Combined effects of VX-770 and VX-809 on several functional abnormalities of F508del-CFTR channels.

PubMed

Kopeikin, Z; Yuksek, Z; Yang, H-Y; Bompadre, S G

2014-09-01

The most common cystic fibrosis-associated mutation, the deletion of phenylalanine 508 (F508del), results in channels with poor membrane expression and impaired function. VX-770, a clinically approved drug for treatment of CF patients carrying the G551D mutation, and VX-809, a corrector shown in vitro to increase membrane expression of mutant channels, are currently undergoing clinical trials, but functional data at the molecular level is still lacking. The effect of VX-770 and VX-809 on the multiple functional defects of F508del-CFTR was assessed via excised inside-out patch-clamp experiments. VX-770 completely restores the low opening-rate of F508del-CFTR, with smaller open-time increase, in temperature-corrected and VX-809-treated channels. The shorter locked-open time of hydrolysis-deficient F508del-CFTR is also prolonged by VX-770. VX-809 does not improve channel function by itself as previously reported. The results from these studies can be interpreted as an equilibrium shift toward the open-channel conformation of F508del-CFTR channels. Copyright © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

In vitro pharmacologic restoration of CFTR-mediated chloride transport with sodium 4-phenylbutyrate in cystic fibrosis epithelial cells containing delta F508-CFTR.

PubMed Central

Rubenstein, R C; Egan, M E; Zeitlin, P L

1997-01-01

The most common cystic fibrosis transmembrane conductance regulator mutation, delta F508-CFTR, is a partially functional chloride channel that is retained in the endoplasmic reticulum and degraded. We hypothesize that a known transcriptional regulator, sodium 4-phenylbutyrate (4PBA), will enable a greater fraction of delta F508-CFTR to escape degradation and appear at the cell surface. Primary cultures of nasal polyp epithelia from CF patients (delta F508 homozygous or heterozygous), or the CF bronchial epithelial cell line IB3-1 (delta F508/W1282X) were exposed to 4PBA for up to 7 d in culture. 4PBA treatment at concentrations of 0.1 and 2 mM resulted in the restoration of forskolin-activated chloride secretion. Protein kinase A-activated, linear, 10 pS chloride channels appeared at the plasma membrane of IB3-1 cells at the tested concentration of 2.5 mM. Treatment of IB3-1 cells with 0.1-1 mM 4PBA and primary nasal epithelia with 5 mM 4PBA also resulted in the appearance of higher molecular mass forms of CFTR consistent with addition and modification of oligosaccharides in the Golgi apparatus, as detected by immunoblotting of whole cell lysates with anti-CFTR antisera. Immunocytochemistry in CF epithelial cells treated with 4PBA was consistent with increasing amounts of delta F508-CFTR. These data indicate that 4PBA is a promising pharmacologic agent for inducing correction of the CF phenotype in CF patients carrying the delta F508 mutation. PMID:9366560

In vitro pharmacologic restoration of CFTR-mediated chloride transport with sodium 4-phenylbutyrate in cystic fibrosis epithelial cells containing delta F508-CFTR.

PubMed

Rubenstein, R C; Egan, M E; Zeitlin, P L

1997-11-15

The most common cystic fibrosis transmembrane conductance regulator mutation, delta F508-CFTR, is a partially functional chloride channel that is retained in the endoplasmic reticulum and degraded. We hypothesize that a known transcriptional regulator, sodium 4-phenylbutyrate (4PBA), will enable a greater fraction of delta F508-CFTR to escape degradation and appear at the cell surface. Primary cultures of nasal polyp epithelia from CF patients (delta F508 homozygous or heterozygous), or the CF bronchial epithelial cell line IB3-1 (delta F508/W1282X) were exposed to 4PBA for up to 7 d in culture. 4PBA treatment at concentrations of 0.1 and 2 mM resulted in the restoration of forskolin-activated chloride secretion. Protein kinase A-activated, linear, 10 pS chloride channels appeared at the plasma membrane of IB3-1 cells at the tested concentration of 2.5 mM. Treatment of IB3-1 cells with 0.1-1 mM 4PBA and primary nasal epithelia with 5 mM 4PBA also resulted in the appearance of higher molecular mass forms of CFTR consistent with addition and modification of oligosaccharides in the Golgi apparatus, as detected by immunoblotting of whole cell lysates with anti-CFTR antisera. Immunocytochemistry in CF epithelial cells treated with 4PBA was consistent with increasing amounts of delta F508-CFTR. These data indicate that 4PBA is a promising pharmacologic agent for inducing correction of the CF phenotype in CF patients carrying the delta F508 mutation.

Results of a phase IIa study of VX-809, an investigational CFTR corrector compound, in subjects with cystic fibrosis homozygous for the F508del-CFTR mutation.

PubMed

Clancy, J P; Rowe, Steven M; Accurso, Frank J; Aitken, Moira L; Amin, Raouf S; Ashlock, Melissa A; Ballmann, Manfred; Boyle, Michael P; Bronsveld, Inez; Campbell, Preston W; De Boeck, Kris; Donaldson, Scott H; Dorkin, Henry L; Dunitz, Jordan M; Durie, Peter R; Jain, Manu; Leonard, Anissa; McCoy, Karen S; Moss, Richard B; Pilewski, Joseph M; Rosenbluth, Daniel B; Rubenstein, Ronald C; Schechter, Michael S; Botfield, Martyn; Ordoñez, Claudia L; Spencer-Green, George T; Vernillet, Laurent; Wisseh, Steve; Yen, Karl; Konstan, Michael W

2012-01-01

VX-809, a cystic fibrosis transmembrane conductance regulator (CFTR) modulator, has been shown to increase the cell surface density of functional F508del-CFTR in vitro. A randomised, double-blind, placebo-controlled study evaluated the safety, tolerability and pharmacodynamics of VX-809 in adult patients with cystic fibrosis (n=89) who were homozygous for the F508del-CFTR mutation. Subjects were randomised to one of four VX-809 28 day dose groups (25, 50, 100 and 200 mg) or matching placebo. The type and incidence of adverse events were similar among VX-809- and placebo-treated subjects. Respiratory events were the most commonly reported and led to discontinuation by one subject in each active treatment arm. Pharmacokinetic data supported a once-daily oral dosing regimen. Pharmacodynamic data suggested that VX-809 improved CFTR function in at least one organ (sweat gland). VX-809 reduced elevated sweat chloride values in a dose-dependent manner (p=0.0013) that was statistically significant in the 100 and 200 mg dose groups. There was no statistically significant improvement in CFTR function in the nasal epithelium as measured by nasal potential difference, nor were there statistically significant changes in lung function or patient-reported outcomes. No maturation of immature F508del-CFTR was detected in the subgroup that provided rectal biopsy specimens. In this study, VX-809 had a similar adverse event profile to placebo for 28 days in F508del-CFTR homozygous patients, and demonstrated biological activity with positive impact on CFTR function in the sweat gland. Additional data are needed to determine how improvements detected in CFTR function secondary to VX-809 in the sweat gland relate to those measurable in the respiratory tract and to long-term measures of clinical benefit. NCT00865904.

Rescue of murine F508del CFTR activity in native intestine by low temperature and proteasome inhibitors.

PubMed

Wilke, Martina; Bot, Alice; Jorna, Huub; Scholte, Bob J; de Jonge, Hugo R

2012-01-01

Most patients with Cystic Fibrosis (CF) carry at least one allele with the F508del mutation, resulting in a CFTR chloride channel protein with a processing, gating and stability defect, but with substantial residual activity when correctly sorted to the apical membranes of epithelial cells. New therapies are therefore aimed at improving the folding and trafficking of F508del CFTR, (CFTR correctors) or at enhancing the open probability of the CFTR chloride channel (CFTR potentiators). Preventing premature breakdown of F508del CFTR is an alternative or additional strategy, which is investigated in this study. We established an ex vivo assay for murine F508del CFTR rescue in native intestinal epithelium that can be used as a pre-clinical test for candidate therapeutics. Overnight incubation of muscle stripped ileum in modified William's E medium at low temperature (26°C), and 4 h or 6 h incubation at 37°C with different proteasome inhibitors (PI: ALLN, MG-132, epoxomicin, PS341/bortezomib) resulted in fifty to hundred percent respectively of the wild type CFTR mediated chloride secretion (forskolin induced short-circuit current). The functional rescue was accompanied by enhanced expression of the murine F508del CFTR protein at the apical surface of intestinal crypts and a gain in the amount of complex-glycosylated CFTR (band C) up to 20% of WT levels. Sustained rescue in the presence of brefeldin A shows the involvement of a post-Golgi compartment in murine F508del CFTR degradation, as was shown earlier for its human counterpart. Our data show that proteasome inhibitors are promising candidate compounds for improving rescue of human F508del CFTR function, in combination with available correctors and potentiators.

Allosteric modulation balances thermodynamic stability and restores function of ΔF508 CFTR

PubMed Central

Aleksandrov, Andrei A.; Kota, Pradeep; Cui, Liying; Jensen, Tim; Alekseev, Alexey E.; Reyes, Santiago; He, Lihua; Gentzsch, Martina; Aleksandrov, Luba A.; Dokholyan, Nikolay V.; Riordan, John R.

2013-01-01

Most cystic fibrosis is caused by a deletion of a single residue (F508) in CFTR that disrupts the folding and biosynthetic maturation of the ion channel protein. Progress towards understanding the underlying mechanisms and overcoming the defect remain 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 the first nucleotide domain (NBD1), including the structurally diverse region (SDR), the gamma phosphate switch loop and the Regulatory Insertion (RI). Molecular Dynamic 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 SDR, 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. PMID:22406676

Increased NF-κB Activity and Decreased Wnt/β-Catenin Signaling Mediate Reduced Osteoblast Differentiation and Function in ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mice.

PubMed

Le Henaff, Carole; Mansouri, Rafik; Modrowski, Dominique; Zarka, Mylène; Geoffroy, Valérie; Marty, Caroline; Tarantino, Nadine; Laplantine, Emmanuel; Marie, Pierre J

2015-07-17

The prevalent human ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with reduced bone formation and bone loss in mice. The molecular mechanisms by which the ΔF508-CFTR mutation causes alterations in bone formation are poorly known. In this study, we analyzed the osteoblast phenotype in ΔF508-CFTR mice and characterized the signaling mechanisms underlying this phenotype. Ex vivo studies showed that the ΔF508-CFTR mutation negatively impacted the differentiation of bone marrow stromal cells into osteoblasts and the activity of osteoblasts, demonstrating that the ΔF508-CFTR mutation alters both osteoblast differentiation and function. Treatment with a CFTR corrector rescued the abnormal collagen gene expression in ΔF508-CFTR osteoblasts. Mechanistic analysis revealed that NF-κB signaling and transcriptional activity were increased in mutant osteoblasts. Functional studies showed that the activation of NF-κB transcriptional activity in mutant osteoblasts resulted in increased β-catenin phosphorylation, reduced osteoblast β-catenin expression, and altered expression of Wnt/β-catenin target genes. Pharmacological inhibition of NF-κB activity or activation of canonical Wnt signaling rescued Wnt target gene expression and corrected osteoblast differentiation and function in bone marrow stromal cells and osteoblasts from ΔF508-CFTR mice. Overall, the results show that the ΔF508-CFTR mutation impairs osteoblast differentiation and function as a result of overactive NF-κB and reduced Wnt/β-catenin signaling. Moreover, the data indicate that pharmacological inhibition of NF-κB or activation of Wnt/β-catenin signaling can rescue the abnormal osteoblast differentiation and function induced by the prevalent ΔF508-CFTR mutation, suggesting novel therapeutic strategies to correct the osteoblast dysfunctions in cystic fibrosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Increased NF-κB Activity and Decreased Wnt/β-Catenin Signaling Mediate Reduced Osteoblast Differentiation and Function in ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mice*

PubMed Central

Le Henaff, Carole; Mansouri, Rafik; Modrowski, Dominique; Zarka, Mylène; Geoffroy, Valérie; Marty, Caroline; Tarantino, Nadine; Laplantine, Emmanuel; Marie, Pierre J.

2015-01-01

The prevalent human ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with reduced bone formation and bone loss in mice. The molecular mechanisms by which the ΔF508-CFTR mutation causes alterations in bone formation are poorly known. In this study, we analyzed the osteoblast phenotype in ΔF508-CFTR mice and characterized the signaling mechanisms underlying this phenotype. Ex vivo studies showed that the ΔF508-CFTR mutation negatively impacted the differentiation of bone marrow stromal cells into osteoblasts and the activity of osteoblasts, demonstrating that the ΔF508-CFTR mutation alters both osteoblast differentiation and function. Treatment with a CFTR corrector rescued the abnormal collagen gene expression in ΔF508-CFTR osteoblasts. Mechanistic analysis revealed that NF-κB signaling and transcriptional activity were increased in mutant osteoblasts. Functional studies showed that the activation of NF-κB transcriptional activity in mutant osteoblasts resulted in increased β-catenin phosphorylation, reduced osteoblast β-catenin expression, and altered expression of Wnt/β-catenin target genes. Pharmacological inhibition of NF-κB activity or activation of canonical Wnt signaling rescued Wnt target gene expression and corrected osteoblast differentiation and function in bone marrow stromal cells and osteoblasts from ΔF508-CFTR mice. Overall, the results show that the ΔF508-CFTR mutation impairs osteoblast differentiation and function as a result of overactive NF-κB and reduced Wnt/β-catenin signaling. Moreover, the data indicate that pharmacological inhibition of NF-κB or activation of Wnt/β-catenin signaling can rescue the abnormal osteoblast differentiation and function induced by the prevalent ΔF508-CFTR mutation, suggesting novel therapeutic strategies to correct the osteoblast dysfunctions in cystic fibrosis. PMID:26060255

F508del-CFTR rescue: a matter of cell stress response.

PubMed

Nieddu, Erika; Pollarolo, Benedetta; Merello, Luisa; Schenone, Silvia; Mazzei, Mauro

2013-01-01

Cystic fibrosis (CF) is a common inherited fatal disease affecting 70,000 people worldwide, with a median predicted age of survival of approximately 38 years. The deletion of Phenylalanine in position 508 of the Cystic Fibrosis Transmembrane conductance Regulator (F508del-CFTR) is the most common mutation in CF patients: the deleted protein, not properly folded, is degraded. To date no commercial drugs are available. Low temperature, some osmolytes and conditions able to induce heat shock protein 70 (Hsp70) expression and heat shock cognate 70 (Hsc70) inhibition result in F508del-CFTR rescue, hence restoring its physiological function: this review sheds light on the correlation between these several evidences. Interestingly, all these approaches have a role in the cell stress response (CSR), a set of cell reactions to stress. In addition, unpredictably, F508del-CFTR rescue has to be considered in the frame of CSR: entities that induce - or are induced during - the CSR are, in general, also able to correct trafficking defect of CFTR. Specifically, the low temperature induces, by definition, a CSR; osmolytes, such as glycerol and trimethylamine N-oxide (TMAO), are products of the CSR; pharmacological correctors, such as Matrine and 4-phenylbutirric acid (4PBA), down-regulate the constitutive Hsc70 in favor of an up-regulation of the inducible chaperone Hsp70, another component of the CSR. The identification of a common mechanism of action for different types of correctors could drive the discovery of new active molecules in CF, overcoming methods clinically inapplicable, such as the low temperature.

Generation of ΔF508-CFTR T84 cell lines by CRISPR/Cas9-mediated genome editing.

PubMed

Chung, Woo Young; Song, Myungjae; Park, Jinhong; Namkung, Wan; Lee, Jinu; Kim, Hyongbum; Lee, Min Goo; Kim, Joo Young

2016-12-01

To provide a simple method to make a stable ΔF508-CFTR-expressing T84 cell line that can be used as an efficient screening model system for ΔF508-CFTR rescue. CFTR knockout cell lines were generated by Cas9 with a single-guide RNA (sgRNA) targeting exon 1 of the CFTR genome, which produced indels that abolished CFTR protein expressions. Next, stable ΔF508-CFTR expression was achieved by genome integration of ΔF508-CFTR via the lentivirus infection system. Finally, we showed functional rescue of ΔF508-CFTR not only by growing the cells at a low temperature, but also incubating with VX-809, a ΔF508-CFTR corrector, in the established T84 cells expressing ΔF508-CFTR. This cell system provides an appropriate screening platform for rescue of ΔF508-CFTR, especially related to protein folding, escaped from endoplasmic-reticulum-associated protein degradation, and membrane transport.

Tezacaftor/Ivacaftor in Subjects with Cystic Fibrosis and F508del/F508del-CFTR or F508del/G551D-CFTR.

PubMed

Donaldson, Scott H; Pilewski, Joseph M; Griese, Matthias; Cooke, Jon; Viswanathan, Lakshmi; Tullis, Elizabeth; Davies, Jane C; Lekstrom-Himes, Julie A; Wang, Linda T

2018-01-15

Tezacaftor (formerly VX-661) is an investigational small molecule that improves processing and trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) in vitro, and improves CFTR function alone and in combination with ivacaftor. To evaluate the safety and efficacy of tezacaftor monotherapy and of tezacaftor/ivacaftor combination therapy in subjects with cystic fibrosis homozygous for F508del or compound heterozygous for F508del and G551D. This was a randomized, placebo-controlled, double-blind, multicenter, phase 2 study (NCT01531673). Subjects homozygous for F508del received tezacaftor (10 to 150 mg) every day alone or in combination with ivacaftor (150 mg every 12 h) in a dose escalation phase, as well as in a dosage regimen testing phase. Subjects compound heterozygous for F508del and G551D, taking physician-prescribed ivacaftor, received tezacaftor (100 mg every day). Primary endpoints were safety through Day 56 and change in sweat chloride from baseline through Day 28. Secondary endpoints included change in percent predicted FEV 1 (ppFEV 1 ) from baseline through Day 28 and pharmacokinetics. The incidence of adverse events was similar across treatment arms. Tezacaftor (100 mg every day)/ivacaftor (150 mg every 12 h) resulted in a 6.04 mmol/L decrease in sweat chloride and 3.75 percentage point increase in ppFEV 1 in subjects homozygous for F508del, and a 7.02 mmol/L decrease in sweat chloride and 4.60 percentage point increase in ppFEV 1 in subjects compound heterozygous for F508del and G551D from baseline through Day 28 (P < 0.05 for all). These results support continued clinical development of tezacaftor (100 mg every day) in combination with ivacaftor (150 mg every 12 h) in subjects with cystic fibrosis. Clinical trial registered with www.clinicaltrials.gov (NCT01531673).

Increased efficacy of VX-809 in different cellular systems results from an early stabilization effect of F508del-CFTR.

PubMed

Farinha, Carlos M; Sousa, Marisa; Canato, Sara; Schmidt, André; Uliyakina, Inna; Amaral, Margarida D

2015-08-01

Cystic fibrosis (CF), the most common recessive autosomal disease among Caucasians, is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, F508del, leads to CFTR impaired plasma membrane trafficking. Therapies modulating CFTR basic defect are emerging, such as VX-809, a corrector of F508del-CFTR traffic which just succeeded in a Phase III clinical trial. We recently showed that VX-809 is additive to two other correctors (VRT-325 and compound 4a). Here, we aimed to determine whether the differential rescuing by these compounds results from cell-specific factors or rather from distinct effects at the early biogenesis and/or processing. The rescuing efficiencies of the above three correctors were first compared in different cellular models (primary respiratory cells, cystic fibrosis bronchial epithelial and baby hamster kidney [BHK] cell lines) by functional approaches: micro-Ussing chamber and iodide efflux. Next, biochemical methods (metabolic labeling, pulse-chase and immunoprecipitation) were used to determine their impact on CFTR biogenesis / processing. Functional analyses revealed that VX-809 has the greatest rescuing efficacy and that the relative efficiencies of the three compounds are essentially maintained in all three cellular models tested. Nevertheless, biochemical data show that VX-809 significantly stabilizes F508del-CFTR immature form, an effect that is not observed for C3 nor C4. VX-809 and C3 also significantly increase accumulation of immature CFTR. Our data suggest that VX-809 increases the stability of F508del-CFTR immature form at an early phase of its biogenesis, thus explaining its increased efficacy when inducing its rescue.

Increased efficacy of VX-809 in different cellular systems results from an early stabilization effect of F508del-CFTR

PubMed Central

Farinha, Carlos M; Sousa, Marisa; Canato, Sara; Schmidt, André; Uliyakina, Inna; Amaral, Margarida D

2015-01-01

Cystic fibrosis (CF), the most common recessive autosomal disease among Caucasians, is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, F508del, leads to CFTR impaired plasma membrane trafficking. Therapies modulating CFTR basic defect are emerging, such as VX-809, a corrector of F508del-CFTR traffic which just succeeded in a Phase III clinical trial. We recently showed that VX-809 is additive to two other correctors (VRT-325 and compound 4a). Here, we aimed to determine whether the differential rescuing by these compounds results from cell-specific factors or rather from distinct effects at the early biogenesis and/or processing. The rescuing efficiencies of the above three correctors were first compared in different cellular models (primary respiratory cells, cystic fibrosis bronchial epithelial and baby hamster kidney [BHK] cell lines) by functional approaches: micro-Ussing chamber and iodide efflux. Next, biochemical methods (metabolic labeling, pulse-chase and immunoprecipitation) were used to determine their impact on CFTR biogenesis / processing. Functional analyses revealed that VX-809 has the greatest rescuing efficacy and that the relative efficiencies of the three compounds are essentially maintained in all three cellular models tested. Nevertheless, biochemical data show that VX-809 significantly stabilizes F508del-CFTR immature form, an effect that is not observed for C3 nor C4. VX-809 and C3 also significantly increase accumulation of immature CFTR. Our data suggest that VX-809 increases the stability of F508del-CFTR immature form at an early phase of its biogenesis, thus explaining its increased efficacy when inducing its rescue. PMID:26171232

5'-adenosine monophosphate mediated cooling treatment enhances ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) stability in vivo.

PubMed

Zhang, Yueqiang; O'Brien, William G; Zhao, Zhaoyang; Lee, Cheng Chi

2015-09-04

Gene mutations that produce misprocessed proteins are linked to many human disorders. Interestingly, some misprocessed proteins retained their biological function when stabilized by low temperature treatment of cultured cells in vitro. Here we investigate whether low temperature treatment in vivo can rescue misfolded proteins by applying 5'-AMP mediated whole body cooling to a Cystic Fibrosis (CF) mouse model carrying a mutant cystic fibrosis transmembrane conductance regulator (CFTR) with a deletion of the phenylalanine residue in position 508 (ΔF508-CFTR). Low temperature treatment of cultured cells was previously shown to be able to alleviate the processing defect of ΔF508-CFTR, enhancing its plasma membrane localization and its function in mediating chloride ion transport. Here, we report that whole body cooling enhanced the retention of ΔF508-CFTR in intestinal epithelial cells. Functional analysis based on β-adrenergic dependent salivary secretion and post-natal mortality rate revealed a moderate but significant improvement in treated compared with untreated CF mice. Our findings demonstrate that temperature sensitive processing of mutant proteins can be responsive to low temperature treatment in vivo.

Association between F508 deletion in CFTR and chronic pancreatitis risk.

PubMed

Zhao, Dong; Xu, Yanzhen; Li, Jiatong; Fu, Shien; Xiao, Feifan; Song, Xiaowei; Xie, Zhibin; Jiang, Min; He, Yan; Liu, Chengwu; Wen, Qiongxian; Yang, Xiaoli

2017-09-01

The cystic fibrosis transmembrane conductance regulator (CFTR) has been reported to influence individual susceptibility to chronic pancreatitis (CP), but the results of previous studies are controversial. We performed a study to demonstrate the relationship between CFTR and CP. We searched PubMed, Scopus, and Embase for studies of patients with CP. Seven studies from 1995 to 2016 were identified, and included 64,832 patients. Pooled prevalence and 95% confidence intervals (CIs) were calculated. F508 deletion in CFTR was significantly positively associated with CP risk in the overall analysis (odds ratio [OR]=3.20, 95% CI: 2.30-4.44, I 2 =31.7%). In subgroup analysis stratified by ethnicity, F508 deletion was significantly associated with CP risk in Indian populations, using a fixed effects model (ORs=5.45, 95% CI: 2.52-11.79, I 2 =0.0%), and in non-Indian populations, using a random effects model (ORs=3.59, 95% CI: 1.73-7.48, I 2 =60.9%). At the same time, we found that Indians with F508 deletion had much higher CP prevalence than non-Indians. Interestingly, F508 deletion was also associated with CP and idiopathic CP risk in subgroup analysis stratified by aeitiology, using the fixed effects model. Based on current evidence, F508 deletion is a risk factor for CP, and Indians with F508 deletion have much higher CP morbidity. Copyright © 2017 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

Probing Conformational Rescue Induced by a Chemical Corrector of F508del-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mutant*

PubMed Central

Yu, Wilson; Chiaw, Patrick Kim; Bear, Christine E.

2011-01-01

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that cause loss of function of the CFTR channel on the apical surface of epithelial cells. The major CF-causing mutation, F508del-CFTR, is misfolded, retained in the endoplasmic reticulum, and degraded. Small molecule corrector compounds have been identified using high throughput screens, which partially rescue the trafficking defect of F508del-CFTR, allowing a fraction of the mutant protein to escape endoplasmic reticulum retention and traffic to the plasma membrane, where it exhibits partial function as a cAMP-regulated chloride channel. A subset of such corrector compounds binds directly to the mutant protein, prompting the hypothesis that they rescue the biosynthetic defect by inducing improved protein conformation. We tested this hypothesis directly by evaluating the consequences of a corrector compound on the conformation of each nucleotide binding domain (NBD) in the context of the full-length mutant protein in limited proteolytic digest studies. Interestingly, we found that VRT-325 was capable of partially restoring compactness in NBD1. However, VRT-325 had no detectable effect on the conformation of the second half of the molecule. In comparison, ablation of the di-arginine sequence, R553XR555 (F508del-KXK-CFTR), modified protease susceptibility of NBD1, NBD2, and the full-length protein. Singly, each intervention led to a partial correction of the processing defect. Together, these interventions restored processing of F508del-CFTR to near wild type. Importantly, however, a defect in NBD1 conformation persisted, as did a defect in channel activation after the combined interventions. Importantly, this defect in channel activation can be fully corrected by the addition of the potentiator, VX-770. PMID:21602569

Anchored PDE4 regulates chloride conductance in wild-type and ΔF508-CFTR human airway epithelia

PubMed Central

Blanchard, Elise; Zlock, Lorna; Lao, Anna; Mika, Delphine; Namkung, Wan; Xie, Moses; Scheitrum, Colleen; Gruenert, Dieter C.; Verkman, Alan S.; Finkbeiner, Walter E.; Conti, Marco; Richter, Wito

2014-01-01

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that impair its expression and/or chloride channel function. Here, we provide evidence that type 4 cyclic nucleotide phosphodiesterases (PDE4s) are critical regulators of the cAMP/PKA-dependent activation of CFTR in primary human bronchial epithelial cells. In non-CF cells, PDE4 inhibition increased CFTR activity under basal conditions (ΔISC 7.1 μA/cm2) and after isoproterenol stimulation (increased ΔISC from 13.9 to 21.0 μA/cm2) and slowed the return of stimulated CFTR activity to basal levels by >3-fold. In cells homozygous for ΔF508-CFTR, the most common mutation found in CF, PDE4 inhibition alone produced minimal channel activation. However, PDE4 inhibition strongly amplified the effects of CFTR correctors, drugs that increase expression and membrane localization of CFTR, and/or CFTR potentiators, drugs that increase channel gating, to reach ∼25% of the chloride conductance observed in non-CF cells. Biochemical studies indicate that PDE4s are anchored to CFTR and mediate a local regulation of channel function. Taken together, our results implicate PDE4 as an important determinant of CFTR activity in airway epithelia, and support the use of PDE4 inhibitors to potentiate the therapeutic benefits of CFTR correctors and potentiators.—Blanchard, E., Zlock, L., Lao, A., Mika, D., Namkung, W., Xie, M., Scheitrum, C., Gruenert, D.C., Verkman, A.S., Finkbeiner, W.E., Conti, M., Richter, W. Anchored PDE4 regulates chloride conductance in wild type and ΔF508-CFTR human airway epithelia. PMID:24200884

Evidence against Resveratrol as a viable therapy for the rescue of defective ΔF508 CFTR

PubMed Central

Jai, Ying; Shah, Kalpit; Bridges, Robert J.; Bradbury, Neil A.

2015-01-01

BACKGROUND Resveratrol, a natural phenolic compound, has been reported to rescue mutant ΔF508 CFTR in expression systems and primary epithelial cells. Although this implies a therapeutic benefit to patients with CF, investigations were performed using resveratrol concentrations greatly in excess of those achievable in plasma. We evaluated the efficacy of resveratrol as a CFTR corrector in relevant primary airway cells, using physiologically achievable resveratrol concentrations. METHODS Cells expressing wt or ΔF508 CFTR were exposed to chronic or acute resveratrol. CFTR mRNA and protein expression were monitored. The effects of resveratrol on primary ΔF508 human airway cells were evaluated by equivalent current analysis using modified Ussing chambers. RESULTS Consistent with previously published data in heterologous expression systems, high doses of resveratrol increased CFTR expression; however physiologically relevant concentrations were without effect. In contrast to heterologous expression systems, resveratrol was unable to increase mutant CFTR channel activity in primary airway cells. Elevated amiloride-sensitive currents, indicative of sodium transport and characteristically elevated in CF airway cells, were also unaffected by resveratrol CONCLUSIONS High concentrations of resveratrol can increase CFTR mRNA and protein in some cell types. In addition, acute resveratrol exposure can stimulate CFTR mediated chloride secretion, probably by increasing cellular cAMP levels. Resveratrol at physiologically achievable levels yielded no benefit in primary ΔF508 airway cells, either in terms of amiloride-sensitive currents of CFTR currents. PMID:26342647

SYVN1, NEDD8, and FBXO2 Proteins Regulate ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Ubiquitin-mediated Proteasomal Degradation.

PubMed

Ramachandran, Shyam; Osterhaus, Samantha R; Parekh, Kalpaj R; Jacobi, Ashley M; Behlke, Mark A; McCray, Paul B

2016-12-02

We previously reported that delivery of a microRNA-138 mimic or siRNA against SIN3A to cultured cystic fibrosis (ΔF508/ΔF508) airway epithelia partially restored ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR)-mediated cAMP-stimulated Cl - conductance. We hypothesized that dissecting this microRNA-138/SIN3A-regulated gene network would identify individual proteins contributing to the rescue of ΔF508-CFTR function. Among the genes in the network, we rigorously validated candidates using functional CFTR maturation and electrolyte transport assays in polarized airway epithelia. We found that depletion of the ubiquitin ligase SYVN1, the ubiquitin/proteasome system regulator NEDD8, or the F-box protein FBXO2 partially restored ΔF508-CFTR-mediated Cl - transport in primary cultures of human cystic fibrosis airway epithelia. Moreover, knockdown of SYVN1, NEDD8, or FBXO2 in combination with corrector compound 18 further potentiated rescue of ΔF508-CFTR-mediated Cl - conductance. This study provides new knowledge of the CFTR biosynthetic pathway. It suggests that SYVN1 and FBXO2 represent two distinct multiprotein complexes that may degrade ΔF508-CFTR in airway epithelia and identifies a new role for NEDD8 in regulating ΔF508-CFTR ubiquitination. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Synergy-based small-molecule screen using a human lung epithelial cell line yields ΔF508-CFTR correctors that augment VX-809 maximal efficacy.

PubMed

Phuan, Puay-Wah; Veit, Guido; Tan, Joseph; Roldan, Ariel; Finkbeiner, Walter E; Lukacs, Gergely L; Verkman, A S

2014-07-01

The most prevalent cystic fibrosis transmembrane conductance regulator (CFTR) mutation causing cystic fibrosis, ΔF508, impairs folding of nucleotide binding domain (NBD) 1 and stability of the interface between NBD1 and the membrane-spanning domains. The interfacial stability defect can be partially corrected by the investigational drug VX-809 (3-[6-[[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl]amino]-3-methyl-2-pyridinyl]-benzoic acid) or the R1070W mutation. Second-generation ΔF508-CFTR correctors are needed to improve on the modest efficacy of existing cystic fibrosis correctors. We postulated that a second corrector targeting a distinct folding/interfacial defect might act in synergy with VX-809 or the R1070W suppressor mutation. A biochemical screen for ΔF508-CFTR cell surface expression was developed in a human lung epithelium-derived cell line (CFBE41o(-)) by expressing chimeric CFTRs with a horseradish peroxidase (HRP) in the fourth exofacial loop in either the presence or absence of R1070W. Using a luminescence readout of HRP activity, screening of approximately 110,000 small molecules produced nine novel corrector scaffolds that increased cell surface ∆F508-CFTR expression by up to 200% in the presence versus absence of maximal VX-809. Further screening of 1006 analogs of compounds identified from the primary screen produced 15 correctors with an EC50 < 5 µM. Eight chemical scaffolds showed synergy with VX-809 in restoring chloride permeability in ∆F508-expressing A549 cells. An aminothiazole increased chloride conductance in human bronchial epithelial cells from a ΔF508 homozygous subject beyond that of maximal VX-809. Mechanistic studies suggested that NBD2 is required for the aminothiazole rescue. Our results provide proof of concept for synergy screening to identify second-generation correctors, which, when used in combination, may overcome the "therapeutic ceiling" of first-generation correctors. Copyright © 2014 by The

Compounds that correct F508del-CFTR trafficking can also correct other protein trafficking diseases: an in vitro study using cell lines

PubMed Central

2013-01-01

Background Many genetic diseases are due to defects in protein trafficking where the mutant protein is recognized by the quality control systems, retained in the endoplasmic reticulum (ER), and degraded by the proteasome. In many cases, the mutant protein retains function if it can be trafficked to its proper cellular location. We have identified structurally diverse correctors that restore the trafficking and function of the most common mutation causing cystic fibrosis, F508del-CFTR. Most of these correctors do not act directly as ligands of CFTR, but indirectly on other pathways to promote folding and correction. We hypothesize that these proteostasis regulators may also correct other protein trafficking diseases. Methods To test our hypothesis, we used stable cell lines or transient transfection to express 2 well-studied trafficking disease mutations in each of 3 different proteins: the arginine-vasopressin receptor 2 (AVPR2, also known as V2R), the human ether-a-go-go-related gene (KCNH2, also known as hERG), and finally the sulfonylurea receptor 1 (ABCC8, also known as SUR1). We treated cells expressing these mutant proteins with 9 structurally diverse F508del-CFTR correctors that function through different cellular mechanisms and assessed whether correction occurred via immunoblotting and functional assays. Results were deemed significantly different from controls by a one-way ANOVA (p < 0.05). Results Here we show that F508del-CFTR correctors RDR1, KM60 and KM57 also correct some mutant alleles of other protein trafficking diseases. We also show that one corrector, the cardiac glycoside ouabain, was found to alter the glycosylation of all mutant alleles tested. Conclusions Correctors of F508del-CFTR trafficking might have broader applications to other protein trafficking diseases. PMID:23316740

Functional genomic responses to cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR(delta508) in the lung.

PubMed

Xu, Yan; Liu, Cong; Clark, Jean C; Whitsett, Jeffrey A

2006-04-21

Cystic fibrosis (CF), a common lethal pulmonary disorder in Caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) that disturbs fluid homeostasis and host defense in target organs. The effects of CFTR and delta508-CFTR were assessed in transgenic mice that 1) lack CFTR expression (Cftr-/-); 2) express the human delta508 CFTR (CFTR(delta508)); 3) overexpress the normal human CFTR (CFTR(tg)) in respiratory epithelial cells. Genes were selected from Affymetrix Murine Gene-Chips analysis and subjected to functional classification, k-means clustering, promoter cis-elements/modules searching, literature mining, and pathway exploring. Genomic responses to Cftr-/- were not corrected by expression of CFTR(delta508). Genes regulating host defense, inflammation, fluid and electrolyte transport were similarly altered in Cftr-/- and CFTR(delta508) mice. CFTR(delta508) induced a primary disturbance in expression of genes regulating redox and antioxidant systems. Genomic responses to CFTR(tg) were modest and were not associated with lung pathology. CFTR(tg) and CFTR(delta508) induced genes encoding heat shock proteins and other chaperones but did not activate the endoplasmic reticulum-associated degradation pathway. RNAs encoding proteins that directly interact with CFTR were identified in each of the CFTR mouse models, supporting the hypothesis that CFTR functions within a multiprotein complex whose members interact at the level of protein-protein interactions and gene expression. Promoters of genes influenced by CFTR shared common regulatory elements, suggesting that their co-expression may be mediated by shared regulatory mechanisms. Genes and pathways involved in the response to CFTR may be of interest as modifiers of CF.

The ΔF508 Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Is Associated With Progressive Insulin Resistance and Decreased Functional β-Cell Mass in Mice.

PubMed

Fontés, Ghislaine; Ghislain, Julien; Benterki, Isma; Zarrouki, Bader; Trudel, Dominique; Berthiaume, Yves; Poitout, Vincent

2015-12-01

Cystic fibrosis (CF) is the result of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CF-related diabetes affects 50% of adult CF patients. How CFTR deficiency predisposes to diabetes is unknown. Herein, we examined the impact of the most frequent cftr mutation in humans, deletion of phenylalanine at position 508 (ΔF508), on glucose homeostasis in mice. We compared ΔF508 mutant mice with wild-type (WT) littermates. Twelve-week-old male ΔF508 mutants had lower body weight, improved oral glucose tolerance, and a trend toward higher insulin tolerance. Glucose-induced insulin secretion was slightly diminished in ΔF508 mutant islets, due to reduced insulin content, but ΔF508 mutant islets were not more sensitive to proinflammatory cytokines than WT islets. Hyperglycemic clamps confirmed an increase in insulin sensitivity with normal β-cell function in 12- and 18-week-old ΔF508 mutants. In contrast, 24-week-old ΔF508 mutants exhibited insulin resistance and reduced β-cell function. β-Cell mass was unaffected at 11 weeks of age but was significantly lower in ΔF508 mutants versus controls at 24 weeks. This was not associated with gross pancreatic pathology. We conclude that the ΔF508 CFTR mutation does not lead to an intrinsic β-cell secretory defect but is associated with insulin resistance and a β-cell mass deficit in aging mutants. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

A synonymous codon change alters the drug sensitivity of ΔF508 cystic fibrosis transmembrane conductance regulator

PubMed Central

Bali, Vedrana; Lazrak, Ahmed; Guroji, Purushotham; Fu, Lianwu; Matalon, Sadis; Bebok, Zsuzsanna

2016-01-01

Synonymous mutations, such as I507-ATC→ATT, in deletion of Phe508 in cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR), the most frequent disease-associated mutant of CFTR, may affect protein biogenesis, structure, and function and contribute to an altered disease phenotype. Small-molecule drugs are being developed to correct ΔF508 CFTR. To understand correction mechanisms and the consequences of synonymous mutations, we analyzed the effect of mechanistically distinct correctors, corrector 4a (C4) and lumacaftor (VX-809), on I507-ATT and I507-ATC ΔF508 CFTR biogenesis and function. C4 stabilized I507-ATT ΔF508 CFTR band B, but without considerable biochemical and functional correction. VX-809 biochemically corrected ∼10% of both of the variants, leading to stable, forskolin+3-isobutyl-1-methylxanthine (IBMX)-activated whole-cell currents in the presence of the corrector. Omitting VX-809 during whole-cell recordings led to a spontaneous decline of the currents, suggesting posttranslational stabilization by VX-809. Treatment of cells with the C4+VX-809 combination resulted in enhanced rescue and 2-fold higher forskolin+IBMX–activated currents of both I507-ATT and I507-ATC ΔF508 CFTR, compared with VX-809 treatment alone. The lack of an effect of C4 on I507-ATC ΔF508 CFTR, but its additive effect in combination with VX-809, implies that C4 acted on VX-809–modified I507-ATC ΔF508 CFTR. Our results suggest that binding of C4 and VX-809 to ΔF508 CFTR is conformation specific and provide evidence that synonymous mutations can alter the drug sensitivity of proteins.—Bali, V., Lazrak, A., Guroji, P., Fu, L., Matalon, S., Bebok, Z. A synonymous codon change alters the drug sensitivity of ΔF508 cystic fibrosis transmembrane conductance regulator. PMID:26336913

Molecular basis of cystic fibrosis in Lithuania: incomplete CFTR mutation detection by PCR-based screening protocols.

PubMed

Giannattasio, S; Bobba, A; Jurgelevicius, V; Vacca, R A; Lattanzio, P; Merafina, R S; Utkus, A; Kucinskas, V; Marra, E

2006-01-01

Mutational analysis of the cystic fibrosis transmembrane regulator (CFTR) gene was performed in 98 unrelated CF chromosomes from 49 Lithuanian CF patients through a combined approach in which the p.F508del mutation was first screened by allele-specific PCR while CFTR mutations in nonp.F508del chromosomes have been screened for by denaturing gradient gel electrophoresis analysis. A CFTR mutation was characterized in 62.2% of CF chromosomes, two of which (2.0%) have been previously shown to carry a large gene deletion CFTRdele2,3(21 kb). The most frequent Lithuanian CF mutation is p.F508del (52.0%). Seven CFTR mutations, p.N1303K (2.0%), p.R75Q (1.0%), p.G314R (1.0%), p.R553X (4.2%), p.W1282X (1.0%), and g.3944delGT (1.0%), accounted for 10.1% of Lithuanian CF chromosomes. It was not possible to characterize 35.8% of the CF Lithuanian chromosomes. Analysis of intron 8 (TG)mTn and M470V polymorphic loci did not permit the characterization of the CFTR dysfunction underlying the CF phenotype in the patients for which no CFTR mutation was identified. Thus, screening of the eight CFTR mutations identified in this study and of the large deletion CFTRdele2,3(21 kb) allows the implementation of an early molecular or confirmatory CF diagnosis for 65% of Lithuanian CF chromosomes.

Small molecule correctors of F508del-CFTR discovered by structure-based virtual screening

NASA Astrophysics Data System (ADS)

Kalid, Ori; Mense, Martin; Fischman, Sharon; Shitrit, Alina; Bihler, Hermann; Ben-Zeev, Efrat; Schutz, Nili; Pedemonte, Nicoletta; Thomas, Philip J.; Bridges, Robert J.; Wetmore, Diana R.; Marantz, Yael; Senderowitz, Hanoch

2010-12-01

Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, tenfold higher than hit rates obtained in corresponding high-throughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of our knowledge this is the first report of structure-based discovery of CFTR modulators.

Cystic fibrosis Δf508 mutation screening in Brazilian women with altered fertility.

PubMed

Brunoro, G V F; Wolfgramm, E V; Louro, I D; Degasperi, I I; Busatto, V C W; Perrone, A M S; Batitucci, M C P

2011-10-01

Cystic Fibrosis (CF) is an autosomal recessive disease, caused by mutations in the Cystic Fibrosis Transmembrane Regulator gene (CFTR). The most frequent mutation in CF is ΔF508. The disease is clinically characterized by elevated concentrations of sweat chlorides and abnormally thick mucus. It affects organs such as lung, pancreas, gastrointestinal and reproductive tract. Women with CF commonly present delayed puberty and amenorrhea due to malnutrition. Our objective was to screen the presence of ΔF508 mutation in 24 women with altered fertility. Nine of these women presented reduced fertility without a known cause, four showed polycystic ovaries and two had early menopause. One woman with early menopause was a carrier of the ΔF508 mutation. Our study demonstrates that it is possible that the frequency of CF mutations among patients with altered fertility may be higher than expected. Previous data showed that fibrocystic women can show reduced fertility, maternal mortality associated with pregnancy and increased incidence of spontaneous abortion. We therefore recommend that women with reduced fertility undertake genetic tests for a better evaluation of pregnancy risks and clinical monitoring.

Amniotic fluid digestive enzyme analysis is useful for identifying CFTR gene mutations of unclear significance.

PubMed

Oca, Florine; Dreux, Sophie; Gérard, Bénédicte; Simon-Bouy, Brigitte; de Becdelièvre, Alix; Ferec, Claude; Girodon, Emmanuelle; Muller, Françoise

2009-12-01

The large number of CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] mutations and the existence of variants of unclear significance complicate the prenatal diagnosis of cystic fibrosis (CF). The aim of this study was to determine whether the pattern of amniotic fluid digestive enzymes (AF-DEs) could be correlated with the severity of CFTR mutations. The AF-DE pattern (gamma-glutamyltranspeptidase, aminopeptidase M, and the intestinal isoform of alkaline phosphatase) was retrospectively analyzed in 43 AF samples. All fetuses presented 2 CFTR mutations, which were classified according to the severity of the disease: CF/CF (n = 38); CF/CFTR-related disorders (n = 1); and CF/unknown variant (n = 4). The relationships between clinical CF status, CFTR mutations, and AF-DE pattern were studied. Of 38 severely affected CF fetuses, an "obstructive" AF-DE pattern was observed in 15 of 15 samples collected before 22 weeks, irrespective of the CFTR mutation (diagnostic sensitivity, 100%; diagnostic specificity, 99.8%). In the 23 fetuses evaluated after 22 weeks, the AF-DE pattern was abnormal in 7 cases and noncontributive in 16 (diagnostic sensitivity, 30.4%; diagnostic specificity, 99.8%). Of the 5 questionable cases (F508del/N1224K, F508del/L73F, 3849+10kbC>T/G1127E, F508del/S1235R, F508del/G622D), all were CF symptom free at 2-4 years of follow-up. The AF-DE pattern (<22 weeks) was typical in 3 cases but abnormal in the last 2 cases. AF-DE analysis is of value for prenatal CF diagnosis in classic forms of CF and could be helpful in nonclassic CF.

Highly preferential association of NonF508del CF mutations with the M470 allele.

PubMed

Ciminelli, B M; Bonizzato, A; Bombieri, C; Pompei, F; Gabaldo, M; Ciccacci, C; Begnini, A; Holubova, A; Zorzi, P; Piskackova, T; Macek, M; Castellani, C; Modiano, G; Pignatti, P F

2007-01-01

On the basis of previous findings on random individuals, we hypothesized a preferential association of CF causing mutations with the M allele of the M470V polymorphic site of the CFTR gene. We have determined the M/V-CF mutation haplotype in a series of 201 North East Italian and 73 Czech CF patients who were not F508del homozygotes, as F508del was already known to be fully associated with the M allele. Out of 358 not F508del CF genes, 84 carried the V allele and 274 the less common M allele. In the N-E Italian population, MM subjects have a risk of carrying a CF causing mutation 6.9x greater than VV subjects when F508del is excluded and 15.4x when F508del is included. In the Czech population a similar, although less pronounced, association is observed. Besides the possible biological significance of this association, the possibility of exploiting it for a pilot screening program has been explored in a local North East Italian population for which CF patients were characterized for their CF mutation. General M470V genotyping followed by common CF mutation screening limited to couples in which each partner carries at least one M allele would need testing only 39% of the couples, which contribute 89% of the total risk, with a cost benefit.

Cysteamine re-establishes the clearance of Pseudomonas aeruginosa by macrophages bearing the cystic fibrosis-relevant F508del-CFTR mutation.

PubMed

Ferrari, Eleonora; Monzani, Romina; Villella, Valeria R; Esposito, Speranza; Saluzzo, Francesca; Rossin, Federica; D'Eletto, Manuela; Tosco, Antonella; De Gregorio, Fabiola; Izzo, Valentina; Maiuri, Maria C; Kroemer, Guido; Raia, Valeria; Maiuri, Luigi

2017-01-12

Cystic fibrosis (CF), the most common lethal monogenic disease in Caucasians, is characterized by recurrent bacterial infections and colonization, mainly by Pseudomonas aeruginosa, resulting in unresolved airway inflammation. CF is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which functions as a chloride channel in epithelial cells, macrophages, and other cell types. Impaired bacterial handling by macrophages is a feature of CF airways, although it is still debated how defective CFTR impairs bacterial killing. Recent evidence indicates that a defective autophagy in CF macrophages leads to alterations of bacterial clearance upon infection. Here we use bone marrow-derived macrophages from transgenic mice to provide the genetic proof that defective CFTR compromises both uptake and clearance of internalized Pseudomonas aeruginosa. We demonstrate that the proteostasis regulator cysteamine, which rescues the function of the most common F508del-CFTR mutant and hence reduces lung inflammation in CF patients, can also repair the defects of CF macrophages, thus restoring both bacterial internalization and clearance through a process that involves upregulation of the pro-autophagic protein Beclin 1 and re-establishment of the autophagic pathway. Altogether these results indicate that cysteamine restores the function of several distinct cell types, including that of macrophages, which might contribute to its beneficial effects on CF.

