abnormal in CFTR?/? animals. Use of ZFN based gene disruption successfully generated a CF animal model that recapitulates many aspects of human disease, and may be useful for modeling other CF genotypes, including CFTR processing defects, premature truncation alleles, and channel gating abnormalities.
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Allelic Variants Relate to Shifts in Faecal Microbiota of Cystic Fibrosis Patients  [PDF]
Serena Schippa, Valerio Iebba, Floriana Santangelo, Antonella Gagliardi, Riccardo Valerio De Biase, Antonella Stamato, Serenella Bertasi, Marco Lucarelli, Maria Pia Conte, Serena Quattrucci
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0061176
Abstract: Introduction In this study we investigated the effects of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene variants on the composition of faecal microbiota, in patients affected by Cystic Fibrosis (CF). CFTR mutations (F508del is the most common) lead to a decreased secretion of chloride/water, and to mucus sticky secretions, in pancreas, respiratory and gastrointestinal tracts. Intestinal manifestations are underestimated in CF, leading to ileum meconium at birth, or small bowel bacterial overgrowth in adult age. Methods Thirty-six CF patients, fasting and under no-antibiotic treatment, were CFTR genotyped on both alleles. Faecal samples were subjected to molecular microbial profiling through Temporal Temperature Gradient Electrophoresis and species-specific PCR. Ecological parameters and multivariate algorithms were employed to find out if CFTR variants could be related to the microbiota structure. Results Patients were classified by two different criteria: 1) presence/absence of F508del mutation; 2) disease severity in heterozygous and homozygous F508del patients. We found that homozygous-F508del and severe CF patients exhibited an enhanced dysbiotic faecal microbiota composition, even within the CF cohort itself, with higher biodiversity and evenness. We also found, by species-specific PCR, that potentially harmful species (Escherichia coli and Eubacterium biforme) were abundant in homozygous-F508del and severe CF patients, while beneficial species (Faecalibacterium prausnitzii, Bifidobacterium spp., and Eubacterium limosum) were reduced. Conclusions This is the first report that establishes a link among CFTR variants and shifts in faecal microbiota, opening the way to studies that perceive CF as a ‘systemic disease’, linking the lung and the gut in a joined axis.
Relating the Disease Mutation Spectrum to the Evolution of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)  [PDF]
Lavanya Rishishwar, Neha Varghese, Eishita Tyagi, Stephen C. Harvey, I. King Jordan, Nael A. McCarty
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0042336
Abstract: Cystic fibrosis (CF) is the most common genetic disease among Caucasians, and accordingly the cystic fibrosis transmembrane conductance regulator (CFTR) protein has perhaps the best characterized disease mutation spectrum with more than 1,500 causative mutations having been identified. In this study, we took advantage of that wealth of mutational information in an effort to relate site-specific evolutionary parameters with the propensity and severity of CFTR disease-causing mutations. To do this, we devised a scoring scheme for known CFTR disease-causing mutations based on the Grantham amino acid chemical difference matrix. CFTR site-specific evolutionary constraint values were then computed for seven different evolutionary metrics across a range of increasing evolutionary depths. The CFTR mutational scores and the various site-specific evolutionary constraint values were compared in order to evaluate which evolutionary measures best reflect the disease-causing mutation spectrum. Site-specific evolutionary constraint values from the widely used comparative method PolyPhen2 show the best correlation with the CFTR mutation score spectrum, whereas more straightforward conservation based measures (ConSurf and ScoreCons) show the greatest ability to predict individual CFTR disease-causing mutations. While far greater than could be expected by chance alone, the fraction of the variability in mutation scores explained by the PolyPhen2 metric (3.6%), along with the best set of paired sensitivity (58%) and specificity (60%) values for the prediction of disease-causing residues, were marginal. These data indicate that evolutionary constraint levels are informative but far from determinant with respect to disease-causing mutations in CFTR. Nevertheless, this work shows that, when combined with additional lines of evidence, information on site-specific evolutionary conservation can and should be used to guide site-directed mutagenesis experiments by more narrowly defining the set of target residues, resulting in a potential savings of both time and money.
