[Show abstract][Hide abstract] ABSTRACT: Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial anion channel. Since the identification of the disease in 1938 and up until 2012, CF patients have been treated exclusively with medications aimed at bettering their respiratory, digestive, inflammatory and infectious symptoms. The identification of the CFTR gene in 1989 gave hopes of rapidly finding a cure for the disease, for which over 1,950 mutations have been identified. Since 2012, recent approaches have enabled the identification of small molecules targeting either the CFTR protein directly or its key processing steps, giving rise to novel promising therapeutic tools.
This review presents the current CFTR mutation classifications according to their clinical consequences and to their effect on the structure and function of the CFTR channel. How these classifications are essential in the establishment of mutation-targeted therapeutic strategies is then discussed. The future of CFTR-targeted treatment lies in combinatory therapies that will enable CF patients to receive a customized treatment.
The international journal of biochemistry & cell biology 07/2014; 52. DOI:10.1016/j.biocel.2014.02.023 · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cystic Fibrosis may be revealed by nasal polyposis (NP) starting early in life. We performed CFTR DNA and mRNA analyses in the family of a 12y-old boy presenting with NP and a normal sweat test. Routine DNA analysis only showed the heterozygous c.2551C>T (p.Arg851*) mutation in the child and the father. mRNA analysis showed partial exon skipping due to c.2551C>T and a significant increase of total CFTR mRNA in the patient and the mother, which was attributable to the heterozygous c.-2954G>A variant in the distant promoter region, as demonstrated by in vitro luciferase assays. 5'RACE analysis showed the presence of a novel transcript, where the canonical exon 1 was replaced by an alternative exon called 1a-L (1a-Long). This case report could represent the first description of a CFTR related-disorder associated with the presence of a 5' alternative, probably non functional transcript, similar to those of fetal origin. This article is protected by copyright. All rights reserved.
Human Mutation 07/2014; 35(7). DOI:10.1002/humu.22548 · 5.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial anion channel. Morbidity is mainly due to lung disease, which is characterized by chronic neutrophilic inflammation. Deregulation of inflammatory pathways is observed in the airways of CF patients, as evidenced by exaggerated NF-κB activity, causing an increase in the local release of pro-inflammatory cytokines such as IL-8. COMMD1, a pleiotropic protein, was recently shown to interact with CFTR and to promote CFTR cell surface expression. The effect of COMMD1 on the NF-κB pathway was assessed in CF and non-CF bronchial epithelial cells by knockdown and overexpression experiments. Results showed that (i) COMMD1 knockdown induced NF-κB-dependent transcription, (ii) COMMD1 overexpression inhibited NF-κB activity and was associated with a decrease in IL-8 transcript level and protein secretion. These data demonstrate the anti-inflammatory properties of COMMD1 in bronchial epithelial cells and open new therapeutic avenues in CF.
The international journal of biochemistry & cell biology 07/2013; 45(11). DOI:10.1016/j.biocel.2013.07.012 · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: With the increased number of identified nucleotide sequence variations in genes, the current challenge is to classify them as disease-causing or neutral. These variants of unknown clinical significance can alter multiple processes, from gene transcription to RNA splicing or protein function. Using an approach combining several in silico tools, we identified some exons presenting weaker splicing motifs than other exons in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. These exons exhibit higher rates of basal skipping than exons harbouring no identifiable weak splicing signals using minigene assays. We then screened 19 described mutations in three different exons, and identified exon-skipping substitutions. These substitutions induced higher skipping levels in exons having one or more weak splicing motifs. Indeed, this level remained under 2% for exons with strong splicing motifs and could reach 40% for exons having at least one weak motif. Further analysis revealed a functional exon splicing enhancer within exon 3 that was associated with the SR protein SF2/ASF and whose disruption induced exon skipping. Exon skipping was confirmed in vivo in two nasal epithelial cell brushing samples. Our approach, which point out exons with some splicing signals weaknesses, will help spot splicing mutations of clinical relevance.
Human Mutation 06/2013; 34(6). DOI:10.1002/humu.22300 · 5.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The correction of premature termination codons (PTCs) by agents that promote readthrough represents a promising emerging tool for the treatment of many genetic diseases. The efficiency of the treatment, however, varies depending on the stop codon itself and the amount of correctible transcripts related to the efficiency of nonsense-mediated decay (NMD). In the current study, a screen by in vitro minigene assay of all six PTCs described in exon 15 of the CFTR gene demonstrated alternative splicing to differing degrees for five of them. Of the five, PTC mutations c.2537G>A (p.Trp846*(UAG) ) and c.2551C>T (p.Arg851*) cause the greatest proportion of transcripts lacking exon 15; both mutations altering exonic splicing regulatory elements. In order to increase the amount of full-length transcripts, different pharmacological treatments were performed showing both negative and positive effects on exon inclusion for the same mutation. Therefore, the total amount of transcripts together with the splicing profile should be assessed to anticipate and improve efficacy of readthrough therapy.