Cysteamine re-establishes the clearance of Pseudomonas aeruginosa by macrophages bearing the cystic fibrosis-relevant F508del-CFTR mutation

PubMed Central

Ferrari, Eleonora; Monzani, Romina; Villella, Valeria R; Esposito, Speranza; Saluzzo, Francesca; Rossin, Federica; D'Eletto, Manuela; Tosco, Antonella; De Gregorio, Fabiola; Izzo, Valentina; Maiuri, Maria C; Kroemer, Guido; Raia, Valeria; Maiuri, Luigi

2017-01-01

Cystic fibrosis (CF), the most common lethal monogenic disease in Caucasians, is characterized by recurrent bacterial infections and colonization, mainly by Pseudomonas aeruginosa, resulting in unresolved airway inflammation. CF is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which functions as a chloride channel in epithelial cells, macrophages, and other cell types. Impaired bacterial handling by macrophages is a feature of CF airways, although it is still debated how defective CFTR impairs bacterial killing. Recent evidence indicates that a defective autophagy in CF macrophages leads to alterations of bacterial clearance upon infection. Here we use bone marrow-derived macrophages from transgenic mice to provide the genetic proof that defective CFTR compromises both uptake and clearance of internalized Pseudomonas aeruginosa. We demonstrate that the proteostasis regulator cysteamine, which rescues the function of the most common F508del-CFTR mutant and hence reduces lung inflammation in CF patients, can also repair the defects of CF macrophages, thus restoring both bacterial internalization and clearance through a process that involves upregulation of the pro-autophagic protein Beclin 1 and re-establishment of the autophagic pathway. Altogether these results indicate that cysteamine restores the function of several distinct cell types, including that of macrophages, which might contribute to its beneficial effects on CF. PMID:28079883

Targeting autophagy as a novel strategy for facilitating the therapeutic action of potentiators on ΔF508 cystic fibrosis transmembrane conductance regulator.

PubMed

Luciani, Alessandro; Villella, Valeria Rachela; Esposito, Speranza; Gavina, Manuela; Russo, Ilaria; Silano, Marco; Guido, Stefano; Pettoello-Mantovani, Massimo; Carnuccio, Rosa; Scholte, Bob; De Matteis, Antonella; Maiuri, Maria Chiara; Raia, Valeria; Luini, Alberto; Kroemer, Guido; Maiuri, Luigi

2012-11-01

Channel activators (potentiators) of cystic fibrosis (CF) transmembrane conductance regulator (CFTR), can be used for the treatment of the small subset of CF patients that carry plasma membrane-resident CFTR mutants. However, approximately 90% of CF patients carry the misfolded ΔF508-CFTR and are poorly responsive to potentiators, because ΔF508-CFTR is intrinsically unstable at the plasma membrane (PM) even if rescued by pharmacological correctors. We have demonstrated that human and mouse CF airways are autophagy deficient due to functional sequestration of BECN1 and that the tissue transglutaminase-2 inhibitor, cystamine, or antioxidants restore BECN1-dependent autophagy and reduce SQSTM1/p62 levels, thus favoring ΔF508-CFTR trafficking to the epithelial surface. Here, we investigated whether these treatments could facilitate the beneficial action of potentiators on ΔF508-CFTR homozygous airways. Cystamine or the superoxide dismutase (SOD)/catalase-mimetic EUK-134 stabilized ΔF508-CFTR at the plasma membrane of airway epithelial cells and sustained the expression of CFTR at the epithelial surface well beyond drug withdrawal, overexpressing BECN1 and depleting SQSTM1. This facilitates the beneficial action of potentiators in controlling inflammation in ex vivo ΔF508-CFTR homozygous human nasal biopsies and in vivo in mouse ΔF508-CFTR lungs. Direct depletion of Sqstm1 by shRNAs in vivo in ΔF508-CFTR mice synergized with potentiators in sustaining surface CFTR expression and suppressing inflammation. Cystamine pre-treatment restored ΔF508-CFTR response to the CFTR potentiators genistein, Vrx-532 or Vrx-770 in freshly isolated brushed nasal epithelial cells from ΔF508-CFTR homozygous patients. These findings delineate a novel therapeutic strategy for the treatment of CF patients with the ΔF508-CFTR mutation in which patients are first treated with cystamine and subsequently pulsed with CFTR potentiators.

Low free drug concentration prevents inhibition of F508del CFTR functional expression by the potentiator VX-770 (ivacaftor).

PubMed

Matthes, Elizabeth; Goepp, Julie; Carlile, Graeme W; Luo, Yishan; Dejgaard, Kurt; Billet, Arnaud; Robert, Renaud; Thomas, David Y; Hanrahan, John W

2016-02-01

The most common cystic fibrosis (CF) mutation F508del inhibits the gating and surface expression of CFTR, a plasma membrane anion channel. Optimal pharmacotherapies will probably require both a 'potentiator' to increase channel open probability and a 'corrector' that improves folding and trafficking of the mutant protein and its stability at the cell surface. Interaction between CF drugs has been reported but remains poorly understood. CF bronchial epithelial cells were exposed to the corrector VX-809 (lumacaftor) and potentiator VX-770 (ivacaftor) individually or in combination. Functional expression of CFTR was assayed as the forskolin-stimulated short-circuit current (Isc ) across airway epithelial monolayers expressing F508del CFTR. The potentiated Isc response during forskolin stimulation was increased sixfold after pretreatment with VX-809 alone and reached ~11% that measured across non-CF monolayers. VX-770 (100 nM) and genistein (50 μM) caused similar levels of potentiation, which were not additive and were abolished by the CFTR inhibitor CFTRinh -172. The unbound fraction of VX-770 in plasma was 0.13 ± 0.04%, which together with previous measurements in patients given 250 mg p.o. twice daily, suggests a peak free plasma concentration of 1.5-8.5 nM. Chronic exposure to high VX-770 concentrations (>1 μM) inhibited functional correction by VX-809 but not in the presence of physiological protein levels (20-40 mg·mL(-1) ). Chronic exposure to a low concentration of VX-770 (100 nM) together with VX-809 (1 μM) also did not reduce the forskolin-stimulated Isc , relative to cells chronically exposed to VX-809 alone, provided it was assayed acutely using the same, clinically relevant concentration of potentiator. Chronic exposure to clinically relevant concentrations of VX-770 did not reduce F508del CFTR function. Therapeutic benefit of VX-770 + VX-809 (Orkambi) is probably limited by the efficacy of VX-809 rather than by inhibition by VX-770. © 2015

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, P.W.; Hamosh, A.; Macek, M. Jr.

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 ({le}40 mmol/liter). One patient carried two CF mutations ({Delta}F508/R347H), and five were found tomore » carry one CF mutation (four {Delta}F508; one R117H). The frequency of the {Delta}F508 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. 54 refs., 2 tabs.« less

ERp29 Regulates ΔF508 and Wild-type Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Trafficking to the Plasma Membrane in Cystic Fibrosis (CF) and Non-CF Epithelial Cells*

PubMed Central

Suaud, Laurence; Miller, Katelyn; Alvey, Lora; Yan, Wusheng; Robay, Amal; Kebler, Catherine; Kreindler, James L.; Guttentag, Susan; Hubbard, Michael J.; Rubenstein, Ronald C.

2011-01-01

Sodium 4-phenylbutyrate (4PBA) improves the intracellular trafficking of ΔF508-CFTR in cystic fibrosis (CF) epithelial cells. The underlying mechanism is uncertain, but 4PBA modulates the expression of some cytosolic molecular chaperones. To identify other 4PBA-regulated proteins that might regulate ΔF508-CFTR trafficking, we performed a differential display RT-PCR screen on IB3-1 CF bronchiolar epithelial cells exposed to 4PBA. One transcript up-regulated by 4PBA encoded ERp29, a luminal resident of the endoplasmic reticulum (ER) thought to be a novel molecular chaperone. We tested the hypothesis that ERp29 is a 4PBA-regulated ER chaperone that influences ΔF508-CFTR trafficking. ERp29 mRNA and protein expression was significantly increased (∼1.5-fold) in 4PBA-treated IB3-1 cells. In Xenopus oocytes, ERp29 overexpression increased the functional expression of both wild-type and ΔF508-CFTR over 3-fold and increased wild-type cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane expression. In CFBE41o− WT-CFTR cells, expression of and short circuit currents mediated by CFTR decreased upon depletion of ERp29 as did maturation of newly synthesized CFTR. In IB3-1 cells, ΔF508-CFTR co-immunoprecipitated with endogenous ERp29, and overexpression of ERp29 led to increased ΔF508-CFTR expression at the plasma membrane. These data suggest that ERp29 is a 4PBA-regulated ER chaperone that regulates WT-CFTR biogenesis and can promote ΔF508-CFTR trafficking in CF epithelial cells. PMID:21525008

ERp29 regulates DeltaF508 and wild-type cystic fibrosis transmembrane conductance regulator (CFTR) trafficking to the plasma membrane in cystic fibrosis (CF) and non-CF epithelial cells.

PubMed

Suaud, Laurence; Miller, Katelyn; Alvey, Lora; Yan, Wusheng; Robay, Amal; Kebler, Catherine; Kreindler, James L; Guttentag, Susan; Hubbard, Michael J; Rubenstein, Ronald C

2011-06-17

Sodium 4-phenylbutyrate (4PBA) improves the intracellular trafficking of ΔF508-CFTR in cystic fibrosis (CF) epithelial cells. The underlying mechanism is uncertain, but 4PBA modulates the expression of some cytosolic molecular chaperones. To identify other 4PBA-regulated proteins that might regulate ΔF508-CFTR trafficking, we performed a differential display RT-PCR screen on IB3-1 CF bronchiolar epithelial cells exposed to 4PBA. One transcript up-regulated by 4PBA encoded ERp29, a luminal resident of the endoplasmic reticulum (ER) thought to be a novel molecular chaperone. We tested the hypothesis that ERp29 is a 4PBA-regulated ER chaperone that influences ΔF508-CFTR trafficking. ERp29 mRNA and protein expression was significantly increased (∼1.5-fold) in 4PBA-treated IB3-1 cells. In Xenopus oocytes, ERp29 overexpression increased the functional expression of both wild-type and ΔF508-CFTR over 3-fold and increased wild-type cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane expression. In CFBE41o- WT-CFTR cells, expression of and short circuit currents mediated by CFTR decreased upon depletion of ERp29 as did maturation of newly synthesized CFTR. In IB3-1 cells, ΔF508-CFTR co-immunoprecipitated with endogenous ERp29, and overexpression of ERp29 led to increased ΔF508-CFTR expression at the plasma membrane. These data suggest that ERp29 is a 4PBA-regulated ER chaperone that regulates WT-CFTR biogenesis and can promote ΔF508-CFTR trafficking in CF epithelial cells.

Defective Fluid Secretion from Submucosal Glands of Nasal Turbinates from CFTR-/- and CFTRΔF508/ΔF508 Pigs

PubMed Central

Cho, Hyung-Ju; Joo, Nam Soo; Wine, Jeffrey J.

2011-01-01

Background Cystic fibrosis (CF), caused by reduced CFTR function, includes severe sinonasal disease which may predispose to lung disease. Newly developed CF pigs provide models to study the onset of CF pathophysiology. We asked if glands from pig nasal turbinates have secretory responses similar to those of tracheal glands and if CF nasal glands show reduced fluid secretion. Methodology/Principal Findings Unexpectedly, we found that nasal glands differed from tracheal glands in five ways, being smaller, more numerous (density per airway surface area), more sensitive to carbachol, more sensitive to forskolin, and nonresponsive to Substance P (a potent agonist for pig tracheal glands). Nasal gland fluid secretion from newborn piglets (12 CF and 12 controls) in response to agonists was measured using digital imaging of mucus bubbles formed under oil. Secretion rates were significantly reduced in all conditions tested. Fluid secretory rates (Controls vs. CF, in pl/min/gland) were as follows: 3 µM forskolin: 9.2±2.2 vs. 0.6±0.3; 1 µM carbachol: 143.5±35.5 vs. 52.2±10.3; 3 µM forskolin + 0.1 µM carbachol: 25.8±5.8 vs. CF 4.5±0.9. We also compared CFΔF508/ΔF508 with CFTR-/- piglets and found significantly greater forskolin-stimulated secretion rates in the ΔF508 vs. the null piglets (1.4±0.8, n = 4 vs. 0.2±0.1, n = 7). An unexpected age effect was also discovered: the ratio of secretion to 3 µM forskolin vs. 1 µM carbachol was ∼4 times greater in adult than in neonatal nasal glands. Conclusions/Significance These findings reveal differences between nasal and tracheal glands, show defective fluid secretion in nasal glands of CF pigs, reveal some spared function in the ΔF508 vs. null piglets, and show unexpected age-dependent differences. Reduced nasal gland fluid secretion may predispose to sinonasal and lung infections. PMID:21935358

Targeting autophagy as a novel strategy for facilitating the therapeutic action of potentiators on ΔF508 cystic fibrosis transmembrane conductance regulator

PubMed Central

Luciani, Alessandro; Villella, Valeria Rachela; Esposito, Speranza; Gavina, Manuela; Russo, Ilaria; Silano, Marco; Guido, Stefano; Pettoello-Mantovani, Massimo; Carnuccio, Rosa; Scholte, Bob; De Matteis, Antonella; Maiuri, Maria Chiara; Raia, Valeria; Luini, Alberto; Kroemer, Guido; Maiuri, Luigi

2012-01-01

Channel activators (potentiators) of cystic fibrosis (CF) transmembrane conductance regulator (CFTR), can be used for the treatment of the small subset of CF patients that carry plasma membrane-resident CFTR mutants. However, approximately 90% of CF patients carry the misfolded ΔF508-CFTR and are poorly responsive to potentiators, because ΔF508-CFTR is intrinsically unstable at the plasma membrane (PM) even if rescued by pharmacological correctors. We have demonstrated that human and mouse CF airways are autophagy deficient due to functional sequestration of BECN1 and that the tissue transglutaminase-2 inhibitor, cystamine, or antioxidants restore BECN1-dependent autophagy and reduce SQSTM1/p62 levels, thus favoring ΔF508-CFTR trafficking to the epithelial surface. Here, we investigated whether these treatments could facilitate the beneficial action of potentiators on ΔF508-CFTR homozygous airways. Cystamine or the superoxide dismutase (SOD)/catalase-mimetic EUK-134 stabilized ΔF508-CFTR at the plasma membrane of airway epithelial cells and sustained the expression of CFTR at the epithelial surface well beyond drug withdrawal, overexpressing BECN1 and depleting SQSTM1. This facilitates the beneficial action of potentiators in controlling inflammation in ex vivo ΔF508-CFTR homozygous human nasal biopsies and in vivo in mouse ΔF508-CFTR lungs. Direct depletion of Sqstm1 by shRNAs in vivo in ΔF508-CFTR mice synergized with potentiators in sustaining surface CFTR expression and suppressing inflammation. Cystamine pre-treatment restored ΔF508-CFTR response to the CFTR potentiators genistein, Vrx-532 or Vrx-770 in freshly isolated brushed nasal epithelial cells from ΔF508-CFTR homozygous patients. These findings delineate a novel therapeutic strategy for the treatment of CF patients with the ΔF508-CFTR mutation in which patients are first treated with cystamine and subsequently pulsed with CFTR potentiators. PMID:22874563

Epithelial IgG and its relationship to the loss of F508 in the common mutant form of the cystic fibrosis transmembrane conductance regulator.

PubMed

Treharne, Kate J; Cassidy, Diane; Goddard, Catharine; Colledge, William H; Cassidy, Andrew; Mehta, Anil

2009-08-06

The most debilitating feature of cystic fibrosis (CF) disease is uncontrolled inflammation of respiratory epithelium. The relationship between the commonest mutated form of CFTR (F508del or DeltaF508) and inflammation has not yet been elucidated. Here, we present a new paradigm suggesting that CFTR can interact with intra-epithelial IgG, establishing a direct link between normal CFTR and the immune system. Further, our data show that the amino-acid sequence local to F508 can bind IgG with high affinity, dependent on F508, such that loss of F508 abolishes this link both in vitro and in the intact cell.

Lumacaftor/ivacaftor, a novel agent for the treatment of cystic fibrosis patients who are homozygous for the F580del CFTR mutation.

PubMed

Bulloch, Marilyn N; Hanna, Cameron; Giovane, Richard

2017-10-01

Cystic Fibrosis (CF) is an autosomal recessive disease affecting up to 90,000 people worldwide. Approximately 73% of patients are homozygous for the F508del cystic fibrosis transmembrane conductance regulator [CFTR] mutation. Traditionally treatment has only included supportive care. Therefore, there is a need for safe and effective novel therapies targeting the underlying molecular defects seen with CF. Areas covered: In 2016, the Food and Drug Administration and the European Commission approved LUM/IVA (Orkambi), a CFTR modulator that includes both a CFTR corrector and potentiator, for CF patients homozygous for the F508del CFTR mutation. This article reviews the pharmacologic features, clinical efficacy, and safety of LUM/IVA and summarize the available pre-clinical and clinical data of LUM/IVA use. Expert commentary: LUM/IVA showed modest, but significant improvements from baseline in percent predicted FEV 1 (ppFEV 1 ) as well as a reduction in pulmonary exacerbations by 35% It was shown to be safe for short- and long-term use. Currently, LUM/IVA is the only oral agent in its class available and represents a milestone the development of therapies for the management of CF. Nonetheless, pharmacoeconomic data are necessary to justify its high cost before is use becomes standard of care.

Identification of the M1101K mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and complete detection of cystic fibrosis mutations in the Hutterite population

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zielenski, J.; Markiewicz, D.; Fujiwara, M.

1993-03-01

The Hutterite population is a genetic isolate with an increased incidence of cystic fibrosis (CF). Previously the authors identified three CF haplotypes defined by polymorphisms flanking the CF transmembrane conductance regulator (CFTR) gene. [Delta]F508 was present on one of the haplotypes in only 35% of CF chromosomes. They hypothesized that the other two CF haplotypes, one of which was the most common and the other of which is rare, each harbored different non-[Delta]F508 mutations. Single-strand conformation polymorphism analysis detected a missense mutation, M1101K, in both chromosomes of a Hutterite patient carrying the two non-[Delta]F508 haplotypes. M1101K appears to have originatedmore » on an uncommon CFTR allele and to be infrequent outside the Hutterite population. The presence of M1101K on two haplotypes is likely the result of a CFTR intragenic recombination which occurred since the founding, 10-12 generations ago, of the Hutterite population. The crossover was located between exons 14a and 17b, an interval of approximately 15 kbp. [Delta]F508 and M1101K accounted for all of the CF mutations in patients from 16 CF families representing the three subdivisions of the Hutterite population. 38 refs., 3 figs., 1 tab.« less

Fatty Acid Cysteamine Conjugates as Novel and Potent Autophagy Activators That Enhance the Correction of Misfolded F508del-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

PubMed

Vu, Chi B; Bridges, Robert J; Pena-Rasgado, Cecilia; Lacerda, Antonio E; Bordwell, Curtis; Sewell, Abby; Nichols, Andrew J; Chandran, Sachin; Lonkar, Pallavi; Picarella, Dominic; Ting, Amal; Wensley, Allison; Yeager, Maisy; Liu, Feng

2017-01-12

A depressed autophagy has previously been reported in cystic fibrosis patients with the common F508del-CFTR mutation. This report describes the synthesis and preliminary biological characterization of a novel series of autophagy activators involving fatty acid cysteamine conjugates. These molecular entities were synthesized by first covalently linking cysteamine to docosahexaenoic acid. The resulting conjugate 1 synergistically activated autophagy in primary homozygous F508del-CFTR human bronchial epithelial (hBE) cells at submicromolar concentrations. When conjugate 1 was used in combination with the corrector lumacaftor and the potentiator ivacaftor, it showed an additive effect, as measured by the increase in the chloride current in a functional assay. In order to obtain a more stable form for oral dosing, the sulfhydryl group in conjugate 1 was converted into a functionalized disulfide moiety. The resulting conjugate 5 is orally bioavailable in the mouse, rat, and dog and allows a sustained delivery of the biologically active conjugate 1.

High-Content Surface and Total Expression siRNA Kinase Library Screen with VX-809 Treatment Reveals Kinase Targets that Enhance F508del-CFTR Rescue.

PubMed

Perkins, Lydia A; Fisher, Gregory W; Naganbabu, Matharishwan; Schmidt, Brigitte F; Mun, Frederick; Bruchez, Marcel P

2018-03-05

The most promising F508del-CFTR corrector, VX-809, has been unsuccessful as an effective, stand-alone treatment for CF patients, but the rescue effect in combination with other drugs may confer an acceptable level of therapeutic benefit. Targeting cellular factors that modify trafficking may act to enhance the cell surface density of F508-CFTR with VX-809 correction. Our goal is to identify druggable kinases that enhance F508del-CFTR rescue and stabilization at the cell surface beyond that achievable with the VX-809 corrector alone. To achieve this goal, we implemented a new high-throughput screening paradigm that quickly and quantitatively measures surface density and total protein in the same cells. This allowed for rapid screening for increased surface targeting and proteostatic regulation. The assay utilizes fluorogen-activating-protein (FAP) technology with cell excluded and cell permeant fluorogenic dyes in a quick, wash-free fluorescent plate reader format on live cells to first measure F508del-CFTR expressed on the surface and then the total amount of F508del-CFTR protein present. To screen for kinase targets, we used Dharmacon's ON-TARGET plus SMARTpool siRNA Kinase library (715 target kinases) with and without 10 μM VX-809 treatment in triplicate at 37 °C. We identified several targets that had a significant interaction with VX-809 treatment in enhancing surface density with siRNA knockdown. Select small-molecule inhibitors of the kinase targets demonstrated augmented surface expression with VX-809 treatment.

A small-molecule modulator interacts directly with deltaPhe508-CFTR to modify its ATPase activity and conformational stability.

PubMed

Wellhauser, Leigh; Kim Chiaw, Patrick; Pasyk, Stan; Li, Canhui; Ramjeesingh, Mohabir; Bear, Christine E

2009-06-01

The deletion of Phe-508 (DeltaPhe508) constitutes the most prevalent of a number of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) that cause cystic fibrosis (CF). This mutation leads to CFTR misfolding and retention in the endoplasmic reticulum, as well as impaired channel activity. The biosynthetic defect can be partially overcome by small-molecule "correctors"; once at the cell surface, small-molecule "potentiators" enhance the channel activity of DeltaPhe508-CFTR. Certain compounds, such as VRT-532, exhibit both corrector and potentiator functions. In the current studies, we confirmed that the inherent chloride channel activity of DeltaPhe508-CFTR (after biosynthetic rescue) is potentiated in studies of intact cells and membrane vesicles. It is noteworthy that we showed that the ATPase activity of the purified and reconstituted mutant protein is directly modulated by binding of VRT-532 [4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol] ATP turnover by reconstituted DeltaPhe508-CFTR is decreased by VRT-532 treatment, an effect that may account for the increase in channel open time induced by this compound. To determine whether the modification of DeltaPhe508-CFTR function caused by direct VRT-532 binding is associated with structural changes, we evaluated the effect of VRT-532 binding on the protease susceptibility of the major mutant. We found that binding of VRT-532 to DeltaPhe508-CFTR led to a minor but significant decrease in the trypsin susceptibility of the full-length mutant protein and a fragment encompassing the second half of the protein. These findings suggest that direct binding of this small molecule induces and/or stabilizes a structure that promotes the channel open state and may underlie its efficacy as a corrector of DeltaPhe508-CFTR.

Restoration of R117H CFTR folding and function in human airway cells through combination treatment with VX-809 and VX-770

PubMed Central

Gentzsch, Martina; Ren, Hong Y.; Houck, Scott A.; Quinney, Nancy L.; Cholon, Deborah M.; Sopha, Pattarawut; Chaudhry, Imron G.; Das, Jhuma; Dokholyan, Nikolay V.; Randell, Scott H.

2016-01-01

Cystic fibrosis (CF) is a lethal recessive genetic disease caused primarily by the F508del mutation in the CF transmembrane conductance regulator (CFTR). The potentiator VX-770 was the first CFTR modulator approved by the FDA for treatment of CF patients with the gating mutation G551D. Orkambi is a drug containing VX-770 and corrector VX809 and is approved for treatment of CF patients homozygous for F508del, which has folding and gating defects. At least 30% of CF patients are heterozygous for the F508del mutation with the other allele encoding for one of many different rare CFTR mutations. Treatment of heterozygous F508del patients with VX-809 and VX-770 has had limited success, so it is important to identify heterozygous patients that respond to CFTR modulator therapy. R117H is a more prevalent rare mutation found in over 2,000 CF patients. In this study we investigated the effectiveness of VX-809/VX-770 therapy on restoring CFTR function in human bronchial epithelial (HBE) cells from R117H/F508del CF patients. We found that VX-809 stimulated more CFTR activity in R117H/F508del HBEs than in F508del/F508del HBEs. R117H expressed exclusively in immortalized HBEs exhibited a folding defect, was retained in the ER, and degraded prematurely. VX-809 corrected the R117H folding defect and restored channel function. Because R117 is involved in ion conductance, VX-770 acted additively with VX-809 to restore CFTR function in chronically treated R117H/F508del cells. Although treatment of R117H patients with VX-770 has been approved, our studies indicate that Orkambi may be more beneficial for rescue of CFTR function in these patients. PMID:27402691

Restoration of R117H CFTR folding and function in human airway cells through combination treatment with VX-809 and VX-770.

PubMed

Gentzsch, Martina; Ren, Hong Y; Houck, Scott A; Quinney, Nancy L; Cholon, Deborah M; Sopha, Pattarawut; Chaudhry, Imron G; Das, Jhuma; Dokholyan, Nikolay V; Randell, Scott H; Cyr, Douglas M

2016-09-01

Cystic fibrosis (CF) is a lethal recessive genetic disease caused primarily by the F508del mutation in the CF transmembrane conductance regulator (CFTR). The potentiator VX-770 was the first CFTR modulator approved by the FDA for treatment of CF patients with the gating mutation G551D. Orkambi is a drug containing VX-770 and corrector VX809 and is approved for treatment of CF patients homozygous for F508del, which has folding and gating defects. At least 30% of CF patients are heterozygous for the F508del mutation with the other allele encoding for one of many different rare CFTR mutations. Treatment of heterozygous F508del patients with VX-809 and VX-770 has had limited success, so it is important to identify heterozygous patients that respond to CFTR modulator therapy. R117H is a more prevalent rare mutation found in over 2,000 CF patients. In this study we investigated the effectiveness of VX-809/VX-770 therapy on restoring CFTR function in human bronchial epithelial (HBE) cells from R117H/F508del CF patients. We found that VX-809 stimulated more CFTR activity in R117H/F508del HBEs than in F508del/F508del HBEs. R117H expressed exclusively in immortalized HBEs exhibited a folding defect, was retained in the ER, and degraded prematurely. VX-809 corrected the R117H folding defect and restored channel function. Because R117 is involved in ion conductance, VX-770 acted additively with VX-809 to restore CFTR function in chronically treated R117H/F508del cells. Although treatment of R117H patients with VX-770 has been approved, our studies indicate that Orkambi may be more beneficial for rescue of CFTR function in these patients. Copyright © 2016 the American Physiological Society.

Spectrum of CFTR gene mutations in Ecuadorian cystic fibrosis patients: the second report of the p.H609R mutation.

PubMed

Ortiz, Sofía C; Aguirre, Santiago J; Flores, Sofía; Maldonado, Claudio; Mejía, Juan; Salinas, Lilian

2017-11-01

High heterogeneity in the CFTR gene mutations disturbs the molecular diagnosis of cystic fibrosis (CF). In order to improve the diagnosis of CF in our country, the present study aims to define a panel of common CFTR gene mutations by sequencing 27 exons of the gene in Ecuadorian Cystic Fibrosis patients. Forty-eight Ecuadorian individuals with suspected/confirmed CF diagnosis were included. Twenty-seven exons of CFTR gene were sequenced to find sequence variations. Prevalence of pathogenic variations were determined and compared with other countries' data. We found 70 sequence variations. Eight of these are CF-causing mutations: p.F508del, p.G85E, p.G330E, p.A455E, p.G970S, W1098X, R1162X, and N1303K. Also this study is the second report of p.H609R in Ecuadorian population. Mutation prevalence differences between Ecuadorian population and other Latin America countries were found. The panel of mutations suggested as an initial screening for the Ecuadorian population with cystic fibrosis should contain the mutations: p.F508del, p.G85E, p.G330E, p.A455E, p.G970S, W1098X, R1162X, and N1303K. © 2017 NETLAB Laboratorios Especializados. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

CFTR gene mutations in isolated chronic obstructive pulmonary disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pignatti, P.F.; Bombien, C.; Marigo, C.

1994-09-01

In order to identify a possible hereditary predisposition to the development of chronic obstructive pulmonary disease (COPD), we have looked for the presence of cystic fibrosis transmembrane regulator (CFTR) gene DNA sequence modifications in 28 unrelated patients with no signs of cystic fibrosis. The known mutations in Italian CF patients, as well as the most frequent worldwide CF mutations, were investigated. In addition, a denaturing gradient gel electrophoresis analysis of about half of the coding sequence of the gene in 56 chromosomes from the patients and in 102 chromosomes from control individuals affected by other pulmonary diseases and from normalmore » controls was performed. Nine different CFTR gene mutations and polymorphisms were found in seven patients, a highly significant increase over controls. Two of the patients were compound heterozygotes. Two frequent CF mutations were detected: deletion F508 and R117H; two rare CF mutations: R1066C and 3667ins4; and five CF sequence variants: R75Q (which was also described as a disease-causing mutation in male sterility cases due to the absence of the vasa deferentia), G576A, 2736 A{r_arrow}G, L997F, and 3271+18C{r_arrow}T. Seven (78%) of the mutations are localized in transmembrane domains. Six (86%) of the patients with defined mutations and polymorphisms had bronchiectasis. These results indicate that CFTR gene mutations and sequence alterations may be involved in the etiopathogenesis of some cases of COPD.« less

Targeting the Intracellular Environment in Cystic Fibrosis: Restoring Autophagy as a Novel Strategy to Circumvent the CFTR Defect

PubMed Central

Villella, Valeria Rachela; Esposito, Speranza; Bruscia, Emanuela M.; Maiuri, Maria Chiara; Raia, Valeria; Kroemer, Guido; Maiuri, Luigi

2013-01-01

Cystic fibrosis (CF) patients harboring the most common deletion mutation of the CF transmembrane conductance regulator (CFTR), F508del, are poor responders to potentiators of CFTR channel activity which can be used to treat a small subset of CF patients who genetically carry plasma membrane (PM)-resident CFTR mutants. The misfolded F508del-CFTR protein is unstable in the PM even if rescued by pharmacological agents that prevent its intracellular retention and degradation. CF is a conformational disease in which defective CFTR induces an impressive derangement of general proteostasis resulting from disabled autophagy. In this review, we discuss how rescuing Beclin 1 (BECN1), a major player of autophagosome formation, either by means of direct gene transfer or indirectly by administration of proteostasis regulators, could stabilize F508del-CFTR at the PM. We focus on the relationship between the improvement of peripheral proteostasis and CFTR PM stability in F508del-CFTR homozygous bronchial epithelia or mouse lungs. Moreover, this article reviews recent pre-clinical evidence indicating that targeting the intracellular environment surrounding the misfolded mutant CFTR instead of protein itself could constitute an attractive therapeutic option to sensitize patients carrying the F508del-CFTR mutation to the beneficial action of CFTR potentiators on lung inflammation. PMID:23346057

Activation of 3-Phosphoinositide-dependent Kinase 1 (PDK1) and Serum- and Glucocorticoid-induced Protein Kinase 1 (SGK1) by Short-chain Sphingolipid C4-ceramide Rescues the Trafficking Defect of ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (ΔF508-CFTR)*

PubMed Central

Caohuy, Hung; Yang, Qingfeng; Eudy, Yvonne; Ha, Thien-An; Xu, Andrew E.; Glover, Matthew; Frizzell, Raymond A.; Jozwik, Catherine; Pollard, Harvey B.

2014-01-01

Cystic fibrosis (CF) is due to a folding defect in the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, ΔF508, prevents CFTR from trafficking to the apical plasma membrane. Here we show that activation of the PDK1/SGK1 signaling pathway with C4-ceramide (C4-CER), a non-toxic small molecule, functionally corrects the trafficking defect in both cultured CF cells and primary epithelial cell explants from CF patients. The mechanism of C4-CER action involves a series of mutual autophosphorylation and phosphorylation events between PDK1 and SGK1. Detailed mechanistic studies indicate that C4-CER initially induces autophosphorylation of SGK1 at Ser422. SGK1[Ser(P)422] and C4-CER coincidently bind PDK1 and permit PDK1 to autophosphorylate at Ser241. Then PDK1[Ser(P)241] phosphorylates SGK1[Ser(P)422] at Thr256 to generate fully activated SGK1[Ser422, Thr(P)256]. SGK1[Ser(P)422,Thr(P)256] phosphorylates and inactivates the E3 ubiquitin ligase Nedd4-2. ΔF508-CFTR is thus free to traffic to the plasma membrane. Importantly, C4-CER-mediated activation of both PDK1 and SGK1 is independent of the PI3K/Akt/mammalian target of rapamycin signaling pathway. Physiologically, C4-CER significantly increases maturation and stability of ΔF508-CFTR (t½ ∼10 h), enhances cAMP-activated chloride secretion, and suppresses hypersecretion of interleukin-8 (IL-8). We suggest that candidate drugs for CF directed against the PDK1/SGK1 signaling pathway, such as C4-CER, provide a novel therapeutic strategy for a life-limiting disorder that affects one child, on average, each day. PMID:25384981

Increasing the Endoplasmic Reticulum Pool of the F508del Allele of the Cystic Fibrosis Transmembrane Conductance Regulator Leads to Greater Folding Correction by Small Molecule Therapeutics.

PubMed

Chung, W Joon; Goeckeler-Fried, Jennifer L; Havasi, Viktoria; Chiang, Annette; Rowe, Steven M; Plyler, Zackery E; Hong, Jeong S; Mazur, Marina; Piazza, Gary A; Keeton, Adam B; White, E Lucile; Rasmussen, Lynn; Weissman, Allan M; Denny, R Aldrin; Brodsky, Jeffrey L; Sorscher, Eric J

2016-01-01

Small molecules that correct the folding defects and enhance surface localization of the F508del mutation in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) comprise an important therapeutic strategy for cystic fibrosis lung disease. However, compounds that rescue the F508del mutant protein to wild type (WT) levels have not been identified. In this report, we consider obstacles to obtaining robust and therapeutically relevant levels of F508del CFTR. For example, markedly diminished steady state amounts of F508del CFTR compared to WT CFTR are present in recombinant bronchial epithelial cell lines, even when much higher levels of mutant transcript are present. In human primary airway cells, the paucity of Band B F508del is even more pronounced, although F508del and WT mRNA concentrations are comparable. Therefore, to augment levels of "repairable" F508del CFTR and identify small molecules that then correct this pool, we developed compound library screening protocols based on automated protein detection. First, cell-based imaging measurements were used to semi-quantitatively estimate distribution of F508del CFTR by high content analysis of two-dimensional images. We evaluated ~2,000 known bioactive compounds from the NIH Roadmap Molecular Libraries Small Molecule Repository in a pilot screen and identified agents that increase the F508del protein pool. Second, we analyzed ~10,000 compounds representing diverse chemical scaffolds for effects on total CFTR expression using a multi-plate fluorescence protocol and describe compounds that promote F508del maturation. Together, our findings demonstrate proof of principle that agents identified in this fashion can augment the level of endoplasmic reticulum (ER) resident "Band B" F508del CFTR suitable for pharmacologic correction. As further evidence in support of this strategy, PYR-41-a compound that inhibits the E1 ubiquitin activating enzyme-was shown to synergistically enhance F508del rescue by C18, a small

Distribution of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mutations in a Cohort of Patients Residing in Palestine.

PubMed

Siryani, Issa; Jama, Mohamed; Rumman, Nisreen; Marzouqa, Hiyam; Kannan, Moein; Lyon, Elaine; Hindiyeh, Musa

2015-01-01

Cystic fibrosis (CF) is an autosomal recessive inherited life-threatening disorder that causes severe damage to the lungs and the digestive system. In Palestine, mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) that contributes to the clinical presentation of CF are ill defined. A cohort of thirty three clinically diagnosed CF patients from twenty one different Palestinian families residing in the central and southern part of Palestine were incorporated in this study. Sweat chloride testing was performed using the Sweat Chek Conductivity Analyzer (ELITECH Group, France) to confirm the clinical diagnosis of CF. In addition, nucleic acid from the patients' blood samples was extracted and the CFTR mutation profiles were assessed by direct sequencing of the CFTR 27 exons and the intron-exon boundaries. For patient's DNA samples where no homozygous or two heterozygous CFTR mutations were identified by exon sequencing, DNA samples were tested for deletions or duplications using SALSA MLPA probemix P091-D1 CFTR assay. Sweat chloride testing confirmed the clinical diagnosis of CF in those patients. All patients had NaCl conductivity >60 mmol/l. In addition, nine different CFTR mutations were identified in all 21 different families evaluated. These mutations were c.1393-1G>A, F508del, W1282X, G85E, c.313delA, N1303K, deletion exons 17a-17b-18, deletion exons 17a-17b and Q1100P. c.1393-1G>A was shown to be the most frequent occurring mutation among tested families. We have profiled the underling mutations in the CFTR gene of a cohort of 21 different families affected by CF. Unlike other studies from the Arab countries where F508del was reported to be the most common mutation, in southern/central Palestine, the c.1393-1G>A appeared to be the most common. Further studies are needed per sample size and geographic distribution to account for other possible CFTR genetic alterations and their frequencies. Genotype/phenotype assessments are also

A pilot clinical trial of oral sodium 4-phenylbutyrate (Buphenyl) in deltaF508-homozygous cystic fibrosis patients: partial restoration of nasal epithelial CFTR function.

PubMed

Rubenstein, R C; Zeitlin, P L

1998-02-01

Sodium 4-phenylbutyrate (Buphenyl, 4PBA) is a new FDA approved drug for management of urea cycle disorders. We have previously presented data suggesting that 4PBA, at clinically achievable concentrations, induces CFTR channel function on the plasma membrane of deltaF508-expressing cystic fibrosis (CF) airway epithelial cells in vitro (Rubenstein, R. C., and P. L. Zeitlin, 1997. J. Clin. Invest. 100:2457-2463). We hypothesized that 4PBA would induce epithelial CFTR function in vivo in individuals homozygous for deltaF508-CFTR. A randomized, double-blind, placebo-controlled trial in 18 deltaF508-homozygous patients with CF was performed with the maximum approved adult dose of 4PBA, 19 grams p.o. divided t.i.d., given for 1 wk. Nasal potential difference (NPD) response patterns and sweat chloride concentrations were determined before and after study drug treatment, and 4PBA and metabolites were assayed in plasma and urine at the end of study drug treatment. Subjects in the 4PBA group demonstrated small, but statistically significant improvements of the NPD response to perfusion of an isoproterenol/amiloride/chloride-free solution; this measure reflects epithelial CFTR function and is highly discriminatory between patients with and without CF. Subjects who had received 4PBA did not demonstrate significantly reduced sweat chloride concentrations or alterations in the amiloride-sensitive NPD. Side effects due to drug therapy were minimal and comparable in the two groups. These data are consistent with 4PBA therapy inducing CFTR function in the nasal epithelia of deltaF508-homozygous CF patients.

Matrine in association with FD-2 stimulates F508del-cystic fibrosis transmembrane conductance regulator activity in the presence of corrector VX809

PubMed Central

Marengo, Barbara; Speciale, Andrea; Senatore, Lisa; Garibaldi, Silvano; Musumeci, Francesca; Nieddu, Erika; Pollarolo, Benedetta; Pronzato, Maria Adelaide; Schenone, Silvia; Mazzei, Mauro; Domenicotti, Cinzia

2017-01-01

Cystic fibrosis is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and the predominant mutation is termed Phe508del (F508del). Therapy for F508del-CFTR patients is based on the use of Orkambi®, a combination of VX809 and VX770. However, though Orkambi leads to an improvement in the lung function of patients, a progressive reduction in its efficacy has been observed. In order to overcome this effect, the aim of the present study was to investigate the role of matrine and the in-house compound FD-2 in increasing the action of VX809 and VX770. Fischer rat thyroid cells overexpressing F508del-CFTR were treated with matrine, VX809 (corrector) and/or with a number of potentiators (VX770, FD-1 and FD-2). The results demonstrated that matrine was able to stimulate CFTR activity and, in association with FD-2, increased the functionality of the channel in the presence of VX809. Based on these results, it may be hypothesized that FD-2 may be a novel and more effective potentiator compared with VX770. PMID:29039559

Prevalence of meconium ileus marks the severity of mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene.

PubMed

Dupuis, Annie; Keenan, Katherine; Ooi, Chee Y; Dorfman, Ruslan; Sontag, Marci K; Naehrlich, Lutz; Castellani, Carlo; Strug, Lisa J; Rommens, Johanna M; Gonska, Tanja

2016-04-01

Meconium ileus (MI) is a perinatal complication in cystic fibrosis (CF), which is only minimally influenced by environmental factors. We derived and examined MI prevalence (MIP) scores to assess CFTR phenotype-phenotype correlation for severe mutations. MIP scores were established using a Canadian CF population (n = 2,492) as estimates of the proportion of patients with MI among all patients carrying the same CFTR mutation, focusing on patients with p.F508del as the second allele. Comparisons were made to the registries from the US CF Foundation (n = 43,432), Italy (Veneto/Trentino/Alto Adige regions) (n = 1,788), and Germany (n = 3,596). The prevalence of MI varied among the different registries (13-21%). MI was predominantly prevalent in patients with pancreatic insufficiency carrying "severe" CFTR mutations. In this severe spectrum MIP scores further distinguished between mutation types, for example, G542X (0.31) with a high, F508del (0.22) with a moderate, and G551D (0.08) with a low MIP score. Higher MIP scores were associated with more severe clinical phenotypes, such as a lower forced expiratory volume in 1 second (P = 0.01) and body mass index z score (P = 0.04). MIP scores can be used to rank CFTR mutations according to their clinical severity and provide a means to expand delineation of CF phenotypes.Genet Med 18 4, 333-340.

Short communication: novel truncating mutations in the CFTR gene causing a severe form of cystic fibrosis in Italian patients.

PubMed

Lenarduzzi, S; Morgutti, M; Crovella, S; Coiana, A; Rosatelli, M C

2014-11-14

Cystic fibrosis (CF) is a common recessive genetic disease caused by mutations in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. More than 1800 different mutations have been described to date. Here, we report 3 novel mutations in CFTR in 3 Italian CF patients. To detect and identify 36 frequent mutations in Caucasians, we used the INNO-LiPA CFTR19 and INNO-LiPA CFTR17+Tn Update kits (Innogenetics; Ghent, Belgium). Our first analysis did not reveal both of the responsible mutations; thus, direct sequencing of the CFTR gene coding region was performed. The 3 patients were compound heterozygous. In one allele, the F508del (c.1521_1523delCTT, p.PHE508del) mutation in exon 11 was observed in each case. For the second allele, in patient No.1, direct sequencing revealed an 11-base pair deletion (GAGGCGATACT) in exon 14 (c.2236_2246del; pGlu746Alafs*29). In patient No. 2, direct sequencing revealed a nonsense mutation at nucleotide 3892 (c.3892G>T) in exon 24. In patient No. 3, direct sequencing revealed a deletion of cytosine in exon 27 (c.4296delC; p.Asn1432Lysfs*16). These 3 novel mutations indicate the production of a truncated protein, which consequently results in a non-functional polypeptide.

Lumacaftor-Ivacaftor in Patients with Cystic Fibrosis Homozygous for Phe508del CFTR.

PubMed

Wainwright, Claire E; Elborn, J Stuart; Ramsey, Bonnie W; Marigowda, Gautham; Huang, Xiaohong; Cipolli, Marco; Colombo, Carla; Davies, Jane C; De Boeck, Kris; Flume, Patrick A; Konstan, Michael W; McColley, Susanna A; McCoy, Karen; McKone, Edward F; Munck, Anne; Ratjen, Felix; Rowe, Steven M; Waltz, David; Boyle, Michael P

2015-07-16

Cystic fibrosis is a life-limiting disease that is caused by defective or deficient cystic fibrosis transmembrane conductance regulator (CFTR) protein activity. Phe508del is the most common CFTR mutation. We conducted two phase 3, randomized, double-blind, placebo-controlled studies that were designed to assess the effects of lumacaftor (VX-809), a CFTR corrector, in combination with ivacaftor (VX-770), a CFTR potentiator, in patients 12 years of age or older who had cystic fibrosis and were homozygous for the Phe508del CFTR mutation. In both studies, patients were randomly assigned to receive either lumacaftor (600 mg once daily or 400 mg every 12 hours) in combination with ivacaftor (250 mg every 12 hours) or matched placebo for 24 weeks. The primary end point was the absolute change from baseline in the percentage of predicted forced expiratory volume in 1 second (FEV1) at week 24. A total of 1108 patients underwent randomization and received study drug. The mean baseline FEV1 was 61% of the predicted value. In both studies, there were significant improvements in the primary end point in both lumacaftor-ivacaftor dose groups; the difference between active treatment and placebo with respect to the mean absolute improvement in the percentage of predicted FEV1 ranged from 2.6 to 4.0 percentage points (P<0.001), which corresponded to a mean relative treatment difference of 4.3 to 6.7% (P<0.001). Pooled analyses showed that the rate of pulmonary exacerbations was 30 to 39% lower in the lumacaftor-ivacaftor groups than in the placebo group; the rate of events leading to hospitalization or the use of intravenous antibiotics was lower in the lumacaftor-ivacaftor groups as well. The incidence of adverse events was generally similar in the lumacaftor-ivacaftor and placebo groups. The rate of discontinuation due to an adverse event was 4.2% among patients who received lumacaftor-ivacaftor versus 1.6% among those who received placebo. These data show that lumacaftor in

Assessing the residual CFTR gene expression in human nasal epithelium cells bearing CFTR splicing mutations causing cystic fibrosis

PubMed Central

Masvidal, Laia; Igreja, Susana; Ramos, Maria D; Alvarez, Antoni; de Gracia, Javier; Ramalho, Anabela; Amaral, Margarida D; Larriba, Sara; Casals, Teresa

2014-01-01

The major purpose of the present study was to quantify correctly spliced CFTR transcripts in human nasal epithelial cells from cystic fibrosis (CF) patients carrying the splicing mutations c.580-1G>T (712-1G>T) and c.2657+5G>A (2789+5G>A) and to assess the applicability of this model in CFTR therapeutic approaches. We performed the relative quantification of CFTR mRNA by reverse transcription quantitative PCR (RT-qPCR) of these splicing mutations in four groups (wild type, CF-F508del controls, CF patients and CF carriers) of individuals. In addition, in vitro assays using minigene constructs were performed to evaluate the effect of a new CF complex allele c.[2657+5G>A; 2562T>G]. Ex vivo qPCR data show that the primary consequence of both mutations at the RNA level is the skipping of their neighboring exon (6 and 16, respectively). The CFTR minigenes results mimicked the ex vivo data, as exon 16 skipping is the main aberrant transcript, and the correctly spliced transcript level was observed in a similar proportion when the c.2657+5G>A mutation is present. In summary, we provide evidence that ex vivo quantitative transcripts analysis using RT/qPCR is a robust technology that could be useful for measuring the efficacy of therapeutic approaches that attempt to achieve an increase in CFTR gene expression. PMID:24129438

The novel complex allele [A238V;F508del] of the CFTR gene: clinical phenotype and possible implications for cystic fibrosis etiological therapies.

PubMed

Diana, Anna; Polizzi, Angela Maria; Santostasi, Teresa; Ratclif, Luigi; Pantaleo, Maria Giuseppina; Leonetti, Giuseppina; Iusco, Danila Rosa; Gallo, Crescenzio; Conese, Massimo; Manca, Antonio

2016-06-01

Few mutations in cis have been annotated for F508del homozygous patients. Southern Italy patients who at a first analysis appeared homozygous for the F508del mutation (n=63) or compound heterozygous for the F508del and another mutation in the cystic fibrosis transmembrane conductance regulator gene (n=155) were searched for the A238V mutation in exon 6. The allelic frequency of the complex allele [A238V;F508del] was 0.04. When the whole data set was used (comprised also of 56 F508del/F508del and 34 F508del/other mutation controls), no differences reached the statistical significance in the clinical parameters, except chloride concentrations which were lower in [A238V;F508del]/other mutation compared with F508del/other mutation (P=0.03). The two study groups presented less complications than the control groups. Within the minimal data set (34 F508del/F508del, 27 F508del/other mutation, 4 [A238V;F508del]/F508del cases and 5 [A238V;F508del]/other mutation cases); that is, presenting all the variables in each patient, forced expiratory volume in 1 s and forced vital capacity presented a trend to lower levels in the study groups in comparison with the F508del/F508del group, and C-reactive protein approximated statistically significant higher levels in the [A238V;F508del]/other mutation as compared with F508del/F508del patients (P=0.09). The analysis of statistical dependence among the variables showed a significant anticorrelation between chloride and body mass index in the [A238V;F508del]/other mutation group. In conclusion, the complex allele [A238V;F508del] seems to be associated with less general complications than in the control groups, on the other hand possibly giving a worse pulmonary phenotype and higher systemic/local inflammatory response. These findings have implications for the correct recruitment and clinical response of F508del patients in the clinical trials testing the new etiological drugs for cystic fibrosis.

Ethnic heterogeneity and cystic fibrosis transmembrane regulator (CFTR) mutation frequencies in Chicago-area CF families

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ober, C.; Lester, L.A.; Mott, C.