'CFTR-opathies': disease phenotypes associated with cystic fibrosis transmembrane regulator gene mutations
Peadar G Noone, Michael R Knowles
Respiratory Research , 2001, DOI: 10.1186/rr82
Abstract: Cystic fibrosis (CF) is a recessive genetic disease that is caused by mutations on both CFTR alleles, resulting in abnormal sweat electrolytes, sino-pulmonary disease, male infertility, and pancreatic exocrine insufficiency in 95% of patients [1,2]. In its classic form, the disease is easily diagnosed early in life, through a combination of clinical evaluation and laboratory testing (including sweat testing, and CFTR mutation analysis) [3]. Depending on the ethnic background of the populations tested, common genetic mutations are identified in the majority of cases of CF. In the USA, two-thirds of patients carry at least one copy of the ΔF508 mutation, with approximately 50% of CF patients being homozygous for this mutation [4].A wide spectrum of molecular abnormalities may occur in the CFTR gene, and uncommon mutations that result in partial (residual) CFTR function may be associated with nonclassic presentations of disease. Overall, 7% of CF patients are not diagnosed until age 10 years, with a proportion not diagnosed until after age 15 years; some of these patients present a considerable challenge in establishing a diagnosis of CF. Moreover, the phenotype in these patients may vary widely [5,6]. The focus of the present review is on nonclassic phenotypes associated with mutations in the CFTR gene, which may manifest as male infertility (congenital bilateral absence of the vas deferens [CBAVD]), mild pulmonary disease and idiopathic chronic pancreatitis (ICP). These phenotypes are included within the definition of 'atypical CF'.CFTR is a transmembrane spanning protein with multiple activities that are related to normal epithelial cell function [2]. Mutations in CFTR result in abnormalities in epithelial ion and water transport, which are associated with derangements in airway mucociliary clearance and other cellular functions related to normal cell biology [7]. Depending on the molecular abnormality, the defect in CFTR may be the equivalent of that associated wit
Analysis of mutations in the cystic fibrosis transmembrane regulator (CFTR) gene in patients with obstructive azoospermia
Bernardino, Andrea L.F.;Lima, Cintia E.;Zatz, Mayana;
Genetics and Molecular Biology , 2003, DOI: 10.1590/S1415-47572003000100001
Abstract: congenital bilateral absence of the vas deferens (cbavd) accounts for 1%-2% of sterility in men. a high incidence of mutations, as well as the involvement of the 5t variant of the t tract length in intron 8 of the cystic fibrosis conductance regulator (cftr) gene, have been previously described in males with cbavd. herein we report the screening for mutations and for the 5t variant of the cftr gene in 17 patients with cbavd and three others with non-cabvd obstructive azoospermia. in the cbavd group, three patients (15%) were compound heterozygotes for mutations, and five patients (25%) had a mutation in one allele and the 5t variant in the other; the 5t variant was also present in two other patients, one of them being homozygous. the most frequent mutation was df508, present on five chromosomes (12.5%). a novel missense mutation (a399d) was detected in a japanese cbvad patient. our results yield further evidence for a strong association between male obstructive azoospermia caused by cbavd and mutation/5t variant in the cftr gene. the search for cftr mutations in such patients is thus recommended for genetic counseling of couples who undergo assisted fertilization due to cbavd.
Analysis of mutations in the cystic fibrosis transmembrane regulator (CFTR) gene in patients with obstructive azoospermia  [cached]
Bernardino Andrea L.F.,Lima Cintia E.,Zatz Mayana
Genetics and Molecular Biology , 2003,
Abstract: Congenital bilateral absence of the vas deferens (CBAVD) accounts for 1%-2% of sterility in men. A high incidence of mutations, as well as the involvement of the 5T variant of the T tract length in intron 8 of the cystic fibrosis conductance regulator (CFTR) gene, have been previously described in males with CBAVD. Herein we report the screening for mutations and for the 5T variant of the CFTR gene in 17 patients with CBAVD and three others with non-CABVD obstructive azoospermia. In the CBAVD group, three patients (15%) were compound heterozygotes for mutations, and five patients (25%) had a mutation in one allele and the 5T variant in the other; the 5T variant was also present in two other patients, one of them being homozygous. The most frequent mutation was DF508, present on five chromosomes (12.5%). A novel missense mutation (A399D) was detected in a Japanese CBVAD patient. Our results yield further evidence for a strong association between male obstructive azoospermia caused by CBAVD and mutation/5T variant in the CFTR gene. The search for CFTR mutations in such patients is thus recommended for genetic counseling of couples who undergo assisted fertilization due to CBAVD.
The Mitochondrial Complex I Activity Is Reduced in Cells with Impaired Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Function  [PDF]
Angel G. Valdivieso, Mariángeles Clauzure, María C. Marín, Guillermo L. Taminelli, María M. Massip Copiz, Francisco Sánchez, Gustavo Schulman, María L. Teiber, Tomás A. Santa-Coloma
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0048059
Abstract: Cystic fibrosis (CF) is a frequent and lethal autosomal recessive disease. It results from different possible mutations in the CFTR gene, which encodes the CFTR chloride channel. We have previously studied the differential expression of genes in CF and CF corrected cell lines, and found a reduced expression of MTND4 in CF cells. MTND4 is a mitochondrial gene encoding the MTND4 subunit of the mitochondrial Complex I (mCx-I). Since this subunit is essential for the assembly and activity of mCx-I, we have now studied whether the activity of this complex was also affected in CF cells. By using Blue Native-PAGE, the in-gel activity (IGA) of the mCx-I was found reduced in CFDE and IB3-1 cells (CF cell lines) compared with CFDE/6RepCFTR and S9 cells, respectively (CFDE and IB3-1 cells ectopically expressing wild-type CFTR). Moreover, colon carcinoma T84 and Caco-2 cells, which express wt-CFTR, either treated with CFTR inhibitors (glibenclamide, CFTR(inh)-172 or GlyH101) or transfected with a CFTR-specific shRNAi, showed a significant reduction on the IGA of mCx-I. The reduction of the mCx-I activity caused by CFTR inhibition under physiological or pathological conditions may have a profound impact on mitochondrial functions of CF and non-CF cells.