Human Mutation 02/2013; 34(2). DOI:10.1002/humu.22236 · 5.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The identification by CFTR mRNA studies of a new deep-intronic splicing mutation, c.870-1113_1110delGAAT, in one patient of our series with mild CF symptoms and in three CF patients of an Italian study, led us to evaluate the mutation frequency and phenotype/genotype correlations.
266 patients with CF and related disorders and having at least one undetected mutation, were tested at the gDNA level in three French reference laboratories.
In total, the mutation was found in 13 unrelated patients (5% of those already carrying a mutation) plus 4 siblings, including one homozygote and 12 heterozygotes having a severe CF mutation. The sweat test was positive in 10/14 documented cases, the diagnosis was delayed after 20 years in 9/15 and pancreatic insufficiency was present in 5/16.
c.870-1113_1110delGAAT should be considered as CF-causing with phenotype variability and overall delayed diagnosis. Its frequency highlights the potential of mRNA studies.
Journal of cystic fibrosis: official journal of the European Cystic Fibrosis Society 07/2011; 10(6):479-82. DOI:10.1016/j.jcf.2011.06.011 · 3.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The CFTR (cystic fibrosis transmembrane conductance regulator) protein is a large polytopic protein whose biogenesis is inefficient. To better understand the regulation of CFTR processing and trafficking, we conducted a genetic screen that identified COMMD1 as a new CFTR partner. COMMD1 is a protein associated with multiple cellular pathways, including the regulation of hepatic copper excretion, sodium uptake through interaction with ENaC (epithelial sodium channel) and NF-kappaB signaling. In this study, we show that COMMD1 interacts with CFTR in cells expressing both proteins endogenously. This interaction promotes CFTR cell surface expression as assessed by biotinylation experiments in heterologously expressing cells through regulation of CFTR ubiquitination. In summary, our data demonstrate that CFTR is protected from ubiquitination by COMMD1, which sustains CFTR expression at the plasma membrane. Thus, increasing COMMD1 expression may provide an approach to simultaneously inhibit ENaC absorption and enhance CFTR trafficking, two major issues in cystic fibrosis.
PLoS ONE 03/2011; 6(3):e18334. DOI:10.1371/journal.pone.0018334 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of the mild to asymptomatic phenotype observed in cystic fibrosis patients homozygous for the E831X mutation (2623G>T) in the CFTR gene. Analyses performed on nasal epithelial cell mRNA detected three distinct isoforms, a considerably more complex situation than expected for a single nucleotide substitution. Structure-function studies and in silico analyses provided the first experimental evidence of an indel of a stop codon by alternative splicing at a NAGNAG acceptor site. In addition to contributing to proteome plasticity, alternative splicing at a NAGNAG tandem site can thus remove a disease-causing UAG stop codon. This molecular study reveals a naturally occurring mechanism where the effect of either modifier genes or epigenetic factors could be suspected. This finding is of importance for genetic counseling as well as for deciding appropriate therapeutic strategies.
[Show abstract][Hide abstract] ABSTRACT: Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. The mutations G551D and G1349D, which affect the nucleotide-binding domains (NBDs) of CFTR protein, reduce channel activity. This defect can be corrected pharmacologically by small molecules called potentiators. CF mutations residing in the intracellular loops (ICLs), connecting the transmembrane segments of CFTR, may also reduce channel activity. We have investigated the extent of loss of function caused by ICL mutations and the sensitivity to pharmacological stimulation. We found that E193K and G970R (in ICL1 and ICL3, respectively) cause a severe loss of CFTR channel activity that can be rescued by the same potentiators that are effective on NBD mutations. We compared potency and efficacy of three different potentiators for E193K, G970R, and G551D. The 1,4-dihydropyridine felodipine and the phenylglycine PG-01 [2-[(2-1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopropylphenyl)-2-phenylacetamide] were strongly effective on the three CFTR mutants. The efficacy of sulfonamide SF-01 [6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid cycloheptylamide], another CFTR potentiator, was instead significantly lower than felodipine and PG-01 for the E193K and G970R mutations, and almost abolished for G551D. Furthermore, SF-01 modified the response of G551D and G970R to the other two potentiators, an effect that may be explained by an allosteric antagonistic effect. Our results indicate that CFTR potentiators correct the basic defect caused by CF mutations residing in different CFTR domains. However, there are differences among potentiators, with felodipine and PG-01 having a wider pharmacological activity, and SF-01 being more mutation specific. Our observations are useful in the prioritization and development of drugs targeting the CF basic defect.