1992-12-01

The identification of a common mutation, [Delta]F508, in the CFTR gene allowed, for the first time, the detection of cystic fibrosis (CF) carriers in the general population. Further genetic studies revealed >100 additional disease-causing mutations in this gene, few of which occur on >1% of CF chromosomes in any ethnic group. Prior to establishing counseling guidelines and carrier risk assessments, the authors sought to establish the frequencies of the CFTR mutations that are present in CF families living in the Chicago are, a region notable for its ethnic heterogeneity. Their sample included 283 unrelated CF carriers, with the following ethnicmore » composition: 78% non-Ashkenazi Caucasians, 5% Ashkenazi, 9% African-American, 3% Mexican, 0.3% Native American, and 5% mixed ancestry. When a panel of 10 mutations ([Delta]F508, [Delta]I507, G542X, G551D, R553X, S549N, R1162X, W1282X, N1303K, and 1717-1G[r arrow]A) was used, detection rates ranged from 75% in non-Ashkenazi Caucasians to 40% in African-Americans. These data suggest that the goal of screening for 90%-95% of CF mutations may be unrealistic in this and other, similar US populations. 22 refs., 1 tab.« less

Missense variations in the cystic fibrosis gene: Heteroduplex formation in the F508C mutation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Macek, M. Jr.; Ladanyi, L.; Buerger, J.

1992-11-01

Kobayashi et al. (1990) have described missense variations in the conserved region of exon 10 of the cystic fibrosis (CF) transmembrane conductance regulator gene. In their paper, two [Delta]F508/F508C compound heterozygous individuals were reported. Clinical and epithelial physiological studies in both cases were normal, suggesting that the substitution of cysteine for phenylalanine at position 508, the F508C mutation, is benign. However, Kerem et al. reported a patient with this substitution who had typical symptoms of CF. In routine [Delta]F508 mutation screening by visualization of the 3-bp deletion on a 12% polyacrylamide gel the authors detected an abnormal heteroduplex in themore » father of a CF patient of German origin. Subsequent direct sequencing of the PCR product confirmed that this clinically normal father is a compound heterozygote for the [Delta]F508/F508C mutations. This heteroduplex is slightly different from the usual heteroduplex in [Delta]F508/F508C heteroduplex was not published, it is likely that similar cases can be overseen during the widely performed [Delta]F508 mutation screening by PAGE. Detection of more cases, such as the one presented here, together with careful, standardized clinical examination of the proband, would be valuable to verify the nature of this mutation. 4 refs., 1 fig.« less

Two Small Molecules Restore Stability to a Subpopulation of the Cystic Fibrosis Transmembrane Conductance Regulator with the Predominant Disease-causing Mutation.

PubMed

Meng, Xin; Wang, Yiting; Wang, Xiaomeng; Wrennall, Joe A; Rimington, Tracy L; Li, Hongyu; Cai, Zhiwei; Ford, Robert C; Sheppard, David N

2017-03-03

Cystic fibrosis (CF) is caused by mutations that disrupt the plasma membrane expression, stability, and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl - channel. Two small molecules, the CFTR corrector lumacaftor and the potentiator ivacaftor, are now used clinically to treat CF, although some studies suggest that they have counteracting effects on CFTR stability. Here, we investigated the impact of these compounds on the instability of F508del-CFTR, the most common CF mutation. To study individual CFTR Cl - channels, we performed single-channel recording, whereas to assess entire CFTR populations, we used purified CFTR proteins and macroscopic CFTR Cl - currents. At 37 °C, low temperature-rescued F508del-CFTR more rapidly lost function in cell-free membrane patches and showed altered channel gating and current flow through open channels. Compared with purified wild-type CFTR, the full-length F508del-CFTR was about 10 °C less thermostable. Lumacaftor partially stabilized purified full-length F508del-CFTR and slightly delayed deactivation of individual F508del-CFTR Cl - channels. By contrast, ivacaftor further destabilized full-length F508del-CFTR and accelerated channel deactivation. Chronic (prolonged) co-incubation of F508del-CFTR-expressing cells with lumacaftor and ivacaftor deactivated macroscopic F508del-CFTR Cl - currents. However, at the single-channel level, chronic co-incubation greatly increased F508del-CFTR channel activity and temporal stability in most, but not all, cell-free membrane patches. We conclude that chronic lumacaftor and ivacaftor co-treatment restores stability in a small subpopulation of F508del-CFTR Cl - channels but that the majority remain destabilized. A fuller understanding of these effects and the characterization of the small F508del-CFTR subpopulation might be crucial for CF therapy development. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A novel treatment of cystic fibrosis acting on-target: cysteamine plus epigallocatechin gallate for the autophagy-dependent rescue of class II-mutated CFTR.

PubMed

Tosco, A; De Gregorio, F; Esposito, S; De Stefano, D; Sana, I; Ferrari, E; Sepe, A; Salvadori, L; Buonpensiero, P; Di Pasqua, A; Grassia, R; Leone, C A; Guido, S; De Rosa, G; Lusa, S; Bona, G; Stoll, G; Maiuri, M C; Mehta, A; Kroemer, G; Maiuri, L; Raia, V

2016-08-01

We previously reported that the combination of two safe proteostasis regulators, cysteamine and epigallocatechin gallate (EGCG), can be used to improve deficient expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients homozygous for the CFTR Phe508del mutation. Here we provide the proof-of-concept that this combination treatment restored CFTR function and reduced lung inflammation (P<0.001) in Phe508del/Phe508del or Phe508del/null-Cftr (but not in Cftr-null mice), provided that such mice were autophagy-competent. Primary nasal cells from patients bearing different class II CFTR mutations, either in homozygous or compound heterozygous form, responded to the treatment in vitro. We assessed individual responses to cysteamine plus EGCG in a single-centre, open-label phase-2 trial. The combination treatment decreased sweat chloride from baseline, increased both CFTR protein and function in nasal cells, restored autophagy in such cells, decreased CXCL8 and TNF-α in the sputum, and tended to improve respiratory function. These positive effects were particularly strong in patients carrying Phe508del CFTR mutations in homozygosity or heterozygosity. However, a fraction of patients bearing other CFTR mutations failed to respond to therapy. Importantly, the same patients whose primary nasal brushed cells did not respond to cysteamine plus EGCG in vitro also exhibited deficient therapeutic responses in vivo. Altogether, these results suggest that the combination treatment of cysteamine plus EGCG acts 'on-target' because it can only rescue CFTR function when autophagy is functional (in mice) and improves CFTR function when a rescuable protein is expressed (in mice and men). These results should spur the further clinical development of the combination treatment.

Increased folding and channel activity of a rare cystic fibrosis mutant with CFTR modulators

PubMed Central

Grove, Diane E.; Houck, Scott A.

2011-01-01

Cystic fibrosis (CF) is a lethal recessive genetic disease caused by mutations in the CFTR gene. The gene product is a PKA-regulated anion channel that is important for fluid and electrolyte transport in the epithelia of lung, gut, and ducts of the pancreas and sweat glands. The most common CFTR mutation, ΔF508, causes a severe, but correctable, folding defect and gating abnormality, resulting in negligible CFTR function and disease. There are also a large number of rare CF-related mutations where disease is caused by CFTR misfolding. Yet the extent to which defective biogenesis of these CFTR mutants can be corrected is not clear. CFTRV232D is one such mutant that exhibits defective folding and trafficking. CFTRΔF508 misfolding is difficult to correct, but defective biogenesis of CFTRV232D is corrected to near wild-type levels by small-molecule folding correctors in development as CF therapeutics. To determine if CFTRV232D protein is competent as a Cl− channel, we utilized single-channel recordings from transfected human embryonic kidney (HEK-293) cells. After PKA stimulation, CFTRV232D channels were detected in patches with a unitary Cl− conductance indistinguishable from that of CFTR. Yet the frequency of detecting CFTRV232D channels was reduced to ∼20% of patches compared with 60% for CFTR. The folding corrector Corr-4a increased the CFTRV232D channel detection rate and activity to levels similar to CFTR. CFTRV232D-corrected channels were inhibited with CFTRinh-172 and stimulated fourfold by the CFTR channel potentiator VRT-532. These data suggest that CF patients with rare mutations that cause CFTR misfolding, such as CFTRV232D, may benefit from treatment with folding correctors and channel potentiators in development to restore CFTRΔF508 function. PMID:21642448

A novel treatment of cystic fibrosis acting on-target: cysteamine plus epigallocatechin gallate for the autophagy-dependent rescue of class II-mutated CFTR

PubMed Central

Tosco, A; De Gregorio, F; Esposito, S; De Stefano, D; Sana, I; Ferrari, E; Sepe, A; Salvadori, L; Buonpensiero, P; Di Pasqua, A; Grassia, R; Leone, C A; Guido, S; De Rosa, G; Lusa, S; Bona, G; Stoll, G; Maiuri, M C; Mehta, A; Kroemer, G; Maiuri, L; Raia, V

2016-01-01

We previously reported that the combination of two safe proteostasis regulators, cysteamine and epigallocatechin gallate (EGCG), can be used to improve deficient expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients homozygous for the CFTR Phe508del mutation. Here we provide the proof-of-concept that this combination treatment restored CFTR function and reduced lung inflammation (P<0.001) in Phe508del/Phe508del or Phe508del/null-Cftr (but not in Cftr-null mice), provided that such mice were autophagy-competent. Primary nasal cells from patients bearing different class II CFTR mutations, either in homozygous or compound heterozygous form, responded to the treatment in vitro. We assessed individual responses to cysteamine plus EGCG in a single-centre, open-label phase-2 trial. The combination treatment decreased sweat chloride from baseline, increased both CFTR protein and function in nasal cells, restored autophagy in such cells, decreased CXCL8 and TNF-α in the sputum, and tended to improve respiratory function. These positive effects were particularly strong in patients carrying Phe508del CFTR mutations in homozygosity or heterozygosity. However, a fraction of patients bearing other CFTR mutations failed to respond to therapy. Importantly, the same patients whose primary nasal brushed cells did not respond to cysteamine plus EGCG in vitro also exhibited deficient therapeutic responses in vivo. Altogether, these results suggest that the combination treatment of cysteamine plus EGCG acts ‘on-target' because it can only rescue CFTR function when autophagy is functional (in mice) and improves CFTR function when a rescuable protein is expressed (in mice and men). These results should spur the further clinical development of the combination treatment. PMID:27035618

Curcumin/poly(2-methyl-2-oxazoline-b-tetrahydrofuran-b-2-methyl-2-oxazoline) formulation: An improved penetration and biological effect of curcumin in F508del-CFTR cell lines.

PubMed

Gonçalves, Cristine; Gomez, Jean-Pierre; Même, William; Rasolonjatovo, Bazoly; Gosset, David; Nedellec, Steven; Hulin, Philippe; Huin, Cécile; Le Gall, Tony; Montier, Tristan; Lehn, Pierre; Pichon, Chantal; Guégan, Philippe; Cheradame, Hervé; Midoux, Patrick

2017-08-01

Neutral amphiphilic triblock ABA copolymers are of great interest to solubilize hydrophobic drugs. We reported that a triblock ABA copolymer consisting of methyl-2-oxazoline (MeOx) and tetrahydrofuran (THF) (MeOx 6 -THF 19 -MeOx 6 ) (TBCP2) can solubilize curcumin (Cur) a very hydrophobic molecule exhibiting multiple therapeutic effects but whose insolubility and low stability in water is a major drawback for clinical applications. Here, we provide evidences by flow cytometry and confocal microscopy that Cur penetration in normal and ΔF508-CFTR human airway epithelial cell lines is facilitated by TBCP2. When used on ΔF508-CFTR cell lines, the Cur/TBCP2 formulation promotes the restoration of the expression of the CFTR protein in the plasma membrane. Furthermore, patch-clamp and MQAE fluorescence experiments show that this effect is associated with a correction of a Cl - selective current at the membrane surface of F508del-CFTR cells. The results show the great potential of the neutral amphiphilic triblock copolymer MeOx 6 -THF 19 -MeOx 6 as carrier for curcumin in a Cystic Fibrosis context. We anticipate that other MeOx n -THF m -MeOx n copolymers could have similar behaviours for other highly insoluble therapeutic drugs or cosmetic active ingredients. Copyright © 2017 Elsevier B.V. All rights reserved.

CFTR Mutations Spectrum and the Efficiency of Molecular Diagnostics in Polish Cystic Fibrosis Patients

PubMed Central

Ziętkiewicz, Ewa; Rutkiewicz, Ewa; Pogorzelski, Andrzej; Klimek, Barbara; Voelkel, Katarzyna; Witt, Michał

2014-01-01

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR). In light of the strong allelic heterogeneity and regional specificity of the mutation spectrum, the strategy of molecular diagnostics and counseling in CF requires genetic tests to reflect the frequency profile characteristic for a given population. The goal of the study was to provide an updated comprehensive estimation of the distribution of CFTR mutations in Polish CF patients and to assess the effectiveness of INNOLiPA_CFTR tests in Polish population. The analyzed cohort consisted of 738 patients with the clinically confirmed CF diagnosis, prescreened for molecular defects using INNOLiPA_CFTR panels from Innogenetics. A combined efficiency of INNOLiPA CFTR_19 and CFTR_17_TnUpdate tests was 75.5%; both mutations were detected in 68.2%, and one mutation in 14.8% of the affected individuals. The group composed of all the patients with only one or with no mutation detected (109 and 126 individuals, respectively) was analyzed further using a mutation screening approach, i.e. SSCP/HD (single strand conformational polymorphism/heteroduplex) analysis of PCR products followed by sequencing of the coding sequence. As a result, 53 more mutations were found in 97 patients. The overall efficiency of the CF allele detection was 82.5% (7.0% increase compared to INNOLiPA tests alone). The distribution of the most frequent mutations in Poland was assessed. Most of the mutations repetitively found in Polish patients had been previously described in other European populations. The most frequent mutated allele, F508del, represented 54.5% of Polish CF chromosomes. Another eight mutations had frequencies over 1%, 24 had frequencies between 1 and 0.1%; c.2052-2053insA and c.3468+2_3468+3insT were the most frequent non-INNOLiPA mutations. Mutation distribution described herein is also relevant to the Polish diaspora. Our study also demonstrates that the reported efficiency of

CFTR mutations spectrum and the efficiency of molecular diagnostics in Polish cystic fibrosis patients.

PubMed

Ziętkiewicz, Ewa; Rutkiewicz, Ewa; Pogorzelski, Andrzej; Klimek, Barbara; Voelkel, Katarzyna; Witt, Michał

2014-01-01

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR). In light of the strong allelic heterogeneity and regional specificity of the mutation spectrum, the strategy of molecular diagnostics and counseling in CF requires genetic tests to reflect the frequency profile characteristic for a given population. The goal of the study was to provide an updated comprehensive estimation of the distribution of CFTR mutations in Polish CF patients and to assess the effectiveness of INNOLiPA_CFTR tests in Polish population. The analyzed cohort consisted of 738 patients with the clinically confirmed CF diagnosis, prescreened for molecular defects using INNOLiPA_CFTR panels from Innogenetics. A combined efficiency of INNOLiPA CFTR_19 and CFTR_17_TnUpdate tests was 75.5%; both mutations were detected in 68.2%, and one mutation in 14.8% of the affected individuals. The group composed of all the patients with only one or with no mutation detected (109 and 126 individuals, respectively) was analyzed further using a mutation screening approach, i.e. SSCP/HD (single strand conformational polymorphism/heteroduplex) analysis of PCR products followed by sequencing of the coding sequence. As a result, 53 more mutations were found in 97 patients. The overall efficiency of the CF allele detection was 82.5% (7.0% increase compared to INNOLiPA tests alone). The distribution of the most frequent mutations in Poland was assessed. Most of the mutations repetitively found in Polish patients had been previously described in other European populations. The most frequent mutated allele, F508del, represented 54.5% of Polish CF chromosomes. Another eight mutations had frequencies over 1%, 24 had frequencies between 1 and 0.1%; c.2052-2053insA and c.3468+2_3468+3insT were the most frequent non-INNOLiPA mutations. Mutation distribution described herein is also relevant to the Polish diaspora. Our study also demonstrates that the reported efficiency of

Mechanistic Approaches to Improve Correction of the Most Common Disease-Causing Mutation in Cystic Fibrosis

PubMed Central

Bali, Vedrana; Lazrak, Ahmed; Guroji, Purushotham; Matalon, Sadis; Bebok, Zsuzsanna

2016-01-01

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to deletion of the phenylalanine at position 508 (ΔF508) in the CFTR protein and causes multiple folding and functional defects. Contrary to large-scale efforts by industry and academia, no significant therapeutic benefit has been achieved with a single “corrector”. Therefore, investigations concentrate on drug combinations. Orkambi (Vertex Pharmaceuticals), the first FDA-approved drug for treatment of cystic fibrosis (CF) caused by this mutation, is a combination of a corrector (VX-809) that facilitates ΔF508 CFTR biogenesis and a potentiator (VX-770), which improves its function. Yet, clinical trials utilizing this combination showed only modest therapeutic benefit. The low efficacy Orkambi has been attributed to VX-770-mediated destabilization of VX-809-rescued ΔF508 CFTR. Here we report that the negative effects of VX-770 can be reversed by increasing the half-life of the endoplasmic reticulum (ER) form (band B) of ΔF508 CFTR with another corrector (Corr-4a.) Although Corr-4a alone has only minimal effects on ΔF508 CFTR rescue, it increases the half-life of ΔF508 CFTR band B when it is present during half-life measurements. Our data shows that stabilization of band B ΔF508 CFTR with Corr-4a and simultaneous rescue with VX-809, leads to a >2-fold increase in cAMP-activated, CFTRinh-172-inhibited currents compared to VX-809 alone, or VX-809+VX-770. The negative effects of VX-770 and the Corr-4a protection are specific to the native I507-ATT ΔF508 CFTR without affecting the inherently more stable, synonymous variant I507-ATC ΔF508 CFTR. Our studies emphasize that stabilization of ΔF508 CFTR band B in the ER might improve its functional rescue by Orkambi. PMID:27214033

General anesthetic octanol and related compounds activate wild-type and delF508 cystic fibrosis chloride channels

PubMed Central

Marcet, Brice; Becq, Frédéric; Norez, Caroline; Delmas, Patrick; Verrier, Bernard

2004-01-01

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is defective during cystic fibrosis (CF). Activators of the CFTR Cl− channel may be useful for therapy of CF. Here, we demonstrate that a range of general anesthetics like normal-alkanols (n-alkanols) and related compounds can stimulate the Cl− channel activity of wild-type CFTR and delF508-CFTR mutant. The effects of n-alkanols like octanol on CFTR activity were measured by iodide (125I) efflux and patch-clamp techniques on three distinct cellular models: (1) CFTR-expressing Chinese hamster ovary cells, (2) human airway Calu-3 epithelial cells and (3) human airway JME/CF15 epithelial cells which express the delF508-CFTR mutant. Our data show for the first time that n-alkanols activate both wild-type CFTR and delF508-CFTR mutant. Octanol stimulated 125I efflux in a dose-dependent manner in CFTR-expressing cells (wild-type and delF508) but not in cell lines lacking CFTR. 125I efflux and Cl− currents induced by octanol were blocked by glibenclamide but insensitive to 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, as expected for a CFTR Cl− current. CFTR activation by octanol was neither due to cell-to-cell uncoupling properties of octanol nor to an intracellular cAMP increase. CFTR activation by octanol requires phosphorylation by protein kinase-A (PKA) since it was prevented by H-89, a PKA inhibitor. n-Alkanols chain length was an important determinant for channel activation, with rank order of potencies: 1-heptanol<1-octanol<2-octanol<1-decanol. Our findings may be of valuable interest for developing novel therapeutic strategies for CF. PMID:14967738

General anesthetic octanol and related compounds activate wild-type and delF508 cystic fibrosis chloride channels.

PubMed

Marcet, Brice; Becq, Frédéric; Norez, Caroline; Delmas, Patrick; Verrier, Bernard

2004-03-01

1. Cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is defective during cystic fibrosis (CF). Activators of the CFTR Cl(-) channel may be useful for therapy of CF. Here, we demonstrate that a range of general anesthetics like normal-alkanols (n-alkanols) and related compounds can stimulate the Cl(-) channel activity of wild-type CFTR and delF508-CFTR mutant. 2. The effects of n-alkanols like octanol on CFTR activity were measured by iodide ((125)I) efflux and patch-clamp techniques on three distinct cellular models: (1). CFTR-expressing Chinese hamster ovary cells, (2). human airway Calu-3 epithelial cells and (3). human airway JME/CF15 epithelial cells which express the delF508-CFTR mutant. 3. Our data show for the first time that n-alkanols activate both wild-type CFTR and delF508-CFTR mutant. Octanol stimulated (125)I efflux in a dose-dependent manner in CFTR-expressing cells (wild-type and delF508) but not in cell lines lacking CFTR. (125)I efflux and Cl(-) currents induced by octanol were blocked by glibenclamide but insensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, as expected for a CFTR Cl(-) current. 4. CFTR activation by octanol was neither due to cell-to-cell uncoupling properties of octanol nor to an intracellular cAMP increase. CFTR activation by octanol requires phosphorylation by protein kinase-A (PKA) since it was prevented by H-89, a PKA inhibitor. 5. n-Alkanols chain length was an important determinant for channel activation, with rank order of potencies: 1-heptanol<1-octanol<2-octanol<1-decanol. Our findings may be of valuable interest for developing novel therapeutic strategies for CF.

Structure and Dynamics of NBD1 from CFTR Characterized Using Crystallography and Hydrogen/Deuterium Exchange Mass Spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lewis, H.A.; Wang, C.; Zhao, X.

2012-04-30

The {Delta}F508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and {Delta}F508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because {Delta}F508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and {Delta}F508 constructs, and the {Delta}F508 structure contained additional mutationsmore » that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide {sup 1}H/{sup 2}H exchange rates in matched F508 and {Delta}F508 constructs reveal that {Delta}F508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the {Delta}F508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-{Delta}F508 structures but completely solvent exposed in all {Delta}F508 structures. These results reinforce the importance of the perturbation {Delta}F508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that

mRNA-based detection of rare CFTR mutations improves genetic diagnosis of cystic fibrosis in populations with high genetic heterogeneity.

PubMed

Felício, V; Ramalho, A S; Igreja, S; Amaral, M D

2017-03-01

Even with advent of next generation sequencing complete sequencing of large disease-associated genes and intronic regions is economically not feasible. This is the case of cystic fibrosis transmembrane conductance regulator (CFTR), the gene responsible for cystic fibrosis (CF). Yet, to confirm a CF diagnosis, proof of CFTR dysfunction needs to be obtained, namely by the identification of two disease-causing mutations. Moreover, with the advent of mutation-based therapies, genotyping is an essential tool for CF disease management. There is, however, still an unmet need to genotype CF patients by fast, comprehensive and cost-effective approaches, especially in populations with high genetic heterogeneity (and low p.F508del incidence), where CF is now emerging with new diagnosis dilemmas (Brazil, Asia, etc). Herein, we report an innovative mRNA-based approach to identify CFTR mutations in the complete coding and intronic regions. We applied this protocol to genotype individuals with a suspicion of CF and only one or no CFTR mutations identified by routine methods. It successfully detected multiple intronic mutations unlikely to be detected by CFTR exon sequencing. We conclude that this is a rapid, robust and inexpensive method to detect any CFTR coding/intronic mutation (including rare ones) that can be easily used either as primary approach or after routine DNA analysis. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Simple image-based no-wash method for quantitative detection of surface expressed CFTR

PubMed Central

Larsen, Mads Breum; Hu, Jennifer; Frizzell, Raymond A.; Watkins, Simon C.

2016-01-01

Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians. It is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, which encodes an apical membrane anion channel that is required for regulating the volume and composition of epithelial secretions. The most common CFTR mutation, present on at least one allele in >90% of CF patients, deletes phenylalanine at position 508 (F508del), which causes the protein to misfold. Endoplasmic reticulum (ER) quality control elicits the degradation of mutant CFTR, compromising its trafficking to the epithelial cell apical membrane. The absence of functional CFTR leads to depletion of airway surface liquid, impaired clearance of mucus and bacteria from the lung, and predisposes to recurrent infections. Ultimately, respiratory failure results from inflammation and bronchiectasis. Although high throughput screening has identified small molecules that can restore the anion transport function of F508del CFTR, they correct less than 15% of WT CFTR activity, yielding insufficient clinical benefit. To date, most primary CF drug discovery assays have employed measurements of CFTR’s anion transport function, a method that depends on the recruitment of a functional CFTR to the cell surface, involves multiple wash steps, and relies on a signal that saturates rapidly. Screening efforts have also included assays for detection of extracellularly HA-tagged or HRP-tagged CFTR, which require multiple washing steps. We have recently developed tools and cell lines that report the correction of mutant CFTR trafficking by currently available small molecules, and have extended this assay to the 96-well format. This new and simple no-wash assay of F508del CFTR at the cell surface may permit the discovery of more efficacious drugs, and hopefully thereby prevent the catastrophic effects of this disease. In addition, the modular design of this platform should make it useful for other diseases where loss

Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770

PubMed Central

Van Goor, Fredrick; Hadida, Sabine; Grootenhuis, Peter D. J.; Burton, Bill; Cao, Dong; Neuberger, Tim; Turnbull, Amanda; Singh, Ashvani; Joubran, John; Hazlewood, Anna; Zhou, Jinglan; McCartney, Jason; Arumugam, Vijayalaksmi; Decker, Caroline; Yang, Jennifer; Young, Chris; Olson, Eric R.; Wine, Jeffery J.; Frizzell, Raymond A.; Ashlock, Melissa; Negulescu, Paul

2009-01-01

Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), a protein kinase A (PKA)-activated epithelial anion channel involved in salt and fluid transport in multiple organs, including the lung. Most CF mutations either reduce the number of CFTR channels at the cell surface (e.g., synthesis or processing mutations) or impair channel function (e.g., gating or conductance mutations) or both. There are currently no approved therapies that target CFTR. Here we describe the in vitro pharmacology of VX-770, an orally bioavailable CFTR potentiator in clinical development for the treatment of CF. In recombinant cells VX-770 increased CFTR channel open probability (Po) in both the F508del processing mutation and the G551D gating mutation. VX-770 also increased Cl− secretion in cultured human CF bronchial epithelia (HBE) carrying the G551D gating mutation on one allele and the F508del processing mutation on the other allele by ≈10-fold, to ≈50% of that observed in HBE isolated from individuals without CF. Furthermore, VX-770 reduced excessive Na+ and fluid absorption to prevent dehydration of the apical surface and increased cilia beating in these epithelial cultures. These results support the hypothesis that pharmacological agents that restore or increase CFTR function can rescue epithelial cell function in human CF airway. PMID:19846789

Additive effect of multiple pharmacological chaperones on maturation of CFTR processing mutants

PubMed Central

Wang, Ying; Loo, Tip W.; Bartlett, M. Claire; Clarke, David M.

2007-01-01

The most common cause of CF (cystic fibrosis) is the deletion of Phe508 (ΔF508) in the CFTR [CF TM (transmembrane) conductance regulator] chloride channel. One major problem with ΔF508 CFTR is that the protein is defective in folding so that little mature protein is delivered to the cell surface. Expression of ΔF508 CFTR in the presence of small molecules known as correctors or pharmacological chaperones can increase the level of mature protein. Unfortunately, the efficiency of corrector-induced maturation of ΔF508 CFTR is probably too low to have therapeutic value and approaches are needed to increase maturation efficiency. We postulated that expression of ΔF508 CFTR in the presence of multiple correctors that bound to different sites may have an additive effect on maturation. In support of this mechanism, we found that expression of P-glycoprotein (CFTR's sister protein) processing mutants in the presence of two compounds that bind to different sites (rhodamine B and Hoechst 33342) had an additive effect on maturation. Therefore we tested whether expression of ΔF508 CFTR in the presence of combinations of three different classes of corrector molecules would increase its maturation efficiency. It was found that the combination of the quinazoline VRT-325 together with the thiazole corr-2b or bisaminomethylbithiazole corr-4a doubled the steady-state maturation efficiency of ΔF508 CFTR (approx. 40% of total CFTR was mature protein) compared with expression in the presence of a single compound. The additive effect of the correctors on ΔF508 CFTR maturation suggests that they directly interact at different sites of the protein. PMID:17535157

CFTR DeltaF508 mutation detection from dried blood samples in the first trimester of pregnancy: a possible routine prenatal screening strategy for cystic fibrosis?

PubMed

Konialis, Christopher P; Hagnefelt, Birgitta; Kazamia, Constantina; Karapanou, Sophia; Pangalos, Constantinos

2007-01-01

The implementation and evaluation of a proposed wide-scale prenatal screening strategy, based on DNA isolated from dried blood spots in the first trimester of pregnancy, for the early detection of pregnancies at risk for cystic fibrosis (CF). The screening was performed in conjunction with routine biochemical marker screening for Down's syndrome risk in the first trimester of pregnancy. DNA was isolated from 1,233 dried blood spots and analyzed for the presence of the CF transmembrane regulator DeltaF508 mutation. Women carriers were offered and accepted the option for additional full testing of their partners in order to assess the risk for the fetus. All 1,233 samples were successfully analyzed, identifying 23 DeltaF508 carriers, corresponding to a DeltaF508 carrier rate of approximately 1/55 (1.8%). All partners of the women carriers were further tested without revealing any need for further prenatal testing in this group. This study reveals the relatively high frequency of the DeltaF508 CF mutation in the Greek population. More importantly, we demonstrate that the proposed prenatal screening strategy, based on the ease and cost-effectiveness of the analysis for the detection of a single common mutation, can be considered as a feasible and practical approach for wide-scale prenatal screening for CF, following the sequential model. It is applied early on in pregnancy, allowing for the timely management of families at risk for the corresponding genetic disorders. Finally, it can easily be extended to include screening for other common genetic disorders in specific population groups.

Correctors and Potentiators Rescue Function of the Truncated W1282X-Cystic Fibrosis Transmembrane Regulator (CFTR) Translation Product*♦

PubMed Central

Haggie, Peter M.; Phuan, Puay-Wah; Tan, Joseph-Anthony; Xu, Haijin; Avramescu, Radu G.; Perdomo, Doranda; Zlock, Lorna; Nielson, Dennis W.; Finkbeiner, Walter E.; Lukacs, Gergely L.; Verkman, Alan S.

2017-01-01

W1282X is the fifth most common cystic fibrosis transmembrane regulator (CFTR) mutation that causes cystic fibrosis. Here, we investigated the utility of a small molecule corrector/potentiator strategy, as used for ΔF508-CFTR, to produce functional rescue of the truncated translation product of the W1282X mutation, CFTR1281, without the need for read-through. In transfected cell systems, certain potentiators and correctors, including VX-809 and VX-770, increased CFTR1281 activity. To identify novel correctors and potentiators with potentially greater efficacy on CFTR1281, functional screens were done of ∼30,000 synthetic small molecules and drugs/nutraceuticals in CFTR1281-transfected cells. Corrector scaffolds of 1-arylpyrazole-4-arylsulfonyl-piperazine and spiro-piperidine-quinazolinone classes were identified with up to ∼5-fold greater efficacy than VX-809, some of which were selective for CFTR1281, whereas others also corrected ΔF508-CFTR. Several novel potentiator scaffolds were identified with efficacy comparable with VX-770; remarkably, a phenylsulfonamide-pyrrolopyridine acted synergistically with VX-770 to increase CFTR1281 function ∼8-fold over that of VX-770 alone, normalizing CFTR1281 channel activity to that of wild type CFTR. Corrector and potentiator combinations were tested in primary cultures and conditionally reprogrammed cells generated from nasal brushings from one W1282X homozygous subject. Although robust chloride conductance was seen with correctors and potentiators in homozygous ΔF508 cells, increased chloride conductance was not found in W1282X cells despite the presence of adequate transcript levels. Notwithstanding the negative data in W1282X cells from one human subject, we speculate that corrector and potentiator combinations may have therapeutic efficacy in cystic fibrosis caused by the W1282X mutation, although additional studies are needed on human cells from W1282X subjects. PMID:27895116

Cystic fibrosis transmembrane conductance regulator (CFTR) allelic variants relate to shifts in faecal microbiota of cystic fibrosis patients.

PubMed

Schippa, Serena; Iebba, Valerio; Santangelo, Floriana; Gagliardi, Antonella; De Biase, Riccardo Valerio; Stamato, Antonella; Bertasi, Serenella; Lucarelli, Marco; Conte, Maria Pia; Quattrucci, Serena

2013-01-01

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. 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. 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. 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.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Allelic Variants Relate to Shifts in Faecal Microbiota of Cystic Fibrosis Patients

PubMed Central

Santangelo, Floriana; Gagliardi, Antonella; De Biase, Riccardo Valerio; Stamato, Antonella; Bertasi, Serenella; Lucarelli, Marco

2013-01-01

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. PMID:23613805

Pharmacokinetics and safety of cavosonstat (N91115) in healthy and cystic fibrosis adults homozygous for F508DEL-CFTR.

PubMed

Donaldson, Scott H; Solomon, George M; Zeitlin, Pamela L; Flume, Patrick A; Casey, Alicia; McCoy, Karen; Zemanick, Edith T; Mandagere, Arun; Troha, Janice M; Shoemaker, Steven A; Chmiel, James F; Taylor-Cousar, Jennifer L

2017-05-01

Cavosonstat (N91115), an orally bioavailable inhibitor of S-nitrosoglutathione reductase, promotes cystic fibrosis transmembrane conductance regulator (CFTR) maturation and plasma membrane stability, with a mechanism of action complementary to CFTR correctors and potentiators. A Phase I program evaluated pharmacokinetics, drug-drug interactions and safety of cavosonstat in healthy and cystic fibrosis (CF) subjects homozygous for F508del-CFTR. Exploratory outcomes included changes in sweat chloride in CF subjects. Cavosonstat was rapidly absorbed and demonstrated linear and predictable pharmacokinetics. Exposure was unaffected by a high-fat meal or rifampin-mediated effects on drug metabolism and transport. Cavosonstat was well tolerated, with no dose-limiting toxicities or significant safety findings. At the highest dose, significant reductions from baseline in sweat chloride were observed (-4.1mmol/L; P=0.032) at day 28. The favorable safety and clinical profile warrant further study of cavosonstat in CF. ClinicalTrials.gov Numbers: NCT02275936, NCT02013388, NCT02500667. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Orkambi® and amplifier co-therapy improves function from a rare CFTR mutation in gene-edited cells and patient tissue.

PubMed

Molinski, Steven V; Ahmadi, Saumel; Ip, Wan; Ouyang, Hong; Villella, Adriana; Miller, John P; Lee, Po-Shun; Kulleperuma, Kethika; Du, Kai; Di Paola, Michelle; Eckford, Paul Dw; Laselva, Onofrio; Huan, Ling Jun; Wellhauser, Leigh; Li, Ellen; Ray, Peter N; Pomès, Régis; Moraes, Theo J; Gonska, Tanja; Ratjen, Felix; Bear, Christine E

2017-09-01

The combination therapy of lumacaftor and ivacaftor (Orkambi ® ) is approved for patients bearing the major cystic fibrosis (CF) mutation: ΔF508 It has been predicted that Orkambi ® could treat patients with rarer mutations of similar "theratype"; however, a standardized approach confirming efficacy in these cohorts has not been reported. Here, we demonstrate that patients bearing the rare mutation: c.3700 A>G, causing protein misprocessing and altered channel function-similar to ΔF508-CFTR, are unlikely to yield a robust Orkambi ® response. While  in silico  and biochemical studies confirmed that this mutation could be corrected and potentiated by lumacaftor and ivacaftor, respectively, this combination led to a minor in vitro response in patient-derived tissue. A CRISPR/Cas9-edited bronchial epithelial cell line bearing this mutation enabled studies showing that an "amplifier" compound, effective in increasing the levels of immature CFTR protein, augmented the Orkambi ® response. Importantly, this "amplifier" effect was recapitulated in patient-derived nasal cultures-providing the first evidence for its efficacy in augmenting Orkambi ® in tissues harboring a rare CF-causing mutation. We propose that this multi-disciplinary approach, including creation of CRISPR/Cas9-edited cells to profile modulators together with validation using primary tissue, will facilitate therapy development for patients with rare CF mutations. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Correctors and Potentiators Rescue Function of the Truncated W1282X-Cystic Fibrosis Transmembrane Regulator (CFTR) Translation Product.

PubMed

Haggie, Peter M; Phuan, Puay-Wah; Tan, Joseph-Anthony; Xu, Haijin; Avramescu, Radu G; Perdomo, Doranda; Zlock, Lorna; Nielson, Dennis W; Finkbeiner, Walter E; Lukacs, Gergely L; Verkman, Alan S

2017-01-20

W1282X is the fifth most common cystic fibrosis transmembrane regulator (CFTR) mutation that causes cystic fibrosis. Here, we investigated the utility of a small molecule corrector/potentiator strategy, as used for ΔF508-CFTR, to produce functional rescue of the truncated translation product of the W1282X mutation, CFTR 1281 , without the need for read-through. In transfected cell systems, certain potentiators and correctors, including VX-809 and VX-770, increased CFTR 1281 activity. To identify novel correctors and potentiators with potentially greater efficacy on CFTR 1281 , functional screens were done of ∼30,000 synthetic small molecules and drugs/nutraceuticals in CFTR 1281 -transfected cells. Corrector scaffolds of 1-arylpyrazole-4-arylsulfonyl-piperazine and spiro-piperidine-quinazolinone classes were identified with up to ∼5-fold greater efficacy than VX-809, some of which were selective for CFTR 1281 , whereas others also corrected ΔF508-CFTR. Several novel potentiator scaffolds were identified with efficacy comparable with VX-770; remarkably, a phenylsulfonamide-pyrrolopyridine acted synergistically with VX-770 to increase CFTR 1281 function ∼8-fold over that of VX-770 alone, normalizing CFTR 1281 channel activity to that of wild type CFTR. Corrector and potentiator combinations were tested in primary cultures and conditionally reprogrammed cells generated from nasal brushings from one W1282X homozygous subject. Although robust chloride conductance was seen with correctors and potentiators in homozygous ΔF508 cells, increased chloride conductance was not found in W1282X cells despite the presence of adequate transcript levels. Notwithstanding the negative data in W1282X cells from one human subject, we speculate that corrector and potentiator combinations may have therapeutic efficacy in cystic fibrosis caused by the W1282X mutation, although additional studies are needed on human cells from W1282X subjects. © 2017 by The American Society for

Comparison of cystic fibrosis transmembrane conductance regulator (CFTR) and ciliary beat frequency activation by the CFTR Modulators Genistein, VRT-532, and UCCF-152 in primary sinonasal epithelial cultures.

PubMed

Conger, Bryant T; Zhang, Shaoyan; Skinner, Daniel; Hicks, Stephen B; Sorscher, Eric J; Rowe, Steven M; Woodworth, Bradford A

2013-08-01

Pharmacologic activation of mucociliary clearance (MCC) represents an emerging therapeutic strategy for patients with chronic rhinosinusitis, even in the absence of congenital mutations of the CFTR gene. Drug discovery efforts have identified small molecules that activate the cystic fibrosis transmembrane conductance regulator (CFTR), including potentiators under development for treatment of cystic fibrosis. To evaluate the properties of CFTR modulators and their effects on ciliary beat frequency (CBF) in human sinonasal epithelium (HSNE). Primary HSNE cultures (wild type and F508del/F508del) were used to compare stimulation of CFTR-mediated Cl- conductance and CBF by the CFTR modulators genistein, VRT-532, and UCCF-152. Increase in CFTR-dependent anion transport and CBF. HSNE cultures were analyzed using pharmacologic manipulation of ion transport (change in short-circuit current [∆ISC]) and high-speed digital imaging (CBF). Activation of CFTR-dependent anion transport was significantly different among agonists (P < .001), with genistein exerting the greatest effect (mean [SD] ∆ISC, genistein, 23.1 [1.8] μA/cm2² > VRT-532, 8.1 [1.0] μA/cm² > UCCF-152, 3.4 [1.4] μA/cm² > control, 0.7 [0.2] μA/cm²; Tukey-Kramer P < .05) in the absence of forskolin. Genistein and UCCF-152 augmented CBF (under submerged conditions) significantly better (Tukey-Kramer P < .05) than cells treated with VRT-532 or dimethyl sulfoxide vehicle control (mean [SD] fold change over baseline, genistein, 1.63 [0.06]; UCCF-152, 1.56 [0.06]; VRT-532, 1.38 [0.08]; control, 1.27 [0.02]). Activation of CBF was blunted in F508del/F508del HSNE cultures. The degree of CBF stimulation was not dependent on the magnitude of Cl- secretion, suggesting that different mechanisms of action may underlie MCC activation by these small molecule potentiators. Agents that activate both CFTR-dependent ISC and CBF are particularly attractive as therapeutics because they may address 2

CFTR rescue with VX-809 and VX-770 favors the repair of primary airway epithelial cell cultures from patients with class II mutations in the presence of Pseudomonas aeruginosa exoproducts.

PubMed

Adam, Damien; Bilodeau, Claudia; Sognigbé, Laura; Maillé, Émilie; Ruffin, Manon; Brochiero, Emmanuelle

2018-04-13

Progressive airway damage due to bacterial infections, especially with Pseudomonas aeruginosa remains the first cause of morbidity and mortality in CF patients. Our previous work revealed a repair delay in CF airway epithelia compared to non-CF. This delay was partially prevented after CFTR correction (with VRT-325) in the absence of infection. Our goals were now to evaluate the effect of the Orkambi combination (CFTR VX-809 corrector + VX-770 potentiator) on the repair of CF primary airway epithelia, in infectious conditions. Primary airway epithelial cell cultures from patients with class II mutations were mechanically injured and wound healing rates and transepithelial resistances were monitored after CFTR rescue, in the absence and presence of P. aeruginosa exoproducts. Our data revealed that combined treatment with VX-809 and VX-770 elicited a greater beneficial impact on airway epithelial repair than VX-809 alone, in the absence of infection. The treatment with Orkambi was effective not only in airway epithelial cell cultures from patients homozygous for the F508del mutation but also from heterozygous patients carrying F508del and another class II mutation (N1303 K, I507del). The stimulatory effect of the Orkambi treatment was prevented by CFTR inhibition with GlyH101. Finally, Orkambi combination elicited a slight but significant improvement in airway epithelial repair and transepithelial resistance, despite the presence of P. aeruginosa exoproducts. Our findings indicate that Orkambi may favor airway epithelial integrity in CF patients with class II mutations. Complementary approaches would however be needed to further improve CFTR rescue and airway epithelial repair. Copyright © 2018 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

CFTR RECRUITMENT TO PHAGOSOMES IN NEUTROPHILS

PubMed Central

Zhou, Yun; Song, Kejing; Painter, Richard G.; Aiken, Martha; Reiser, Jakob; Stanton, Bruce A.; Nauseef, William M.; Wang, Guoshun

2013-01-01

Optimal microbicidal activity of human polymorphonuclear leukocytes (PMN) relies on generation of toxic agents such as hypochlorous acid (HOCl) in phagosomes. HOCl formation requires H2O2 produced by the NADPH oxidase, myeloperoxidase derived from azurophilic granules, and chloride ion. Chloride transport from cytoplasm into phagosomes requires chloride channels which include cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. However, the phagosomal targeting of CFTR in PMN has not been defined. Using human peripheral blood PMN, we determined that ~95–99% of LAMP-1 positive mature phagosomes were CFTR-positive, as judged by immunostaining and flow cytometric analysis. To establish a model cell system to evaluate CFTR phagosomal recruitment, we stably expressed EGFP alone, EGFP-wt-CFTR and EGFP-ΔF508-CFTR fusion proteins in promyelocytic PLB-985 cells, respectively. After differentiation into neutrophil-like cells, CFTR presentation to phagosomes was examined. EGFP-wt-CFTR was observed to associate with phagosomes and co-localize with LAMP-1. Flow cytometric analysis of the isolated phagosomes indicated that such a phagosomal targeting was determined by the CFTR portion of the fusion protein. In contrast, significantly less EGFP-ΔF508-CFTR was found in phagosomes, indicating a defective targeting of the molecule to the organelle. Importantly, CFTR corrector compound VRT-325 facilitated the recruitment of ΔF508-CFTR to phagosomes. These data demonstrate the possibility of pharmacologic correction of impaired recruitment of mutant CFTR, thereby providing a potential means to augment chloride supply to the phagosomes of PMN in patients with cystic fibrosis to enhance their microbicidal function. PMID:23486169

Determination of CFTR densities in erythrocyte plasma membranes using recognition imaging

NASA Astrophysics Data System (ADS)

Ebner, Andreas; Nikova, Dessy; Lange, Tobias; Häberle, Johannes; Falk, Sabine; Dübbers, Angelika; Bruns, Reimer; Hinterdorfer, Peter; Oberleithner, Hans; Schillers, Hermann

2008-09-01

CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated chloride (Cl-) channel that plays an important role in salt and fluid movement across epithelia. Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR. The most predominant mutation, F508del, disturbs CFTR protein trafficking, resulting in a reduced number of CFTR in the plasma membrane. Recent studies indicate that CFTR is not only found in epithelia but also in human erythrocytes. Although considerable attempts have been made to quantify CFTR in cells, conclusions on numbers of CFTR molecules localized in the plasma membrane have been drawn indirectly. AFM has the power to provide the needed information, since both sub-molecular spatial resolution and direct protein recognition via antibody-antigen interaction can be observed. We performed a quantification study of the CFTR copies in erythrocyte membranes at the single molecule level, and compared the difference between healthy donors and CF patients. We detected that the number of CFTR molecules is reduced by 70% in erythrocytes of cystic fibrosis patients.

The cystic fibrosis transmembrane conductance regulator (CFTR) and its stability.

PubMed

Meng, Xin; Clews, Jack; Kargas, Vasileios; Wang, Xiaomeng; Ford, Robert C

2017-01-01

The cystic fibrosis transmembrane conductance regulator (CFTR) is responsible for the disease cystic fibrosis (CF). It is a membrane protein belonging to the ABC transporter family functioning as a chloride/anion channel in epithelial cells around the body. There are over 1500 mutations that have been characterised as CF-causing; the most common of these, accounting for ~70 % of CF cases, is the deletion of a phenylalanine at position 508. This leads to instability of the nascent protein and the modified structure is recognised and then degraded by the ER quality control mechanism. However, even pharmacologically 'rescued' F508del CFTR displays instability at the cell's surface, losing its channel function rapidly and it is rapidly removed from the plasma membrane for lysosomal degradation. This review will, therefore, explore the link between stability and structure/function relationships of membrane proteins and CFTR in particular and how approaches to study CFTR structure depend on its stability. We will also review the application of a fluorescence labelling method for the assessment of the thermostability and the tertiary structure of CFTR.

Detection of CFTR function and modulation in primary human nasal cell spheroids.

PubMed

Brewington, John J; Filbrandt, Erin T; LaRosa, F J; Ostmann, Alicia J; Strecker, Lauren M; Szczesniak, Rhonda D; Clancy, John P

2018-01-01

Expansion of CFTR modulators to patients with rare/undescribed mutations will be facilitated by patient-derived models quantifying CFTR function and restoration. We aimed to generate a personalized model system of CFTR function and modulation using non-surgically obtained nasal epithelial cells (NECs). NECs obtained by curettage from healthy volunteers and CF patients were expanded and grown in 3-dimensional culture as spheroids, characterized, and stimulated with cAMP-inducing agents to activate CFTR. Spheroid swelling was quantified as a proxy for CFTR function. NEC spheroids recapitulated characteristics of pseudostratified respiratory epithelia. When stimulated with forskolin/IBMX, spheroids swelled in the presence of functional CFTR, and shrank in its absence. Spheroid swelling quantified mutant CFTR restoration in F508del homozygous cells using clinically available CFTR modulators. NEC spheroids hold promise for understanding rare CFTR mutations and personalized modulator testing to drive evaluation for CF patients with common, rare or undescribed mutations. Portions of this data have previously been presented in abstract form at the 2016 meetings of the American Thoracic Society and the 2016 North American Cystic Fibrosis Conference. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Comparison of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Ciliary Beat Frequency Activation by the CFTR Modulators Genistein, VRT-532, and UCCF-152 in Primary Sinonasal Epithelial Cultures

PubMed Central

Conger, Bryant T.; Zhang, Shaoyan; Skinner, Daniel; Hicks, Stephen B.; Sorscher, Eric J.; Rowe, Steven M.; Woodworth, Bradford A.

2014-01-01

IMPORTANCE Pharmacologic activation of mucociliary clearance (MCC) represents an emerging therapeutic strategy for patients with chronic rhinosinusitis, even in the absence of congenital mutations of the CFTR gene. Drug discovery efforts have identified small molecules that activate the cystic fibrosis transmembrane conductance regulator (CFTR), including potentiators under development for treatment of cystic fibrosis. OBJECTIVE To evaluate the properties of CFTR modulators and their effects on ciliary beat frequency (CBF) in human sinonasal epithelium (HSNE). DESIGN Primary HSNE cultures (wild type and F508del/F508del) were used to compare stimulation of CFTR-mediated Cl− conductance and CBF by the CFTR modulators genistein, VRT-532, and UCCF-152. MAIN OUTCOMES AND MEASURES Increase in CFTR-dependent anion transport and CBF. RESULTS HSNE cultures were analyzed using pharmacologic manipulation of ion transport (change in short-circuit current [ΔISC]) and high-speed digital imaging (CBF). Activation of CFTR-dependent anion transport was significantly different among agonists (P < .001), with genistein exerting the greatest effect (mean [SD] ΔISC, genistein, 23.1 [1.8] µA/cm2 > VRT-532, 8.1 [1.0] µA/cm2 > UCCF-152, 3.4 [1.4] µA/cm2 > control, 0.7 [0.2] µA/cm2; Tukey-Kramer P < .05) in the absence of forskolin. Genistein and UCCF-152 augmented CBF (under submerged conditions) significantly better (Tukey-Kramer P < .05) than cells treated with VRT-532 or dimethyl sulfoxide vehicle control (mean [SD] fold change over baseline, genistein, 1.63 [0.06]; UCCF-152, 1.56 [0.06]; VRT-532, 1.38 [0.08]; control, 1.27 [0.02]). Activation of CBF was blunted in F508del/F508del HSNE cultures. CONCLUSIONS AND RELEVANCE The degree of CBF stimulation was not dependent on the magnitude of Cl− secretion, suggesting that different mechanisms of action may underlie MCC activation by these small molecule potentiators. Agents that activate both CFTR-dependent ISC and CBF are

Correlation of the level of full-length CFTR transcript with pulmonary phenotype in patients carrying R117H and 1342-1,-2delAG mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamosh, A.; Cutting, G.R.; Oates, R.