Identificación de mutaciones en el gen CFTR en pacientes chilenos con fibrosis quística Identification of cystic fibrosis transmembrane regulator (CFTR) mutations in Chilean patients with cystic fibrosis  [cached]
Gabriela Repetto L,Helena Poggi M,Paul Harris D,Héctor Navarro M
Revista médica de Chile , 2001,
Abstract: Background: Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene, that codes for a chloride channel located in the apical surface of epithelial cells. The main role of this protein is the regulation of chloride transport, and secondarily, of sodium and water to the extracellular space. More than 900 gene mutations have been described, and their relative frequency in different populations depends on their ethnic origin. Aim: To report the findings of Chilean patients with cystic fibrosis, in whom the presence of 20 common mutations was analyzed. Patients and methods: Fifty seven patients with established diagnosis or suspicion of CF were studied. The simultaneous identification of 20 mutations and the normal deltaF508 allele was done using polymerase chain reactions with a commercial assay. Results: Eight mutations were found. Fifty patients fulfilled diagnostic criteria proposed by the Consensus Panel of the CF Foundation and 66% of alleles were identified in this group. F508 mutation was found in 45%. We did not identify mutations in any of the remaining 7 patients. Conclusions: Our results suggest that the majority of undetected mutations are associated with atypical phenotypes or that some patients in this series could have other diseases. We recommend to include mutation analysis in the evaluation of Chilean patients with CF. It is useful to establish prognosis and genetic counselling (Rev Méd Chile 2001; 129: 841-7).
Synergistic Post-Transcriptional Regulation of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) by miR-101 and miR-494 Specific Binding  [PDF]
Francesca Megiorni, Samantha Cialfi, Carlo Dominici, Serena Quattrucci, Antonio Pizzuti
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0026601
Abstract: microRNAs (miRNAs) are a class of regulatory small non-coding molecules that control gene expression at post-transcriptional level. Deregulation of miRNA functions affects a variety of biological processes also involved in the etiology of several human mendelian and complex diseases. Recently, aberrant miRNA expression has been observed in Cystic Fibrosis (CF), an autosomal-recessive genetic disorder caused by mutations in the CFTR gene, in which a genotype-phenotype correlation is not always found. In order to determine miRNA role in CFTR post-transcriptional regulation, we searched for miR-responsive elements in the CFTR 3′-UTR. In silico analysis, performed using different computational on-line programs, identified some putative miRNAs. Both miR-101 and miR-494 synthetic mimics significantly inhibited the expression of a reporter construct containing the 3′-UTR of CFTR in luciferase assays. Interestingly, miR-101/miR-494 combination was able to markedly suppress CFTR activity by approximately 80% (p<0.001). This is one of the first in vitro studies implicating microRNAs as negative regulators of the CFTR gene expression. miRNA aberrant expression and function might explain the wide phenotypic variability observed among CF patients.
Structure and function of the cystic fibrosis transmembrane conductance regulator
Morales, M.M.;Capella, M.A.M.;Lopes, A.G.;
Brazilian Journal of Medical and Biological Research , 1999, DOI: 10.1590/S0100-879X1999000800013
Abstract: cystic fibrosis (cf) is a lethal autosomal recessive genetic disease caused by mutations in the cf transmembrane conductance regulator (cftr). mutations in the cftr gene may result in a defective processing of its protein and alter the function and regulation of this channel. mutations are associated with different symptoms, including pancreatic insufficiency, bile duct obstruction, infertility in males, high sweat cl-, intestinal obstruction, nasal polyp formation, chronic sinusitis, mucus dehydration, and chronic pseudomonas aeruginosa and staphylococcus aureus lung infection, responsible for 90% of the mortality of cf patients. the gene responsible for the cellular defect in cf was cloned in 1989 and its protein product cftr is activated by an increase of intracellular camp. the cftr contains two membrane domains, each with six transmembrane domain segments, two nucleotide-binding domains (nbds), and a cytoplasmic domain. in this review we discuss the studies that have correlated the role of each cftr domain in the protein function as a chloride channel and as a regulator of the outwardly rectifying cl- channels (orccs).
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