Journal of Pharmacology and Experimental Therapeutics 07/2009; 330(3):783-91. DOI:10.1124/jpet.109.154146 · 3.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CFTR (cystic fibrosis transmembrane conductance regulator) is an epithelial Cl- channel inhibited with high affinity and selectivity by the thiazolidinone compound CFTR(inh)-172. In the present study, we provide evidence that CFTR(inh)-172 acts directly on the CFTR. We introduced mutations in amino acid residues of the sixth transmembrane helix of the CFTR protein, a domain that has an important role in the formation of the channel pore. Basic and hydrophilic amino acids at positions 334-352 were replaced with alanine residues and the sensitivity to CFTR(inh)-172 was assessed using functional assays. We found that an arginine-to-alanine change at position 347 reduced the inhibitory potency of CFTR(inh)-172 by 20-30-fold. Mutagenesis of Arg347 to other amino acids also decreased the inhibitory potency, with aspartate producing near total loss of CFTR(inh)-172 activity. The results of the present study provide evidence that CFTR(inh)-172 interacts directly with CFTR, and that Arg347 is important for the interaction.
[Show abstract][Hide abstract] ABSTRACT: Cystic fibrosis is mainly caused by mutations that interfere with the biosynthetic folding of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The aim of this study was to find cellular proteins interacting with CFTR and regulating its processing. We have used a genetic screen in yeast to identify such proteins and identified CSN5 that interacted with the third cytoplasmic loop of CFTR. CSN5 is the 5th component of the COP9 signalosome, a complex of eight subunits that shares significant homologies to the lid subcomplex of the 26S proteasome and controls the stability of many proteins. The present study shows that CSN5 associates with the core-glycosylated form of CFTR and suggests that this association targets misfolded CFTR to the degradative pathway. Identifying CSN5 as a new component of the degradative pathway is an important step towards the goal of unraveling the sorting between misfolded and correctly folded CFTR proteins.
[Show abstract][Hide abstract] ABSTRACT: ClC-2 is a broadly expressed member of the voltage-gated ClC chloride channel family. In this study, we aimed to evaluate the role of the membrane lipid environment in ClC-2 function, and in particular the effect of cholesterol and ClC-2 distribution in membrane microdomains. Detergent-resistant and detergent-soluble microdomains (DSM) were isolated from stably transfected HEK293 cells by a discontinuous OptiPrep gradient. ClC-2 was found concentrated in detergent-insoluble membranes in basal conditions and relocalized to DSM upon cholesterol depletion by methyl-beta-cyclodextrin. As assessed by patch clamp recordings, relocalization was accompanied by acceleration of the activation kinetics of the channel. A similar distribution and activation pattern were obtained when cells were treated with the oxidant tert-butyl hydroperoxide and after ATP depletion. In both cases activation was prevented by cholesterol enrichment of cells. We conclude that the cholesterol environment regulates ClC-2 activity, and we provide evidence that the increase in ClC-2 activity in response to acute oxidative or metabolic stress involves relocalization of this channel to DSM.
[Show abstract][Hide abstract] ABSTRACT: The voltage-dependent ClC-2 chloride channel has been implicated in a variety of physiological functions, including fluid transport across specific epithelia. ClC-2 is activated by hyperpolarization, weakly acidic external pH, intracellular Cl-, and cell swelling. To add more insight into the mechanisms involved in ClC-2 regulation, we searched for associated proteins that may influence ClC-2 activity. With the use of immunoprecipitation of ClC-2 from human embryonic kidney-293 cells stably expressing the channel, followed by electrophoretic separation of coimmunoprecipitated proteins and mass spectrometry identification, Hsp70 and Hsp90 were unmasked as possible ClC-2 interacting partners. Association of Hsp90 with ClC-2 was confirmed in mouse brain. Inhibition of Hsp90 by two specific inhibitors, geldanamycin or radicicol, did not affect total amounts of ClC-2 but did reduce plasma membrane channel abundance. Functional experiments using the whole cell configuration of the patch-clamp technique showed that inhibition of Hsp90 reduced ClC-2 current amplitude and impaired the intracellular Cl- concentration [Cl-]-dependent rightward shift of the fractional conductance. Geldanamycin and radicicol increased both the slow and fast activation time constants in a chloride-dependent manner. Heat shock treatment had the opposite effect. These results indicate that association of Hsp90 with ClC-2 results in greater channel activity due to increased cell surface channel expression, facilitation of channel opening, and enhanced channel sensitivity to intracellular [Cl-]. This association may have important pathophysiological consequences, enabling increased ClC-2 activity in response to cellular stresses such as elevated temperature, ischemia, or oxidative reagents.