The R117H mutation occurs on two chromosome backgrounds, one associated with a 7 thymidine tract (7T-R11H) in the splice-acceptor site of intron 8, the other with a 5 thymidine tract (5T-R117H). We examined exon 9 splicing efficiency in 5 patients of genotype R117H/{delta}F508 and one carrying 1342-1,-2delAG{delta}F508, an obligate exon 9 slice site mutation. Four patients carried R117H on a 7T background -- three adult men with congenital bilateral absence of the vas deferens and one adolescent female with pancreatitis and borderline sweat chloride concentration. The patient with R117H on a 5T background had pancreatic sufficient CF (PS-CF). The 1342-1,-2delAGmore » patient has classic pancreatic insufficient CF (PI-CF). cDNA was synthesized from total RNA extracted from nasal epithlial cells and analyzed for CFTR splicing by 35 cycle PCR using primers in exon 7 and 11. The quantity of full length transcript derived from the R117H or {delta}F508 alleles was assessed by allele-specific oligonucleotide hybridization. While 91.4% of transcript from the 5T-R117H allele was full-length, only 42.2% of CFTR transcript from the 5T-R117H allele was full length. Since CBAVD patients have no lung disease and PS-CF patients do, this indicates that the threshold of developing CF lung disease is crossed when the amount of CFTR transcript bearing R117H is reduced by half. Interestingly, 17.1% of transcript derived from the 1342-1,-2delAG allele (or 8.6% of total CFTR transcript) was normal and full length. This suggests that up to 9% of full length wild-type CFTR transcript may be inadequate to escape the lung disease of CF and that a 9 thymidine tract followed by AAC (the result of the AG deletion) can be used as a splice donor with 2-9% efficiency.« less

Cystic fibrosis gene modifier SLC26A9 modulates airway response to CFTR-directed therapeutics.

PubMed

Strug, Lisa J; Gonska, Tanja; He, Gengming; Keenan, Katherine; Ip, Wan; Boëlle, Pierre-Yves; Lin, Fan; Panjwani, Naim; Gong, Jiafen; Li, Weili; Soave, David; Xiao, Bowei; Tullis, Elizabeth; Rabin, Harvey; Parkins, Michael D; Price, April; Zuberbuhler, Peter C; Corvol, Harriet; Ratjen, Felix; Sun, Lei; Bear, Christine E; Rommens, Johanna M

2016-10-15

Cystic fibrosis is realizing the promise of personalized medicine. Recent advances in drug development that target the causal CFTR directly result in lung function improvement, but variability in response is demanding better prediction of outcomes to improve management decisions. The genetic modifier SLC26A9 contributes to disease severity in the CF pancreas and intestine at birth and here we assess its relationship with disease severity and therapeutic response in the airways. SLC26A9 association with lung disease was assessed in individuals from the Canadian and French CF Gene Modifier consortia with CFTR-gating mutations and in those homozygous for the common Phe508del mutation. Variability in response to a CFTR-directed therapy attributed to SLC26A9 genotype was assessed in Canadian patients with gating mutations. A primary airway model system determined if SLC26A9 shows modification of Phe508del CFTR function upon treatment with a CFTR corrector. In those with gating mutations that retain cell surface-localized CFTR we show that SLC26A9 modifies lung function while this is not the case in individuals homozygous for Phe508del where cell surface expression is lacking. Treatment response to ivacaftor, which aims to improve CFTR-channel opening probability in patients with gating mutations, shows substantial variability in response, 28% of which can be explained by rs7512462 in SLC26A9 (P = 0.0006). When homozygous Phe508del primary bronchial cells are treated to restore surface CFTR, SLC26A9 likewise modifies treatment response (P = 0.02). Our findings indicate that SLC26A9 airway modification requires CFTR at the cell surface, and that a common variant in SLC26A9 may predict response to CFTR-directed therapeutics.

Clinical expression of patients with the D1152H CFTR mutation.

PubMed

Terlizzi, Vito; Carnovale, Vincenzo; Castaldo, Giuseppe; Castellani, Carlo; Cirilli, Natalia; Colombo, Carla; Corti, Fabiola; Cresta, Federico; D'Adda, Alice; Lucarelli, Marco; Lucidi, Vincenzina; Macchiaroli, Annamaria; Madarena, Elisa; Padoan, Rita; Quattrucci, Serena; Salvatore, Donatello; Zarrilli, Federica; Raia, Valeria

2015-07-01

Discordant results were reported on the clinical expression of subjects bearing the D1152H CFTR mutation, and also for the small number of cases reported so far. A retrospective review of clinical, genetic and biochemical data was performed from individuals homozygous or compound heterozygous for the D1152H mutation followed in 12 Italian cystic fibrosis (CF) centers. 89 subjects carrying at least D1152H on one allele were identified. 7 homozygous patients had very mild clinical expression. Over half of the 74 subjects compound heterozygous for D1152H and a I-II-III class mutation had borderline or pathological sweat test and respiratory or gastrointestinal symptoms; one third had pulmonary bacteria colonization and 10/74 cases had complications (i.e. diabetes, allergic bronchopulmonary aspergillosis, and hemoptysis). However, their clinical expression was less severe as compared to a group of CF patients homozygous for the F508del mutation. Finally, 8 subjects compound heterozygous for D1152H and a IV-V class mutation showed very mild disease. The natural history of subjects bearing the D1152H mutation is widely heterogeneous and is influenced by the mutation in trans. Copyright © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

CFTR Genotype and Maximal Exercise Capacity in Cystic Fibrosis: A Cross-sectional Study.

PubMed

Radtke, Thomas; Hebestreit, Helge; Gallati, Sabina; Schneiderman, Jane E; Braun, Julia; Stevens, Daniel; Hulzebos, Erik Hj; Takken, Tim; Boas, Steven R; Urquhart, Don S; Lands, Larry C; Tejero, Sergio; Sovtic, Aleksandar; Dwyer, Tiffany; Petrovic, Milos; Harris, Ryan A; Karila, Chantal; Savi, Daniela; Usemann, Jakob; Mei-Zahav, Meir; Hatziagorou, Elpis; Ratjen, Felix; Kriemler, Susi

2018-02-01

Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in human skeletal muscle cells. Variations of CFTR dysfunction among patients with cystic fibrosis may be an important determinant of maximal exercise capacity in cystic fibrosis. Previous studies on the relationship between CFTR genotype and maximal exercise capacity are scarce and contradictory. This study was designed to explore factors influencing maximal exercise capacity, expressed as peak oxygen uptake (V.O2peak), with a specific focus on CFTR genotype in children and adults with cystic fibrosis. In an international, multicenter, cross-sectional study, we collected data on CFTR genotype and cardiopulmonary exercise tests in patients with cystic fibrosis who were ages 8 years and older. CFTR mutations were classified into functional classes I–V. The final analysis included 726 patients (45% females; age range, 8–61 yr; forced expiratory volume in 1 s, 16 to 123% predicted) from 17 cystic fibrosis centers in North America, Europe, Australia, and Asia, all of whom had both valid maximal cardiopulmonary exercise tests and complete CFTR genotype data. Overall, patients exhibited exercise intolerance (V.O2peak, 77.3 ± 19.1% predicted), but values were comparable among different CFTR classes. We did not detect an association between CFTR genotype functional classes I–III and either V.O2peak (percent predicted) (adjusted β = −0.95; 95% CI, −4.18 to 2.29; P = 0.57) or maximum work rate (Wattmax) (adjusted β = −1.38; 95% CI, −5.04 to 2.27; P = 0.46) compared with classes IV–V. Those with at least one copy of a F508del-CFTR mutation and one copy of a class V mutation had a significantly lower V.O2peak (β = −8.24%; 95% CI, −14.53 to −2.99; P = 0.003) and lower Wattmax (adjusted β = −7.59%; 95% CI, −14.21 to −0.95; P = 0.025) than those with two copies of a class II mutation. On the basis of linear regression analysis adjusted for

Lumacaftor/Ivacaftor in Patients Aged 6-11 Years with Cystic Fibrosis and Homozygous for F508del-CFTR.

PubMed

Milla, Carlos E; Ratjen, Felix; Marigowda, Gautham; Liu, Fang; Waltz, David; Rosenfeld, Margaret

2017-04-01

Combination lumacaftor/ivacaftor has been shown to improve lung function and other endpoints in patients aged 12 years and older with cystic fibrosis and homozygous for F508del-CFTR, but it has not been assessed in younger patients. In this open-label phase III trial, we evaluated the safety, tolerability, pharmacodynamics, and efficacy of lumacaftor/ivacaftor combination therapy in patients aged 6-11 years with cystic fibrosis who were homozygous for F508del-CFTR. Patients (N = 58) received 200 mg lumacaftor/250 mg ivacaftor orally every 12 hours for 24 weeks in addition to their existing cystic fibrosis medications. Lumacaftor/ivacaftor was well tolerated; the safety profile was generally similar to that observed in larger lumacaftor/ivacaftor trials with older patients. Four patients discontinued (two because of drug-related adverse events: elevated liver transaminases, n = 1; rash, n = 1). No safety concerns were associated with spirometry. No significant changes in percent predicted FEV 1 were observed (change from baseline at Week 24, +2.5 percentage points; 95% confidence interval [CI], -0.2 to 5.2; P = 0.0671). At Week 24, significant improvements from baseline were observed in sweat chloride (-24.8 mmol/L; 95% CI, -29.1 to -20.5; P < 0.0001), body mass index z score (+0.15; 95% CI, 0.08 to 0.22; P < 0.0001), Cystic Fibrosis Questionnaire-Revised respiratory domain score (+5.4; 95% CI, 1.4 to 9.4; P = 0.0085), and lung clearance index based on lung volume turnover required to reach 2.5% of starting N 2 concentration (-0.88; 95% CI, -1.40 to -0.37; P = 0.0018). Lumacaftor/ivacaftor was well tolerated in this young population; no new safety concerns were identified. Improvements in lung clearance index, sweat chloride, nutritional status, and health-related quality of life were observed after 24 weeks of treatment. Clinical trial registered with www.clinicaltrials.gov (NCT01897233).

Mechanosensitive activation of CFTR by increased cell volume and hydrostatic pressure but not shear stress.

PubMed

Vitzthum, Constanze; Clauss, Wolfgang G; Fronius, Martin

2015-11-01

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-) channel that is essential for electrolyte and fluid homeostasis. Preliminary evidence indicates that CFTR is a mechanosensitive channel. In lung epithelia, CFTR is exposed to different mechanical forces such as shear stress (Ss) and membrane distention. The present study questioned whether Ss and/or stretch influence CFTR activity (wild type, ∆F508, G551D). Human CFTR (hCFTR) was heterologously expressed in Xenopus oocytes and the response to the mechanical stimulus and forskolin/IBMX (FI) was measured by two-electrode voltage-clamp experiments. Ss had no influence on hCFTR activity. Injection of an intracellular analogous solution to increase cell volume alone did not affect hCFTR activity. However, hCFTR activity was augmented by injection after pre-stimulation with FI. The response to injection was similar in channels carrying the common mutations ∆F508 and G551D compared to wild type hCFTR. Stretch-induced CFTR activation was further assessed in Ussing chamber measurements using Xenopus lung preparations. Under control conditions increased hydrostatic pressure (HP) decreased the measured ion current including activation of a Cl(-) secretion that was unmasked by the CFTR inhibitor GlyH-101. These data demonstrate activation of CFTR in vitro and in a native pulmonary epithelium in response to mechanical stress. Mechanosensitive regulation of CFTR is highly relevant for pulmonary physiology that relies on ion transport processes facilitated by pulmonary epithelial cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Cystic fibrosis in the Basque country: high frequency of mutation delta F508 in patients of Basque origin.

PubMed Central

Casals, T; Vázquez, C; Lázaro, C; Girbau, E; Giménez, F J; Estivill, X

1992-01-01

The Basque population is one of the oldest populations of Europe. It has been suggested that the Basques arose from a population established in western Europe during the late Paleolithic Age. The Basque language (Euskera) is a supposedly pre-Indo-European language that originates from the first settlers of Europe. The variable distribution of the major cystic fibrosis (CF) mutation (delta F508 deletion) in Europe, with higher frequencies of the mutation in northern Europe and lower frequencies in southern Europe, has suggested that the delta F508 mutation was spread by early farmers migrating from the Middle East during the Neolithic period. We have studied 45 CF families from the Basque Country, where the incidence of CF is approximately 1/4,500. The birthplaces of the parents and grandparents have been traced and are distributed according to their origin as Basque or Mixed Basque. The frequency of the delta F508 mutation in the chromosomes of Basque origin is 87%, compared with 58% in those of Mixed Basque origin. The analysis of haplotypes, both with markers closely linked to the CF gene and with intragenic markers, suggests that the delta F508 mutation was not spread by the Indo-European invasions but was already present in Europe more than 10,000 years ago, during the Paleolithic period. PMID:1370875

Efficacy and safety of lumacaftor and ivacaftor in patients aged 6-11 years with cystic fibrosis homozygous for F508del-CFTR: a randomised, placebo-controlled phase 3 trial.

PubMed

Ratjen, Felix; Hug, Christopher; Marigowda, Gautham; Tian, Simon; Huang, Xiaohong; Stanojevic, Sanja; Milla, Carlos E; Robinson, Paul D; Waltz, David; Davies, Jane C

2017-07-01

Lumacaftor and ivacaftor combination treatment showed efficacy in patients aged 12 years or older with cystic fibrosis homozygous for F508del-cystic fibrosis transmembrane conductance regulator (CFTR) in placebo-controlled studies and patients aged 6-11 years with cystic fibrosis homozygous for F508del-CFTR in an open-label study. We report efficacy and safety of lumacaftor and ivacaftor in patients with cystic fibrosis aged 6-11 years homozygous for F508del-CFTR. In this phase 3, randomised, double-blind, placebo-controlled, multicentre study, patients were enrolled at 54 hospitals and medical centres in nine countries (the USA, Australia, Belgium, Canada, Denmark, France, Germany, Sweden, and the UK). Eligible patients weighed at least 15 kg, with a confirmed diagnosis of cystic fibrosis, percent predicted forced expiratory volume in 1 s (FEV 1 ) of 70 or more, and lung clearance index 2·5 (LCI 2·5 ) of 7·5 or more at screening (values less than these thresholds were permitted at day 1). All patients were tested for CFTR genotype at screening; eligible patients had to have the F508del-CFTR mutation on both alleles. Exclusion criteria included any comorbidity or laboratory abnormality that might confound the study results or pose additional risk to the patient. Patients were stratified by weight (<25 kg vs ≥25 kg) and ppFEV 1 severity (<90 vs ≥90) determined at the screening visit, and randomly assigned 1:1 to treatment using an interactive web response system to receive 200 mg lumacaftor and 250 mg ivacaftor every 12 hours or placebo for 24 weeks. Patients, all site personnel including the investigator and the site monitor, and the study team were blinded, with the exception of site personnel needing this information in the event of medical emergency or pregnancy and patient safety and regulatory affairs personnel to meet serious adverse event reporting requirements. The primary endpoint was the mean absolute change in LCI 2·5 from all on-treatment study

Identification of eight novel mutations in a collaborative analysis of a part of the second transmembrane domain of the CFTR gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mercier, B.; Audrezet, M.P.; Guillermit, H.

Cystic fibrosis transmembrane conductance regulator (CFTR), the gene responsible, when mutated, for cystic fibrosis (CF), spans over 230 kb on the long arm of chromosome 7 and is composed of 27 exons. The most common mutation responsible for CF worldwide is the deletion of a phenylalanine amino acid at codon 508 in the first nucleotide-binding fold and accounts for approximately 70% of CF chromosomes studied. More than 250 other mutations have been reported through the CF Genetic Analysis Consortium. The majority of the mutations previously described lie in the two nucleotide-binding folds. To explore exhaustively other regions of the gene,more » particularly exons coding for transmembrane domains, the authors have initiated a collaborative study between different laboratories to screen 369 non-[Delta]F508 CF chromosomes of seven ethnic European populations (Belgian, French, Breton, Irish, Italian, Yugoslavian, Russian). Among these chromosomes carrying an unidentified mutation, 63 were from Brittany, 50 of various French origin, 45 of Irish origin, 56 of Italian origin, 41 of Belgian origin, 2 of Turkish origin, 38 of Yugoslavian origin, 22 of Russian origin, and 52 of Bulgarian origin. Diagnostic criteria for CF included at least one positive sweat test and pulmonary disease with or without pancreatic disease. Using a denaturing gradient gel electrophoresis (DGGE) assay, they have identified eight novel mutations in exon 17b coding for part of the second transmembrane domain of the CFTR and they describe them in this report. 8 refs., 1 fig., 1 tab.« less

Activation of deltaF508 CFTR in a cystic fibrosis respiratory epithelial cell line by 4-phenylbutyrate, genistein and CPX.

PubMed

Andersson, C; Roomans, G M

2000-05-01

The cellular basis of cystic fibrosis (CF) is a defect in a cyclic adenosine monophosphate (cAMP)-activated chloride channel (CF transmembrane conductance regulator) in epithelial cells that leads to decreased chloride ion transport and impaired water transport across the cell membrane. This study investigated whether it was possible to activate the defective chloride channel in cystic fibrosis respiratory epithelial cells with 4-phenylbutyrate (4PBA), genistein and 8-cyclopentyl-1,3-dipropylxanthine (CPX). The CF bronchial epithelial cell line CFBE41o-, which expresses the deltaF508 mutation, was treated with these agents and loss of Cl-, indicating Cl- efflux, measured by X-ray microanalysis. 8-bromo-cAMP alone did not induce Cl- efflux in CFBE41o- cells, but after incubation with 4PBA a significant efflux of Cl- occurred. Stimulation of cells with a combination of genistein and cAMP also induced Cl- efflux, whereas a combination of pretreatment with 4PBA and a combined stimulation with genistein and cAMP induced an even larger Cl- efflux. Cl- efflux could also be stimulated by CPX, but this effect was not enhanced by 4PBA pretreatment. The deltaF508 mutation leads to impaired processing of the cystic fibrosis transmembrane conductance regulator. The increased efflux of chloride after 4-phenylbutyrate treatment can be explained by the fact that 4-phenylbutyrate allows the deltaF508 cystic fibrosis transmembrane conductance regulator to escape degradation and to be transported to the cell surface. Genistein and 8-cyclopentyl-1,3-dipropylxanthine act by stimulating chloride ion efflux by increasing the probability of the cystic fibrosis transmembrane conductance regulator being open. The combination of 4-phenylbutyrate and genistein may be useful in a potential pharmacological therapy for cystic fibrosis patients with the deltaF508 mutation.

A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811 + 1.6kbA {yields} G, produces a new exon: High frequency in spanish cystic fibrosis chromosomes and association with severe phenotype

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chillon, M.; Casals, T.; Gimenez, J.

1995-03-01

mRNA analysis of the cystic fibrosis transmembrane regulator (CFTR) gene in tissues of cystic fibrosis (CF) patients has allowed us to detect a cryptic exon. The new exon involves 49 base pairs between exons 11 and 12 and is due to a point mutation (1811+1.6bA{yields}G) that creates a new donor splice site in intron 11. Semiquantitative mRNA analysis showed that 1811+1.6kbA{r_arrow}G-mRNA was 5-10-fold less abundant than {triangle}F508 mRNA. Mutations 1811+1.6kbA{yields}G was found in 21 Spanish and 1 German CF chromosome(s), making it the fourth-most-frequent mutation (2%) in the Spanish population. Individuals with genotype {triangle}F508/1811+1.6kbA{yields}G have only 1%-3% of normal CFTRmore » mRNA. This loss of 97% of normal CFTR mRNA must be responsible for the pancreatic insufficiency and for the severe CF phenotype in these patients. 30 refs., 3 figs., 2 tabs.« less

VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1

PubMed Central

Ren, Hong Yu; Grove, Diane E.; De La Rosa, Oxana; Houck, Scott A.; Sopha, Pattarawut; Van Goor, Fredrick; Hoffman, Beth J.; Cyr, Douglas M.

2013-01-01

Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR. PMID:23924900

VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1.

PubMed

Ren, Hong Yu; Grove, Diane E; De La Rosa, Oxana; Houck, Scott A; Sopha, Pattarawut; Van Goor, Fredrick; Hoffman, Beth J; Cyr, Douglas M

2013-10-01

Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR.

Channel Gating Regulation by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) First Cytosolic Loop.

PubMed

Ehrhardt, Annette; Chung, W Joon; Pyle, Louise C; Wang, Wei; Nowotarski, Krzysztof; Mulvihill, Cory M; Ramjeesingh, Mohabir; Hong, Jeong; Velu, Sadanandan E; Lewis, Hal A; Atwell, Shane; Aller, Steve; Bear, Christine E; Lukacs, Gergely L; Kirk, Kevin L; Sorscher, Eric J

2016-01-22

In this study, we present data indicating a robust and specific domain interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed that binding kinetics were not altered by the common clinical mutation F508del. Both intrinsic ATPase activity and CFTR channel gating were inhibited severely by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR dysregulation has been implicated in the pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease, acquired rhinosinusitis, pancreatitis, and lethal secretory diarrhea (e.g. cholera). On the basis of clinical relevance of the CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508del CFTR and pharmacologic rescue. Our findings support a targetable mechanism of CFTR regulation in which conformational changes in the NBDs cause reorientation of transmembrane domains via interactions with CL1 and result in channel gating. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Quorum Sensing Down-Regulation Counteracts the Negative Impact of Pseudomonas aeruginosa on CFTR Channel Expression, Function and Rescue in Human Airway Epithelial Cells

PubMed Central

Maillé, Émilie; Ruffin, Manon; Adam, Damien; Messaoud, Hatem; Lafayette, Shantelle L.; McKay, Geoffrey; Nguyen, Dao; Brochiero, Emmanuelle

2017-01-01

The function of cystic fibrosis transmembrane conductance regulator (CFTR) channels is crucial in human airways. However unfortunately, chronic Pseudomonas aeruginosa infection has been shown to impair CFTR proteins in non-CF airway epithelial cells (AEC) and to alter the efficiency of new treatments with CFTR modulators designed to correct the basic CFTR default in AEC from cystic fibrosis (CF) patients carrying the F508del mutation. Our aim was first to compare the effect of laboratory strains, clinical isolates, engineered and natural mutants to determine the role of the LasR quorum sensing system in CFTR impairment, and second, to test the efficiency of a quorum sensing inhibitor to counteract the deleterious impact of P. aeruginosa both on wt-CFTR and on the rescue of F508del-CFTR by correctors. We first report that exoproducts from either the laboratory PAO1 strain or a clinical ≪Early≫ isolate (from an early stage of infection) altered CFTR expression, localization and function in AEC expressing wt-CFTR. Genetic inactivation of the quorum-sensing LasR in PAO1 (PAO1ΔlasR) or in a natural clinical mutant (≪Late≫ CF-adapted clinical isolate) abolished wt-CFTR impairment. PAO1 exoproducts also dampened F508del-CFTR rescue by VRT-325 or Vx-809 correctors in CF cells, whereas PAO1ΔlasR had no impact. Importantly, treatment of P. aeruginosa cultures with a quorum sensing inhibitor (HDMF) prevented the negative effect of P. aeruginosa exoproducts on wt-CFTR and preserved CFTR rescue by correctors in CF AEC. These findings indicate that LasR-interfering strategies could be of benefits to counteract the deleterious effect of P. aeruginosa in infected patients. PMID:29177135

Synergy of cAMP and calcium signaling pathways in CFTR regulation

PubMed Central

Bozoky, Zoltan; Ahmadi, Saumel; Milman, Tal; Kim, Tae Hun; Du, Kai; Di Paola, Michelle; Pasyk, Stan; Pekhletski, Roman; Keller, Jacob P.; Bear, Christine E.; Forman-Kay, Julie D.

2017-01-01

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, leading to defective apical chloride transport. Patients also experience overactivation of inflammatory processes, including increased calcium signaling. Many investigations have described indirect effects of calcium signaling on CFTR or other calcium-activated chloride channels; here, we investigate the direct response of CFTR to calmodulin-mediated calcium signaling. We characterize an interaction between the regulatory region of CFTR and calmodulin, the major calcium signaling molecule, and report protein kinase A (PKA)-independent CFTR activation by calmodulin. We describe the competition between calmodulin binding and PKA phosphorylation and the differential effects of this competition for wild-type CFTR and the major F508del mutant, hinting at potential therapeutic strategies. Evidence of CFTR binding to isolated calmodulin domains/lobes suggests a mechanism for the role of CFTR as a molecular hub. Together, these data provide insights into how loss of active CFTR at the membrane can have additional consequences besides impaired chloride transport. PMID:28242698

S737F is a new CFTR mutation typical of patients originally from the Tuscany region in Italy.

PubMed

Terlizzi, Vito; Di Lullo, Antonella Miriam; Comegna, Marika; Centrone, Claudia; Pelo, Elisabetta; Castaldo, Giuseppe; Raia, Valeria; Braggion, Cesare

2018-01-03

An increasing number of patients have been described as having a number of Cystic Fibrosis Transmembrane conductance Regulator (CFTR) variants for which it lacks a clear genotype-phenotype correlation. We assesses the clinical features of patients bearing the S737F (p.Ser737Phe) CFTR missense variant and evaluated the residual function of CFTR protein on nasal epithelial cells (NEC). A retrospective database was performed from individuals homozygous or compound heterozygous for the S737F variant followed in the Cystic Fibrosis (CF) Centre of Florence. We performed a nasal brushing in cooperating patients and compared the results with those of patients followed in the pediatric CF Centre of Naples. 9/295 (3%) subjects carrying at least S737F CFTR variant on one allele were identified. Patients were diagnosed in 7/9 cases by newborn screening and in two cases for dehydration with hypochloremic metabolic alkalosis; at diagnosis sweat chloride levels (SCL) were in the pathological range in only one case. After a mean follow up of 8,6 years (range 0,5-15,8), SCL were in the pathological range in 8/9 cases (mean age at CF diagnosis: 1,5 years), all patients were pancreatic sufficiency and respiratory function was normal. The gating activity on NEC was 15.6% and 12.7% in two patients compound heterozygous for W1282X and DelE22_24, while it was ranged between 6,2% and 9,8% in CF patients. S737F is a CFTR mutation associated to hypochloremic alkalosis in childhood, mild CF phenotype in teenage years and a residual function of CFTR protein.

The role of cystic fibrosis transmembrane conductance regulator phenylalanine 508 side chain in ion channel gating

PubMed Central

Cui, Liying; Aleksandrov, Luba; Hou, Yue-Xian; Gentzsch, Martina; Chen, Jey-Hsin; Riordan, John R; Aleksandrov, Andrei A

2006-01-01

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel employing the ABC transporter structural motif. Deletion of a single residue (Phe508) in the first nucleotide-binding domain (NBD1), which occurs in most patients with cystic fibrosis, impairs both maturation and function of the protein. However, substitution of the Phe508 with small uncharged amino acids, including cysteine, is permissive for maturation. To explore the possible role of the phenylalanine aromatic side chain in channel gating we introduced a cysteine at this position in cysless CFTR, enabling its selective chemical modification by sulfhydryl reagents. Both cysless and wild-type CFTR ion channels have identical mean open times when activated by different nucleotide ligands. Moreover, both channels could be locked in an open state by introducing an ATPase inhibiting mutation (E1371S). However, the introduction of a single cysteine (F508C) prevented the cysless E1371S channel from maintaining the permanently open state, allowing closing to occur. Chemical modification of cysless E1371S/F508C by sulfhydryl reagents was used to probe the role of the side chain in ion channel function. Specifically, benzyl-methanethiosulphonate modification of this variant restored the gating behaviour to that of cysless E1371S containing the wild-type phenylalanine at position 508. This provides the first direct evidence that a specific interaction of the Phe508 aromatic side chain plays a role in determining the residency time in the closed state. Thus, despite the fact that this aromatic side chain is not essential for CFTR folding, it is important in the ion channel function. PMID:16484308

Cystic fibrosis transmembrane conductance regulator (CFTR) potentiator VX-770 (ivacaftor) opens the defective channel gate of mutant CFTR in a phosphorylation-dependent but ATP-independent manner.

PubMed

Eckford, Paul D W; Li, Canhui; Ramjeesingh, Mohabir; Bear, Christine E

2012-10-26

The cystic fibrosis transmembrane conductance regulator (CFTR) acts as a channel on the apical membrane of epithelia. Disease-causing mutations in the cystic fibrosis gene can lead to CFTR protein misfolding as in the case of the F508del mutation and/or channel dysfunction. Recently, a small molecule, VX-770 (ivacaftor), has shown efficacy in restoring lung function in patients bearing the G551D mutation, and this has been linked to repair of its channel gating defect. However, these studies did not reveal the mechanism of action of VX-770 in detail. Normally, CFTR channel activity is regulated by phosphorylation, ATP binding, and hydrolysis. Hence, it has been hypothesized that VX-770 modifies one or more of these metabolic events. In this study, we examined VX-770 activity using a reconstitution system for purified CFTR protein, a system that enables control of known regulatory factors. We studied the consequences of VX-770 interaction with CFTR incorporated in planar lipid bilayers and in proteoliposomes, using a novel flux-based assay. We found that purified and phosphorylated CFTR was potentiated in the presence of Mg-ATP, suggesting that VX-770 bound directly to the CFTR protein, rather than associated kinases or phosphatases. Interestingly, we also found that VX-770 enhanced the channel activity of purified and mutant CFTR in the nominal absence of Mg-ATP. These findings suggest that VX-770 can cause CFTR channel opening through a nonconventional ATP-independent mechanism. This work sets the stage for future studies of the structural properties that mediate CFTR gating using VX-770 as a probe.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Potentiator VX-770 (Ivacaftor) Opens the Defective Channel Gate of Mutant CFTR in a Phosphorylation-dependent but ATP-independent Manner* ♦

PubMed Central

Eckford, Paul D. W.; Li, Canhui; Ramjeesingh, Mohabir; Bear, Christine E.

2012-01-01

The cystic fibrosis transmembrane conductance regulator (CFTR) acts as a channel on the apical membrane of epithelia. Disease-causing mutations in the cystic fibrosis gene can lead to CFTR protein misfolding as in the case of the F508del mutation and/or channel dysfunction. Recently, a small molecule, VX-770 (ivacaftor), has shown efficacy in restoring lung function in patients bearing the G551D mutation, and this has been linked to repair of its channel gating defect. However, these studies did not reveal the mechanism of action of VX-770 in detail. Normally, CFTR channel activity is regulated by phosphorylation, ATP binding, and hydrolysis. Hence, it has been hypothesized that VX-770 modifies one or more of these metabolic events. In this study, we examined VX-770 activity using a reconstitution system for purified CFTR protein, a system that enables control of known regulatory factors. We studied the consequences of VX-770 interaction with CFTR incorporated in planar lipid bilayers and in proteoliposomes, using a novel flux-based assay. We found that purified and phosphorylated CFTR was potentiated in the presence of Mg-ATP, suggesting that VX-770 bound directly to the CFTR protein, rather than associated kinases or phosphatases. Interestingly, we also found that VX-770 enhanced the channel activity of purified and mutant CFTR in the nominal absence of Mg-ATP. These findings suggest that VX-770 can cause CFTR channel opening through a nonconventional ATP-independent mechanism. This work sets the stage for future studies of the structural properties that mediate CFTR gating using VX-770 as a probe. PMID:22942289

Role of CFTR mutation analysis in the diagnostic algorithm for cystic fibrosis.

PubMed

Ratkiewicz, Michelle; Pastore, Matthew; McCoy, Karen Sharrock; Thompson, Rohan; Hayes, Don; Sheikh, Shahid Ijaz

2017-04-01

The cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation identification is being used with increased frequency to aid in the diagnosis of cystic fibrosis (CF) in those suspected with CF. Aim of this study was to identify diagnostic outcomes when CFTR mutational analysis was used in CF diagnosis. CFTR mutational analysis results were also compared with sweat chloride results. This study was done on all patients at our institution who had CFTR mutation analysis over a sevenyear period since August 2006. A total of 315 patients underwent CFTR mutational analysis. Fifty-one (16.2%) patients had two mutations identified. Among them 32 had positive sweat chloride levels (≥60 mmol/L), while seven had borderline sweat chloride levels (40-59 mmol/L). An additional 70 patients (22.3%) had only one mutation identified. Among them eight had positive sweat chloride levels, and 17 had borderline sweat chloride levels. Fifty-five patients (17.5%) without CFTR mutations had either borderline (n=45) or positive (n=10) sweat chloride results. Three patients with a CF phenotype had negative CFTR analysis but elevated sweat chloride levels. In eighty-three patients (26.4%) CFTR mutational analysis was done without corresponding sweat chloride testing. Although CFTR mutation analysis has improved the diagnostic capability for CF, its use either as the first step or the only test to diagnose CFTR dysfunction should be discouraged and CF diagnostic guidelines need to be followed.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene mutations in North Egyptian population: implications for the genetic diagnosis in Egypt.

PubMed

El-Seedy, A; Pasquet, M C; Shafiek, H; Morsi, T; Kitzis, A; Ladevèze, V

2016-11-30

Cystic fibrosis (CF) occurrence in Arab populations is not common and still remains underidentified. Furthermore, the lack of disease awareness and diagnosis facilities have mislead the identification of cystic fibrosis for decades. The knowledge about cystic fibrosis (CF) in Egypt is very limited, and a few reports have drawn attention to the existence of CF or CFTR-related disorders (CFTR-RDs) in the Egyptian population. Therefore a comprehensive genetic analysis of the CFTR gene was realized in patients of North Egypt. DNA samples of 56 Egyptian patients were screened for the CFTR gene mutations. The 27 exons and their flanking regions of the CFTR gene were amplified by PCR, using the published primer pairs, and were studied by automated direct DNA sequencing to detect disease-causing mutations. Moreover, large duplication/deletion was analysed by MLPA technique. CFTR screening revealed the identification of thirteen mutations including four novel ones: c.92G>A (p.Arg31His), c.2782G>C (p.Ala928Pro), c.3718-24G>A, c.4207A>G (p.Arg1403Gly) and nine previously reported mutations: c.454A>T (p.Met152Leu), c.902A>G (p.Tyr301Cys), c.1418delG, c.2620-15C>G, c.2997_3000delAATT, c.3154T>G (p.Phe1052Val), c.3872A>G (p.Gln1291Arg), c.3877G>A (p.Val1293Ile), c.4242+10T>C. Furthermore, eight polymorphisms were found: c.743+40A>G, c.869+11C>T, c.1408A>G, c.1584G>A, c.2562T>G, c.3870A>G, c.4272C>T, c.4389G>A. These mutations and polymorphisms were not previously described in the Egyptian population except for the c.1408A>G polymorphism. Here we demonstrate the importance of the newly discovered mutations in Egyptian patients and the presence of CF, whereas the p.Phe508del mutation is not detected. The identification of CFTR mutations will become increasingly important in undocumented populations. The current findings will help us expand the mutational spectrum of CF and establish the first panel of the CFTR gene

Analysis of some polymorphic markers of the CFTR gene in cystic fibrosis patients and healthy donors from the Moscow region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Amosenko, F.A.; Sazonova, M.A.; Kapranov, N.I.

1995-04-01

Allelic frequencies of three polymorphic markers in the CFTR gene were estimated on chromosomes derived from cystic fibrosis (CF) patients and healthy donors from Moscow and the Moscow region. These polymorphic markers are tetranucleotide tandem repeats GATT in intron 6B, M470V in exon 10, and T854T in exon 14 (fragment A). Frequencies at allele 1 of the M470V marker, along with allele 2 of GATT and T854T, are two times higher for CF patients without {Delta}F508 mutation than for healthy donors, and there is linkage disequilibrium of these alleles of the polymorphic markers analyzed with the CF gene. Allele 1more » of M470V and T854T markers, as well as allele 2 of the GATT marker (six repeats), are absolutely linked to mutation F508 of the CFTR gene. Using the polymorphic markers studied, family analysis of CF was carried out in two families. 10 refs., 1 fig., 1 tab.« less

Comparative processing and function of human and ferret cystic fibrosis transmembrane conductance regulator.

PubMed

Fisher, John T; Liu, Xiaoming; Yan, Ziying; Luo, Meihui; Zhang, Yulong; Zhou, Weihong; Lee, Ben J; Song, Yi; Guo, Chenhong; Wang, Yujiong; Lukacs, Gergely L; Engelhardt, John F

2012-06-22

The most common cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation is ΔF508, and this causes cystic fibrosis (CF). New CF models in the pig and ferret have been generated that develop lung, pancreatic, liver, and intestinal pathologies that reflect disease in CF patients. Species-specific biology in the processing of CFTR has demonstrated that pig and mouse ΔF508-CFTR proteins are more effectively processed to the apical membrane of airway epithelia than human ΔF508-CFTR. The processing behavior of ferret WT- and ΔF508-CFTR proteins remains unknown, and such information is important to predicting the utility of a ΔF508-CFTR ferret. To this end, we sought to compare processing, membrane stability, and function of human and ferret WT- and ΔF508-CFTR proteins in a heterologous expression system using HT1080, HEK293T, BHK21, and Cos7 cells as well as human and ferret CF polarized airway epithelia. Analysis of the protein processing and stability by metabolic pulse-chase and surface On-Cell Western blots revealed that WT-fCFTR half-life and membrane stability were increased relative to WT-hCFTR. Furthermore, in BHK21, Cos7, and CuFi cells, human and ferret ΔF508-CFTR processing was negligible, whereas low levels of processing of ΔF508-fCFTR could be seen in HT1080 and HEK293T cells. Only the WT-fCFTR, but not ΔF508-fCFTR, produced functional cAMP-inducible chloride currents in both CF human and ferret airway epithelia. Further elucidation of the mechanism responsible for elevated fCFTR protein stability may lead to new therapeutic approaches to augment CFTR function. These findings also suggest that generation of a ferret CFTR(ΔF508/ΔF508) animal model may be useful.

Comparative Processing and Function of Human and Ferret Cystic Fibrosis Transmembrane Conductance Regulator*

PubMed Central

Fisher, John T.; Liu, Xiaoming; Yan, Ziying; Luo, Meihui; Zhang, Yulong; Zhou, Weihong; Lee, Ben J.; Song, Yi; Guo, Chenhong; Wang, Yujiong; Lukacs, Gergely L.; Engelhardt, John F.

2012-01-01

The most common cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation is ΔF508, and this causes cystic fibrosis (CF). New CF models in the pig and ferret have been generated that develop lung, pancreatic, liver, and intestinal pathologies that reflect disease in CF patients. Species-specific biology in the processing of CFTR has demonstrated that pig and mouse ΔF508-CFTR proteins are more effectively processed to the apical membrane of airway epithelia than human ΔF508-CFTR. The processing behavior of ferret WT- and ΔF508-CFTR proteins remains unknown, and such information is important to predicting the utility of a ΔF508-CFTR ferret. To this end, we sought to compare processing, membrane stability, and function of human and ferret WT- and ΔF508-CFTR proteins in a heterologous expression system using HT1080, HEK293T, BHK21, and Cos7 cells as well as human and ferret CF polarized airway epithelia. Analysis of the protein processing and stability by metabolic pulse-chase and surface On-Cell Western blots revealed that WT-fCFTR half-life and membrane stability were increased relative to WT-hCFTR. Furthermore, in BHK21, Cos7, and CuFi cells, human and ferret ΔF508-CFTR processing was negligible, whereas low levels of processing of ΔF508-fCFTR could be seen in HT1080 and HEK293T cells. Only the WT-fCFTR, but not ΔF508-fCFTR, produced functional cAMP-inducible chloride currents in both CF human and ferret airway epithelia. Further elucidation of the mechanism responsible for elevated fCFTR protein stability may lead to new therapeutic approaches to augment CFTR function. These findings also suggest that generation of a ferret CFTRΔF508/ΔF508 animal model may be useful. PMID:22570484

[Frequency of the most common mutations of the CFTR gene in peruvian patients with cystic fibrosis using the ARMS-PCR technique].

PubMed

Aquino, Ruth; Protzel, Ana; Rivera, Juan; Abarca, Hugo; Dueñas, Milagros; Nestarez, Cecilia; Purizaga, Nestor; Diringer, Benoit

2017-01-01

To determine the frequency of the ten most common mutations of the CFTR gene reported in Latin Americausing amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR) in patients with cystic fibrosis (CF) in two referral hospitals in Peru during the year 2014. The frequency of the ten most common mutations of the CFTR gene was assessed in patients of the Hospital Nacional Edgardo Rebagliati Martins and the Instituto Nacional de Salud del Niño, both located in Lima, Peru. Blood samples were collected from 36 patients with CF, and the ARMS-PCR technique was used to determine the presence of these mutations. The study group included 73.5% of patients with a known diagnosis of CF in the country when the study was carried out. ARMS-PCR allowed three of the mutations to be identified in a combined 30.6% of the alleles from patients with CF, and 64.9% of the mutated alleles were not identified. The mutations found were p.Phe508del (22,2%), p.Gly542* (6,9%), and p.Arg1162* (1,4%). There is significant variability in both the frequency and type of mutations present in our study population and in what has been reported in other Latin American countries. It is necessary to perform studies that use complete sequencing technology for the CFTR gene to identify other mutations present in our population.

Genetic deletion of keratin 8 corrects the altered bone formation and osteopenia in a mouse model of cystic fibrosis.

PubMed

Le Henaff, Carole; Faria Da Cunha, Mélanie; Hatton, Aurélie; Tondelier, Danielle; Marty, Caroline; Collet, Corinne; Zarka, Mylène; Geoffroy, Valérie; Zatloukal, Kurt; Laplantine, Emmanuel; Edelman, Aleksander; Sermet-Gaudelus, Isabelle; Marie, Pierre J

2016-04-01

Patients with cystic fibrosis (CF) display low bone mass and alterations in bone formation. Mice carrying the F508del genetic mutation in the cystic fibrosis conductance regulator (Cftr) gene display reduced bone formation and decreased bone mass. However, the underlying molecular mechanisms leading to these skeletal defects are unknown, which precludes the development of an efficient anti-osteoporotic therapeutic strategy. Here we report a key role for the intermediate filament protein keratin 8 (Krt8), in the osteoblast dysfunctions in F508del-Cftr mice. We found that murine and human osteoblasts express Cftr and Krt8 at low levels. Genetic studies showed that Krt8 deletion (Krt8(-/-)) in F508del-Cftr mice increased the levels of circulating markers of bone formation, corrected the expression of osteoblast phenotypic genes, promoted trabecular bone formation and improved bone mass and microarchitecture. Mechanistically, Krt8 deletion in F508del-Cftr mice corrected overactive NF-κB signaling and decreased Wnt-β-catenin signaling induced by the F508del-Cftr mutation in osteoblasts. In vitro, treatment with compound 407, which specifically disrupts the Krt8-F508del-Cftr interaction in epithelial cells, corrected the abnormal NF-κB and Wnt-β-catenin signaling and the altered phenotypic gene expression in F508del-Cftr osteoblasts. In vivo, short-term treatment with 407 corrected the altered Wnt-β-catenin signaling and bone formation in F508del-Cftr mice. Collectively, the results show that genetic or pharmacologic targeting of Krt8 leads to correction of osteoblast dysfunctions, altered bone formation and osteopenia in F508del-Cftr mice, providing a therapeutic strategy targeting the Krt8-F508del-CFTR interaction to correct the abnormal bone formation and bone loss in cystic fibrosis. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Cystic fibrosis transmembrane regulator haplotypes in households of patients with cystic fibrosis.

PubMed

Furgeri, Daniela Tenório; Marson, Fernando Augusto Lima; Correia, Cyntia Arivabeni Araújo; Ribeiro, José Dirceu; Bertuzzo, Carmen Sílvia

2018-01-30

Nearly 2000 mutations in the cystic fibrosis transmembrane regulator (CFTR) gene have been reported. The F508del mutation occurs in approximately 50-65% of patients with cystic fibrosis (CF). However, molecular diagnosis is not always possible. Therefore, silent polymorphisms can be used to label the mutant allele in households of patients with CF. To verify the haplotypes of four polymorphisms at the CFTR locus in households of patients with CF for pre-fertilization, pre-implantation, and prenatal indirect mutation diagnosis to provide better genetic counseling for families and patients with CF and to associate the genotypes/haplotypes with the F508del mutation screening. GATT polymorphism analysis was performed using direct polymerase chain reaction amplification, and the MP6-D9, TUB09 and TUB18 polymorphism analyses were performed using restriction fragment length polymorphism. Nine haplotypes were found in 37 CFTR alleles, and of those, 24 were linked with the F508del mutation and 13 with other CFTR mutations. The 6 (GATT), C (MP6-D9), G (TUB09), and C (TUB18) haplotypes showed the highest prevalence (48%) of the mutant CFTR allele and were linked to the F508del mutation (64%). In 43% of households analyzed, at least one informative polymorphism can be used for the indirect diagnostic test. CFTR polymorphisms are genetic markers that are useful for identifying the mutant CFTR alleles in households of patients with CF when it is not possible to establish the complete CFTR genotype. Moreover, the polymorphisms can be used for indirect CFTR mutation identification in cases of pre-fertilization, pre-implantation and prenatal analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Modeling cystic fibrosis disease progression in patients with the rare CFTR mutation P67L.

PubMed

MacKenzie, Isobel E R; Paquette, Valerie; Gosse, Frances; George, Sheenagh; Chappe, Frederic; Chappe, Valerie

2017-05-01

The progression of cystic fibrosis (CF) in patients with the rare mutation P67L was examined to determine if it induced a milder form of CF compared to the common severe ΔF508 mutation. Parameters of lung function, level of bacterial infection, nutritional status and hospitalization were used to represent CF progression. Age at diagnosis and pancreatic status were used to assess CF presentation. Analysis of data from the CF Canada Registry collected over a 15-year period included 266 ΔF508/ΔF508 homozygote patients from CF clinics in Atlantic Canada and 26 compound heterozygote patients with the rare P67L mutation from clinics across Canada. Late age at diagnosis, high incidence of pancreatic sufficiency, maintained Body Mass Index (BMI) with age, delayed life-threatening bacterial infection, and fewer days in hospital were observed for P67L heterozygote patients included in this study. Although the decline of lung function did not differ from ΔF508 homozygotes, the fact that a greater proportion of P67L heterozygotes live to an older age suggests that lung function is not the primary factor determining CF progression for P67L heterozygote patients. The P67L mutation is associated with a mild disease, even when combined with the severe ΔF508 mutation. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Distribution of CFTR mutations in Eastern Hungarians: relevance to genetic testing and to the introduction of newborn screening for cystic fibrosis.

PubMed

Ivady, Gergely; Madar, Laszlo; Nagy, Bela; Gonczi, Ferenc; Ajzner, Eva; Dzsudzsak, Erika; Dvořáková, Lenka; Gombos, Eva; Kappelmayer, Janos; Macek, Milan; Balogh, Istvan

2011-05-01

The aim of this study was characterization of an updated distribution of CFTR mutations in a representative cohort of 40 CF patients with the classical form of the disease drawn from Eastern Hungary. Due to the homogeneity of the Hungarian population our data are generally applicable to other regions of the country, including the sizeable diaspora. We utilized the recommended "cascade" CFTR mutation screening approach, initially using a commercial assay, followed by examination of the common "Slavic" deletion CFTRdele2,3(21kb). Subsequently, the entire CFTR coding region of the CFTR gene was sequenced in patients with yet unidentified mutations. The Elucigene CF29(Tm) v2 assay detected 81.25% of all CF causing mutations. An addition of the CFTRdele2,3(21kb) increased the mutation detection rate to 86.25%. DNA sequencing enabled us to identify mutations on 79/80 CF alleles. Mutations [CFTRdele2,3(21kb), p.Gln685ThrfsX4 (2184insA) were found at an unusually high frequency, each comprising 5.00% of all CF alleles. We have identified common CF causing mutations in the Hungarian population with the most common mutations (p.Phe508del, p.Asn1303Lys, CFTRdele2,3(21kb), 2184insA, p.Gly542X, and p.Leu101X), comprising over 93.75% of all CF alleles. Obtained data are applicable to the improvement of DNA diagnostics in Hungary and beyond, and are the necessary prerequisite for the introduction of a nationwide "two tier" CF newborn screening program. Copyright © 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Proinflammatory effect of sodium 4-phenylbutyrate in deltaF508-cystic fibrosis transmembrane conductance regulator lung epithelial cells: involvement of extracellular signal-regulated protein kinase 1/2 and c-Jun-NH2-terminal kinase signaling.

PubMed

Roque, Telma; Boncoeur, Emilie; Saint-Criq, Vinciane; Bonvin, Elise; Clement, Annick; Tabary, Olivier; Jacquot, Jacky

2008-09-01

Sodium 4-phenylbutyrate (4-PBA) has attracted a great deal of attention in cystic fibrosis (CF) pathology due to its capacity to traffic DeltaF508-cystic fibrosis transmembrane conductance regulator (CFTR) to the cell membrane and restore CFTR chloride function at the plasma membrane of CF lung cells in vitro and in vivo. Using two different DeltaF508-CFTR lung epithelial cell lines (CFBE41o- and IB3-1 cells, characterized with DeltaF508-homozygous and heterozygous genotype, respectively) in vitro, 4-PBA induced an increase of proinflammatory cytokine interleukin (IL)-8 production in a concentration-dependent manner. This 4-PBA-induced IL-8 production was associated with a strong reduction of proteasome and nuclear factor-kappaB transcriptional activities in the two DeltaF508-CFTR lung cells either in a resting state or after tumor necrosis factor-alpha stimulation. In contrast, a strong increase of activator protein-1 transcriptional activity was observed. The inhibition of extracellular signal-regulated protein kinase 1/2 (ERK1/2) by 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126) and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) and c-Jun-NH(2)-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) by anthra[1,9-cd] pyrazol-6 (2H)-one (SP600125), respectively, was associated with a reduction (2-3.5-fold) of IL-8 production in both DeltaF508-CFTR lung cell lines treated with 4-PBA. No significant change of IL-8 production was observed after an inhibition of p38 MAPK with 4-[4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl] phenol (SB202190). Therefore, we suggest that inhibition of both ERK1/2 and JNK signaling may be a means to strongly reduce 4-PBA-induced IL-8 production in combination with 4-PBA treatment to restore CFTR Cl(-) channel function in lung epithelial cells of patients with CF.

Is congenital bilateral absence of vas deferens a primary form of cystic fibrosis? Analyses of the CFTR gene in 67 patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mercier, B.; Verlingue, C.; Audrezet, M.P.

1995-01-01

Congenital bilateral absence of the vas deferens (CBAVD) is an important cause of sterility in men. Although the genetic basis of this condition is still unclear, it has been shown recently that some of these patients carry mutations in their cystic fibrosis transmembrane conductance regulator (CFTR) genes. To extend this observation, we have analyzed the entire coding sequence of the CFTR gene in a cohort of 67 men with CBAVD, who are otherwise healthy. We have identified four novel missense mutations (A800G, G149R, R258G, and E193K). We have shown that 42% of subjects were carriers of one CFTR allele andmore » that 24% are compound heterozygous for CFTR alleles. Thus, we have been unable to identify 76% of these patients as carrying two CFTR mutations. Furthermore, we have described the segregation of CFTR haplotypes in the family of one CBAVD male; in this family are two male siblings, with identical CFTR loci but displaying different phenotypes, one of them being fertile and the other sterile. The data presented in this family, indicating a discordance between the CBAVD phenotype and a marked carrier ({delta}F508) chromosome, support the involvement of another gene(s), in the etiology of CBAVD. 35 refs., 2 figs., 1 tab.« less

From the endoplasmic reticulum to the plasma membrane: mechanisms of CFTR folding and trafficking.

PubMed

Farinha, Carlos M; Canato, Sara

2017-01-01

CFTR biogenesis starts with its co-translational insertion into the membrane of endoplasmic reticulum and folding of the cytosolic domains, towards the acquisition of a fully folded compact native structure. Efficiency of this process is assessed by the ER quality control system that allows the exit of folded proteins but targets unfolded/misfolded CFTR to degradation. If allowed to leave the ER, CFTR is modified at the Golgi and reaches the post-Golgi compartments to be delivered to the plasma membrane where it functions as a cAMP- and phosphorylation-regulated chloride/bicarbonate channel. CFTR residence at the membrane is a balance of membrane delivery, endocytosis, and recycling. Several adaptors, motor, and scaffold proteins contribute to the regulation of CFTR stability and are involved in continuously assessing its structure through peripheral quality control systems. Regulation of CFTR biogenesis and traffic (and its dysregulation by mutations, such as the most common F508del) determine its overall activity and thus contribute to the fine modulation of chloride secretion and hydration of epithelial surfaces. This review covers old and recent knowledge on CFTR folding and trafficking from its synthesis to the regulation of its stability at the plasma membrane and highlights how several of these steps can be modulated to promote the rescue of mutant CFTR.

Four novel cystic fibrosis mutations in splice junction sequences affecting the CFTR nucleotide binding folds

DOE Office of Scientific and Technical Information (OSTI.GOV)

Doerk, T.; Wulbrand, U.; Tuemmler, B.

1993-03-01

Single cases of the four novel splice site mutations 1525[minus]1 G [r arrow] A (intron 9), 3601[minus]2 A [r arrow] G (intron 18), 3850[minus]3 T [r arrow] G (intron 19), and 4374+1 G [r arrow] T (intron 23) were detected in the CFTR gene of cystic fibrosis patients of Indo-Iranian, Turkish, Polish, and Germany descent. The nucleotide substitutions at the +1, [minus]1, and [minus]2 positions all destroy splice sites and lead to severe disease alleles associated with features typical of gastrointestinal and pulmonary cystic fibrosis disease. The 3850[minus]3 T-to-G change was discovered in a very mildly affected 33-year-old [Delta]F508 compoundmore » heterozygote, suggesting that the T-to-G transversion at the less conserved [minus]3 position of the acceptor splice site may retain some wildtype function. 13 refs., 1 fig., 2 tabs.« less

Novel short chain fatty acids restore chloride secretion in cystic fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nguyen, Toan D.; Kim, Ug-Sung; Perrine, Susan P.

2006-03-31

Phenylalanine deletion at position 508 of the cystic fibrosis transmembrane conductance regulator ({delta}F508-CFTR), the most common mutation in cystic fibrosis (CF), causes a misfolded protein exhibiting partial chloride conductance and impaired trafficking to the plasma membrane. 4-Phenylbutyrate corrects defective {delta}F508-CFTR trafficking in vitro, but is not clinically efficacious. From a panel of short chain fatty acid derivatives, we showed that 2,2-dimethyl-butyrate (ST20) and {alpha}-methylhydrocinnamic acid (ST7), exhibiting high oral bioavailability and sustained plasma levels, correct the {delta}F508-CFTR defect. Pre-incubation ({>=}6 h) of CF IB3-1 airway cells with {>=}1 mM ST7 or ST20 restored the ability of 100 {mu}M forskolin tomore » stimulate an {sup 125}I{sup -} efflux. This efflux was fully inhibited by NPPB, DPC, or glibenclamide, suggesting mediation through CFTR. Partial inhibition by DIDS suggests possible contribution from an additional Cl{sup -} channel regulated by CFTR. Thus, ST7 and ST20 offer treatment potential for CF caused by the {delta}F508 mutation.« less

Assembly and misassembly of cystic fibrosis transmembrane conductance regulator: folding defects caused by deletion of F508 occur before and after the calnexin-dependent association of membrane spanning domain (MSD) 1 and MSD2.

PubMed

Rosser, Meredith F N; Grove, Diane E; Chen, Liling; Cyr, Douglas M

2008-11-01

Cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic membrane protein that functions as a Cl(-) channel and consists of two membrane spanning domains (MSDs), two cytosolic nucleotide binding domains (NBDs), and a cytosolic regulatory domain. Cytosolic 70-kDa heat shock protein (Hsp70), and endoplasmic reticulum-localized calnexin are chaperones that facilitate CFTR biogenesis. Hsp70 functions in both the cotranslational folding and posttranslational degradation of CFTR. Yet, the mechanism for calnexin action in folding and quality control of CFTR is not clear. Investigation of this question revealed that calnexin is not essential for CFTR or CFTRDeltaF508 degradation. We identified a dependence on calnexin for proper assembly of CFTR's membrane spanning domains. Interestingly, efficient folding of NBD2 was also found to be dependent upon calnexin binding to CFTR. Furthermore, we identified folding defects caused by deletion of F508 that occurred before and after the calnexin-dependent association of MSD1 and MSD2. Early folding defects are evident upon translation of the NBD1 and R-domain and are sensed by the RMA-1 ubiquitin ligase complex.

21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... regulator (CFTR) gene mutation detection system. 866.5900 Section 866.5900 Food and Drugs FOOD AND DRUG...) gene mutation detection system. (a) Identification. The CFTR gene mutation detection system is a device used to simultaneously detect and identify a panel of mutations and variants in the CFTR gene. It is...

21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... regulator (CFTR) gene mutation detection system. 866.5900 Section 866.5900 Food and Drugs FOOD AND DRUG...) gene mutation detection system. (a) Identification. The CFTR gene mutation detection system is a device used to simultaneously detect and identify a panel of mutations and variants in the CFTR gene. It is...

21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... regulator (CFTR) gene mutation detection system. 866.5900 Section 866.5900 Food and Drugs FOOD AND DRUG...) gene mutation detection system. (a) Identification. The CFTR gene mutation detection system is a device used to simultaneously detect and identify a panel of mutations and variants in the CFTR gene. It is...

21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... regulator (CFTR) gene mutation detection system. 866.5900 Section 866.5900 Food and Drugs FOOD AND DRUG...) gene mutation detection system. (a) Identification. The CFTR gene mutation detection system is a device used to simultaneously detect and identify a panel of mutations and variants in the CFTR gene. It is...

Cystic fibrosis transmembrane conductance regulator modulators: precision medicine in cystic fibrosis.

PubMed

Burgener, Elizabeth B; Moss, Richard B

2018-06-01

The aim of this study was to describe the newest development in cystic fibrosis (CF) care, CF transmembrane conductance regulator (CFTR) modulator therapies. Phase II results showing CFTR modulator triple therapies are more effective than current CFTR modulators. CFTR modulator therapy targets the protein defective in CF and boosts its function, but the drug must match mutation pathobiology. Ivacaftor, a CFTR potentiator, was the first modulator approved in 2012, with impressive improvement in lung function and other measures of disease in patients with gating and other residual function mutations (∼10% of CF patients). In 2015, the combination of lumacaftor, a CFTR corrector, and ivacaftor was approved for patients homozygous for the F508del mutation (∼40-50% of the CF population) with positive but less impressive clinical response and 10-20% incidence of intolerance. A next-generation CFTR corrector, tezacaftor, with ivacaftor equally effective and better tolerated than lumacaftor, has also received US Food and Drug Administration approval. Novel CFTR correctors, entering Phase 3 trials in triple modulator combination with tezacaftor-ivacaftor, appear substantially more effective for patients who are homozygous for the F508del mutation and can provide benefit for patients with a single F508del mutation. This offers promise of effective CFTR modulator therapy for nearly 90% of CF patients.

Nasal potential difference in cystic fibrosis considering severe CFTR mutations.

PubMed

Ng, Ronny Tah Yen; Marson, Fernando Augusto de Lima; Ribeiro, Jose Dirceu; Ribeiro, Antonio Fernando; Bertuzzo, Carmen Silvia; Ribeiro, Maria Angela Gonçalves de Oliveira; Severino, Silvana Dalge; Sakano, Eulalia

2015-01-01

The gold standard for diagnosing cystic fibrosis (CF) is a sweat chloride value above 60 mEq/L. However, this historical and important tool has limitations; other techniques should be studied, including the nasal potential difference (NPD) test. CFTR gene sequencing can identify CFTR mutations, but this method is time-consuming and too expensive to be used in all CF centers. The present study compared CF patients with two classes I-III CFTR mutations (10 patients) (G1), CF patients with classes IV-VI CFTR mutations (five patients) (G2), and 21 healthy subjects (G3). The CF patients and healthy subjects also underwent the NPD test. A statistical analysis was performed using the Mann-Whitney, Kruskal-Wallis, χ(2), and Fisher's exact tests, α = 0.05. No differences were observed between the CF patients and healthy controls for the PDMax, Δamiloride, and Δchloride + free + amiloride markers from the NPD test. For the finger value, a difference between G2 and G3 was described. The Wilschanski index values were different between G1 and G3. In conclusion, our data showed that NPD is useful for CF diagnosis when classes I-III CFTR mutations are screened. However, if classes IV-VI are considered, the NPD test showed an overlap in values with healthy subjects.

Nasal Potential Difference in Cystic Fibrosis considering Severe CFTR Mutations

PubMed Central

Ng, Ronny Tah Yen; Marson, Fernando Augusto de Lima; Ribeiro, Jose Dirceu; Ribeiro, Antonio Fernando; Bertuzzo, Carmen Silvia; Ribeiro, Maria Angela Gonçalves de Oliveira; Severino, Silvana Dalge; Sakano, Eulalia

2015-01-01

The gold standard for diagnosing cystic fibrosis (CF) is a sweat chloride value above 60 mEq/L. However, this historical and important tool has limitations; other techniques should be studied, including the nasal potential difference (NPD) test. CFTR gene sequencing can identify CFTR mutations, but this method is time-consuming and too expensive to be used in all CF centers. The present study compared CF patients with two classes I-III CFTR mutations (10 patients) (G1), CF patients with classes IV-VI CFTR mutations (five patients) (G2), and 21 healthy subjects (G3). The CF patients and healthy subjects also underwent the NPD test. A statistical analysis was performed using the Mann-Whitney, Kruskal-Wallis, χ 2, and Fisher's exact tests, α = 0.05. No differences were observed between the CF patients and healthy controls for the PDMax, Δamiloride, and Δchloride + free + amiloride markers from the NPD test. For the finger value, a difference between G2 and G3 was described. The Wilschanski index values were different between G1 and G3. In conclusion, our data showed that NPD is useful for CF diagnosis when classes I-III CFTR mutations are screened. However, if classes IV-VI are considered, the NPD test showed an overlap in values with healthy subjects. PMID:25667564

Structural effects of extracellular loop mutations in CFTR helical hairpins.

PubMed

Chang, Yuan-Heng; Stone, Tracy A; Chin, Stephanie; Glibowicka, Mira; Bear, Christine E; Deber, Charles M

2018-05-01

Missense mutations constitute 40% of 2000 cystic fibrosis-phenotypic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) database, yet the precise mechanism as to how a point mutation can render the entire 1480-residue CFTR protein dysfunctional is not well-understood. Here we investigate the structural effects of two CF-phenotypic mutations - glutamic acid to glycine at position 217 (E217G) and glutamine to arginine at position 220 (Q220R) - in the extracellular (ECL2) loop region of human CFTR using helical hairpin constructs derived from transmembrane (TM) helices 3 and 4 of the first membrane domain. We systematically replaced the wild type (WT) residues E217 and Q220 with the subset of missense mutations that could arise through a single nucleotide change in their respective codons. Circular dichroism spectra of E217G revealed that a significant increase in helicity vs. WT arises in the membrane-mimetic environment of sodium dodecylsulfate (SDS) micelles, while this mutant showed a similar gel shift to WT on SDS-PAGE gels. In contrast, the CF-mutant Q220R showed similar helicity but an increased gel shift vs. WT. These structural variations are compared with the maturation levels of the corresponding mutant full-length CFTRs, which we found are reduced to approx. 50% for E217G and 30% for Q220R vs. WT. The overall results with CFTR hairpins illustrate the range of impacts that single mutations can evoke in intramolecular protein-protein and/or protein-lipid interactions - and the levels to which corresponding mutations in full-length CFTR may be flagged by quality control mechanisms during biosynthesis. Copyright © 2018 Elsevier B.V. All rights reserved.

Cftr gene targeting in mouse embryonic stem cells mediated by Small Fragment Homologous Replacement (SFHR).

PubMed

Sangiuolo, Federica; Scaldaferri, Maria Lucia; Filareto, Antonio; Spitalieri, Paola; Guerra, Lorenzo; Favia, Maria; Caroppo, Rosa; Mango, Ruggiero; Bruscia, Emanuela; Gruenert, Dieter C; Casavola, Valeria; De Felici, Massimo; Novelli, Giuseppe

2008-01-01

Different gene targeting approaches have been developed to modify endogenous genomic DNA in both human and mouse cells. Briefly, the process involves the targeting of a specific mutation in situ leading to the gene correction and the restoration of a normal gene function. Most of these protocols with therapeutic potential are oligonucleotide based, and rely on endogenous enzymatic pathways. One gene targeting approach, "Small Fragment Homologous Replacement (SFHR)", has been found to be effective in modifying genomic DNA. This approach uses small DNA fragments (SDF) to target specific genomic loci and induce sequence and subsequent phenotypic alterations. This study shows that SFHR can stably introduce a 3-bp deletion (deltaF508, the most frequent cystic fibrosis (CF) mutation) into the Cftr (CF Transmembrane Conductance Regulator) locus in the mouse embryonic stem (ES) cell genome. After transfection of deltaF508-SDF into murine ES cells, SFHR-mediated modification was evaluated at the molecular levels on DNA and mRNA obtained from transfected ES cells. About 12% of transcript corresponding to deleted allele was detected, while 60% of the electroporated cells completely lost any measurable CFTR-dependent chloride efflux. The data indicate that the SFHR technique can be used to effectively target and modify genomic sequences in ES cells. Once the SFHR-modified ES cells differentiate into different cell lineages they can be useful for elucidating tissue-specific gene function and for the development of transplantation-based cellular and therapeutic protocols.

Regulatory domain phosphorylation to distinguish the mechanistic basis underlying acute CFTR modulators

PubMed Central

Pyle, Louise C.; Ehrhardt, Annette; Mitchell, Lisa High; Fan, LiJuan; Ren, Aixia; Naren, Anjaparavanda P.; Li, Yao; Clancy, J. P.; Bolger, Graeme B.; Sorscher, Eric J.

2011-01-01

Modulator compounds intended to overcome disease-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) show significant promise in clinical testing for cystic fibrosis. However, the mechanism(s) of action underlying these compounds are not fully understood. Activation of CFTR ion transport requires PKA-regulated phosphorylation of the regulatory domain (R-D) and dimerization of the nucleotide binding domains. Using a newly developed assay, we evaluated nine compounds including both CFTR potentatiators and activators discovered via various high-throughput screening strategies to acutely augment CFTR activity. We found considerable differences in the effects on R-D phosphorylation. Some (including UCCF-152) stimulated robust phosphorylation, and others had little effect (e.g., VRT-532 and VX-770). We then compared CFTR activation by UCCF-152 and VRT-532 in Ussing chamber studies using two epithelial models, CFBE41o− and Fischer rat thyroid cells, expressing various CFTR forms. UCCF-152 activated wild-type-, G551D-, and rescued F508del-CFTR currents but did not potentiate cAMP-mediated CFTR activation. In contrast, VRT-532 moderately activated CFTR short-circuit current and strongly potentiated forskolin-mediated current. Combined with the result that UCCF-152, but not VRT-532 or VX-770, acts by increasing CFTR R-D phosphorylation, these findings indicate that potentiation of endogenous cAMP-mediated activation of mutant CFTR is not due to a pathway involving augmented R-D phosphorylation. This study presents an assay useful to distinguish preclinical compounds by a crucial mechanism underlying CFTR activation, delineates two types of compound able to acutely augment CFTR activity (e.g., activators and potentiators), and demonstrates that a number of different mechanisms can be successfully employed to activate mutant CFTR. PMID:21724857

Non-native Conformers of Cystic Fibrosis Transmembrane Conductance Regulator NBD1 Are Recognized by Hsp27 and Conjugated to SUMO-2 for Degradation.

PubMed

Gong, Xiaoyan; Ahner, Annette; Roldan, Ariel; Lukacs, Gergely L; Thibodeau, Patrick H; Frizzell, Raymond A

2016-01-22

A newly identified pathway for selective degradation of the common mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), F508del, is initiated by binding of the small heat shock protein, Hsp27. Hsp27 collaborates with Ubc9, the E2 enzyme for protein SUMOylation, to selectively degrade F508del CFTR via the SUMO-targeted ubiquitin E3 ligase, RNF4 (RING finger protein 4) (1). Here, we ask what properties of CFTR are sensed by the Hsp27-Ubc9 pathway by examining the ability of NBD1 (locus of the F508del mutation) to mimic the disposal of full-length (FL) CFTR. Similar to FL CFTR, F508del NBD1 expression was reduced 50-60% by Hsp27; it interacted preferentially with the mutant and was modified primarily by SUMO-2. Mutation of the consensus SUMOylation site, Lys(447), obviated Hsp27-mediated F508del NBD1 SUMOylation and degradation. As for FL CFTR and NBD1 in vivo, SUMO modification using purified components in vitro was greater for F508del NBD1 versus WT and for the SUMO-2 paralog. Several findings indicated that Hsp27-Ubc9 targets the SUMOylation of a transitional, non-native conformation of F508del NBD1: (a) its modification decreased as [ATP] increased, reflecting stabilization of the nucleotide-binding domain by ligand binding; (b) a temperature-induced increase in intrinsic fluorescence, which reflects formation of a transitional NBD1 conformation, was followed by its SUMO modification; and (c) introduction of solubilizing or revertant mutations to stabilize F508del NBD1 reduced its SUMO modification. These findings indicate that the Hsp27-Ubc9 pathway recognizes a non-native conformation of mutant NBD1, which leads to its SUMO-2 conjugation and degradation by the ubiquitin-proteasome system. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Ivacaftor treatment of cystic fibrosis patients with the G551D mutation: a review of the evidence.

PubMed

Kotha, Kavitha; Clancy, John P

2013-10-01

Cystic fibrosis (CF) is a recessive disorder caused by mutations in the gene that encodes the CF transmembrane conductance regulator (CFTR) protein. CFTR protein is a chloride and bicarbonate channel that is critical for normal epithelial ion transport and hydration of epithelial surfaces. Current CF care is supportive, but recent breakthroughs have occurred with the advent of novel therapeutic strategies that assist the function of mutant CFTR proteins. The development and key clinical trial results of ivacaftor, a small molecule that targets gating defects in disease-causing CFTR mutations including G551D CFTR, are summarized in this review. The G551D mutation is reasonably common in the CF patient population and produces a CFTR protein that localizes normally to the plasma membrane, but fails to open in response to cellular cues. Ivacaftor treatment produces dramatic improvements in lung function, weight, lung disease stability, patient-reported outcomes, and CFTR biomarkers in patients with CF harboring the G551D CFTR mutation compared with placebo controls and patients with two copies of the common F508del CFTR mutation. The unprecedented success of ivacaftor treatment for the G551D CF patient population has generated excitement in the CF care community regarding the expansion of its use to other CF patient populations with primary or secondary gating defects.

Quantitation of normal CFTR mRNA in CF patients with splice-site mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Z.; Olsen, J.C.; Silverman, L.M.

Previously we identified two mutations in introns of the CFTR gene associated with partially active splice sites and unusual clinical phenotypes. One mutation in intron 19 (3849+10 kb C to T) is common in CF patients with normal sweat chloride values; an 84 bp sequence from intron 19, which contains a stop codon, is inserted between exon 19 and exon 20 in most nasal CFTR transcripts. The other mutation in intron 14B (2789+5 G to A) is associated with elevated sweat chloride levels, but mild pulmonary disease; exon 14B (38 bp) is spliced out of most nasal CFTR transcipts. Themore » remaining CFTR cDNA sequences, other than the 84 bp insertion of exon 14B deletion, are identical to the published sequence. To correlate genotype and phenotype, we used quantitative RT-PCR to determine the levels of normally-spliced CFTR mRNA in nasal epithelia from these patients. CFTR cDNA was amplified (25 cycles) by using primers specific for normally-spliced species, {gamma}-actin cDNA was amplified as a standard.« less

Disease-causing Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator Determine the Functional Responses of Alveolar Macrophages*

PubMed Central

Deriy, Ludmila V.; Gomez, Erwin A.; Zhang, Guangping; Beacham, Daniel W.; Hopson, Jessika A.; Gallan, Alexander J.; Shevchenko, Pavel D.; Bindokas, Vytautas P.; Nelson, Deborah J.

2009-01-01

Alveolar macrophages (AMs) play a major role in host defense against microbial infections in the lung. To perform this function, these cells must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Recently, we demonstrated that murine alveolar macrophages employ the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel as a determinant in lysosomal acidification (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933–944). Lysosomes and phagosomes in murine cftr−/− AMs failed to acidify, and the cells were deficient in bacterial killing compared with wild type controls. Cystic fibrosis is caused by mutations in CFTR and is characterized by chronic lung infections. The information about relationships between the CFTR genotype and the disease phenotype is scarce both on the organismal and cellular level. The most common disease-causing mutation, ΔF508, is found in 70% of patients with cystic fibrosis. The mutant protein fails to fold properly and is targeted for proteosomal degradation. G551D, the second most common mutation, causes loss of function of the protein at the plasma membrane. In this study, we have investigated the impact of CFTR ΔF508 and G551D on a set of core intracellular functions, including organellar acidification, granule secretion, and microbicidal activity in the AM. Utilizing primary AMs from wild type, cftr−/−, as well as mutant mice, we show a tight correlation between CFTR genotype and levels of lysosomal acidification, bacterial killing, and agonist-induced secretory responses, all of which would be expected to contribute to a significant impact on microbial clearance in the lung. PMID:19837664

Investigating CFTR and KCa3.1 Protein/Protein Interactions

PubMed Central

Trinh, Nguyen Thu Ngan; Luo, Yishan; Wiseman, Paul W.; Hanrahan, John W.; Brochiero, Emmanuelle; Sauvé, Rémy

2016-01-01

In epithelia, Cl- channels play a prominent role in fluid and electrolyte transport. Of particular importance is the cAMP-dependent cystic fibrosis transmembrane conductance regulator Cl- channel (CFTR) with mutations of the CFTR encoding gene causing cystic fibrosis. The bulk transepithelial transport of Cl- ions and electrolytes needs however to be coupled to an increase in K+ conductance in order to recycle K+ and maintain an electrical driving force for anion exit across the apical membrane. In several epithelia, this K+ efflux is ensured by K+ channels, including KCa3.1, which is expressed at both the apical and basolateral membranes. We show here for the first time that CFTR and KCa3.1 can physically interact. We first performed a two-hybrid screen to identify which KCa3.1 cytosolic domains might mediate an interaction with CFTR. Our results showed that both the N-terminal fragment M1-M40 of KCa3.1 and part of the KCa3.1 calmodulin binding domain (residues L345-A400) interact with the NBD2 segment (G1237-Y1420) and C- region of CFTR (residues T1387-L1480), respectively. An association of CFTR and F508del-CFTR with KCa3.1 was further confirmed in co-immunoprecipitation experiments demonstrating the formation of immunoprecipitable CFTR/KCa3.1 complexes in CFBE cells. Co-expression of KCa3.1 and CFTR in HEK cells did not impact CFTR expression at the cell surface, and KCa3.1 trafficking appeared independent of CFTR stimulation. Finally, evidence is presented through cross-correlation spectroscopy measurements that KCa3.1 and CFTR colocalize at the plasma membrane and that KCa3.1 channels tend to aggregate consequent to an enhanced interaction with CFTR channels at the plasma membrane following an increase in intracellular Ca2+ concentration. Altogether, these results suggest 1) that the physical interaction KCa3.1/CFTR can occur early during the biogenesis of both proteins and 2) that KCa3.1 and CFTR form a dynamic complex, the formation of which depends on

Rescuing mutant CFTR: a multi-task approach to a better outcome in treating cystic fibrosis.

PubMed

Amaral, Margarida D; Farinha, Carlos M

2013-01-01

Correcting multiple defects of mutant CFTR with small molecule compounds has been the goal of an increasing number of recent Cystic Fibrosis (CF) drug discovery programmes. However, the mechanism of action (MoA) by which these molecules restore mutant CFTR is still poorly understood, in particular of CFTR correctors, i.e., compounds rescuing to the cells surface the most prevalent mutant in CF patients--F508del-CFTR. However, there is increasing evidence that to fully restore the multiple defects associated with F508del-CFTR, different small molecules with distinct corrective properties may be required. Towards this goal, a better insight into MoA of correctors is needed and several constraints should be addressed. The methodological approaches to achieve this include: 1) testing the combined effect of compounds with that of other (non-pharmacological) rescuing strategies (e.g., revertants or low temperature); 2) assessing effects in multiple cellular models (non-epithelial vs epithelial, non-human vs human, immortalized vs primary cultures, polarized vs non polarized, cells vs tissues); 3) assessing compound effects on isolated CFTR domains (e.g., compound binding by surface plasmon resonance, assessing effects on domain folding and aggregation); and finally 4) assessing compounds specificity in rescuing different CFTR mutants and other mutant proteins. These topics are reviewed and discussed here so as to provide a state-of-the art review on how to combine multiple ways of rescuing mutant CFTR to the ultimate benefit of CF patients.

Electrophysiological Evidence for the Presence of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Mouse Sperm

PubMed Central

Dulce, Figueiras Fierro; José, Acevedo Juan; Pablo, Martínez; Escoffier, Jessica; Sepúlveda, Francisco V.; Enrique, Balderas; Gerardo, Orta; Pablo, Visconti; Alberto, Darszon

2014-01-01

Mammalian sperm must undergo a maturational process, named capacitation, in the female reproductive tract to fertilize the egg. Sperm capacitation is regulated by a cAMP/PKA pathway and involves increases in intracellular Ca2+, pH, Cl−, protein tyrosine phosphorylation, and in mouse and some other mammals a membrane potential hyperpolarization. The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl− channel modulated by cAMP/PKA and ATP, was detected in mammalian sperm and proposed to modulate capacitation. Our whole-cell patch-clamp recordings from testicular mouse sperm now reveal a Cl− selective component to membrane current that is ATP-dependent, stimulated by cAMP, cGMP and genistein (a CFTR agonist, at low concentrations), and inhibited by DPC and CFTRinh-172, two well-known CFTR antagonists. Furthermore, the Cl− current component activated by cAMP and inhibited by CFTRinh-172 is absent in recordings on testicular sperm from mice possessing the CFTR ΔF508 loss-of-function mutation, indicating that CFTR is responsible for this component. A Cl− selective like current component displaying CFTR characteristics was also found in wild type epididymal sperm bearing the cytoplasmatic droplet. Capacitated sperm treated with CFTRinh-172 undergo a shape change, suggesting that CFTR is involved in cell volume regulation. These findings indicate that functional CFTR channels are present in mouse sperm and their biophysical properties are consistent with their proposed participation in capacitation. PMID:22833409

Screening for F508del as a first step in the molecular diagnosis of cystic fibrosis.

PubMed

Marson, Fernando Augusto de Lima; Bertuzzo, Carmen Silvia; Ribeiro, Maria Ângela Gonçalves de Oliveira; Ribeiro, Antônio Fernando; Ribeiro, José Dirceu

2013-01-01

To determine the relevance of screening for the F508del mutation of the cystic fibrosis transmembrane conductance regulator gene as a first step in the genetic diagnosis of cystic fibrosis (CF) by associating the genotype with various clinical variables. We evaluated 180 CF patients regarding the F508del mutation. The clinical data were obtained from the medical records of the patients and from interviews with their parents or legal guardians. Of the 180 patients studied, 65 (36.1%) did not carry the F508del mutation (group 0 [G0]), 67 (37.2%) were F508del heterozygous (G1), and 48 (26.7%) were F508del homozygous (G2). All three groups showed associations with the clinical variables. Homozygosis was associated with younger patients, younger age at CF diagnosis, and younger age at the first isolation of Pseudomonas aeruginosa (PA), as well as with higher prevalence of pancreatic insufficiency (PI) and non-mucoid PA (NMPA) colonization. In comparison with G1+G2 patients, G0 patients were older; first experienced clinical symptoms, digestive disease, and pulmonary disease at an older age; were older at CF diagnosis and at first PA isolation; and had a lower prevalence of PI and meconium ileus, as well as of colonization by NMPA, mucoid PA, and Burkholderia cepacia. In G1 patients, values were intermediate for age at CF diagnosis; age at first PA isolation, first pulmonary symptoms, and first clinical manifestations; MPA colonization; and OR for PI. The identification of F508del in 63.9% of the patients studied showed that this can be a useful tool as a first step in the genetic diagnosis of CF. The F508del genotype was associated with clinical severity of the disease, especially with the variables related to CF onset.

Assessment of safety and efficacy of long-term treatment with combination lumacaftor and ivacaftor therapy in patients with cystic fibrosis homozygous for the F508del-CFTR mutation (PROGRESS): a phase 3, extension study.

PubMed

Konstan, Michael W; McKone, Edward F; Moss, Richard B; Marigowda, Gautham; Tian, Simon; Waltz, David; Huang, Xiaohong; Lubarsky, Barry; Rubin, Jaime; Millar, Stefanie J; Pasta, David J; Mayer-Hamblett, Nicole; Goss, Christopher H; Morgan, Wayne; Sawicki, Gregory S

2017-02-01

The 24-week safety and efficacy of lumacaftor/ivacaftor combination therapy was shown in two randomised controlled trials (RCTs)-TRAFFIC and TRANSPORT-in patients with cystic fibrosis who were aged 12 years or older and homozygous for the F508del-CFTR mutation. We aimed to assess the long-term safety and efficacy of extended lumacaftor/ivacaftor therapy in this group of patients in PROGRESS, the long-term extension of TRAFFIC and TRANSPORT. PROGRESS was a phase 3, parallel-group, multicentre, 96-week study of patients who completed TRAFFIC or TRANSPORT in 191 sites in 15 countries. Patients were eligible if they were at least 12 years old with cystic fibrosis and homozygous for the F508del-CFTR mutation. Exclusion criteria included any comorbidity or laboratory abnormality that, in the opinion of the investigator, might confound the results of the study or pose an additional risk in administering the study drug to the participant, history of drug intolerance, and history of poor compliance with the study drug. Patients who previously received active treatment in TRANSPORT or TRAFFIC remained on the same dose in PROGRESS. Patients who had received placebo in TRANSPORT or TRAFFIC were randomly assigned (1:1) to receive lumacaftor (400 mg every 12 h)/ivacaftor (250 mg every 12 h) or lumacaftor (600 mg once daily)/ivacaftor (250 mg every 12 h). The primary outcome was to assess the long-term safety of combined therapy. The estimated annual rate of decline in percent predicted FEV 1 (ppFEV 1 ) in treated patients was compared with that of a matched registry cohort. Efficacy analyses were based on modified intention-to-treat, such that data were included for all patients who were randomly assigned and received at least one dose of study drug. This study is registered with ClinicalTrials.gov, number NCT01931839. Between Oct 24, 2013, and April 7, 2016, 1030 patients from the TRANSPORT and TRAFFIC studies enrolled in PROGRESS, and 1029 received at least one dose of study

Mutation detection by mismatch binding protein, MutS, in amplified DNA: Application to the cystic fibrosis gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lishanski, A.; Ostrander, E.A.; Rine, J.

1994-03-29

An experimental strategy for detecting heterozygosity in genomic DNA has been developed based on preferential binding of Escherichia coli MutS protein to DNA molecules containing mismatched bases. The binding was detected by a gel mobility-shift assay. This approach was tested by using as a model the most commonly occurring mutations within the cystic fibrosis (CFTR) gene. Genomic DNA samples were amplified with 5{prime}-end-labeled primers that bracket the site of the {Delta}F508 3-bp deletion in exon 10 of the CFTR gene. The renatured PCR products from homozygotes produced homoduplexes; the PCR products from heterozygotes produced heteroduplexes and homoduplexes (1:1). MutS proteinmore » bound more strongly to heteroduplexes that correspond to heterozygous carriers of {Delta}F508 and contain a CTT or a GAA loop in one of the strands than to homoduplexes corresponding to homozygotes. The ability of MutS protein to detect heteroduplexes in PCR-amplified DNA extended to fragments {approximately} 500 bp long. The method was also able to detect carriers of the point mutations in exon 11 of the CFTR gene by a preferential binding of MutS to single-base mismatches in PCR-amplified DNA.« less

A New Targeted CFTR Mutation Panel Based on Next-Generation Sequencing Technology.

PubMed

Lucarelli, Marco; Porcaro, Luigi; Biffignandi, Alice; Costantino, Lucy; Giannone, Valentina; Alberti, Luisella; Bruno, Sabina Maria; Corbetta, Carlo; Torresani, Erminio; Colombo, Carla; Seia, Manuela

2017-09-01

Searching for mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) is a key step in the diagnosis of and neonatal and carrier screening for cystic fibrosis (CF), and it has implications for prognosis and personalized therapy. The large number of mutations and genetic and phenotypic variability make this search a complex task. Herein, we developed, validated, and tested a laboratory assay for an extended search for mutations in CFTR using a next-generation sequencing-based method, with a panel of 188 CFTR mutations customized for the Italian population. Overall, 1426 dried blood spots from neonatal screening, 402 genomic DNA samples from various origins, and 1138 genomic DNA samples from patients with CF were analyzed. The assay showed excellent analytical and diagnostic operative characteristics. We identified and experimentally validated 159 (of 188) CFTR mutations. The assay achieved detection rates of 95.0% and 95.6% in two large-scale case series of CF patients from central and northern Italy, respectively. These detection rates are among the highest reported so far with a genetic test for CF based on a mutation panel. This assay appears to be well suited for diagnostics, neonatal and carrier screening, and assisted reproduction, and it represents a considerable advantage in CF genetic counseling. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Mutations of the cystic fibrosis gene, but not cationic trypsinogen gene, are associated with recurrent or chronic idiopathic pancreatitis.

PubMed

Ockenga, J; Stuhrmann, M; Ballmann, M; Teich, N; Keim, V; Dörk, T; Manns, M P

2000-08-01

We investigated whether mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and cationic trypsinogen gene are associated with recurrent acute, or chronic idiopathic pancreatitis. Twenty patients with idiopathic pancreatitis (11 women, nine men; mean age, 30 yr) were studied for the presence of a CFTR mutation by screening the genomic DNA for more than 30 mutations and variants in the CFTR gene. Selected mutations of the cationic trypsinogen gene were screened by Afl III restriction digestion or by a mutation-specific polymerase chain reaction (PCR). In each patient exons 1, 2, and 3 of the cationic trypsinogen gene were sequenced. Patients with a CFTR mutation underwent evaluation of further functional electrophysiological test (intestinal current measurement). No mutation of the cationic trypsinogen gene was detected. A CFTR mutation was detected in 6/20 (30.0%) patients. Three patients (15.0%) had a cystic fibrosis (CF) mutation on one chromosome (deltaF508, I336K, Y1092X), which is known to cause phenotypical severe cystic fibrosis. One patient was heterozygous for the 5T allele. In addition, two possibly predisposing CFTR variants (R75Q, 1716G-->A) were detected on four patients, one of these being a compound heterozygous for the missense mutation I336K and R75Q. No other family member (maternal I336K; paternal R75Q; sister I1336K) developed pancreatitis. An intestinal current measurement in rectum samples of patients with a CFTR mutation revealed no CF-typical constellations. CFTR mutations are associated with recurrent acute, or chronic idiopathic pancreatitis, whereas mutations of the cationic trypsinogen mutation do not appear to be a frequent pathogenetic factor.

Non-invasive prenatal diagnosis (NIPD) of cystic fibrosis: an optimized protocol using MEMO fluorescent PCR to detect the p.Phe508del mutation.

PubMed

Guissart, C; Dubucs, C; Raynal, C; Girardet, A; Tran Mau Them, F; Debant, V; Rouzier, C; Boureau-Wirth, A; Haquet, E; Puechberty, J; Bieth, E; Dupin Deguine, D; Khau Van Kien, P; Brechard, M P; Pritchard, V; Koenig, M; Claustres, M; Vincent, M C

2017-03-01

Analysis of cell-free foetal DNA (cff-DNA) in maternal plasma is very promising for early diagnosis of monogenic diseases; in particular, cystic fibrosis (CF). However, NIPD of single-gene disorders has been limited by the availability of suitable technical platforms and the need to set up patient or disease-specific custom-made approaches. To make research applications more readily accessible to the clinic, we offer a simple assay combining two independent methods to determine the presence or absence of paternally inherited foetal allele p.Phe508del (the most frequent mutation in CF patients worldwide). The first method detects the presence or absence of a p.Phe508del allele by Mutant Enrichment with 3'-Modified Oligonucleotide PCR coupled to Fragment Length Analysis (MEMO-PCR-FLA). The second method detects the p.Phe508del allele with classical Multiplex Fluorescent PCR including five intragenic and extragenic STR markers of the CFTR locus and a specific SRY sequence. We collected 24 plasma samples from 23 women carrying foetuses at risk for CF and tested each sample using both methods. Our new procedures were successfully applied to 10 couples where fathers carried the p.Phe508del mutation and mothers were carrying a different mutation in the CFTR gene. These simple tests provided clear positive or negative results from the maternal plasma of the pregnant women. We confirmed the presence of cff-DNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. All results were correlated with chorionic villus sampling or amniocentesis analyses. This NIPD approach, easily set up in any clinical laboratory where prenatal diagnosis is routinely performed, offers many advantages over current methods: it is simple, rapid, and cost-effective. It opens up the possibility for testing a large number of couples with offspring at risk for CF. Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lewis, Hal A.; Zhao, Xun; Wang, Chi

Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformationmore » but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.« less

The HDAC inhibitor SAHA does not rescue CFTR membrane expression in Cystic Fibrosis.

PubMed

Bergougnoux, Anne; Petit, Aurélie; Knabe, Lucie; Bribes, Estelle; Chiron, Raphaël; De Sario, Albertina; Claustres, Mireille; Molinari, Nicolas; Vachier, Isabelle; Taulan-Cadars, Magali; Bourdin, Arnaud

2017-07-01

The development of suitable Cystic Fibrosis (CF) models for preclinical bench tests of therapeutic candidates is challenging. Indeed, the validation of molecules to rescue the p.Phe508del-CFTR channel (encoded by the Cystic Fibrosis Transmembrane conductance Regulator gene carrying the p.Phe508del mutation) requires taking into account their overall effects on the epithelium. Suberoylanilide Hydroxamic Acid (SAHA), a histone deacetylase inhibitor (HDACi), was previously shown to be a CFTR corrector via proteostasis modulation in CFTR-deficient immortalized cells. Here, we tested SAHA effects on goblet cell metaplasia using an ex vivo model based on the air-liquid interface (ALI) culture of differentiated airway epithelial cells obtained by nasal scraping from CF patients and healthy controls. Ex vivo epithelium grew successfully in ALI cultures with significant rise in the expression of CFTR and of markers of airway epithelial differentiation compared to monolayer cell culture. SAHA decreased CFTR transcript and protein levels in CF and non-CF epithelia. Whereas SAHA induced lysine hyperacetylation, it did not change histone modifications at the CFTR promoter. SAHA reduced MUC5AC and MUC5B expression and inhibited goblet epithelial cell differentiation. Similar effects were obtained in CF and non-CF epithelia. All the effects were fully reversible within five days from SAHA withdrawal. We conclude that, ex vivo, SAHA modulate the structure of airway epithelia without specific effect on CFTR gene and protein suggesting that HDACi cannot be useful for CF treatment. Copyright © 2017. Published by Elsevier Ltd.

Frequency of 8 CFTR gene mutations in cystic fibrosis patients in Minas Gerais, Brazil, diagnosed by neonatal screening.

PubMed

Perone, C; Medeiros, G S; del Castillo, D M; de Aguiar, M J B; Januário, J N

2010-02-01

The nature and frequency of cystic fibrosis mutations in Brazil is not uniform due to the highly varied ethnic composition of the population. The average frequency of the F508del mutation has been reported to be 48.6%. Other common mutations in Brazil are G542X, R1162X, and N1303K. The aim of this study was to analyze the frequency of 8 mutations (F508del, G542X, R1162X, N1303K, W1282X, G85E, 3120+1G>A, and 711+1G>T) in a sample of 111 newborn patients with cystic fibrosis diagnosed by the Cystic Fibrosis Neonatal Screening Program of Minas Gerais State. The mutations were tested by allele-specific oligonucleotide PCR with specially designed primers. An allele frequency of 48.2% was observed for the F508del mutation, and allele frequencies of 5.41, 4.50, 4.05, and 3.60% were found for the R1162X, G542X, 3120+1G>A, and G85E mutations, respectively. The genotypes obtained were in Hardy-Weinberg equilibrium. These data demonstrate that the 8-mutation panel studied here has extensive coverage (68%) for the cystic fibrosis mutations in Minas Gerais. These data improve our knowledge of cystic fibrosis in Brazil, particularly in this region. In addition, this investigation contributed to the establishment of a sensitive and population-specific mutation panel, which can be helpful for molecular diagnosis of cystic fibrosis.

A Genotypic-Oriented View of CFTR Genetics Highlights Specific Mutational Patterns Underlying Clinical Macrocategories of Cystic Fibrosis

PubMed Central

Lucarelli, Marco; Bruno, Sabina Maria; Pierandrei, Silvia; Ferraguti, Giampiero; Stamato, Antonella; Narzi, Fabiana; Amato, Annalisa; Cimino, Giuseppe; Bertasi, Serenella; Quattrucci, Serena; Strom, Roberto

2015-01-01

Cystic fibrosis (CF) is a monogenic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The genotype–phenotype relationship in this disease is still unclear, and diagnostic, prognostic and therapeutic challenges persist. We enrolled 610 patients with different forms of CF and studied them from a clinical, biochemical, microbiological and genetic point of view. Overall, there were 125 different mutated alleles (11 with novel mutations and 10 with complex mutations) and 225 genotypes. A strong correlation between mutational patterns at the genotypic level and phenotypic macrocategories emerged. This specificity appears to largely depend on rare and individual mutations, as well as on the varying prevalence of common alleles in different clinical macrocategories. However, 19 genotypes appeared to underlie different clinical forms of the disease. The dissection of the pathway from the CFTR mutated genotype to the clinical phenotype allowed to identify at least two components of the variability usually found in the genotype–phenotype relationship. One component seems to depend on the genetic variation of CFTR, the other component on the cumulative effect of variations in other genes and cellular pathways independent from CFTR. The experimental dissection of the overall biological CFTR pathway appears to be a powerful approach for a better comprehension of the genotype–phenotype relationship. However, a change from an allele-oriented to a genotypic-oriented view of CFTR genetics is mandatory, as well as a better assessment of sources of variability within the CFTR pathway. PMID:25910067

Instability of the insertional mutation in CftrTgH(neoim)Hgu cystic fibrosis mouse model

PubMed Central

Charizopoulou, Nikoletta; Jansen, Silke; Dorsch, Martina; Stanke, Frauke; Dorin, Julia R; Hedrich, Hans-Jürgen; Tümmler, Burkhard

2004-01-01

Background A major boost to the cystic fibrosis disease research was given by the generation of various mouse models using gene targeting in embryonal stem cells. Moreover, the introduction of the same mutation on different inbred strains generating congenic strains facilitated the search for modifier genes. From the original CftrTgH(neoim)Hgu CF mouse model we have generated using strict brother × sister mating two inbred CftrTgH(neoim)Hgu mouse lines (CF/1 and CF/3). Thereafter, the insertional mutation was introgressed from CF/3 into three inbred backgrounds (C57BL/6, BALB/c, DBA/2J) generating congenic animals. In every backcross cycle germline transmission of the insertional mutation was monitored by direct probing the insertion via Southern RFLP. In order to bypass this time consuming procedure we devised an alternative PCR based protocol whereby mouse strains are differentiated at the Cftr locus by Cftr intragenic microsatellite genotypes that are tightly linked to the disrupted locus. Results Using this method we were able to identify animals carrying the insertional mutation based upon the differential haplotypic backgrounds of the three inbred strains and the mutant CftrTgH(neoim)Hgu at the Cftr locus. Moreover, this method facilitated the identification of the precise vector excision from the disrupted Cftr locus in two out of 57 typed animals. This reversion to wild type status took place without any loss of sequence revealing the instability of insertional mutations during the production of congenic animals. Conclusions We present intragenic microsatellite markers as a tool for fast and efficient identification of the introgressed locus of interest in the recipient strain during congenic animal breeding. Moreover, the same genotyping method allowed the identification of a vector excision event, posing questions on the stability of insertional mutations in mice. PMID:15102331

Structural stability of purified human CFTR is systematically improved by mutations in nucleotide binding domain 1.

PubMed

Yang, Zhengrong; Hildebrandt, Ellen; Jiang, Fan; Aleksandrov, Andrei A; Khazanov, Netaly; Zhou, Qingxian; An, Jianli; Mezzell, Andrew T; Xavier, Bala M; Ding, Haitao; Riordan, John R; Senderowitz, Hanoch; Kappes, John C; Brouillette, Christie G; Urbatsch, Ina L

2018-05-01

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is an ABC transporter containing two transmembrane domains forming a chloride ion channel, and two nucleotide binding domains (NBD1 and NBD2). CFTR has presented a formidable challenge to obtain monodisperse, biophysically stable protein. Here we report a comprehensive study comparing effects of single and multiple NBD1 mutations on stability of both the NBD1 domain alone and on purified full length human CFTR. Single mutations S492P, A534P, I539T acted additively, and when combined with M470V, S495P, and R555K cumulatively yielded an NBD1 with highly improved structural stability. Strategic combinations of these mutations strongly stabilized the domain to attain a calorimetric T m  > 70 °C. Replica exchange molecular dynamics simulations on the most stable 6SS-NBD1 variant implicated fluctuations, electrostatic interactions and side chain packing as potential contributors to improved stability. Progressive stabilization of NBD1 directly correlated with enhanced structural stability of full-length CFTR protein. Thermal unfolding of the stabilized CFTR mutants, monitored by changes in intrinsic fluorescence, demonstrated that Tm could be shifted as high as 67.4 °C in 6SS-CFTR, more than 20 °C higher than wild-type. H1402S, an NBD2 mutation, conferred CFTR with additional thermal stability, possibly by stabilizing an NBD-dimerized conformation. CFTR variants with NBD1-stabilizing mutations were expressed at the cell surface in mammalian cells, exhibited ATPase and channel activity, and retained these functions to higher temperatures. The capability to produce enzymatically active CFTR with improved structural stability amenable to biophysical and structural studies will advance mechanistic investigations and future cystic fibrosis drug development. Copyright © 2018 Elsevier B.V. All rights reserved.

Treatment burden in patients with at least one class IV or V CFTR mutation.

PubMed

Dewulf, Jonas; Vermeulen, François; Wanyama, Simeon; Thomas, Muriel; Proesmans, Marijke; Dupont, Lieven; De Boeck, Kris

2015-12-01

CFTR mutations are grouped according to disease-causing mechanism. Several studies demonstrated that patients having at least one mutation of class IV/V, present with a milder phenotype, but little is known about their relative treatment burden. We compared treatment burden between patients with two class I, II, or III mutations and patients with at least one mutation of class IV/V in the 2010 database of the Belgian CF Registry. We calculated a "Treatment Burden Index" (TBI) by assigning long term therapies to categories low, medium and high intensity, for differential weighing in the total score. There were 779 patients with two known class I/II/III mutations and 94 patients with at least one class IV/V mutation. Compared to class I/II/III, class IV/V patients had a lower median number of clinic visits (4 vs. 5; P < 0.001), a lower risk of hospitalization (24.7% vs. 50.8%; P < 0.001) and intravenous antibiotic treatment (23.5% vs. 46.0%; P < 0.001) and a lower median TBI (6 vs. 9; P < 0.001). These differences remained significant when only class IV/V patients with pancreatic insufficiency (n = 31) were considered. This study clearly demonstrates the significantly lower treatment burden in patients with CF and at least one class IV/V mutation compared to patients with two class I/II/III mutations and contributes to providing better individual counseling at time of diagnosis. © 2015 Wiley Periodicals, Inc.

Renal proximal tubule function is preserved in Cftrtm2camΔF508 cystic fibrosis mice

PubMed Central

Kibble, J D; Balloch, K J D; Neal, A M; Hill, C; White, S; Robson, L; Green, R; Taylor, C J

2001-01-01

Changes in proximal tubule function have been reported in cystic fibrosis patients. The aim of this study was to investigate proximal tubule function in the Cftrtm2camΔF508 cystic fibrosis (CF) mouse model. A range of techniques were used including renal clearance studies, in situ microperfusion, RT-PCR and whole-cell patch clamping. Renal Na+ clearance was similar in wild-type (1.4 ± 0.3 μl min−1, number of animals, N= 12) and CF mice (1.6 ± 0.4 μl min−1, N= 7) under control conditions. Acute extracellular volume expansion resulted in significant natriuresis in wild-type (7.0 ± 0.8 μl min−1, N= 8) and CF mice (9.3 ± 1.4 μl min−1, N= 9); no difference between genotypes was observed. In situ microperfusion revealed that fluid absorptive rate (Jv) was similar under control conditions between wild-type (2.2 ± 0.4 nl mm−1 min−1, n= 10) and CF mice (1.9 ± 0.3 nl mm−1 min−1, n= 11). Addition of a forskolin-dibutyryl cAMP (db-cAMP) cocktail to the perfusate caused no significant change in Jv in either wild-type (2.6 ± 0.7 nl mm−1 min−1, n= 10) or Cftrtm2camΔF508 mice (2.0 ± 0.5 nl mm−1 min−1, n= 10). CFTR expression was confirmed in samples of outer cortex using RT-PCR. However, no evidence for functional CFTR was obtained when outer cortical cells were stimulated with protein kinase A or forskolin-db-cAMP using whole-cell patch clamping. In conclusion, no functional deficit in proximal tubule function was found in Cftrtm2camΔF508 mice. This may be a consequence of a lack of whole-cell cAMP-dependent Cl− conductance in mouse proximal tubule cells. PMID:11306663

A new compound heterozygous CFTR mutation in a Chinese family with cystic fibrosis.

PubMed

Xie, Yingjun; Huang, Xueqiong; Liang, Yujian; Xu, Lingling; Pei, Yuxin; Cheng, Yucai; Zhang, Lidan; Tang, Wen

2017-11-01

Cystic fibrosis (CF) is the most common autosomal recessive disease among Caucasians but is rarer in the Chinese population, because mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. To elucidate the causative role of a novel compound heterozygous mutation of CF. In this study, clinical samples were obtained from two siblings with recurrent airway infections, clubbed fingers, salt-sweat and failure to gain weight in a non-consanguineous Chinese family. Next-generation sequencing was performed on the 27 coding exons of CFTR in both children, with confirmation by Sanger sequencing. Next-generation sequencing showed the same compound heterozygous CFTR mutation (c.865A>T p.Arg289X and c.3651_3652insAAAT p.Tyr1219X) in both children. As this mutation is consistent with the clinical manifestations of CF and no other mutations were detected after scanning the gene sequence, we suggest that the CF phenotype is caused by compound heterozygosity for c.865A>T and c.3651_3652insAAAT. As c865A>T is not currently listed in the "Cystic Fibrosis Mutation Database", this information about CF in a Chinese population is of interest. © 2015 John Wiley & Sons Ltd.

Chlorogenic Acid Activates CFTR-Mediated Cl- Secretion in Mice and Humans: Therapeutic Implications for Chronic Rhinosinusitis

PubMed Central

Illing, Elisa; Cho, Do-Yeon; Zhang, Shaoyan; Skinner, Daniel F.; Dunlap, Quinn A.; Sorscher, Eric J.; Woodworth, Bradford A.

2016-01-01

Objectives Salubrious effects of the green coffee bean are purportedly secondary to high concentrations of chlorogenic acid. Chlorogenic acid has a molecular structure similar to bioflavonoids that activate transepithelial Cl- transport in sinonasal epithelia. In contrast to flavonoids, the drug is freely soluble in water. The objective of this study is to evaluate the Cl- secretory capability of chlorogenic acid and its potential as a therapeutic activator of mucus clearance in sinus disease. Study Design Basic research Setting Laboratory Subjects and Methods Chlorogenic acid was tested on primary murine nasal septal epithelial(MNSE)[CFTR+/+ and transgenic CFTR-/-] and human sinonasal epithelial(HSNE)[CFTR+/+ and F508del/F508del] cultures under pharmacologic conditions in Ussing chambers to evaluate effects on transepithelial Cl- transport. Cellular cAMP, phosphorylation of the CFTR regulatory domain(R-D), and CFTR mRNA transcription were also measured. Results Chlorogenic acid stimulated transepithelial Cl- secretion [(change in short-circuit current(ΔISC=μA/cm2)] in MNSE(13.1+/-0.9 vs. 0.1+/-0.1, p<0.05) and HSNE(34.3+/-0.9 vs. 0.0+/-0.1, p<0.05). The drug had a long duration until peak effect at 15-30 minutes after application. Significant inhibition with INH-172, as well as absent stimulation in cultures lacking functional CFTR, suggests effects are dependent on CFTR-mediated pathways. However, the absence of elevated cellular cAMP and phosphorylation the CFTR R-D indicates chlorogenic acid does not work through a PKA-dependent mechanism. Conclusion Chlorogenic acid is a water soluble agent that promotes CFTR-mediated Cl- transport in mouse and human sinonasal epithelium. Translating activators of mucociliary transport to clinical use provides a new therapeutic approach to sinus disease. Further in vivo evaluation is planned. PMID:26019132

Chlorogenic Acid Activates CFTR-Mediated Cl- Secretion in Mice and Humans: Therapeutic Implications for Chronic Rhinosinusitis.

PubMed

Illing, Elisa A; Cho, Do-Yeon; Zhang, Shaoyan; Skinner, Daniel F; Dunlap, Quinn A; Sorscher, Eric J; Woodworth, Bradford A

2015-08-01

Salubrious effects of the green coffee bean are purportedly secondary to high concentrations of chlorogenic acid. Chlorogenic acid has a molecular structure similar to bioflavonoids that activate transepithelial Cl(-) transport in sinonasal epithelia. In contrast to flavonoids, the drug is freely soluble in water. The objective of this study is to evaluate the Cl(-) secretory capability of chlorogenic acid and its potential as a therapeutic activator of mucus clearance in sinus disease. Basic research. Laboratory. Chlorogenic acid was tested on primary murine nasal septal epithelial (MNSE) (CFTR(+/+) and transgenic CFTR(-/-)) and human sinonasal epithelial (HSNE) (CFTR(+/+) and F508del/F508del) cultures under pharmacologic conditions in Ussing chambers to evaluate effects on transepithelial Cl(-) transport. Cellular cyclic adenosine monophosphate (cAMP), phosphorylation of the CFTR regulatory domain (R-D), and CFTR mRNA transcription were also measured. Chlorogenic acid stimulated transepithelial Cl(-) secretion (change in short-circuit current [ΔISC = µA/cm(2)]) in MNSE (13.1 ± 0.9 vs 0.1 ± 0.1; P < .05) and HSNE (34.3 ± 0.9 vs 0.0 ± 0.1; P < .05). The drug had a long duration until peak effect at 15 to 30 minutes after application. Significant inhibition with INH-172 as well as absent stimulation in cultures lacking functional CFTR suggest effects are dependent on CFTR-mediated pathways. However, the absence of elevated cellular cAMP and phosphorylation the CFTR R-D indicates chlorogenic acid does not work through a PKA-dependent mechanism. Chlorogenic acid is a water-soluble agent that promotes CFTR-mediated Cl(-) transport in mouse and human sinonasal epithelium. Translating activators of mucociliary transport to clinical use provides a new therapeutic approach to sinus disease. Further in vivo evaluation is planned. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2015.

Analysis of the CFTR gene in Venezuelan cystic fibrosis patients, identification of six novel cystic fibrosis-causing genetic variants.

PubMed

Sánchez, Karen; de Mendonca, Elizabeth; Matute, Xiorama; Chaustre, Ismenia; Villalón, Marlene; Takiff, Howard

2016-01-01

The mutations in the CFTR gene found in patients with cystic fibrosis (CF) have geographic differences, but there are scant data on their prevalence in Venezuelan patients. This study determined the frequency of common CFTR gene mutations in a group of Venezuelan patients with CF. The 27 exons of the CFTR gene from 110 Venezuelan patients in the National CF Program were amplified and sequenced. A total of 36 different mutations were identified, seven with frequencies greater than 1%: p.Phe508del (27.27%), p.Gly542* (3.18%), c.2988+1G>A (3.18%), p.Arg334Trp (1.36%), p.Arg1162* (1.36%), c.1-8G>C (1.36%), and p.[Gly628Arg;Ser1235Arg](1.36). In 40% of patients, all with a clinical diagnosis of CF, no mutations were found. This report represents the largest cohort of Venezuelan patients with CF ever examined, and includes a wider mutation panel than has been previously studied in this population. Mutations common in Southern European populations predominate, and several new mutations were discovered, but no mutations were found in 40% of the cohort.

Analysis of the CFTR gene in Venezuelan cystic fibrosis patients, identification of six novel cystic fibrosis-causing genetic variants

PubMed Central

Sánchez, Karen; de Mendonca, Elizabeth; Matute, Xiorama; Chaustre, Ismenia; Villalón, Marlene; Takiff, Howard

2016-01-01

The mutations in the CFTR gene found in patients with cystic fibrosis (CF) have geographic differences, but there are scant data on their prevalence in Venezuelan patients. This study determined the frequency of common CFTR gene mutations in a group of Venezuelan patients with CF. The 27 exons of the CFTR gene from 110 Venezuelan patients in the National CF Program were amplified and sequenced. A total of 36 different mutations were identified, seven with frequencies greater than 1%: p.Phe508del (27.27%), p.Gly542* (3.18%), c.2988+1G>A (3.18%), p.Arg334Trp (1.36%), p.Arg1162* (1.36%), c.1-8G>C (1.36%), and p.[Gly628Arg;Ser1235Arg](1.36). In 40% of patients, all with a clinical diagnosis of CF, no mutations were found. This report represents the largest cohort of Venezuelan patients with CF ever examined, and includes a wider mutation panel than has been previously studied in this population. Mutations common in Southern European populations predominate, and several new mutations were discovered, but no mutations were found in 40% of the cohort. PMID:27022295

A Little CFTR Goes a Long Way: CFTR-Dependent Sweat Secretion from G551D and R117H-5T Cystic Fibrosis Subjects Taking Ivacaftor

PubMed Central

Char, Jessica E.; Wolfe, Marlene H.; Cho, Hyung-ju; Park, Il-Ho; Jeong, Jin Hyeok; Frisbee, Eric; Dunn, Colleen; Davies, Zoe; Milla, Carlos; Moss, Richard B.; Thomas, Ewart A. C.; Wine, Jeffrey J.

2014-01-01

To determine if oral dosing with the CFTR-potentiator ivacaftor (VX-770, Kalydeco) improves CFTR-dependent sweating in CF subjects carrying G551D or R117H-5T mutations, we optically measured sweat secretion from 32–143 individually identified glands in each of 8 CF subjects; 6 F508del/G551D, one G551D/R117H-5T, and one I507del/R117H-5T. Two subjects were tested only (−) ivacaftor, 3 only (+) ivacaftor and 3 (+/−) ivacaftor (1–5 tests per condition). The total number of gland measurements was 852 (−) ivacaftor and 906 (+) ivacaftor. A healthy control was tested 4 times (51 glands). For each gland we measured both CFTR-independent (M-sweat) and CFTR-dependent (C-sweat); C-sweat was stimulated with a β-adrenergic cocktail that elevated [cAMP]i while blocking muscarinic receptors. Absent ivacaftor, almost all CF glands produced M-sweat on all tests, but only 1/593 glands produced C-sweat (10 tests, 5 subjects). By contrast, 6/6 subjects (113/342 glands) produced C-sweat in the (+) ivacaftor condition, but with large inter-subject differences; 3–74% of glands responded with C/M sweat ratios 0.04%–2.57% of the average WT ratio of 0.265. Sweat volume losses cause proportionally larger underestimates of CFTR function at lower sweat rates. The losses were reduced by measuring C/M ratios in 12 glands from each subject that had the highest M-sweat rates. Remaining losses were estimated from single channel data and used to correct the C/M ratios, giving estimates of CFTR function (+) ivacaftor  = 1.6%–7.7% of the WT average. These estimates are in accord with single channel data and transcript analysis, and suggest that significant clinical benefit can be produced by low levels of CFTR function. PMID:24520399

Binding screen for cystic fibrosis transmembrane conductance regulator correctors finds new chemical matter and yields insights into cystic fibrosis therapeutic strategy.

PubMed

Hall, Justin D; Wang, Hong; Byrnes, Laura J; Shanker, Suman; Wang, Kelong; Efremov, Ivan V; Chong, P Andrew; Forman-Kay, Julie D; Aulabaugh, Ann E

2016-02-01

The most common mutation in cystic fibrosis (CF) patients is deletion of F508 (ΔF508) in the first nucleotide binding domain (NBD1) of the CF transmembrane conductance regulator (CFTR). ΔF508 causes a decrease in the trafficking of CFTR to the cell surface and reduces the thermal stability of isolated NBD1; it is well established that both of these effects can be rescued by additional revertant mutations in NBD1. The current paradigm in CF small molecule drug discovery is that, like revertant mutations, a path may exist to ΔF508 CFTR correction through a small molecule chaperone binding to NBD1. We, therefore, set out to find small molecule binders of NBD1 and test whether it is possible to develop these molecules into potent binders that increase CFTR trafficking in CF-patient-derived human bronchial epithelial cells. Several fragments were identified that bind NBD1 at either the CFFT-001 site or the BIA site. However, repeated attempts to improve the affinity of these fragments resulted in only modest gains. Although these results cannot prove that there is no possibility of finding a high-affinity small molecule binder of NBD1, they are discouraging and lead us to hypothesize that the nature of these two binding sites, and isolated NBD1 itself, may not contain the features needed to build high-affinity interactions. Future work in this area may, therefore, require constructs including other domains of CFTR in addition to NBD1, if high-affinity small molecule binding is to be achieved. © 2016 The Protein Society.

Correctors of the Major Cystic Fibrosis Mutant Interact through Membrane-Spanning Domains.

PubMed

Laselva, Onofrio; Molinski, Steven; Casavola, Valeria; Bear, Christine E

2018-06-01

The most common cystic fibrosis causing mutation is deletion of phenylalanine at position 508 (F508del), a mutation that leads to protein misassembly with defective processing. Small molecule corrector compounds: VX-809 or Corr-4a (C4) partially restores processing of the major mutant. These two prototypical corrector compounds cause an additive effect on F508del/cystic fibrosis transmembrane conductance regulator (CFTR) processing, and hence were proposed to act through distinct mechanisms: VX-809 stabilizing the first membrane-spanning domain (MSD) 1, and C4 acting on the second half of the molecule [consisting of MSD2 and/or nucleotide binding domain (NBD) 2]. We confirmed the effect of VX-809 in enhancing the stability of MSD1 and showed that it also allosterically modulates MSD2 when coexpressed with MSD1. We showed for the first time that C4 stabilizes the second half of the CFTR protein through its action on MSD2. Given the allosteric effect of VX-809 on MSD2, we were prompted to test the hypothesis that the two correctors interact in the full-length mutant protein. We did see evidence supporting their interaction in the full-length F508del-CFTR protein bearing secondary mutations targeting domain:domain interfaces. Disruption of the MSD1:F508del-NBD1 interaction (R170G) prevented correction by both compounds, pointing to the importance of this interface in processing. On the other hand, stabilization of the MSD2:F508del-NBD1 interface (by introducing R1070W) led to a synergistic effect of the compound combination on the total abundance of both the immature and mature forms of the protein. Together, these findings suggest that the two correctors interact in stabilizing the complex of MSDs in F508del-CFTR. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Potentiators (specific therapies for class III and IV mutations) for cystic fibrosis.

PubMed

Patel, Sanjay; Sinha, Ian P; Dwan, Kerry; Echevarria, Carlos; Schechter, Michael; Southern, Kevin W

2015-03-26

placebo in people with cystic fibrosis. In a post hoc change we excluded trials combining CFTR potentiators with other mutation-specific therapies. These will be considered in a separate review. The authors independently extracted data and assessed the risk of bias in included trials; they contacted trial authors for additional data. Meta-analyses were undertaken on outcomes at a number of time points. We included four randomised controlled trials (n = 378), lasting from 28 days to 48 weeks, comparing the potentiator ivacaftor to placebo. Trials differed in terms of design and participant eligibility criteria, which limited the meta-analyses. The phase 2 trial (n = 19) and two phase 3 trials (adult trial (n = 167), paediatric trial (n = 52)), recruited participants with the G551D mutation (class III). The fourth trial (n = 140) enrolled participants homozygous for the ΔF508 mutation (class II).Risks of bias in the trials were moderate. Random sequence generation, allocation concealment and blinding of trial personnel were well-documented. Participant blinding was less clear throughout all trials; in three trials, some participant data were excluded from the analysis. Selective outcome reporting was apparent in three trials. All trials were sponsored by industry and supported by other non-pharmaceutical funding bodies.No trial reported any deaths. Significantly higher quality of life scores in the respiratory domain were reported by the adult phase 3 G551D trial at 24 weeks, mean difference 8.10 (95% confidence interval (CI) 4.77 to 11.43) and 48 weeks, mean difference 8.60 (95% CI 5.27 to 11.93); but not by the paediatric phase 3 G551D trial. The adult phase 3 G551D trial reported improvements in relative change from baseline in forced expiratory volume at one second at 24 weeks, mean difference 16.90% (95% CI 13.60 to 20.20) and 48 weeks, mean difference 16.80% (95% CI 13.50 to 20.10); as did the paediatric G551D trial at 24 weeks, mean difference 17.4% (P < 0

EG-VEGF, BV8, and their receptor expression in human bronchi and their modification in cystic fibrosis: Impact of CFTR mutation (delF508).

PubMed

Chauvet, Sylvain; Traboulsi, Wael; Thevenon, Laura; Kouadri, Amal; Feige, Jean-Jacques; Camara, Boubou; Alfaidy, Nadia; Benharouga, Mohamed

2015-08-01

Enhanced lung angiogenesis has been reported in cystic fibrosis (CF). Recently, two highly homologous ligands, endocrine gland vascular endothelial growth factor (EG-VEGF) and mammalian Bv8, have been described as new angiogenic factors. Both ligands bind and activate two closely related G protein-coupled receptors, the prokineticin receptor (PROKR) 1 and 2. Yet, the expression, regulation, and potential role of EG-VEGF, BV8, and their receptors in normal and CF lung are still unknown. The expression of the receptors and their ligands was examined using molecular, biochemical, and immunocytochemistry analyses in lungs obtained from CF patients vs. control and in normal and CF bronchial epithelial cells. Cystic fibrosis transmembrane conductance regulator (CFTR) activity was evaluated in relation to both ligands, and concentrations of EG-VEGF were measured by ELISA. At the mRNA level, EG-VEGF, BV8, and PROKR2 gene expression was, respectively, approximately five, four, and two times higher in CF lungs compared with the controls. At the cellular level, both the ligands and their receptors showed elevated expressions in the CF condition. Similar results were observed at the protein level. The EG-VEGF secretion was apical and was approximately two times higher in CF compared with the normal epithelial cells. This secretion was increased following the inhibition of CFTR chloride channel activity. More importantly, EG-VEGF and BV8 increased the intracellular concentration of Ca(2+) and cAMP and stimulated CFTR-chloride channel activity. Altogether, these data suggest local roles for epithelial BV8 and EG-VEGF in the CF airway peribronchial vascular remodeling and highlighted the role of CFTR activity in both ligand biosynthesis and secretion. Copyright © 2015 the American Physiological Society.

Role of Mutant CFTR in Hypersusceptibility of Cystic Fibrosis Patients to Lung Infections

NASA Astrophysics Data System (ADS)

Pier, Gerald B.; Grout, Martha; Zaidi, Tanweer S.; Olsen, John C.; Johnson, Larry G.; Yankaskas, James R.; Goldberg, Joanna B.

1996-01-01

Cystic fibrosis (CF) patients are hypersusceptible to chronic Pseudomonas aeruginosa lung infections. Cultured human airway epithelial cells expressing the ΔF508 allele of the cystic fibrosis transmembrane conductance regulator (CFTR) were defective in uptake of P. aeruginosa compared with cells expressing the wild-type allele. Pseudomonas aeruginosa lipopolysaccharide (LPS)-core oligosaccharide was identified as the bacterial ligand for epithelial cell ingestion; exogenous oligosaccharide inhibited bacterial ingestion in a neonatal mouse model, resulting in increased amounts of bacteria in the lungs. CFTR may contribute to a host-defense mechanism that is important for clearance of P. aeruginosa from the respiratory tract.

Breakthrough Therapies: Cystic Fibrosis (CF) Potentiators and Correctors

PubMed Central

Solomon, George M.; Marshall, Susan G.; Ramsey, Bonnie W.; Rowe, Steven M.

2015-01-01

Cystic Fibrosis is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene resulting in abnormal protein function. Recent advances of targeted molecular therapies and high throughput screening have resulted in multiple drug therapies that target many important mutations in the CFTR protein. In this review, we provide the latest results and current progress of CFTR modulators for the treatment of cystic fibrosis, focusing on potentiators of CFTR channel gating and Phe508del processing correctors for the Phe508del CFTR mutation. Special emphasis is placed on the molecular basis underlying these new therapies and emerging results from the latest clinical trials. The future directions for augmenting the rescue of Phe508del with CFTR modulators is also emphasized. PMID:26097168

Oxidative stress modulates the expression of genes involved in cell survival in ΔF508 cystic fibrosis airway epithelial cells.

PubMed

Voisin, Grégory; Bouvet, Guillaume F; Legendre, Pierre; Dagenais, André; Massé, Chantal; Berthiaume, Yves

2014-09-01

Although cystic fibrosis (CF) pathophysiology is explained by a defect in CF transmembrane conductance regulator (CFTR) protein, the broad spectrum of disease severity is the consequence of environmental and genetic factors. Among them, oxidative stress has been demonstrated to play an important role in the evolution of this disease, with susceptibility to oxidative damage, decline of pulmonary function, and impaired lung antioxidant defense. Although oxidative stress has been implicated in the regulation of inflammation, its molecular outcomes in CF cells remain to be evaluated. To address the question, we compared the gene expression profile in NuLi-1 cells with wild-type CFTR and CuFi-1 cells homozygous for ΔF508 mutation cultured at air-liquid interface. We analyzed the transcriptomic response of these cell lines with microarray technology, under basal culture conditions and after 24 h oxidative stress induced by 15 μM 2,3-dimethoxy-1,4-naphtoquinone. In the absence of oxidative conditions, CuFi-1 gene profiling showed typical dysregulated inflammatory responses compared with NuLi-1. In the presence of oxidative conditions, the transcriptome of CuFi-1 cells reflected apoptotic transcript modulation. These results were confirmed in the CFBE41o- and corrCFBE41o- cell lines as well as in primary culture of human CF airway epithelial cells. Altogether, our data point to the influence of oxidative stress on cell survival functions in CF and identify several genes that could be implicated in the inflammation response observed in CF patients. Copyright © 2014 the American Physiological Society.

Mutation analysis of the cystic fibrosis transmembrane regulator gene in native American populations of the southwest

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grebe, T.A.; Doane, W.W.; Norman, R.A.

1992-10-01

The authors report DNA and clinical analysis of cystic fibrosis (CF) in two previously unstudied, genetically isolated populations: Pueblo and Navajo Native Americans. Direct mutation analysis of six mutations of the CFTR gene - namely, [Delta]F508, G542X, G551D, R553X, N1303K, and W1282X - was performed on PCR-amplified genomic DNA extracted from blood samples. Haplotype analyses with marker/enzyme pairs XV2c/TaqI and KM29/PstI were performed as well. Of the 12 affected individuals studied, no [Delta]F508 mutation was detected; only one G542X mutation was found. None of the other mutations was detected. All affected individuals have either an AA, AC, or CC haplotype,more » except for the one carrying the G542X mutation, who has the haplotye AB. Clinically, six of the affected individuals examined exhibit growth deficiency, and five (all from the Zuni Pueblo) have a severe CF phenotype. Four of the six Zunis with CF are also microcephalic, a finding not previously noted in CF patients. The DNA data have serious implications for risk assessment of CF carrier status for these people. 14 refs., 3 tabs.« less

Patterns of haplotypes for 92 cystic fibrosis mutations: Variability, association and recurrence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morral, N.; Llevadot, R.; Estivill, X.

1994-09-01

Most CFTR mutations are very uncommon among the cystic fibrosis population, with frequencies of less than 1%, and many are found only in specific areas. We have analyzed 92 CF mutations for several markers (4 microsatellites and 3 other polymorphisms) scattered in the CFTR gene. Haplotypes associated with these mutations can be used as a framework in the screening of chromosomes carrying unknown mutations. The association between mutation and haplotype reduces the number of mutations it is necessary to search for to a maximum of 16 for the same haplotype. Only mutations {triangle}F508, G542X and N1303K are associated with moremore » than one haplotype as a result of slippage at more than one microsatellite loci, suggesting that these three are the most ancient CF mutations. Recurrence has been found for at least 7 mutations: H199Y, R347P, L558S, R553X, 2184insA, 3272-26A{r_arrow}G, 3849+10kbC{r_arrow}T and R1162X. Also microsatellite analysis of chromosomes of several ethnic origins (Czech, Italian, Russian, Slovac and Spanish) suggested that possibility of three or more independent origins for mutations R334W, R347P, R1162X, and 3849+10kbC{r_arrow}T, which was confirmed by analysis of markers flanking these mutations.« less

Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells

NASA Astrophysics Data System (ADS)

Rich, Devra P.; Anderson, Matthew P.; Gregory, Richard J.; Cheng, Seng H.; Paul, Sucharita; Jefferson, Douglas M.; McCann, John D.; Klinger, Katherine W.; Smith, Alan E.; Welsh, Michael J.

1990-09-01

The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in cultured cystic fibrosis airway epithelial cells and Cl- channel activation assessed in single cells using a fluorescence microscopic assay and the patch-clamp technique. Expression of CFTR, but not of a mutant form of CFTR (ΔF508), corrected the Cl- channel defect. Correction of the phenotypic defect demonstrates a causal relationship between mutations in the CFTR gene and defective Cl- transport which is the hallmark of the disease.

Effect of the F508del genotype on outcomes of endoscopic sinus surgery in children with cystic fibrosis.

PubMed

Do, Bao Anh Julie; Lands, Larry C; Saint-Martin, Christine; Mascarella, Marco A; Manoukian, John J; Daniel, Sam J; Nguyen, Lily H P

2014-07-01

Numerous authors have sought to describe genotype-phenotype correlations in cystic fibrosis (CF), notably to pancreatic insufficiency and lung disease. However, few studies have focused on the association between the F508del genotype and response to sinus surgery. The objective of this study is to assess the effect of the F508del genotype on sinonasal disease severity and outcomes following functional endoscopic sinus surgery (FESS) in a pediatric population. A retrospective chart review of 153 children with CF seen at a tertiary care pediatric hospital from 1995 to 2008 was performed. Patients were classified into one of three groups according to F508del genotype, either as homozygous, heterozygous or not carrying a F508del mutation. The sinonasal disease phenotype of the three groups was compared based on clinical and radiological findings, extent of endoscopic sinus surgery and rate of revision surgery. The relationship between the F508del genotype and pancreatic insufficiency was confirmed (p<0.05). There was no association between the F508del genotype and increased need for FESS (p=0.75). Moreover, no association was established between F508del homozygosity and presence of nasal polyps, Lund-Mackay score, extent of surgery or length of postoperative hospitalization. The rates of revision surgery did not differ significantly among the three genotypes analyzed (p=0.59). There is no clear association between the F508del genotype and an increased need for FESS, extent of surgery, or revision surgery. Given the phenotypic variability of sinonasal disease in patients with CF, a prospective study is needed to better understand outcomes following FESS and the contribution of gene modifiers to this effect. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Nuclease-mediated double-strand break (DSB) enhancement of small fragment homologous recombination (SFHR) gene modification in human-induced pluripotent stem cells (hiPSCs).

PubMed

Sargent, R Geoffrey; Suzuki, Shingo; Gruenert, Dieter C

2014-01-01

Recent developments in methods to specifically modify genomic DNA using sequence-specific endonucleases and donor DNA have opened the door to a new therapeutic paradigm for cell and gene therapy of inherited diseases. Sequence-specific endonucleases, in particular transcription activator-like (TAL) effector nucleases (TALENs), have been coupled with polynucleotide small/short DNA fragments (SDFs) to correct the most common mutation in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, a 3-base-pair deletion at codon 508 (delF508), in induced pluripotent stem (iPS) cells. The studies presented here describe the generation of candidate TALENs and their co-transfection with wild-type (wt) CFTR-SDFs into CF-iPS cells homozygous for the delF508 mutation. Using an allele-specific PCR (AS-PCR)-based cyclic enrichment protocol, clonal populations of corrected CF-iPS cells were isolated and expanded.

The magnitude of ivacaftor effects on fluid secretion via R117H-CFTR channels: Human in vivo measurements

PubMed Central

Char, Jessica E.; Dunn, Colleen; Davies, Zoe; Milla, Carlos; Moss, Richard B.; Wine, Jeffrey J.

2017-01-01

We optically measured effects of orally available ivacaftor (Kalydeco®) on sweat rates of identified glands in 3 R117H subjects, each having a unique set of additional mutations, and compared them with 5 healthy control subjects tested contemporaneously. We injected β-adrenergic agonists intradermally to stimulate CFTR-dependent ‘C-sweat’ and methacholine to stimulate ‘M-sweat’, which persists in CF subjects. We focused on an R117H-7T/F508del subject who produced quantifiable C-sweat off ivacaftor and was available for 1 blinded, 3 off ivacaftor, and 3 on ivacaftor tests, allowing us to estimate in vivo fold-increase in sweat rates produced by ivacaftor’s effect on the open probability (PO) of R117H-CFTR. Measured sweat rates must be corrected for sweat losses. With estimated sweat losses of 0.023 to 0.08 nl·gland-1·min-1, ivacaftor increased the average C-sweat rates 3–7 fold, and estimated function as % of WT were 4.1–12% off ivacaftor and 21.9–32% on ivacaftor (larger values reflect increased loss estimates). Based on single tests, an R117H-7T/ R117H-7T subject showed 6–9% WT function off ivacaftor and 28–43% on ivacaftor. Repeat testing of an R117H-5T/F508del subject detected only trace responding to ivacaftor. We conclude that in vivo, R117H PO is strongly increased by ivacaftor, but channel number, mainly determined by variable deletion of exon 10, has a marked influence on outcomes. PMID:28419121

Cystic fibrosis transmembrane conductance regulator protein (CFTR) expression in the developing human brain: comparative immunohistochemical study between patients with normal and mutated CFTR.

PubMed

Marcorelles, Pascale; Friocourt, Gaëlle; Uguen, Arnaud; Ledé, Françoise; Férec, Claude; Laquerrière, Annie

2014-11-01

Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein has recently been shown to be expressed in the human adult central nervous system (CNS). As CFTR expression has also been documented during embryonic development in several organs, such as the respiratory tract, the intestine and the male reproductive system, suggesting a possible role during development we decided to investigate the expression of CFTR in the human developing CNS. In addition, as some, although rare, neurological symptoms have been reported in patients with CF, we compared the expression of normal and mutated CFTR at several fetal stages. Immunohistochemistry was performed on brain and spinal cord samples of foetuses between 13 and 40 weeks of gestation and compared with five patients with cystic fibrosis (CF) of similar ages. We showed in this study that CFTR is only expressed in neurons and has an early and widespread distribution during development. Although we did not observe any cerebral abnormality in patients with CF, we observed a slight delay in the maturation of several brain structures. We also observed different expression and localization of CFTR depending on the brain structure or the cell maturation stage. Our findings, along with a literature review on the neurological phenotypes of patients with CF, suggest that this gene may play previously unsuspected roles in neuronal maturation or function. © The Author(s) 2014.

Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations.

PubMed

Vernon, Robert M; Chong, P Andrew; Lin, Hong; Yang, Zhengrong; Zhou, Qingxian; Aleksandrov, Andrei A; Dawson, Jennifer E; Riordan, John R; Brouillette, Christie G; Thibodeau, Patrick H; Forman-Kay, Julie D

2017-08-25

Characterization of the second nucleotide-binding domain (NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR) has lagged behind research into the NBD1 domain, in part because NBD1 contains the F508del mutation, which is the dominant cause of cystic fibrosis. Research on NBD2 has also been hampered by the overall instability of the domain and the difficulty of producing reagents. Nonetheless, multiple disease-causing mutations reside in NBD2, and the domain is critical for CFTR function, because channel gating involves NBD1/NBD2 dimerization, and NBD2 contains the catalytically active ATPase site in CFTR. Recognizing the paucity of structural and biophysical data on NBD2, here we have defined a bioinformatics-based method for manually identifying stabilizing substitutions in NBD2, and we used an iterative process of screening single substitutions against thermal melting points to both produce minimally mutated stable constructs and individually characterize mutations. We present a range of stable constructs with minimal mutations to help inform further research on NBD2. We have used this stabilized background to study the effects of NBD2 mutations identified in cystic fibrosis (CF) patients, demonstrating that mutants such as N1303K and G1349D are characterized by lower stability, as shown previously for some NBD1 mutations, suggesting a potential role for NBD2 instability in the pathology of CF. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Changes of CFTR functional measurements and clinical improvements in cystic fibrosis patients with non p.Gly551Asp gating mutations treated with ivacaftor.

PubMed

Mesbahi, Myriam; Shteinberg, Michal; Wilschanski, Michael; Hatton, Aurelie; Nguyen-Khoa, Thao; Friedman, Hannah; Cohen, Michael; Escabasse, Virginie; Le Bourgeois, Muriel; Lucidi, Vicenzina; Sermet-Gaudelus, Isabelle; Bassinet, Laurence; Livnat, Galit

2017-01-01

Ivacaftor, a CFTR potentiator, has been found to improve CFTR function and clinical outcomes in patients with cystic fibrosis (CF) gating mutations. We investigated the effects of ivacaftor on CFTR functional measurement in CF patients carrying gating mutations other than p.Gly551Asp. Two siblings aged 13 and 12 carrying the p.Ser549Asn mutation, two sisters (45 and 43years old) compound heterozygotes for p.Asp1152His and p.Gly1244Glu, a 37year old man homozygous for the p.Gly1244Glu mutation, and a 7year old girl with p.Arg352Gln and p.Gly1244Glu mutations commenced treatment with ivacaftor. NPD was performed in all the patients and approached normal for four patients who had also clinical improvement (p.Ser549Asn compound heterozygotes, and p.Asp1152His/p.Gly1244Glu siblings). Beta-adrenergic sweat chloride secretion performed in thep.Asp1152His/p.Gly1244Glu patients improved significantly. The p.Gly1244Glu mutation homozygous patient, who had undergone an ileal resection with ileostomy and enterocutaneous fistula, did not respond clinically to ivacaftor and did not modify his sweat test. These results highlight the importance of different CFTR activity measurements to explore CFTR modulator efficacy. Copyright © 2016. Published by Elsevier B.V.

Cystic fibrosis transmembrane conductance regulator gene mutations: do they play a role in the aetiology of allergic bronchopulmonary aspergillosis?

PubMed

Eaton, T E; Weiner Miller, P; Garrett, J E; Cutting, G R

2002-05-01

Previous work suggests that cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations may be implicated in the aetiology of allergic bronchopulmonary aspergilosis (ABPA). To compare the frequency of CF gene mutations in asthmatics with ABPA of varying severity with asthmatics who were skin prick test (SPT)-positive to Aspergillus fumigatus (Af) without evidence of ABPA and asthmatics SPT-negative to Af. Thirty-one Caucasian patients with ABPA were identified, together with asthmatics SPT positive to Af without evidence of ABPA (n = 23) and SPT negative to Af (n = 28). Genomic DNA was tested for 16 CF mutations accounting for approximately 85% of CF alleles in Caucasian New Zealanders. Four (12.9%) ABPA patients were found to be carriers of a CF mutation (DeltaF508 n = 3, R117H n = 1), one (4.3%) asthmatic SPT positive to Af without ABPA (DeltaF508), and one (3.6%) asthmatic SPT negative to Af (R117H). All patients with a CF mutation had normal sweat chloride (< 40 mM). There was no significant difference between the frequency of CF mutations in the ABPA patients and asthmatics without ABPA. However, the frequency of CF mutations in the ABPA patients was significantly different (P = 0.0125) to the expected carrier rate in the general population. These results lend further support to a possible link between CF mutations and ABPA.

Optimizing nasal potential difference analysis for CFTR modulator development: assessment of ivacaftor in CF subjects with the G551D-CFTR mutation.

PubMed

Rowe, Steven M; Liu, Bo; Hill, Aubrey; Hathorne, Heather; Cohen, Morty; Beamer, John R; Accurso, Frank J; Dong, Qunming; Ordoñez, Claudia L; Stone, Anne J; Olson, Eric R; Clancy, John P

2013-01-01

Nasal potential difference (NPD) is used as a biomarker of the cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) activity. We evaluated methods to detect changes in chloride and sodium transport by NPD based on a secondary analysis of a Phase II CFTR-modulator study. Thirty-nine subjects with CF who also had the G551D-CFTR mutation were randomized to receive ivacaftor (Kalydeco™; also known as VX-770) in four doses or placebo twice daily for at least 14 days. All data were analyzed by a single investigator who was blinded to treatment assignment. We compared three analysis methods to determine the best approach to quantify changes in chloride and sodium transport: (1) the average of both nostrils; (2) the most-polarized nostril at each visit; and (3) the most-polarized nostril at screening carried forward. Parameters of ion transport included the PD change with zero chloride plus isoproterenol (CFTR activity), the basal PD, Ringer's PD, and change in PD with amiloride (measurements of ENaC activity), and the delta NPD (measuring CFTR and ENaC activity). The average and most-polarized nostril at each visit were most sensitive to changes in chloride and sodium transport, whereas the most-polarized nostril at screening carried forward was less discriminatory. Based on our findings, NPD studies should assess both nostrils rather than a single nostril. We also found that changes in CFTR activity were more readily detected than changes in ENaC activity, and that rigorous standardization was associated with relatively good within-subject reproducibility in placebo-treated subjects (± 2.8 mV). Therefore, we have confirmed an assay of reasonable reproducibility for detecting chloride-transport improvements in response to CFTR modulation.

Newborn screening for cystic fibrosis in Wisconsin: comparison of biochemical and molecular methods.

PubMed

Gregg, R G; Simantel, A; Farrell, P M; Koscik, R; Kosorok, M R; Laxova, A; Laessig, R; Hoffman, G; Hassemer, D; Mischler, E H; Splaingard, M

1997-06-01

To evaluate neonatal screening for cystic fibrosis (CF), including study of the screening procedures and characteristics of false-positive infants, over the past 10 years in Wisconsin. An important objective evolving from the original design has been to compare use of a single-tier immunoreactive trypsinogen (IRT) screening method with that of a two-tier method using IRT and analyses of samples for the most common cystic fibrosis transmembrane regulator (CFTR) (DeltaF508) mutation. We also examined the benefit of including up to 10 additional CFTR mutations in the screening protocol. From 1985 to 1994, using either the IRT or IRT/DNA protocol, 220 862 and 104 308 neonates, respectively, were screened for CF. For the IRT protocol, neonates with an IRT >/=180 ng/mL were considered positive, and the standard sweat chloride test was administered to determine CF status. For the IRT/DNA protocol, samples from the original dried-blood specimen on the Guthrie card of neonates with an IRT >/=110 ng/mL were tested for the presence of the DeltaF508 CFTR allele, and if the DNA test revealed one or two DeltaF508 alleles, a sweat test was obtained. Both screening procedures had very high specificity. The sensitivity tended to be higher with the IRT/DNA protocol, but the differences were not statistically significant. The positive predictive value of the IRT/DNA screening protocol was 15.2% compared with 6.4% if the same samples had been screened by the IRT method. Assessment of the false-positive IRT/DNA population revealed that the two-tier method eliminates the disproportionate number of infants with low Apgar scores and also the high prevalence of African-Americans identified previously in our study of newborns with high IRT levels. We found that 55% of DNA-positive CF infants were homozygous for DeltaF508 and 40% had one DeltaF508 allele. Adding analyses for 10 more CFTR mutations has only a small effect on the sensitivity but is likely to add significantly to the cost of

Mild CFTR mutations and genetic predisposition to lactase persistence in cystic fibrosis

PubMed Central

Mądry, Edyta; Fidler, Ewa; Sobczyńska-Tomaszewska, Agnieszka; Lisowska, Aleksandra; Krzyżanowska, Patrycja; Pogorzelski, Andrzej; Minarowski, Łukasz; Oralewska, Beata; Mojs, Ewa; Sapiejka, Ewa; Marciniak, Ryszard; Sands, Dorota; Korzon-Burakowska, Anna; Kwiecień, Jarosław; Walkowiak, Jarosław

2011-01-01

Taking into account the reported incidence of hypolactasia in cystic fibrosis (CF) and the possible impact of milk products on nutritional status we aimed to assess the genetic predisposition to adult-type hypolactasia (ATH) and its incidence in CF. Single nucleotide polymorphism upstream of the lactase gene (LCT) was assessed in 289 CF patients. In subject with −13910C/C genotype (C/C) predisposing to ATH, hydrogen-methane breath test (BT) with lactose loading was conducted and clinical symptoms typical for lactose malabsorption were assessed. The percentage of CF patients with C/C was similar to that observed in healthy subjects (HS) (31.5 vs 32.5% ). Eleven out of 52 (24.5%) CF C/C patients had abnormal BT results. The recalculated frequency of lactose malabsorption was similar for the entire CF and HS populations (6.9 vs 7.2%). Similarly as in the control group, few CF patients have identified and linked to lactose consumption clinical symptoms. The frequency of LCT polymorphic variants in CF patients having and not having severe mutations of CFTR gene showed significant differences. The C allele was more frequent in homozygotes of the severe mutations than in patients carrying at least one mild/unknown mutation (P<0.0028) and in patients with at least one mild mutation (P<0.0377). In conclusion, CF patients carrying mild CFTR mutations seem to have lower genetic predisposition to ATH. Lactose malabsorption due to ATH in CF is not more frequent than in the general population. Symptomatic assessment of lactose malabsorption in CF is not reliable. PMID:21407263

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia*

PubMed Central

Dong, Qian; Ernst, Sarah E.; Ostedgaard, Lynda S.; Shah, Viral S.; Ver Heul, Amanda R.; Welsh, Michael J.; Randak, Christoph O.

2015-01-01

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P1,P5-di(adenosine-5′) pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5′-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5′-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl− channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. PMID:25887396

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

PubMed

Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

2015-05-29

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Activation of the Cystic Fibrosis Transmembrane Conductance Regulator by the Flavonoid Quercetin

PubMed Central

Pyle, Louise C.; Fulton, Jennifer C.; Sloane, Peter A.; Backer, Kyle; Mazur, Marina; Prasain, Jeevan; Barnes, Stephen; Clancy, J. P.; Rowe, Steven M.

2010-01-01

Therapies to correct the ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) folding defect require sensitive methods to detect channel activity in vivo. The β2 adrenergic receptor agonists, which provide the CFTR stimuli commonly used in nasal potential difference assays, may not overcome the channel gating defects seen in ΔF508 CFTR after plasma membrane localization. In this study, we identify an agent, quercetin, that enhances the detection of surface ΔF508 CFTR, and is suitable for nasal perfusion. A screen of flavonoids in CFBE41o− cells stably transduced with ΔF508 CFTR, corrected to the cell surface with low temperature growth, revealed that quercetin stimulated an increase in the short-circuit current. This increase was dose-dependent in both Fisher rat thyroid and CFBE41o− cells. High concentrations inhibited Cl− conductance. In CFBE41o− airway cells, quercetin (20 μg/ml) activated ΔF508 CFTR, whereas the β2 adrenergic receptor agonist isoproterenol did not. Quercetin had limited effects on cAMP levels, but did not produce detectable phosphorylation of the isolated CFTR R-domain, suggesting an activation independent of channel phosphorylation. When perfused in the nares of Cftr+ mice, quercetin (20 μg/ml) produced a hyperpolarization of the potential difference that was absent in Cftr−/− mice. Finally, quercetin-induced, dose-dependent hyperpolarization of the nasal potential difference was also seen in normal human subjects. Quercetin activates CFTR-mediated anion transport in respiratory epithelia in vitro and in vivo, and may be useful in studies intended to detect the rescue of ΔF508 CFTR by nasal potential difference. PMID:20042712

CFTR Cl- channel and CFTR-associated ATP channel: distinct pores regulated by common gates.

PubMed Central

Sugita, M; Yue, Y; Foskett, J K

1998-01-01

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is regulated by phosphorylation of the R domain and ATP hydrolysis at two nucleotide-binding domains (NBDs). It is controversial whether CFTR conducts ATP or whether CFTR might be closely associated with a separate ATP conductance. To characterize ATP channels associated with CFTR, we analyzed Cl- and ATP single channel-currents in excised inside-out membrane patches from MDCK epithelial cells transiently expressing CFTR. With 100 mM ATP in the pipette and 140 mM Cl- in the bath, ATP channels were associated with CFTR Cl- channels in two-thirds of patches that included CFTR. CFTR Cl- channels and CFTR-associated ATP channels had slope conductances of 7.4 pS and 5.2 pS, respectively, and had distinct reversal potentials and sensitivities to channel blockers. CFTR-associated ATP channels exhibited slow gating kinetics that depended on the presence of protein kinase A and cytoplasmic ATP, similar to CFTR Cl- channels. Gating kinetics of the ATP channels as well as the CFTR Cl- channels were similarly affected by non-hydrolyzable ATP analogues and mutations in the CFTR R domain and NBDs. Our results indicate that phosphorylation- and nucleotide-hydrolysis-dependent gating of CFTR is directly involved in gating of an associated ATP channel. However, the permeation pathways for Cl- and ATP are distinct and the ATP conduction pathway is not obligatorily associated with the expression of CFTR. PMID:9463368

Applying Cystic Fibrosis Transmembrane Conductance Regulator Genetics and CFTR2 Data to Facilitate Diagnoses.

PubMed

Sosnay, Patrick R; Salinas, Danieli B; White, Terry B; Ren, Clement L; Farrell, Philip M; Raraigh, Karen S; Girodon, Emmanuelle; Castellani, Carlo

2017-02-01

As a Mendelian disease, genetics plays an integral role in the diagnosis of cystic fibrosis (CF). The identification of 2 disease-causing mutations in the CF transmembrane conductance regulator (CFTR) in an individual with a phenotype provides evidence that the disease is CF. However, not all variations in CFTR always result in CF. Therefore, for CFTR genotype to provide the same level of evidence of CFTR dysfunction as shown by direct tests such as sweat chloride or nasal potential difference, the mutations identified must be known to always result in CF. The use of CFTR genetics in CF diagnosis, therefore, relies heavily on mutation interpretation. Progress that has been made on mutation interpretation and annotation was reviewed at the recent CF Foundation Diagnosis Consensus Conference. A modified Delphi method was used to identify consensus statements on the use of genetic analysis in CF diagnosis. The largest recent advance in CF genetics has come through the Clinical and Functional Translation of CFTR (CFTR2) project. This undertaking seeks to characterize CFTR mutations from patients with CF around the world. The project also established guidelines for the clinical, functional, and population/penetrance criteria that can be used to interpret mutations not yet included in CFTR2's review. The use of CFTR genetics to aid in diagnosis of CF requires that the mutations identified have a known disease liability. The demonstration of 2 in trans mutations known to always result in CF is satisfactory evidence of CFTR dysfunction. However, if the identified mutations are known to be associated with variable outcomes, or have unknown consequence, that genotype may not result in a CF phenotype. In these cases, other tests of CFTR function may help. Copyright © 2016 Elsevier Inc. All rights reserved.

Infertility due to congenital absence of vas deferens in mainly caused by variable exon 9 skipping of the CFTR gene in heterozygous males for cystic fibrosis mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chillon, M.; Casals, T.; Nunes, V.

1994-09-01

About 65% or the individuals with congenital bilateral absence of the vas deferens (CBAVD) have mutations in at least one of the CFTR alleles. We have studied the phenotypic effects of the CFTR gene intron 8 polyT tract 5T allele in 90 CBAVD subjects and in parents of CF patients. This group was compared with normal individuals, and with fathers and mothers of CF patients. Allele 5T was significantly associated with CBAVD (19.6%) when compared to the general population (5.2%) ({chi}{sup 2} = 33.3%; p<<0.0001). It was represented poorly in fathers of CF patients (1.3%). Mutations were identified in onemore » (60%) or both CFTR alleles (8.9%) of CBAVD patients. Heterozygosity for the 5T allele was strongly associated with heterozygosity for CF mutations ({chi}{sup 2} = 10.9; p<0.0004). The strong correlation between allele 5T and CBAVD, together with the low frequency of this allele in fathers of CF patients, demonstrates that variable {Delta}exon 9 produces infertility in males if associated with a CF mutation on the other chromosome. The 30% of CBAVD cases with only one CFTR mutation and without a 5T-allele may be due to other molecular mechanisms involving CFTR, distinct from {Delta}exon 9. Since there is a relatively high proportion of CBAVD without CF mutations (25%), other gene(s), distinct from CFTR, may have a role in the CBAVD phenotype.« less

Association of a nonsense mutation (W1282X), the most common mutation in the Ashkenazi Jewish cystic fibrosis patients in Israel, with presentation of severe disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shoshani, T.; Bashan, N.; Seret, H.

1992-01-01

Only about 30% of the cystic fibrosis chromosomes in the Israeli cystic fibrosis patient populations carry the major CF mutation ({Delta}F508). Since different Jewish ethnic groups tended to live as closed isolates until recent times, high frequencies of specific mutations are expected among the remainder cystic fibrosis chromosomes of these ethnic groups. Genetic factors appear to influence the severity of the disease. It is therefore expected that different mutations will be associated with either severe or mild phenotype. Direct genomic sequencing of exons included in the two nucleotide-binding folds of the putative CFTR protein was performed on 119 Israeli cysticmore » fibrosis patients from 97 families. One sequence alteration which is expected to create a termination at residue 1282 (W1282X) was found in 63 chromosomes. Of 95 chromosomes, 57(60%) are of Ashkenazi origin. In conclusion, the W1282X mutation is the most common cystic fibrosis mutation in the Ashkenazi Jewish patient population in Israel. This nonsense mutation is associated with presentation of severe disease.« less

Strategies for the etiological therapy of cystic fibrosis.

PubMed

Maiuri, Luigi; Raia, Valeria; Kroemer, Guido

2017-11-01

Etiological therapies aim at repairing the underlying cause of cystic fibrosis (CF), which is the functional defect of the cystic fibrosis transmembrane conductance regulator (CFTR) protein owing to mutations in the CFTR gene. Among these, the F508del CFTR mutation accounts for more than two thirds of CF cases worldwide. Two somehow antinomic schools of thought conceive CFTR repair in a different manner. According to one vision, drugs should directly target the mutated CFTR protein to increase its plasma membrane expression (correctors) or improve its ion transport function (potentiators). An alternative strategy consists in modulating the cellular environment and proteostasis networks in which the mutated CFTR protein is synthesized, traffics to its final destination, the plasma membrane, and is turned over. We will analyze distinctive advantages and drawbacks of these strategies in terms of their scientific and clinical dimensions, and we will propose a global strategy for CF research and development based on a reconciliatory approach. Moreover, we will discuss the utility of preclinical biomarkers that may guide the personalized, patient-specific implementation of CF therapies.

Strategies for the etiological therapy of cystic fibrosis

PubMed Central

Maiuri, Luigi; Raia, Valeria; Kroemer, Guido

2017-01-01

Etiological therapies aim at repairing the underlying cause of cystic fibrosis (CF), which is the functional defect of the cystic fibrosis transmembrane conductance regulator (CFTR) protein owing to mutations in the CFTR gene. Among these, the F508del CFTR mutation accounts for more than two thirds of CF cases worldwide. Two somehow antinomic schools of thought conceive CFTR repair in a different manner. According to one vision, drugs should directly target the mutated CFTR protein to increase its plasma membrane expression (correctors) or improve its ion transport function (potentiators). An alternative strategy consists in modulating the cellular environment and proteostasis networks in which the mutated CFTR protein is synthesized, traffics to its final destination, the plasma membrane, and is turned over. We will analyze distinctive advantages and drawbacks of these strategies in terms of their scientific and clinical dimensions, and we will propose a global strategy for CF research and development based on a reconciliatory approach. Moreover, we will discuss the utility of preclinical biomarkers that may guide the personalized, patient-specific implementation of CF therapies. PMID:28937684

Vx-809/Vx-770 treatment reduces inflammatory response to Pseudomonas aeruginosa in primary differentiated cystic fibrosis bronchial epithelial cells.

PubMed

Ruffin, Manon; Roussel, Lucie; Maillé, Émilie; Rousseau, Simon; Brochiero, Emmanuelle

2018-04-01

Cystic fibrosis patients exhibit chronic Pseudomonas aeruginosa respiratory infections and sustained proinflammatory state favoring lung tissue damage and remodeling, ultimately leading to respiratory failure. Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function is associated with MAPK hyperactivation and increased cytokines expression, such as interleukin-8 [chemoattractant chemokine (C-X-C motif) ligand 8 (CXCL8)]. Recently, new therapeutic strategies directly targeting the basic CFTR defect have been developed, and ORKAMBI (Vx-809/Vx-770 combination) is the only Food and Drug Administration-approved treatment for CF patients homozygous for the F508del mutation. Here we aimed to determine the effect of the Vx-809/Vx-770 combination on the induction of the inflammatory response by fully differentiated primary bronchial epithelial cell cultures from CF patients carrying F508del mutations, following exposure to P. aeruginosa exoproducts. Our data unveiled that CFTR functional rescue with Vx-809/Vx-770 drastically reduces CXCL8 (as well as CXCL1 and CXCL2) transcripts and p38 MAPK phosphorylation in response to P. aeruginosa exposure through a CFTR-dependent mechanism. These results suggest that ORKAMBI has anti-inflammatory properties that could decrease lung inflammation and contribute to the observed beneficial impact of this treatment in CF patients.

Molecular genetic analysis of some mutations in the cystic fibrosis gene in Moldova: Characterization of molecular markers and their linkage to various mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gimbovskaya, S.D.; Kalinin, V.N.; Ivashchenko, T.E.

1994-12-01

Sixty-one patients with cystic fibrosis (CF) from Moldova were tested for mutations {Delta}F508, G551D, and R553X. Frequencies of various alleles of the repeated GATT sequence in intron 6B of the GFTR gene, their linkage to other polymorphic markers, and various mutations were determined. The frequency of occurrence of mutation {Delta}F508 was only 25%. An absolute majority of CF patients (80%) had pancreatic insufficiency. Mutations G551D and R553X were not found in our sample. Each of 31 chromosomes with mutation {Delta}F508 carry the 6-GATT allele. Most {open_quotes}non {Delta}F508{close_quotes} (78%) and normal (80%) chromosomes were marked by the 7-GATT allele. Twenty-seven {Delta}F508more » chromosomes (96.4%) belong to haplotype B6, and only one to D6. Most chromosomes with {open_quotes}non {Delta}F508{close_quotes} mutations are associated with haplotypes D7 (26.3%) and C7 (21%). In addition, a significant portion of chromosomes from this subgroup were associated with haplotypes A7 (23.7%), A6 (10.5%), and C6 (2.7%), which are not yet described for mutant chromosomes. The results obtained demonstrate that CF in Moldova is mainly associated with mutations other than {Delta}F508, G551D, and R553X. Severe forms of the disease, with pancreatic insufficiency, are more frequently caused by these mutations; moreover, our data provides strong evidence for the presence of at least seven additional CF mutations in Moldova, apart from {Delta}F508, G551D, and R553X. Some of these are probably not described.« less

Small heat shock proteins target mutant cystic fibrosis transmembrane conductance regulator for degradation via a small ubiquitin-like modifier–dependent pathway

PubMed Central

Ahner, Annette; Gong, Xiaoyan; Schmidt, Bela Z.; Peters, Kathryn W.; Rabeh, Wael M.; Thibodeau, Patrick H.; Lukacs, Gergely L.; Frizzell, Raymond A.

2013-01-01

Small heat shock proteins (sHsps) bind destabilized proteins during cell stress and disease, but their physiological functions are less clear. We evaluated the impact of Hsp27, an sHsp expressed in airway epithelial cells, on the common protein misfolding mutant that is responsible for most cystic fibrosis. F508del cystic fibrosis transmembrane conductance regulator (CFTR), a well-studied protein that is subject to cytosolic quality control, selectively associated with Hsp27, whose overexpression preferentially targeted mutant CFTR to proteasomal degradation. Hsp27 interacted physically with Ubc9, the small ubiquitin-like modifier (SUMO) E2 conjugating enzyme, implying that F508del SUMOylation leads to its sHsp-mediated degradation. Enhancing or disabling the SUMO pathway increased or blocked Hsp27’s ability to degrade mutant CFTR. Hsp27 promoted selective SUMOylation of F508del NBD1 in vitro and of full-length F508del CFTR in vivo, which preferred endogenous SUMO-2/3 paralogues that form poly-chains. The SUMO-targeted ubiquitin ligase (STUbL) RNF4 recognizes poly-SUMO chains to facilitate nuclear protein degradation. RNF4 overexpression elicited F508del degradation, whereas Hsp27 knockdown blocked RNF4’s impact on mutant CFTR. Similarly, the ability of Hsp27 to degrade F508del CFTR was lost during overexpression of dominant-negative RNF4. These findings link sHsp-mediated F508del CFTR degradation to its SUMOylation and to STUbL-mediated targeting to the ubiquitin–proteasome system and thereby implicate this pathway in the disposal of an integral membrane protein. PMID:23155000

Implementation of newborn screening for cystic fibrosis in Norway. Results from the first three years.

PubMed

Lundman, Emma; Gaup, H Junita; Bakkeheim, Egil; Olafsdottir, Edda J; Rootwelt, Terje; Storrøsten, Olav Trond; Pettersen, Rolf D

2016-05-01

Norway introduced newborn screening for cystic fibrosis (CF) March 1, 2012. We present results from the first three years of the national newborn CF screening program. Positive primary screening of immunoreactive trypsinogen (IRT) was followed by DNA testing of the Cystic fibrosis transmembrane conductance regulator (CFTR) gene. Infants with two CFTR mutations were reported for diagnostic follow-up. Of 181,859 infants tested, 1454 samples (0.80%) were assessed for CFTR mutations. Forty children (1:4546) had two CFTR mutations, of which only 21 (1:8660) were confirmed to have a CF diagnosis. The CFTR mutations differed from previously clinically diagnosed CF patients, and p.R117H outnumbered p.F508del as the most frequent mutation. One child with a negative IRT screening test was later clinically diagnosed with CF. The CF screening program identified fewer children with a conclusive CF diagnosis than expected. Our data suggest a revision of the IRT/DNA protocol. Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Elevated Mirc1/Mir17-92 cluster expression negatively regulates autophagy and CFTR (cystic fibrosis transmembrane conductance regulator) function in CF macrophages.

PubMed

Tazi, Mia F; Dakhlallah, Duaa A; Caution, Kyle; Gerber, Madelyn M; Chang, Sheng-Wei; Khalil, Hany; Kopp, Benjamin T; Ahmed, Amr E; Krause, Kathrin; Davis, Ian; Marsh, Clay; Lovett-Racke, Amy E; Schlesinger, Larry S; Cormet-Boyaka, Estelle; Amer, Amal O

2016-11-01

Cystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR function. Macroautophagy/autophagy is a highly regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their macrophages exhibit limited autophagy activity. The study of microRNAs (Mirs), and other noncoding RNAs, continues to offer new therapeutic targets. The objective of this study was to elucidate the role of Mirs in dysregulated autophagy-related genes in CF macrophages, and then target them to restore this host-defense function and improve CFTR channel function. We identified the Mirc1/Mir17-92 cluster as a potential negative regulator of autophagy as CF macrophages exhibit decreased autophagy protein expression and increased cluster expression when compared to wild-type (WT) counterparts. The absence or reduced expression of the cluster increases autophagy protein expression, suggesting the canonical inverse relationship between Mirc1/Mir17-92 and autophagy gene expression. An in silico study for targets of Mirs that comprise the cluster suggested that the majority of the Mirs target autophagy mRNAs. Those targets were validated by luciferase assays. Notably, the ability of macrophages expressing mutant F508del CFTR to transport halide through their membranes is compromised and can be restored by downregulation of these inherently elevated Mirs, via restoration of autophagy. In vivo, downregulation of Mir17 and Mir20a partially restored autophagy expression and hence improved the clearance of Burkholderia cenocepacia. Thus, these data advance our understanding of mechanisms underlying the pathobiology of CF and provide a new therapeutic platform for restoring CFTR function

The c.1364C>A (p.A455E) Mutation in the CFTR Pseudogene Results in an Incorrectly Assigned Carrier Status by a Commonly Used Screening Platform.

PubMed

Deeb, Kristin K; Metcalf, James D; Sesock, Kaitlin M; Shen, Junqing; Wensel, Christine A; Rippel, Larisa I; Smith, Michelle; Chapman, Mark S; Zhang, Shulin

2015-07-01

Cystic fibrosis (CF) is one of the most common recessive conditions among whites, with an estimated carrier frequency of 1 in 25 in the United States. Population-based CF carrier screening was implemented in the United States in 2001. The number of mutations screened by each laboratory may vary; however, the 23 most common CF mutations recommended for screening by the American College of Medical Genetics and American College of Obstetricians and Gynecologists are included in all platforms. The c.1364C>A (p.A455E) mutation located in exon 10 of the CFTR gene is one of the 23 mutations. Because CFTR exon 10 and its flanking intronic regions are duplicated and transposed onto several other chromosomes of the human genome during evolution and function as unprocessed pseudogenes, variations in the CFTR pseudogenes may confound CF screening results for mutations located in exon 10 of the CFTR gene. We report an incorrectly identified carrier status for the c.1364C>A (p.A455E) mutation in a healthy individual using the Hologic InPlex CF assay. Further analysis revealed that the mutation resides in one of the CFTR pseudogenes. Because most commercial kits and laboratory-developed tests for CF carrier screening involve a short amplicon encompassing this mutation, this finding suggests that individuals with the c.1364C>A (p.A455E) mutation may require further investigation to avoid a false assignment of CF carrier status. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Efficacy and safety of lumacaftor/ivacaftor combination therapy in patients with cystic fibrosis homozygous for Phe508del CFTR by pulmonary function subgroup: a pooled analysis.

PubMed

Elborn, J Stuart; Ramsey, Bonnie W; Boyle, Michael P; Konstan, Michael W; Huang, Xiaohong; Marigowda, Gautham; Waltz, David; Wainwright, Claire E

2016-08-01

Lumacaftor/ivacaftor combination therapy has shown clinical benefits in patients with cystic fibrosis homozygous for the Phe508del CFTR mutation; however, pretreatment lung function is a confounding factor that potentially affects the efficacy and safety of this therapy. We aimed to assess the efficacy and safety of lumacaftor/ivacaftor therapy in these patients, defined by specific categories of lung function. Both trials (TRAFFIC and TRANSPORT) included in this pooled analysis were multinational, randomised, double-blind, placebo-controlled, parallel-group, phase 3 studies. Eligible patients from 187 participating centres in North America, Australia, and the European Union (both trials) were aged 12 years or older with a confirmed diagnosis of cystic fibrosis, homozygous for the Phe508del CFTR mutation, and with a percent predicted FEV1 (ppFEV1) of 40-90 at the time of screening. Patients were randomly assigned with an interactive web response system (1:1:1) to receive placebo, lumacaftor (600 mg once daily) plus ivacaftor (250 mg every 12 h), or lumacaftor (400 mg every 12 h) plus ivacaftor (250 mg every 12 h) for 24 weeks. Prespecified subgroup analyses of pooled efficacy and safety data by lung function, as measured by ppFEV1, were done for patients with baseline ppFEV1 (<40 and ≥40) and screening ppFEV1 (<70 and ≥70). The primary endpoint was the absolute change from baseline in ppFEV1 at week 24 analysed in all randomised patients who received at least one dose of study drug. Both trials are registered with ClinicalTrials.gov (TRAFFIC: NCT01807923; TRANSPORT: NCT01807949). Both trials were done between April, 2013, and April, 2014. Of the 1108 patients included in the efficacy analysis, 81 patients had a ppFEV1 that decreased to lower than 40 between screening and baseline and 1016 had a ppFEV1 of 40 or higher at baseline. At screening, 730 had a ppFEV1 of less than 70, and 342 had a ppFEV1 of 70 or higher. Improvements in the absolute change from

CFTR mutation distribution among U.S. Hispanic and African American individuals: evaluation in cystic fibrosis patient and carrier screening populations.

PubMed

Sugarman, Elaine A; Rohlfs, Elizabeth M; Silverman, Lawrence M; Allitto, Bernice A

2004-01-01

We reviewed CFTR mutation distribution among Hispanic and African American individuals referred for CF carrier screening and compared mutation frequencies to those derived from CF patient samples. Results from CFTR mutation analyses received from January 2001 through September 2003, were analyzed for four populations: Hispanic individuals with a CF diagnosis (n = 159) or carrier screening indication (n = 15,333) and African American individuals with a CF diagnosis (n = 108) or carrier screening indication (n = 8,973). All samples were tested for the same 87 mutation panel. In the Hispanic population, 42 mutations were identified: 30 in the patient population (77.5% detection rate) and 33 among carrier screening referrals. Five mutations not included in the ACMG/ACOG carrier screening panel (3876delA, W1089X, R1066C, S549N, 1949del84) accounted for 7.55% detection in patients and 5.58% among carriers. Among African American referrals, 33 different mutations were identified: 21 in the patient population (74.4% detection) and 23 in the carrier screening population. Together, A559T and 711+5G>A were observed at a detection rate of 3.71% in CF patients and 6.38% in carriers. The mutation distribution seen in both the carrier screening populations reflected an increased frequency of mutations with variable expression such as D1152H, R117H, and L206W. A detailed analysis of CFTR mutation distribution in the Hispanic and African American patient and carrier screening populations demonstrates that a diverse group of mutations is most appropriate for diagnostic and carrier screening in these populations. To best serve the increasingly diverse U.S. population, ethnic-specific mutations should be included in mutation panels.

Genomic sequencing in cystic fibrosis newborn screening: what works best, two-tier predefined CFTR mutation panels or second-tier CFTR panel followed by third-tier sequencing?

PubMed

Currier, Robert J; Sciortino, Stan; Liu, Ruiling; Bishop, Tracey; Alikhani Koupaei, Rasoul; Feuchtbaum, Lisa

2017-10-01

PurposeThe purpose of this study was to model the performance of several known two-tier, predefined mutation panels and three-tier algorithms for cystic fibrosis (CF) screening utilizing the ethnically diverse California population.MethodsThe cystic fibrosis transmembrane conductance regulator (CFTR) mutations identified among the 317 CF cases in California screened between 12 August 2008 and 18 December 2012 were used to compare the expected CF detection rates for several two- and three-tier screening approaches, including the current California approach, which consists of a population-specific 40-mutation panel followed by third-tier sequencing when indicated.ResultsThe data show that the strategy of using third-tier sequencing improves CF detection following an initial elevated immunoreactive trypsinogen and detection of only one mutation on a second-tier panel.ConclusionIn a diverse population, the use of a second-tier panel followed by third-tier CFTR gene sequencing provides a better detection rate for CF, compared with the use of a second-tier approach alone, and is an effective way to minimize the referrals of CF carriers for sweat testing. Restricting screening to a second-tier testing to predefined mutation panels, even broad ones, results in some missed CF cases and demonstrates the limited utility of this approach in states that have diverse multiethnic populations.

Respiratory syncytial virus infection disrupts monolayer integrity and function in cystic fibrosis airway cells.

PubMed

Kong, Michele; Maeng, Patrick; Hong, Jeong; Szczesniak, Rhonda; Sorscher, Eric; Sullender, Wayne; Clancy, John Paul

2013-09-19

Respiratory Syncytial Virus (RSV) infection is a common contributor to pulmonary symptoms in children with cystic fibrosis (CF). Here we examined RSV infection in immortalized bronchial epithelial cells (CFBE41o-) expressing wild-type (wt) or F508del cystic fibrosis transmembrane conductance regulator (CFTR), for monolayer integrity and RSV replication. CFBE41o- monolayers expressing wt or F508del CFTR were grown on permeable supports and inoculated with RSV A2 strain. Control experiments utilized UV-inactivated RSV and heat-killed RSV. Monolayer resistance and RSV production was monitored for up to six days post-infection. Within 24 h, a progressive decrease in monolayer resistance was observed in RSV infected F508del CFBE41o- cells, while the monolayer integrity of RSV infected wt CFTR CFBE41o- cells remained stable. RSV replication was necessary to disrupt F508del CFBE41o- monolayers as UV-irradiated and heat killed RSV had no effect on monolayer integrity, with an earlier and much more pronounced peak in RSV titer noted in F508del relative to wt CFTR-expressing cells. RSV infection of wt CFBE41o- monolayers also resulted in blunting of CFTR response. These findings identify an enhanced sensitivity of CFBE41o- cells expressing F508del CFTR to RSV infection, replication and monolayer disruption independent of the cellular immune response, and provide a novel mechanism by which cystic fibrosis airway epithelia are susceptible to RSV-dependent injury.

Next generation diagnostics of cystic fibrosis and CFTR-related disorders by targeted multiplex high-coverage resequencing of CFTR.

PubMed

Trujillano, D; Ramos, M D; González, J; Tornador, C; Sotillo, F; Escaramis, G; Ossowski, S; Armengol, L; Casals, T; Estivill, X

2013-07-01

Here we have developed a novel and much more efficient strategy for the complete molecular characterisation of the cystic fibrosis (CF) transmembrane regulator (CFTR) gene, based on multiplexed targeted resequencing. We have tested this approach in a cohort of 92 samples with previously characterised CFTR mutations and polymorphisms. After enrichment of the pooled barcoded DNA libraries with a custom NimbleGen SeqCap EZ Choice array (Roche) and sequencing with a HiSeq2000 (Illumina) sequencer, we applied several bioinformatics tools to call mutations and polymorphisms in CFTR. The combination of several bioinformatics tools allowed us to detect all known pathogenic variants (point mutations, short insertions/deletions, and large genomic rearrangements) and polymorphisms (including the poly-T and poly-thymidine-guanine polymorphic tracts) in the 92 samples. In addition, we report the precise characterisation of the breakpoints of seven genomic rearrangements in CFTR, including those of a novel deletion of exon 22 and a complex 85 kb inversion which includes two large deletions affecting exons 4-8 and 12-21, respectively. This work is a proof-of-principle that targeted resequencing is an accurate and cost-effective approach for the genetic testing of CF and CFTR-related disorders (ie, male infertility) amenable to the routine clinical practice, and ready to substitute classical molecular methods in medical genetics.

CFTR and/or pancreatitis susceptibility genes mutations as risk factors of pancreatitis in cystic fibrosis patients?

PubMed

Gaitch, Natacha; Hubert, Dominique; Gameiro, Christine; Burgel, Pierre-Régis; Houriez, Florence; Martinez, Brigitte; Honoré, Isabelle; Chapron, Jeanne; Kanaan, Reem; Dusser, Daniel; Girodon, Emmanuelle; Bienvenu, Thierry

2016-01-01

Currently, factors that promote the occurrence of pancreatitis episodes in patients affected with cystic fibrosis (CF) and pancreatic sufficiency (PS) are largely unknown. Six genes involved in pancreatitis or in ion transport into the pancreatic duct were investigated by next generation sequencing in 59 adult CF-PS patients with two identified CF mutations. Data on predisposing environmental factors were also recorded. 19 experienced at least one episode of acute pancreatitis (AP) (AP+) and 40 patients did not (AP-). No influence of environmental factor was evidenced. No specific CFTR genotype was found predictive of pancreatitis. Patients sharing the same CFTR genotype may or may not experience AP episodes. Frequent and rare missense variants were found in 78.9% patients in group AP+ and 67.5% in group AP- but a few of them were pathogenic. AP or recurrent AP (RAP) is a frequent complication in our series of adult CF-PS patients. The majority of mild CFTR mutations found in group AP+ were located in the first transmembrane region. No clear other genetic factor could be found predictive of AP/RAP. Further experiments in large homogenous cohorts of CF-PS patients, including whole genome sequencing, may identify genetic predisposing factors to pancreatitis. Copyright © 2016 IAP and EPC. Published by Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thibodeau, Patrick H.; Brautigam, Chad A.; Machius, Mischa

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near positionmore » 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.« less

Cystic Fibrosis Transmembrane Conductance Regulator is an Epithelial Cell Receptor for Clearance of Pseudomonas aeruginosa from the Lung

NASA Astrophysics Data System (ADS)

Pier, Gerald B.; Grout, Martha; Zaidi, Tanweer S.

1997-10-01

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel, but its relationship to the primary clinical manifestation of CF, chronic Pseudomonas aeruginosa pulmonary infection, is unclear. We report that CFTR is a cellular receptor for binding, endocytosing, and clearing P. aeruginosa from the normal lung. Murine cells expressing recombinant human wild-type CFTR ingested 30-100 times as many P. aeruginosa as cells lacking CFTR or expressing mutant Δ F508 CFTR protein. Purified CFTR inhibited ingestion of P. aeruginosa by human airway epithelial cells. The first extracellular domain of CFTR specifically bound to P. aeruginosa and a synthetic peptide of this region inhibited P. aeruginosa internalization in vivo, leading to increased bacterial lung burdens. CFTR clears P. aeruginosa from the lung, indicating a direct connection between mutations in CFTR and the clinical consequences of CF.

Acute administration of ivacaftor to people with cystic fibrosis and a G551D-CFTR mutation reveals smooth muscle abnormalities

PubMed Central

Adam, Ryan J.; Hisert, Katherine B.; Dodd, Jonathan D.; Grogan, Brenda; Launspach, Janice L.; Barnes, Janel K.; Gallagher, Charles G.; Sieren, Jered P.; Gross, Thomas J.; Fischer, Anthony J.; Cavanaugh, Joseph E.; Hoffman, Eric A.; Singh, Pradeep K.; Welsh, Michael J.; McKone, Edward F.; Stoltz, David A.

2016-01-01

BACKGROUND. Airflow obstruction is common in cystic fibrosis (CF), yet the underlying pathogenesis remains incompletely understood. People with CF often exhibit airway hyperresponsiveness, CF transmembrane conductance regulator (CFTR) is present in airway smooth muscle (ASM), and ASM from newborn CF pigs has increased contractile tone, suggesting that loss of CFTR causes a primary defect in ASM function. We hypothesized that restoring CFTR activity would decrease smooth muscle tone in people with CF. METHODS. To increase or potentiate CFTR function, we administered ivacaftor to 12 adults with CF with the G551D-CFTR mutation; ivacaftor stimulates G551D-CFTR function. We studied people before and immediately after initiation of ivacaftor (48 hours) to minimize secondary consequences of CFTR restoration. We tested smooth muscle function by investigating spirometry, airway distensibility, and vascular tone. RESULTS. Ivacaftor rapidly restored CFTR function, indicated by reduced sweat chloride concentration. Airflow obstruction and air trapping also improved. Airway distensibility increased in airways less than 4.5 mm but not in larger-sized airways. To assess smooth muscle function in a tissue outside the lung, we measured vascular pulse wave velocity (PWV) and augmentation index, which both decreased following CFTR potentiation. Finally, change in distensibility of <4.5-mm airways correlated with changes in PWV. CONCLUSIONS. Acute CFTR potentiation provided a unique opportunity to investigate CFTR-dependent mechanisms of CF pathogenesis. The rapid effects of ivacaftor on airway distensibility and vascular tone suggest that CFTR dysfunction may directly cause increased smooth muscle tone in people with CF and that ivacaftor may relax smooth muscle. FUNDING. This work was funded in part from an unrestricted grant from the Vertex Investigator-Initiated Studies Program. PMID:27158673

RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator

PubMed Central

Smith, Laura; Litman, Paul; Kohli, Ekta; Amick, Joseph; Page, Richard C.; Misra, Saurav

2013-01-01

Mutations in cystic fibrosis transmembrane regulator (CFTR), a chloride channel in the apical membranes of secretory epithelial cells, underlie the fatal genetic disorder cystic fibrosis. Certain CFTR mutations, including the common mutation ΔF508-CFTR, result in greatly decreased levels of active CFTR at the apical membrane. Direct interactions between CFTR and the cytoskeletal adaptors filamin-A (FlnA) and Na+/H+ exchanger regulatory factor 1 (NHERF1) stabilize the expression and localization of CFTR at the plasma membrane. The scaffold protein receptor for activated C kinase 1 (RACK1) also stabilizes CFTR surface expression; however, RACK1 does not interact directly with CFTR and its mechanism of action is unknown. In the present study, we report that RACK1 interacts directly with FlnA in vitro and in a Calu-3 airway epithelial cell line. We mapped the interaction between RACK1 and FlnA to the WD4 and WD6 repeats of RACK1 and to a segment of the large rod domain of FlnA, consisting of immunoglobulin-like repeats 8–15. Disruption of the RACK1-FlnA interaction causes a reduction in CFTR surface levels. Our results suggest that a novel RACK1-FlnA interaction is an important regulator of CFTR surface localization. PMID:23636454

Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface*

PubMed Central

Chin, Stephanie; Yang, Donghe; Miles, Andrew J.; Eckford, Paul D. W.; Molinski, Steven; Wallace, B. A.; Bear, Christine E.

2017-01-01

Cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein that functions as a phosphorylation-regulated anion channel. The interface between its two cytosolic nucleotide binding domains and coupling helices conferred by intracellular loops extending from the channel pore domains has been referred to as a transmission interface and is thought to be critical for the regulated channel activity of CFTR. Phosphorylation of the regulatory domain of CFTR by protein kinase A (PKA) is required for its channel activity. However, it was unclear if phosphorylation modifies the transmission interface. Here, we studied purified full-length CFTR protein using spectroscopic techniques to determine the consequences of PKA-mediated phosphorylation. Synchrotron radiation circular dichroism spectroscopy confirmed that purified full-length wild-type CFTR is folded and structurally responsive to phosphorylation. Intrinsic tryptophan fluorescence studies of CFTR showed that phosphorylation reduced iodide-mediated quenching, consistent with an effect of phosphorylation in burying tryptophans at the transmission interface. Importantly, the rate of phosphorylation-dependent channel activation was compromised by the introduction of disease-causing mutations in either of the two coupling helices predicted to interact with nucleotide binding domain 1 at the interface. Together, these results suggest that phosphorylation modifies the interface between the catalytic and pore domains of CFTR and that this modification facilitates CFTR channel activation. PMID:28003367

Mutation and new polymorphisms insight in introns 11 to 14a of CFTR gene of northern Iranian cystic fibrosis patients.

PubMed

Esmaeili Dooki, Mohammad Reza; Tabaripour, Reza; Rahimi, Razieh; Akhavan-Niaki, Haleh

2015-06-15

Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasians, caused by mutation in cystic fibrosis transmembrane conductance regulator (CFTR). The type and distribution of mutations vary widely between different countries and ethnic groups. We therefore aimed to perform a comprehensive analysis of the CFTR gene in northern Iranian CF patients. Forty northern Iranian CF patients were analyzed for mutations in introns 11 to 14a of their CFTR genes, using sequencing and reverse dot blot methods. Five normal subjects were also analyzed as normal control. One mutation and seven polymorphisms were identified. Of the eighty alleles studied, c.2043delG in exon 13 represented 12.5% of mutant alleles and was associated with two distinct haplotypes. rs1042077T>G, rs4148712delAT, rs4148711T>A and rs3808183 T>C with frequencies varying between 29.2% and 6.9% for the least common allele, as well as three new polymorphisms c.1680-224C>A (11.1%), c.2491-275T>G (14.1%) and c.2491-274C>G (35.9%) were detected. These findings suggest a founder effect for c.2043delG in the Middle East and will assist in genetic counseling, prenatal diagnosis and future screening of CF in Iran. Copyright © 2015 Elsevier B.V. All rights reserved.

Mechanisms of CFTR Functional Variants That Impair Regulated Bicarbonate Permeation and Increase Risk for Pancreatitis but Not for Cystic Fibrosis

PubMed Central

Lewis, Michele D.; Park, Hyun Woo; Brand, Randall E.; Gelrud, Andres; Anderson, Michelle A.; Banks, Peter A.; Conwell, Darwin; Lawrence, Christopher; Romagnuolo, Joseph; Baillie, John; Alkaade, Samer; Cote, Gregory; Gardner, Timothy B.; Amann, Stephen T.; Slivka, Adam; Sandhu, Bimaljit; Aloe, Amy; Kienholz, Michelle L.; Yadav, Dhiraj; Barmada, M. Michael; Bahar, Ivet; Lee, Min Goo; Whitcomb, David C.

2014-01-01

CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<<0.0001). WNK1-SPAK pathway-activated increases in CFTR

Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.

PubMed

LaRusch, Jessica; Jung, Jinsei; General, Ignacio J; Lewis, Michele D; Park, Hyun Woo; Brand, Randall E; Gelrud, Andres; Anderson, Michelle A; Banks, Peter A; Conwell, Darwin; Lawrence, Christopher; Romagnuolo, Joseph; Baillie, John; Alkaade, Samer; Cote, Gregory; Gardner, Timothy B; Amann, Stephen T; Slivka, Adam; Sandhu, Bimaljit; Aloe, Amy; Kienholz, Michelle L; Yadav, Dhiraj; Barmada, M Michael; Bahar, Ivet; Lee, Min Goo; Whitcomb, David C

2014-07-01

CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<0.0001). WNK1-SPAK pathway-activated increases in CFTR

CFTR: A new horizon in the pathomechanism and treatment of pancreatitis

PubMed Central

Hegyi, Péter; Wilschanski, Michael; Muallem, Shmuel; Lukacs, Gergely; Sahin-Tóth, Miklós; Uc, Aliye; Gray, Michael A.; Rakonczay, Zoltán; Maléth, József

2017-01-01

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that conducts chloride and bicarbonate ions across epithelial cell membranes. Mutations in the CFTR gene diminish the ion channel function and lead to impaired epithelial fluid transport in multiple organs such as the lung and the pancreas resulting in cystic fibrosis. Heterozygous carriers of CFTR mutations do not develop cystic fibrosis but exhibit increased risk for pancreatitis and associated pancreatic damage characterized by elevated mucus levels, fibrosis and cyst formation. Importantly, recent studies demonstrated that pancreatitis causing insults, such as alcohol, smoking or bile acids strongly inhibit CFTR function. Furthermore, human studies showed reduced levels of CFTR expression and function in all forms of pancreatitis. These findings indicate that impairment of CFTR is critical in the development of pancreatitis; therefore, correcting CFTR function could be the first specific therapy in pancreatitis. In this review, we summarize recent advances in the field and discuss new possibilities for the treatment of pancreatitis. PMID:26856995

Expression of the cystic fibrosis transmembrane conductance regulator gene in the respiratory tract of normal individuals and individuals with cystic fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trapnell, B.C.; Chinshyan Chu; Paakko, P.K.

1991-08-01

The most common mutation of the cystic fibrosis transmembrane conductance regulator gene, CFTR, associated with the clinical disorder cystic fibrosis (CF) is called {Delta}Phe{sup 508}, a triple-base deletion resulting in loss of phenylalanine at residue 508 of the predicted 1480-amino acid CFTR protein. In the context that the lung is the major site of morbidity and mortality in CF, the authors evaluated airway epithelial cells for CFTR mRNA transcripts in normal individuals, normal-{Delta}Phe{sup 508} heterozygotes, and {Delta}Phe{sup 508} homozygotes to determine if the normal and {Delta}Phe{sup 508} CFTR alleles are expressed in the respiratory epithelium, to what extent they aremore » expressed, and whether there are relative differences in the expression of the normal and abnormal alleles at the mRNA level. Respiratory tract epithelial cells recovered by fiberoptic bronchoscopy with a cytology brush demonstrated CFTR mRNA transcripts with sequences appropriately reflecting the normal and {Delta}Phe{sup 508} CFTR alleles of the various study groups. CFTR gene expression quantified by limited polymerase chain reaction amplification showed that in normal individuals, CFTR mRNA transcripts are expressed in nasal, tracheal, and bronchial epithelial cells.« less

Pharmacological treatment of the ion transport defect in cystic fibrosis.

PubMed

Roomans, G M

2001-01-01

Cystic fibrosis (CF) is a lethal monogenetic disease characterised by impaired water and ion transport over epithelia. The lung pathology is fatal and causes death in 95% of CF patients. The genetic basis of the disease is a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. The most common mutation, DeltaF508, results in a protein that cannot properly be folded in the endoplasmic reticulum, is destroyed and hence does not reach the apical cell membrane. This paper will discuss those pharmacological approaches that are directed at correcting the defect in ion transport. At present, no clinically effective drug is available, although research has defined areas in which progress might be made. These are the following: (1) the drug 4-phenylbutyrate (4PBA) increases the expression of DeltaF508-CFTR in the cell membrane, probably by breaking the association between DeltaF508-CFTR and a chaperone; (2) a number of xanthines, in particular 8-cyclopentyl-1, 3-dipropylxanthine (CPX), are effective in activating CFTR, presumably by direct binding and also possibly by correcting the trafficking defect; (3) the isoflavone genistein can activate both wild-type and mutant CFTR, probably through direct binding to the channel; (4) purinergic agonists (ATP and UTP) can stimulate chloride secretion via a Ca(2+)-dependent chloride channel and in this way compensate for the defect in CFTR, but stable analogues will be required before this type of treatment has clinical significance; (5) treatment with inhaled amiloride may correct the excessive absorption of Na(+) ions and water by airway epithelial cells that appears connected to the defect in CFTR; although clinical tests have not been very successful so far, amiloride analogues with a longer half-life may give better results. The role of CFTR in bicarbonate secretion has not yet been established with certainty, but correction of the defect in bicarbonate secretion may be

[Pseudo-Bartter syndrome as manifestation of cystic fibrosis with DF508 mutation].

PubMed

Galaviz-Ballesteros, María de Jesús; Acosta-Rodríguez-Bueno, Carlos Patricio; Consuelo-Sánchez, Alejandra; Franco-Álvarez, Isidro; Olalla-Mora, Odilo Iván; Vázquez-Frias, Rodrigo

Pseudo Bartter syndrome (PBS) is defined as hypokalaemic hypochloraemic metabolic alkalosis in the absence of renal tubular pathology. Children with cystic fibrosis (CF) are at risk of developing electrolyte abnormalities and even PBS may occur. 5 months old female infant with a history of two events of dehydration with vomit, refusal to eat, chronic cough, polyuria, malnutrition, metabolic alkalosis, hypokalemia, hyponatremia, hypochloremia and acute renal failure. Chronic cough study was performed, discarding pulmonary tuberculosis, gastroesophageal reflux disease and impaired swallowing. PBS was diagnosed due to hypokalaemic hypochloraemic metabolic alkalosis in the absence of renal tubular pathology. CF was corroborated by electrolytes in sweat and through molecular analysis of the delta F508 mutation. This is one of the few reported cases linking PBS and this mutation. In patients with hyponatremic dehydration episodes with hypokalaemic hypochloraemic metabolic alkalosis, PBS should be considered as differential diagnosis. CF could be presented as PBS, mainly in patients younger than 2 years. Copyright © 2016 Hospital Infantil de México Federico Gómez. Publicado por Masson Doyma México S.A. All rights reserved.

Identification of eight mutations and three sequence variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghanem, N.; Costes, B.; Girodon, E.

1994-05-15

To determine cystic fibrosis (CF) defects in a sample of 224 non-[Delta]F508 CF chromosomes, the authors used denaturing gradient gel multiplex analysis of CF transmembrane conductance regulator gene segments, a strategy based on blind exhaustive analysis rather than a search for known mutations. This process allowed detection of 11 novel variations comprising two nonsense mutations (Q890X and W1204X), a splice defect (405 + 4 A [yields] G), a frameshift (3293delA), four presumed missense mutations (S912L, H949Y, L1065P, Q1071P), and three sequence polymorphisms (R31C or 223 C/T, 3471 T/C, and T1220I or 3791 C/T). The authors describe these variations, together withmore » the associated phenotype when defects on both CF chromosomes were identified. 8 refs., 1 fig., 1 tab.« less

CFTR genotype and clinical outcomes of adult patients carried as cystic fibrosis disease.

PubMed

Bonadia, Luciana Cardoso; de Lima Marson, Fernando Augusto; Ribeiro, Jose Dirceu; Paschoal, Ilma Aparecida; Pereira, Monica Corso; Ribeiro, Antonio Fernando; Bertuzzo, Carmen Silvia

2014-05-01

There are nearly 2000 cystic fibrosis transmembrane regulator (CFTR) mutations that cause cystic fibrosis (CF). These mutations are classified into six classes; on the one hand, the first three classes cause severe disease involvement in early childhood, on the other hand, the Class IV, V and VI mutations cause minor severe disease in the same age. Nowadays, with therapeutic advances in CF management and competence of pediatricians, physicians of adults have to deal with two groups of CF patients: (i) adults diagnosed in childhood with severe mutations and (ii) adults who initiated symptoms in adulthood and with Class IV, V and VI mutations. The aim of this study was to analyze adults from a clinical center, treated as CF disease, screening the CFTR genotype and evaluating the clinical characteristics. Thirty patients followed as CF disease at the University Hospital were enrolled. After a complete molecular CFTR negative screening and sweat test levels between 40 and 59mEq/L, five patients were characterized as non-CF disease and were excluded. Molecular screening was performed by CFTR gene sequencing/MLPA or by specific mutation screening. Clinical data was obtained from medical records. The patients were divided into three groups: (1) patients with Class I, II and III mutations in two CFTR alleles; (2) genotype with at least one allele of Class IV, V or VI CFTR mutations and, (3) non-identified CFTR mutation+one patient with one allele with CFTR mutation screened (Class I). There was an association of CFTR class mutation and sodium/chloride concentration in the sweat test (sodium: p=0.040; chloride: p=0.016), onset of digestive symptoms (p=0.012), lung function parameter (SpO2 - p=0.016), Bhalla score (p=0.021), age at diagnosis (p=0.008) and CF-related diabetes (p=0.029). There was an association between Pseudomonas aeruginosa chronic colonization (as clinical marker for the lung disease status) and lung impairment (FEV1% - p=0.027; Bhalla score - p=0.021), CF

Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator

PubMed Central

Chang, Xiu-bao; Mengos, April; Hou, Yue-xian; Cui, Liying; Jensen, Timothy J.; Aleksandrov, Andrei; Riordan, John R.; Gentzsch, Martina

2009-01-01

Summary The epithelial chloride channel CFTR is a glycoprotein that is modified by two N-linked oligosaccharides. The most common mutant CFTR protein in patients with cystic fibrosis, ΔF508, is misfolded and retained by ER quality control. As oligosaccharide moieties of glycoproteins are known to mediate interactions with ER lectin chaperones, we investigated the role of N-linked glycosylation in the processing of wild-type and ΔF508 CFTR. We found that N-glycosylation and ER lectin interactions are not major determinants of trafficking of wild-type and ΔF508 from the ER to the plasma membrane. Unglycosylated CFTR, generated by removal of glycosylation sites or treatment of cells with the N-glycosylation inhibitor tunicamycin, did not bind calnexin, but did traffic to the cell surface and exhibited chloride channel activity. Most importantly, unglycosylated Δ F508 CFTR still could not escape quality control in the early secretory pathway and remained associated with the ER. However, the absence of N-linked oligosaccharides did reduce the stability of wild-type CFTR, causing significantly more-rapid turnover in post-ER compartments. Surprisingly, the individual N-linked carbohydrates do not play equivalent roles and modulate the fate of the wild-type protein in different ways in its early biosynthetic pathway. PMID:18682497

Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator.

PubMed

Chang, Xiu-Bao; Mengos, April; Hou, Yue-Xian; Cui, Liying; Jensen, Timothy J; Aleksandrov, Andrei; Riordan, John R; Gentzsch, Martina

2008-09-01

The epithelial chloride channel CFTR is a glycoprotein that is modified by two N-linked oligosaccharides. The most common mutant CFTR protein in patients with cystic fibrosis, DeltaF508, is misfolded and retained by ER quality control. As oligosaccharide moieties of glycoproteins are known to mediate interactions with ER lectin chaperones, we investigated the role of N-linked glycosylation in the processing of wild-type and DeltaF508 CFTR. We found that N-glycosylation and ER lectin interactions are not major determinants of trafficking of wild-type and DeltaF508 from the ER to the plasma membrane. Unglycosylated CFTR, generated by removal of glycosylation sites or treatment of cells with the N-glycosylation inhibitor tunicamycin, did not bind calnexin, but did traffic to the cell surface and exhibited chloride channel activity. Most importantly, unglycosylated DeltaF508 CFTR still could not escape quality control in the early secretory pathway and remained associated with the ER. However, the absence of N-linked oligosaccharides did reduce the stability of wild-type CFTR, causing significantly more-rapid turnover in post-ER compartments. Surprisingly, the individual N-linked carbohydrates do not play equivalent roles and modulate the fate of the wild-type protein in different ways in its early biosynthetic pathway.

Genotype-phenotype correlation and functional studies in patients with cystic fibrosis bearing CFTR complex alleles.

PubMed

Terlizzi, Vito; Castaldo, Giuseppe; Salvatore, Donatello; Lucarelli, Marco; Raia, Valeria; Angioni, Adriano; Carnovale, Vincenzo; Cirilli, Natalia; Casciaro, Rosaria; Colombo, Carla; Di Lullo, Antonella Miriam; Elce, Ausilia; Iacotucci, Paola; Comegna, Marika; Scorza, Manuela; Lucidi, Vincenzina; Perfetti, Anna; Cimino, Roberta; Quattrucci, Serena; Seia, Manuela; Sofia, Valentina Maria; Zarrilli, Federica; Amato, Felice

2017-04-01

The effect of complex alleles in cystic fibrosis (CF) is poorly defined for the lack of functional studies. To describe the genotype-phenotype correlation and the results of either in vitro and ex vivo studies performed on nasal epithelial cells (NEC) in a cohort of patients with CF carrying cystic fibrosis transmembrane conductance regulator ( CFTR ) complex alleles. We studied 70 homozygous, compound heterozygous or heterozygous for CFTR mutations: p.[Arg74Trp;Val201Met;Asp1270Asn], n=8; p.[Ile148Thr;Ile1023_Val1024del], n=5; p.[Arg117Leu;Leu997Phe], n=6; c.[1210-34TG[12];1210-12T[5];2930C>T], n=3; p.[Arg74Trp;Asp1270Asn], n=4; p.Asp1270Asn, n=2; p.Ile148Thr, n=6; p.Leu997Phe, n=36. In 39 patients, we analysed the CFTR gating activity on NEC in comparison with patients with CF (n=8) and carriers (n=4). Finally, we analysed in vitro the p.[Arg74Trp;Val201Met;Asp1270Asn] complex allele. The p.[Ile148Thr;Ile1023_Val1024del] caused severe CF in five compound heterozygous with a class I-II mutation. Their CFTR activity on NEC was comparable with patients with two class I-II mutations (mean 7.3% vs 6.9%). The p.[Arg74Trp;Asp1270Asn] and the p.Asp1270Asn have scarce functional effects, while p.[Arg74Trp;Val201Met;Asp1270Asn] caused mild CF in four of five subjects carrying a class I-II mutation in trans , or CFTR-related disorders (CFTR-RD) in three having in trans a class IV-V mutation. The p.[Arg74Trp;Val201Met;Asp1270Asn] causes significantly (p<0.001) higher CFTR activity compared with compound heterozygous for class I-II mutations. Furthermore, five of six compounds heterozygous with the p.[Arg117Leu;Leu997Phe] had mild CF, whereas the p.Leu997Phe, in trans with a class I-II CFTR mutation, caused CFTR-RD or a healthy status (CFTR activity: 21.3-36.9%). Finally, compounds heterozygous for the c.[1210-34TG[12];1210-12T[5];2930C>T] and a class I-II mutation had mild CF or CFTR-RD (gating activity: 18.5-19.0%). The effect of complex alleles partially depends on the

Genetic and clinical features of false-negative infants in a neonatal screening programme for cystic fibrosis.

PubMed

Padoan, R; Genoni, S; Moretti, E; Seia, M; Giunta, A; Corbetta, C

2002-01-01

A study was performed on the delayed diagnosis of cystic fibrosis (CF) in infants who had false-negative results in a neonatal screening programme. The genetic and clinical features of false-negative infants in this screening programme were assessed together with the efficiency of the screening procedure in the Lombardia region. In total, 774,687 newborns were screened using a two-step immunoreactive trypsinogen (IRT) (in the years 1990-1992), IRT/IRT + delF508 (1993-1998) or IRT/IRT + polymerase chain reaction (PCR) and oligonucleotide ligation assay (OLA) protocol (1998-1999). Out of 196 CF children born in the 10 y period 15 were false negative on screening (7.6%) and molecular analysis showed a high variability in the genotypes. The cystic fibrosis transmembrane regulator (CFTR) gene mutations identified were delF508, D1152H, R1066C, R334W, G542X, N1303K, F1052V, A120T, 3849 + 10kbC --> T, 2789 + 5G --> A, 5T-12TG and the novel mutation D110E. In three patients no mutation was identified after denaturing gradient gel electrophoresis of the majority of CFTR gene exons. The clinical phenotypes of CF children diagnosed by their symptoms at different ages were very mild. None of them presented with a severe lung disease. The majority of them did not seem to have been damaged by the delayed diagnosis. The combination of IRT assay plus genotype analysis (1998-1999) appears to be a more reliable method of detecting CF than IRT measurement alone or combined with only the delF508 mutation.

Interaction among variants in the SLC gene family (SLC6A14, SLC26A9, SLC11A1, and SLC9A3) and CFTR mutations with clinical markers of cystic fibrosis.

PubMed

Pereira, Stephanie V N; Ribeiro, Jose D; Bertuzzo, Carmen S; Marson, Fernando A L

2018-04-10

Cystic fibrosis (CF) is due to dysfunction of the CFTR channel and function of this channel is, in turn, affected by modifier genes that can impact the clinical phenotype. In this context, we analyzed the interaction among rs3788766*SLC6A14, rs7512462*SLC26A9, rs17235416*SLC11A1, and rs17563161*SLC9A3 variants, CFTR mutations and 40 CF severity markers by the Multifactor Dimensionality Reduction (MDR) model. A total of 164 patients with CF were included in the study. The variants in the modifier genes were identified by real-time PCR and the genotype of the CFTR gene in the diagnostic routine. Analysis of interaction between variants, CFTR mutations groupings and demographic, clinical and laboratory data were performed by the MDR. There were interaction between the rs3788766, rs7512462, rs17235416, and rs17563161 variants, and CFTR mutations with pancreatic insufficiency (PI), onset of digestive symptoms, and presence of mucoid Pseudomonas aeruginosa. Regarding PI, the interaction was observed for CFTR*rs17563161 (P-value = 0.015). Also, for onset of digestive symptoms the interaction was observed for CFTR*rs3788766*rs7512462*rs17235416*rs17563161 (P-value = 0.036). Considering the presence of mucoid P. aeruginosa, the interaction occurred for CFTR*rs3788766*rs7512462*rs17563161 (P-value = 0.035). Interaction between variants in the SLC family genes and the grouping for CFTR mutations were associated with PI, onset of digestive symptoms and mucoid P. aeruginosa, being important to determine one of the factors that may cause the diversity among the patients with CF. © 2018 Wiley Periodicals, Inc.

Specific stabilization of CFTR by phosphatidylserine.

PubMed

Hildebrandt, Ellen; Khazanov, Netaly; Kappes, John C; Dai, Qun; Senderowitz, Hanoch; Urbatsch, Ina L

2017-02-01

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR, ABCC7) is a plasma membrane chloride ion channel in the ABC transporter superfamily. CFTR is a key target for cystic fibrosis drug development, and its structural elucidation would advance those efforts. However, the limited in vivo and in vitro stability of the protein, particularly its nucleotide binding domains, has made structural studies challenging. Here we demonstrate that phosphatidylserine uniquely stimulates and thermally stabilizes the ATP hydrolysis function of purified human CFTR. Among several lipids tested, the greatest stabilization was observed with brain phosphatidylserine, which shifted the Tm for ATPase activity from 22.7±0.8°C to 35.0±0.2°C in wild-type CFTR, and from 26.6±0.7°C to 42.1±0.2°C in a more stable mutant CFTR having deleted regulatory insertion and S492P/A534P/I539T mutations. When ATPase activity was measured at 37°C in the presence of brain phosphatidylserine, Vmax for wild-type CFTR was 240±60nmol/min/mg, a rate higher than previously reported and consistent with rates for other purified ABC transporters. The significant thermal stabilization of CFTR by phosphatidylserine may be advantageous in future structural and biophysical studies of CFTR. Copyright © 2016. Published by Elsevier B.V.

CFTR dysfunction in cystic fibrosis and chronic obstructive pulmonary disease.

PubMed

Fernandez Fernandez, Elena; de Santi, Chiara; De Rose, Virginia; Greene, Catherine M

2018-05-11

Obstructive lung diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are causes of high morbidity and mortality worldwide. CF is a multiorgan genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is characterized by progressive chronic obstructive lung disease. Most cases of COPD are a result of noxious particles, mainly cigarette smoke but also other environmental pollutants. Areas covered: Although the pathogenesis and pathophysiology of CF and COPD differ, they do share key phenotypic features and because of these similarities there is great interest in exploring common mechanisms and/or factors affected by CFTR mutations and environmental insults involved in COPD. Various molecular, cellular and clinical studies have confirmed that CFTR protein dysfunction is common in both the CF and COPD airways. This review provides an update of our understanding of the role of dysfunctional CFTR in both respiratory diseases. Expert Commentary: Drugs developed for people with CF to improve mutant CFTR function and enhance CFTR ion channel activity might also be beneficial in patients with COPD. A move toward personalized therapy using, for example, microRNA modulators in conjunction with CFTR potentiators or correctors, could enhance treatment of both diseases.

Cystic fibrosis transmembrane conductance regulator mutations at a referral center for cystic fibrosis*

PubMed Central

Coutinho, Cyntia Arivabeni de Araújo Correia; Marson, Fernando Augusto de Lima; Ribeiro, Antônio Fernando; Ribeiro, José Dirceu; Bertuzzo, Carmen Silvia

2013-01-01

OBJECTIVE: To determine the frequency of six mutations (F508del, G542X, G551D, R553X, R1162X, and N1303K) in patients with cystic fibrosis (CF) diagnosed, at a referral center, on the basis of abnormal results in two determinations of sweat sodium and chloride concentrations. METHODS: This was a cross-sectional study involving 70 patients with CF. The mean age of the patients was 12.38 ± 9.00 years, 51.43% were female, and 94.29% were White. Mutation screening was performed with polymerase chain reaction (for F508del), followed by enzymatic digestion (for other mutations). Clinical analysis was performed on the basis of gender, age, ethnicity, pulmonary/gastrointestinal symptoms, and Shwachman-Kulczycki (SK) score. RESULTS: All of the patients showed pulmonary symptoms, and 8 had no gastrointestinal symptoms. On the basis of the SK scores, CF was determined to be mild, moderate, and severe in 22 (42.3%), 17 (32.7%), and 13 (25.0%) of the patients, respectively. There was no association between F508del mutation and disease severity by SK score. Of the 140 alleles analyzed, F508del mutation was identified in 70 (50%). Other mutations (G542X, G551D, R553X, R1162X, and N1303K) were identified in 12 (7.93%) of the alleles studied. In F508del homozygous patients with severe disease, the OR was 0.124 (95% CI: 0.005-0.826). CONCLUSIONS: In 50% of the alleles studied, the molecular diagnosis of CF was confirmed by identifying a single mutation (F508del). If we consider the analysis of the six most common mutations in the Brazilian population (including F508del), the molecular diagnosis was confirmed in 58.57% of the alleles studied. PMID:24310628

Cystic fibrosis transmembrane conductance regulator mutations at a referral center for cystic fibrosis.

PubMed

Coutinho, Cyntia Arivabeni de Araújo Correia; Marson, Fernando Augusto de Lima; Ribeiro, Antônio Fernando; Ribeiro, José Dirceu; Bertuzzo, Carmen Silvia

2013-01-01

To determine the frequency of six mutations (F508del, G542X, G551D, R553X, R1162X, and N1303K) in patients with cystic fibrosis (CF) diagnosed, at a referral center, on the basis of abnormal results in two determinations of sweat sodium and chloride concentrations. This was a cross-sectional study involving 70 patients with CF. The mean age of the patients was 12.38 ± 9.00 years, 51.43% were female, and 94.29% were White. Mutation screening was performed with polymerase chain reaction (for F508del), followed by enzymatic digestion (for other mutations). Clinical analysis was performed on the basis of gender, age, ethnicity, pulmonary/gastrointestinal symptoms, and Shwachman-Kulczycki (SK) score. All of the patients showed pulmonary symptoms, and 8 had no gastrointestinal symptoms. On the basis of the SK scores, CF was determined to be mild, moderate, and severe in 22 (42.3%), 17 (32.7%), and 13 (25.0%) of the patients, respectively. There was no association between F508del mutation and disease severity by SK score. Of the 140 alleles analyzed, F508del mutation was identified in 70 (50%). Other mutations (G542X, G551D, R553X, R1162X, and N1303K) were identified in 12 (7.93%) of the alleles studied. In F508del homozygous patients with severe disease, the OR was 0.124 (95% CI: 0.005-0.826). In 50% of the alleles studied, the molecular diagnosis of CF was confirmed by identifying a single mutation (F508del). If we consider the analysis of the six most common mutations in the Brazilian population (including F508del), the molecular diagnosis was confirmed in 58.57% of the alleles studied.

The effect of NO-donors on chloride efflux, intracellular Ca(2+) concentration and mRNA expression of CFTR and ENaC in cystic fibrosis airway epithelial cells.

PubMed

Oliynyk, Igor; Hussain, Rashida; Amin, Ahmad; Johannesson, Marie; Roomans, Godfried M

2013-06-01

Since previous studies showed that the endogenous bronchodilator, S-nitrosglutathione (GSNO), caused a marked increase in CFTR-mediated chloride (Cl(-)) efflux and improved the trafficking of CFTR to the plasma membrane, and that also the nitric oxide (NO)-donor GEA3162 had a similar, but smaller, effect on Cl(-) efflux, it was investigated whether the NO-donor properties of GSNO were relevant for its effect on Cl(-) efflux from airway epithelial cells. Hence, the effect of a number of other NO-donors, sodium nitroprusside (SNP), S-nitroso-N-acetyl-DL-penicillamine (SNAP), diethylenetriamine/nitric oxide adduct (DETA-NO), and diethylenetriamine/nitric oxide adduct (DEA-NONOate) on Cl(-) efflux from CFBE (∆F508/∆F508-CFTR) airway epithelial cells was tested. Cl(-) efflux was determined using the fluorescent N-(ethoxycarbonylmethyl)-6-methoxyquinoliniu bromide (MQAE)-technique. Possible changes in the intracellular Ca(2+) concentration were tested by the fluorescent fluo-4 method in a confocal microscope system. Like previously with GSNO, after 4 h incubation with the NO-donor, an increased Cl(-) efflux was found (in the order SNAP>DETA-NO>SNP). The effect of DEA-NONOate on Cl(-) efflux was not significant, and the compound may have (unspecific) deleterious effects on the cells. Again, as with GSNO, after a short (5 min) incubation, SNP had no significant effect on Cl(-) efflux. None of the NO-donors that had a significant effect on Cl(-) efflux caused significant changes in the intracellular Ca(2+) concentration. After 4 h preincubation, SNP caused a significant increase in the mRNA expression of CFTR. SNAP and DEA-NONOate decreased the mRNA expression of all ENaC subunits significantly. DETA-NO caused a significant decrease only in α-ENaC expression. After a short preincubation, none of the NO-donors had a significant effect, neither on the expression of CFTR, nor on that of the ENaC subunits in the presence and absence of L-cysteine. It can be concluded that

Preconceptional identification of cystic fibrosis carriers in the Sardinian population: A pilot screening program.

PubMed

Coiana, Alessandra; Faa', Valeria; Carta, Daniela; Puddu, Rosalba; Cao, Antonio; Rosatelli, Maria Cristina

2011-05-01

In Sardinia the mutational spectrum of CFTR gene is well defined. A mutation detection rate of 94% can be achieved by screening for 15 CFTR mutations with a frequency higher than 0.5%. The efficiency of this molecular test suggests that Sardinians may represent a suitable population for a preconceptional screening. Five hundred couples of Sardinia descent were screened for 38 mutations using a semi-automated reverse-dot blot and PCR-gel electrophoresis assays. This mutation panel included the 15 most frequent CF alleles in Sardinia. We identified 38 CF carriers, revealing an overall frequency of 1/25 (4%). The most common CF allele was the p.Thr338Ile (T338I) (65%), followed by the p.Phe508del (F508del) (22.5%). We also identified one couple at risk and an asymptomatic female homozygote for the p.Thr338Ile allele. In spite of the low number of the couples tested, the results herein reported demonstrate the efficacy and efficiency of the preconceptional screening program and the high participation rate of the Sardinian population (99%). Copyright © 2010 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Serum zinc concentration in cystic fibrosis patients with CFTR I1234V mutation associated with pancreatic sufficiency.

PubMed

AbdulWahab, Atqah; Abushahin, Ahmed; Allangawi, Mona; Chandra, Prem; Abdel Rahman, Mohamed Osman; Soliman, Ashraf

2017-05-01

To determine serum zinc (Zn) level among cystic fibrosis (CF) patients with homozygous CFTR I1234V mutation associated with pancreatic sufficiency (PS). A cross-sectional study was conducted including both pediatric and adult CF patients. Data on age, weight, height, body mass index (BMI), BMI Z-score, FEV1, and chronic Pseudomonas aeruginosa infection were collected. Serum Zn, albumin, and total proteins were measured and analyzed. Forty-five CF patients with homozygous CFTR I1234V mutation belonging to a large Arab kindred tribe and eight CF patients with other mutations associated with pancreatic insufficiency (PI). Patient's age ranged from 2 to 49 years with a mean age of 15.1 ± 9.1 years and mean plasma Zn of 0.78 ± 0.15 mcg/mL. Seven (13.2%) patients with CFTR I1234V and PS had low Zn levels (<0.6 mcg/mL). Mean age among Zn deficient group was significantly older. The mean FEV1 in the deficient group was found to be insignificant low. Persistent P. aeruginosa colonization was more prevalent in Zn deficient group. BMI Z-scosre of CF patients were positively correlated with Zn levels. Forty-five healthy subjects belonging to the same Arab tribe were selected in order to assess their Zn levels and their mean plasma Zn of 0.84 ± 0.11 mcg/mL (range 0.65-1.1 mcg/mL) with mean age 20.4 ± 10.1 years (range 6-40 years). These findings suggest that Zn deficiency can occur in CF patients with PS. The association of Zn levels and the frequency of P. aeruginosa isolated in CF patients need further investigation. © 2015 The Authors. The Clinical Respiratory Journal Published by John Wiley & Sons Ltd.

Cystic fibrosis transmembrane conductance regulator regulates epithelial cell response to Aspergillus and resultant pulmonary inflammation.

PubMed

Chaudhary, Neelkamal; Datta, Kausik; Askin, Frederic B; Staab, Janet F; Marr, Kieren A

2012-02-01

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) alter epithelial cell (EC) interactions with multiple microbes, such that dysregulated inflammation and injury occur with airway colonization in people with cystic fibrosis (CF). Aspergillus fumigatus frequently colonizes CF airways, but it has been assumed to be an innocent saprophyte; its potential role as a cause of lung disease is controversial. To study the interactions between Aspergillus and EC, and the role of the fungus in evoking inflammatory responses. A. fumigatus expressing green fluorescent protein was developed for in vitro and in vivo models, which used cell lines and mouse tracheal EC. Fungal spores (conidia) are rapidly ingested by ECs derived from bronchial cell lines and murine tracheas, supporting a role for EC in early airway clearance. Bronchial ECs harboring CFTR mutations (ΔF508) or deletion demonstrate impaired uptake and killing of conidia, and ECs with CFTR mutation undergo more conidial-induced apoptosis. Germinated (hyphal) forms of the fungus evoke secretion of inflammatory mediators, with CFTR mutation resulting in increased airway levels of macrophage inflammatory protein 2 and KC, and higher lung monocyte chemotactic protein-1. After A. fumigatus inhalation, CFTR(-/-) mice develop exaggerated lymphocytic inflammation, mucin accumulation, and lung injury. Data demonstrate a critical role for CFTR in mediating EC responses to A. fumigatus. Results suggest that the fungus elicits aberrant pulmonary inflammation in the setting of CFTR mutation, supporting the potential role of antifungals to halt progressive CF lung disease.

Meconium ileus caused by mutations in GUCY2C, encoding the CFTR-activating guanylate cyclase 2C.

PubMed

Romi, Hila; Cohen, Idan; Landau, Daniella; Alkrinawi, Suliman; Yerushalmi, Baruch; Hershkovitz, Reli; Newman-Heiman, Nitza; Cutting, Garry R; Ofir, Rivka; Sivan, Sara; Birk, Ohad S

2012-05-04

Meconium ileus, intestinal obstruction in the newborn, is caused in most cases by CFTR mutations modulated by yet-unidentified modifier genes. We now show that in two unrelated consanguineous Bedouin kindreds, an autosomal-recessive phenotype of meconium ileus that is not associated with cystic fibrosis (CF) is caused by different homozygous mutations in GUCY2C, leading to a dramatic reduction or fully abrogating the enzymatic activity of the encoded guanlyl cyclase 2C. GUCY2C is a transmembrane receptor whose extracellular domain is activated by either the endogenous ligands, guanylin and related peptide uroguanylin, or by an external ligand, Escherichia coli (E. coli) heat-stable enterotoxin STa. GUCY2C is expressed in the human intestine, and the encoded protein activates the CFTR protein through local generation of cGMP. Thus, GUCY2C is a likely candidate modifier of the meconium ileus phenotype in CF. Because GUCY2C heterozygous and homozygous mutant mice are resistant to E. coli STa enterotoxin-induced diarrhea, it is plausible that GUCY2C mutations in the desert-dwelling Bedouin kindred are of selective advantage. Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Sweat chloride and immunoreactive trypsinogen in infants carrying two CFTR mutations and not affected by cystic fibrosis.

PubMed

Castellani, Carlo; Tridello, Gloria; Tamanini, Anna; Assael, Baroukh M

2017-07-01

Newborns with raised immunotrypsinogen levels who have non-pathological sweat chloride values and carry two cystic fibrosis transmembrane regulator ( CFTR ) mutations of which at least one is not acknowledged to be cystic fibrosis (CF)-causing are at risk of developing clinical manifestations consistent with CFTR-related disorders or even CF. It is not known whether newborns with similar genotypes and normal immunoreactive trypsinogen (IRT) may share the same risk. This study found that newborns with these characteristics and normal IRT have lower sweat chloride values than those with raised IRT (p=0.007). Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Is there evidence for correct diagnosis in cystic fibrosis registries?

PubMed

Thomas, Muriel; Lemonnier, Lydie; Gulmans, Vincent; Naehrlich, Lutz; Vermeulen, François; Cuppens, Harry; Castellani, Carlo; Norek, Aleksandra; De Boeck, Kris

2014-05-01

Cystic fibrosis (CF) spans a wide spectrum. Therefore, benchmarking between registries implies comparing similar cohorts. Explore patient characteristics in Belgian (B), French (F), German (G) and Dutch (NL) registries (total N=13,122) and determine whether they fulfill predefined diagnostic criteria. Using as case definition sweat chloride >60mmol/L or 2 CFTR mutations identified, CF diagnosis was not documented in 2.8, 5.7, 6.5 and 21.6% of subjects in the F, B, NL, and G registries. Restricting CFTR mutation interpretation to 124 CF causing mutations in CFTR2, these numbers rose to 10.5, 10.4, 14.5 and 24.3% respectively. Excluding these subjects impacted on outcomes. The impact differed between countries; the largest changes seen were a decrease in % adults from 51.9 to 47.8% in G, a decrease in % pancreas sufficiency from 17.0 to 13.0 in F, an increase in % homozygous for F508del from 55.3 to 63.7 in NL and a decrease of % with sweat chloride ≤60mmol/L from 8.4 to 1.1 in B. CF diagnosis is not documented in 10 to 24% of patients included in CF registries. Excluding these patients for analyses leads to significant changes in outcomes. © 2013.

Impact of Gene Patents and Licensing Practices on Access to Genetic Testing for Cystic Fibrosis

PubMed Central

Chandrasekharan, Subhashini; Heaney, Christopher; James, Tamara; Conover, Chris; Cook-Deegan, Robert

2010-01-01

Cystic fibrosis (CF) is one of the most commonly tested autosomal recessive disorders in the US. Clinical CF is associated with mutations in the CFTR gene, of which the most common mutation among Caucasians, ΔF508, was identified in 1989. The University of Michigan, Johns Hopkins University, and the Hospital for Sick Children, where much of the initial research occurred, hold key patents for CF genetic sequences, mutations and methods for detecting them. Several patents including the one that covers detection of the ΔF508 mutation are jointly held by the University of Michigan and the Hospital for Sick Children in Toronto, with Michigan administering patent licensing in the US. The University of Michigan broadly licenses the ΔF508 patent for genetic testing with over 60 providers of genetic testing to date. Genetic testing is now used in newborn screening, diagnosis, and reproductive decisions. Interviews with key researchers and intellectual property managers, a survey of laboratories’ prices for CF genetic testing, a review of literature on CF tests’ cost effectiveness, and a review of the developing market for CF testing provide no evidence that patents have significantly hindered access to genetic tests for CF or prevented financially cost-effective screening. Current licensing practices for cystic fibrosis (CF) genetic testing appear to facilitate both academic research and commercial testing. More than one thousand different CFTR mutations have been identified, and research continues to determine their clinical significance. Patents have been nonexclusively licensed for diagnostic use, and have been variably licensed for gene transfer and other therapeutic applications. The Cystic Fibrosis Foundation has been engaged in licensing decisions, making CF a model of collaborative and cooperative patenting and licensing practice. PMID:20393308

CFTR allelic heterogeneity in Mexican patients with cystic fibrosis: implications for molecular screening.

PubMed

Chávez-Saldaña, Margarita; Yokoyama, Emiy; Lezana, José Luis; Carnevale, Alessandra; Macías, Miguel; Vigueras, Rosa M; López, Marisol; Orozco, Lorena

2010-01-01

Cystic fibrosis, the most common autosomal recessive disorder, is caused by defects in the CF transmembrane conductance regulator gene (CFTR) that encodes a chloride channel. To date, over 1,800 mutations have been described related to the causative gene of CF, showing a variable frequency among populations. In a previous extensive analysis of the CFTR locus in 97 Mexican patients, 34 different mutations (75% of CF alleles) were found using several strategies for mutation screening; however, 63% had at least an uncharacterized allele. Despite the combined technologies used, there are still a great number of unknown mutations in the Mexican population. Screening of the CFTR gene to provide additional evidence of the mutational wide spectrum responsible for CF in Mexican patients. In this study, the number of unrelated CF patients was increased to 230, 133 new cases and the 97 previously reported to include 63% with at least an uncharacterized allele. Additional tools were used to improve the detection rate of CF mutations, such as a commercial kit for 36 mutations plus a single chain conformational polymorphism method and DNA sequencing. By using a combination of these strategies we characterized 77.7% of all the CF alleles, resulting in a total of 46 different mutations detected, including the identification of 12 additional mutations (p.R334W, p.A455E, c.3120+1G > A, c.3272-26A > G, c.711+1G > T, p.Q552X, p.W1282X, c.IVS8-5T, p.R1162X and p.R347P, p.D1152H and p.T1036N). Although these 12 mutations have been reported in other populations, they have not yet been reported in Mexican patients. This report shows that Mexico has one of the widest spectra of CFTR mutations worldwide. The knowledge of the ethnic and geographic distribution of CFTR mutations in this population will allow the development of more effective methods for diagnosis and treatment.

Diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator gene in patients suspected of having mild or atypical cystic fibrosis *

PubMed Central

Dal'Maso, Vinícius Buaes; Mallmann, Lucas; Siebert, Marina; Simon, Laura; Saraiva-Pereira, Maria Luiza; Dalcin, Paulo de Tarso Roth

2013-01-01

OBJECTIVE: To evaluate the diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in patients suspected of having mild or atypical cystic fibrosis (CF). METHODS: This was a cross-sectional study involving adolescents and adults aged ≥ 14 years. Volunteers underwent clinical, laboratory, and radiological evaluation, as well as spirometry, sputum microbiology, liver ultrasound, sweat tests, and molecular analysis of the CFTR gene. We then divided the patients into three groups by the number of mutations identified (none, one, and two or more) and compared those groups in terms of their characteristics. RESULTS: We evaluated 37 patients with phenotypic findings of CF, with or without sweat test confirmation. The mean age of the patients was 32.5 ± 13.6 years, and females predominated (75.7%). The molecular analysis contributed to the definitive diagnosis of CF in 3 patients (8.1%), all of whom had at least two mutations. There were 7 patients (18.9%) with only one mutation and 26 patients (70.3%) with no mutations. None of the clinical characteristics evaluated was found to be associated with the genetic diagnosis. The most common mutation was p.F508del, which was found in 5 patients. The combination of p.V232D and p.F508del was found in 2 patients. Other mutations identified were p.A559T, p.D1152H, p.T1057A, p.I148T, p.V754M, p.P1290P, p.R1066H, and p.T351S. CONCLUSIONS: The molecular analysis of the CFTR gene coding region showed a limited contribution to the diagnostic investigation of patients suspected of having mild or atypical CF. In addition, there were no associations between the clinical characteristics and the genetic diagnosis. PMID:23670503

Diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator gene in patients suspected of having mild or atypical cystic fibrosis.

PubMed

Dal'Maso, Vinícius Buaes; Mallmann, Lucas; Siebert, Marina; Simon, Laura; Saraiva-Pereira, Maria Luiza; Dalcin, Paulo de Tarso Roth

2013-01-01

To evaluate the diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in patients suspected of having mild or atypical cystic fibrosis (CF). This was a cross-sectional study involving adolescents and adults aged > 14 years. Volunteers underwent clinical, laboratory, and radiological evaluation, as well as spirometry, sputum microbiology, liver ultrasound, sweat tests, and molecular analysis of the CFTR gene. We then divided the patients into three groups by the number of mutations identified (none, one, and two or more) and compared those groups in terms of their characteristics. We evaluated 37 patients with phenotypic findings of CF, with or without sweat test confirmation. The mean age of the patients was 32.5 ± 13.6 years, and females predominated (75.7%). The molecular analysis contributed to the definitive diagnosis of CF in 3 patients (8.1%), all of whom had at least two mutations. There were 7 patients (18.9%) with only one mutation and 26 patients (70.3%) with no mutations. None of the clinical characteristics evaluated was found to be associated with the genetic diagnosis. The most common mutation was p.F508del, which was found in 5 patients. The combination of p.V232D and p.F508del was found in 2 patients. Other mutations identified were p.A559T, p.D1152H, p.T1057A, p.I148T, p.V754M, p.P1290P, p.R1066H, and p.T351S. The molecular analysis of the CFTR gene coding region showed a limited contribution to the diagnostic investigation of patients suspected of having mild or atypical CF. In addition, there were no associations between the clinical characteristics and the genetic diagnosis.

Recent Progress in CFTR Interactome Mapping and Its Importance for Cystic Fibrosis.

PubMed

Lim, Sang Hyun; Legere, Elizabeth-Ann; Snider, Jamie; Stagljar, Igor

2017-01-01

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride channel found in secretory epithelia with a plethora of known interacting proteins. Mutations in the CFTR gene cause cystic fibrosis (CF), a disease that leads to progressive respiratory illness and other complications of phenotypic variance resulting from perturbations of this protein interaction network. Studying the collection of CFTR interacting proteins and the differences between the interactomes of mutant and wild type CFTR provides insight into the molecular machinery of the disease and highlights possible therapeutic targets. This mini review focuses on functional genomics and proteomics approaches used for systematic, high-throughput identification of CFTR-interacting proteins to provide comprehensive insight into CFTR regulation and function.

Cystic fibrosis transmembrane regulator gene (CFTR) is associated with abnormal enamel formation.

PubMed

Arquitt, C K; Boyd, C; Wright, J T

2002-07-01

Cystic fibrosis (CF), a chloride ion transport disorder, is caused by mutations of the cftr gene and is the most common autosomal-recessive heritable disease in Caucasians. CFTR knockout mice have enamel with crystallite defects, retained protein, and hypomineralization, suggesting a role for CFTR in enamel formation and mineralization. This investigation examined CFTR expression and elemental composition in developing murine incisor teeth. RT-PCR showed cftr mRNA expression in the normal mouse apical incisor tissue but not in the CFTR knockout tissue. Elemental analysis by energy-dispersive x-ray spectroscopy showed relatively decreased chloride in secretory-stage CF enamel. Iron and potassium were significantly increased, and calcium was significantly decreased (p value = 0.05) in the CF mature enamel. Abnormal enamel mineralization, ion concentrations, and molecular evidence of cftr mRNA expression by odontogenic cells strongly suggest that CFTR plays an important role in enamel formation.

CFTR-regulated MAPK/NF-κB signaling in pulmonary inflammation in thermal inhalation injury.

PubMed

Dong, Zhi Wei; Chen, Jing; Ruan, Ye Chun; Zhou, Tao; Chen, Yu; Chen, YaJie; Tsang, Lai Ling; Chan, Hsiao Chang; Peng, Yi Zhi

2015-10-30

The mechanism underlying pulmonary inflammation in thermal inhalation injury remains elusive. Cystic fibrosis, also hallmarked with pulmonary inflammation, is caused by mutations in CFTR, the expression of which is temperature-sensitive. We investigated whether CFTR is involved in heat-induced pulmonary inflammation. We applied heat-treatment in 16HBE14o- cells with CFTR knockdown or overexpression and heat-inhalation in rats in vivo. Heat-treatment caused significant reduction in CFTR and, reciprocally, increase in COX-2 at early stages both in vitro and in vivo. Activation of ERK/JNK, NF-κB and COX-2/PGE2 were detected in heat-treated cells, which were mimicked by knockdown, and reversed by overexpression of CFTR or VX-809, a reported CFTR mutation corrector. JNK/ERK inhibition reversed heat-/CFTR-knockdown-induced NF-κB activation, whereas NF-κB inhibitor showed no effect on JNK/ERK. IL-8 was augmented by heat-treatment or CFTR-knockdown, which was abolished by inhibition of NF-κB, JNK/ERK or COX-2. Moreover, in vitro or in vivo treatment with curcumin, a natural phenolic compound, significantly enhanced CFTR expression and reversed the heat-induced increases in COX-2/PGE2/IL-8, neutrophil infiltration and tissue damage in the airway. These results have revealed a CFTR-regulated MAPK/NF-κB pathway leading to COX-2/PGE2/IL-8 activation in thermal inhalation injury, and demonstrated therapeutic potential of curcumin for alleviating heat-induced pulmonary inflammation.

Non-invasive prenatal diagnosis of paternally inherited disorders from maternal plasma: detection of NF1 and CFTR mutations using droplet digital PCR.

PubMed

Gruber, Aurélia; Pacault, Mathilde; El Khattabi, Laila Allach; Vaucouleur, Nicolas; Orhant, Lucie; Bienvenu, Thierry; Girodon, Emmanuelle; Vidaud, Dominique; Leturcq, France; Costa, Catherine; Letourneur, Franck; Anselem, Olivia; Tsatsaris, Vassilis; Goffinet, François; Viot, Géraldine; Vidaud, Michel; Nectoux, Juliette

2018-04-25

To limit risks of miscarriages associated with invasive procedures of current prenatal diagnosis practice, we aim to develop a personalized medicine-based protocol for non-invasive prenatal diagnosis (NIPD) of monogenic disorders relying on the detection of paternally inherited mutations in maternal blood using droplet digital PCR (ddPCR). This study included four couples at risk of transmitting paternal neurofibromatosis type 1 (NF1) mutations and four couples at risk of transmitting compound heterozygous CFTR mutations. NIPD was performed between 8 and 15 weeks of gestation, in parallel to conventional invasive diagnosis. We designed specific hydrolysis probes to detect the paternal mutation and to assess the presence of cell-free fetal DNA by ddPCR. Analytical performances of each assay were determined from paternal sample, an then fetal genotype was inferred from maternal plasma sample. Presence or absence of the paternal mutant allele was correctly determined in all the studied plasma DNA samples. We report an NIPD protocol suitable for implementation in an experienced laboratory of molecular genetics. Our proof-of-principle results point out a high accuracy for early detection of paternal NF1 and CFTR mutations in cell-free DNA, and open new perspectives for extending the technology to NIPD of many other monogenic diseases.

The CF Canada-Sick Kids Program in individual CF therapy: A resource for the advancement of personalized medicine in CF.

PubMed

Eckford, Paul D W; McCormack, Jacqueline; Munsie, Lise; He, Gengming; Stanojevic, Sanja; Pereira, Sergio L; Ho, Karen; Avolio, Julie; Bartlett, Claire; Yang, Jin Ye; Wong, Amy P; Wellhauser, Leigh; Huan, Ling Jun; Jiang, Jia Xin; Ouyang, Hong; Du, Kai; Klingel, Michelle; Kyriakopoulou, Lianna; Gonska, Tanja; Moraes, Theo J; Strug, Lisa J; Rossant, Janet; Ratjen, Felix; Bear, Christine E

2018-04-20

Therapies targeting certain CFTR mutants have been approved, yet variations in clinical response highlight the need for in-vitro and genetic tools that predict patient-specific clinical outcomes. Toward this goal, the CF Canada-Sick Kids Program in Individual CF Therapy (CFIT) is generating a "first of its kind", comprehensive resource containing patient-specific cell cultures and data from 100 CF individuals that will enable modeling of therapeutic responses. The CFIT program is generating: 1) nasal cells from drug naïve patients suitable for culture and the study of drug responses in vitro, 2) matched gene expression data obtained by sequencing the RNA from the primary nasal tissue, 3) whole genome sequencing of blood derived DNA from each of the 100 participants, 4) induced pluripotent stem cells (iPSCs) generated from each participant's blood sample, 5) CRISPR-edited isogenic control iPSC lines and 6) prospective clinical data from patients treated with CF modulators. To date, we have recruited 57 of 100 individuals to CFIT, most of whom are homozygous for F508del (to assess in-vitro: in-vivo correlations with respect to ORKAMBI response) or heterozygous for F508del and a minimal function mutation. In addition, several donors are homozygous for rare nonsense and missense mutations. Nasal epithelial cell cultures and matched iPSC lines are available for many of these donors. This accessible resource will enable development of tools that predict individual outcomes to current and emerging modulators targeting F508del-CFTR and facilitate therapy discovery for rare CF causing mutations. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis

PubMed Central

2010-01-01

Background Previous observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis. Methods Electrochemical detection of cholesterol at the plasma membrane is achieved with capillary microelectrodes with a modified platinum coil that accepts covalent attachment of cholesterol oxidase. Modified electrodes absent cholesterol oxidase serves as a baseline control. Cholesterol synthesis is determined by deuterium incorporation into lipids over time. Incorporation into cholesterol specifically is determined by mass spectrometry analysis. All mice used in the study are on a C57Bl/6 background and are between 6 and 8 weeks of age. Results Membrane cholesterol measurements are elevated in both R117H and ΔF508 mouse nasal epithelium compared to age-matched sibling wt controls demonstrating a genotype correlation to membrane cholesterol detection. Expression of wt CFTR in CF epithelial cells reverts membrane cholesterol to WT levels further demonstrating the impact of CFTR on these processes. In wt epithelial cell, the addition of the CFTR inhibitors, Gly H101 or CFTRinh-172, for 24 h surprisingly results in an initial drop in membrane cholesterol measurement followed by a rebound at 72 h suggesting a feedback mechanism may be driving the increase in membrane cholesterol. De novo cholesterol synthesis contributes to membrane cholesterol accessibility. Conclusions The data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in de novo cholesterol synthesis to restore membrane content. PMID:20487541

Murine and human CFTR exhibit different sensitivities to CFTR potentiators

PubMed Central

Cui, Guiying

2015-01-01

Development of therapeutic molecules with clinical efficacy as modulators of defective CFTR includes efforts to identify potentiators that can overcome or repair the gating defect in mutant CFTR channels. This has taken a great leap forward with the identification of the potentiator VX-770, now available to patients as “Kalydeco.” Other small molecules with different chemical structure also are capable of potentiating the activity of either wild-type or mutant CFTR, suggesting that there are features of the protein that may be targeted to achieve stimulation of channel activity by structurally diverse compounds. However, neither the mechanisms by which these compounds potentiate mutant CFTR nor the site(s) where these compounds bind have been identified. This knowledge gap partly reflects the lack of appropriate experimental models to provide clues toward the identification of binding sites. Here, we have compared the channel behavior and response to novel and known potentiators of human CFTR (hCFTR) and murine (mCFTR) expressed in Xenopus oocytes. Both hCFTR and mCFTR were blocked by GlyH-101 from the extracellular side, but mCFTR activity was increased with GlyH-101 applied directly to the cytoplasmic side. Similarly, glibenclamide only exhibited a blocking effect on hCFTR but both blocked and potentiated mCFTR in excised membrane patches and in intact oocytes. The clinically used CFTR potentiator VX-770 transiently increased hCFTR by ∼13% but potentiated mCFTR significantly more strongly. Our results suggest that mCFTR pharmacological sensitivities differ from hCFTR, which will provide a useful tool for identifying the binding sites and mechanism for these potentiators. PMID:26209275

Cystic Fibrosis Transmembrane Conductance Regulator Regulates Epithelial Cell Response to Aspergillus and Resultant Pulmonary Inflammation

PubMed Central

Chaudhary, Neelkamal; Datta, Kausik; Askin, Frederic B.; Staab, Janet F.

2012-01-01

Rationale: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) alter epithelial cell (EC) interactions with multiple microbes, such that dysregulated inflammation and injury occur with airway colonization in people with cystic fibrosis (CF). Aspergillus fumigatus frequently colonizes CF airways, but it has been assumed to be an innocent saprophyte; its potential role as a cause of lung disease is controversial. Objectives: To study the interactions between Aspergillus and EC, and the role of the fungus in evoking inflammatory responses. Methods: A. fumigatus expressing green fluorescent protein was developed for in vitro and in vivo models, which used cell lines and mouse tracheal EC. Measurements and Main Results: Fungal spores (conidia) are rapidly ingested by ECs derived from bronchial cell lines and murine tracheas, supporting a role for EC in early airway clearance. Bronchial ECs harboring CFTR mutations (ΔF508) or deletion demonstrate impaired uptake and killing of conidia, and ECs with CFTR mutation undergo more conidial-induced apoptosis. Germinated (hyphal) forms of the fungus evoke secretion of inflammatory mediators, with CFTR mutation resulting in increased airway levels of macrophage inflammatory protein 2 and KC, and higher lung monocyte chemotactic protein-1. After A. fumigatus inhalation, CFTR−/− mice develop exaggerated lymphocytic inflammation, mucin accumulation, and lung injury. Conclusions: Data demonstrate a critical role for CFTR in mediating EC responses to A. fumigatus. Results suggest that the fungus elicits aberrant pulmonary inflammation in the setting of CFTR mutation, supporting the potential role of antifungals to halt progressive CF lung disease. PMID:22135344

Whole-gene CFTR sequencing combined with digital RT-PCR improves genetic diagnosis of cystic fibrosis.

PubMed

Straniero, Letizia; Soldà, Giulia; Costantino, Lucy; Seia, Manuela; Melotti, Paola; Colombo, Carla; Asselta, Rosanna; Duga, Stefano

2016-12-01

Despite extensive screening, 1-5% of cystic fibrosis (CF) patients lack a definite molecular diagnosis. Next-generation sequencing (NGS) is making affordable genetic testing based on the identification of variants in extended genomic regions. In this frame, we analyzed 23 CF patients and one carrier by whole-gene CFTR resequencing: 4 were previously characterized and served as controls; 17 were cases lacking a complete diagnosis after a full conventional CFTR screening; 3 were consecutive subjects referring to our centers, not previously submitted to any screening. We also included in the custom NGS design the coding portions of the SCNN1A, SCNN1B and SCNN1G genes, encoding the subunits of the sodium channel ENaC, which were found to be mutated in CF-like patients. Besides 2 novel SCNN1B missense mutations, we identified 22 previously-known CFTR mutations, including 2 large deletions (whose breakpoints were precisely mapped), and novel deep-intronic variants, whose role on splicing was excluded by ex-vivo analyses. Finally, for 2 patients, compound heterozygotes for a CFTR mutation and the intron-9c.1210-34TG [11-12] T 5 allele-known to be associated with decreased CFTR mRNA levels-the molecular diagnosis was implemented by measuring the residual level of wild-type transcript by digital reverse transcription polymerase chain reaction performed on RNA extracted from nasal brushing.

Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFTR-related disorders--updated European recommendations.

PubMed

Dequeker, Els; Stuhrmann, Manfred; Morris, Michael A; Casals, Teresa; Castellani, Carlo; Claustres, Mireille; Cuppens, Harry; des Georges, Marie; Ferec, Claude; Macek, Milan; Pignatti, Pier-Franco; Scheffer, Hans; Schwartz, Marianne; Witt, Michal; Schwarz, Martin; Girodon, Emmanuelle

2009-01-01

The increasing number of laboratories offering molecular genetic analysis of the CFTR gene and the growing use of commercial kits strengthen the need for an update of previous best practice guidelines (published in 2000). The importance of organizing regional or national laboratory networks, to provide both primary and comprehensive CFTR mutation screening, is stressed. Current guidelines focus on strategies for dealing with increasingly complex situations of CFTR testing. Diagnostic flow charts now include testing in CFTR-related disorders and in fetal bowel anomalies. Emphasis is also placed on the need to consider ethnic or geographic origins of patients and individuals, on basic principles of risk calculation and on the importance of providing accurate laboratory reports. Finally, classification of CFTR mutations is reviewed, with regard to their relevance to pathogenicity and to genetic counselling.

Ion Channel Modulators in Cystic Fibrosis.

PubMed

Gentzsch, Martina; Mall, Marcus A

2018-05-08

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and remains one of the most common life-shortening genetic diseases affecting the lung and other organs. CFTR functions as a cAMP-dependent anion channel that transports chloride and bicarbonate across epithelial surfaces and disruption of these ion transport processes plays a central role in the pathogenesis of CF. These findings provided the rationale for pharmacological modulation of ion transport, either by targeting mutant CFTR or alternative ion channels that can compensate for CFTR dysfunction, as a promising therapeutic approach. High throughput screening has supported the development of CFTR modulator compounds. CFTR correctors are designed to improve defective protein processing, trafficking and cell surface expression, whereas potentiators increase the activity of mutant CFTR at the cell surface. The approval of the first potentiator ivacaftor for the treatment of patients with specific CFTR mutations and, more recently the corrector lumacaftor in combination with ivacaftor for patients homozygous for the common F508del mutation, were major breakthroughs on the path to causal therapies for all patients with CF. In this review, we focus on recent developments and remaining challenges of CFTR-directed therapies, as well as modulators of other ion channels such as alternative chloride channels and the epithelial sodium channel (ENaC) as additional targets in CF lung disease. Further, we discuss how patient-derived precision medicine models may aid the translation of emerging next generation ion channel modulators from the laboratory to the clinic and tailor their use for optimal therapeutic benefits in individual patients with CF. Copyright © 2018. Published by Elsevier Inc.

Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis

PubMed Central

Sun, Xingshen; Sui, Hongshu; Fisher, John T.; Yan, Ziying; Liu, Xiaoming; Cho, Hyung-Ju; Joo, Nam Soo; Zhang, Yulong; Zhou, Weihong; Yi, Yaling; Kinyon, Joann M.; Lei-Butters, Diana C.; Griffin, Michelle A.; Naumann, Paul; Luo, Meihui; Ascher, Jill; Wang, Kai; Frana, Timothy; Wine, Jeffrey J.; Meyerholz, David K.; Engelhardt, John F.

2010-01-01

Cystic fibrosis (CF) is a recessive disease that affects multiple organs. It is caused by mutations in CFTR. Animal modeling of this disease has been challenging, with species- and strain-specific differences in organ biology and CFTR function influencing the emergence of disease pathology. Here, we report the phenotype of a CFTR-knockout ferret model of CF. Neonatal CFTR-knockout ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport and submucosal gland fluid secretion; variably penetrant meconium ileus (MI); pancreatic, liver, and vas deferens disease; and a predisposition to lung infection in the early postnatal period. Severe malabsorption by the gastrointestinal (GI) tract was the primary cause of death in CFTR-knockout kits that escaped MI. Elevated liver function tests in CFTR-knockout kits were corrected by oral administration of ursodeoxycholic acid, and the addition of an oral proton-pump inhibitor improved weight gain and survival. To overcome the limitations imposed by the severe intestinal phenotype, we cloned 4 gut-corrected transgenic CFTR-knockout kits that expressed ferret CFTR specifically in the intestine. One clone passed feces normally and demonstrated no detectable ferret CFTR expression in the lung or liver. The animals described in this study are likely to be useful tools for dissecting CF disease pathogenesis and developing treatments. PMID:20739752

Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis.

PubMed

Sun, Xingshen; Sui, Hongshu; Fisher, John T; Yan, Ziying; Liu, Xiaoming; Cho, Hyung-Ju; Joo, Nam Soo; Zhang, Yulong; Zhou, Weihong; Yi, Yaling; Kinyon, Joann M; Lei-Butters, Diana C; Griffin, Michelle A; Naumann, Paul; Luo, Meihui; Ascher, Jill; Wang, Kai; Frana, Timothy; Wine, Jeffrey J; Meyerholz, David K; Engelhardt, John F

2010-09-01

Cystic fibrosis (CF) is a recessive disease that affects multiple organs. It is caused by mutations in CFTR. Animal modeling of this disease has been challenging, with species- and strain-specific differences in organ biology and CFTR function influencing the emergence of disease pathology. Here, we report the phenotype of a CFTR-knockout ferret model of CF. Neonatal CFTR-knockout ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport and submucosal gland fluid secretion; variably penetrant meconium ileus (MI); pancreatic, liver, and vas deferens disease; and a predisposition to lung infection in the early postnatal period. Severe malabsorption by the gastrointestinal (GI) tract was the primary cause of death in CFTR-knockout kits that escaped MI. Elevated liver function tests in CFTR-knockout kits were corrected by oral administration of ursodeoxycholic acid, and the addition of an oral proton-pump inhibitor improved weight gain and survival. To overcome the limitations imposed by the severe intestinal phenotype, we cloned 4 gut-corrected transgenic CFTR-knockout kits that expressed ferret CFTR specifically in the intestine. One clone passed feces normally and demonstrated no detectable ferret CFTR expression in the lung or liver. The animals described in this study are likely to be useful tools for dissecting CF disease pathogenesis and developing treatments.

Cirrhosis could be associated with severe mutations of the cystic fibrosis gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lenaerts, C.; Piussan, C.; Soto, B.

1994-09-01

Previous studies failed to demonstrate a genetic predisposition to liver disease in cystic fibrosis. In order to characterize patients with cirrhosis defined on the basis of either hepatosplenomegaly, portal hypertension or liver biopsy, we analyzed a total number of 110 cirrhotic CF patients from different CF centers in France. Of them, 71 are males, which is not different from the overall CF french population. All but 2 are pancreatic insufficient. A history of meconium ileus {plus_minus} meconium ileus equivalent seems to be a risk factor for cirrhosis since these complications are present in 29% of the cirrhotic patients vs. 19%more » in the non-cirrhotic population (p = 0.03). This confirms our previous data in a postmortem study. Genotype analysis was performed in all the patients. {Delta}F508 represents 70% of the identified mutations with a higher proportion of {Delta}F508 +/+ in the cirrhotic than in the non-cirrhotic population (52% vs. 42%, p=0.003), 35% {Delta}F508 +/- vs. 42% and 13% {Delta}F508 -/- vs. 16%. Sixty percent of the other mutations associated with cirrhosis are identified, usually in {Delta}F508 +/- and include 1303 N-K, 542 G-X, 1078 del T, 1282 W-X, 1313 Q-X, 827 E-X, 1061 G-R, 1301 N-H, 14 K-X, 1717-1 G-A, 1918 delGC, 2183 A-G, 2184 delA, 405+1 G-A, 507 {Delta}l, 574 delA, 621+1 G-T, 85 G-E and 1303 N-K/other, 227 L-R/other. None of the cirrhotic patients bear one of the dominant missense mutations regarded as mild with respect to pancreatic function (117 R-H, 334 R-W, 347 R-P, 455 A-E, 574 P-H) or both the {Delta}F508 and the 5512 G-A mutations associated with a decreased risk of meconium ileus. Cirrhosis could thus be linked to the presence of 2 of the severe mutations of the CF gene associated with pancreatic insufficiency.« less

A PDZ-interacting domain in CFTR is an apical membrane polarization signal

PubMed Central

Moyer, Bryan D.; Denton, Jerod; Karlson, Katherine H.; Reynolds, Donna; Wang, Shusheng; Mickle, John E.; Milewski, Michal; Cutting, Garry R.; Guggino, William B.; Li, Min; Stanton, Bruce A.

1999-01-01

Polarization of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, to the apical plasma membrane of epithelial cells is critical for vectorial transport of chloride in a variety of epithelia, including the airway, pancreas, intestine, and kidney. However, the motifs that localize CFTR to the apical membrane are unknown. We report that the last 3 amino acids in the COOH-terminus of CFTR (T-R-L) comprise a PDZ-interacting domain that is required for the polarization of CFTR to the apical plasma membrane in human airway and kidney epithelial cells. In addition, the CFTR mutant, S1455X, which lacks the 26 COOH-terminal amino acids, including the PDZ-interacting domain, is mispolarized to the lateral membrane. We also demonstrate that CFTR binds to ezrin-radixin-moesin–binding phosphoprotein 50 (EBP50), an apical membrane PDZ domain–containing protein. We propose that COOH-terminal deletions of CFTR, which represent about 10% of CFTR mutations, result in defective vectorial chloride transport, partly by altering the polarized distribution of CFTR in epithelial cells. Moreover, our data demonstrate that PDZ-interacting domains and PDZ domain–containing proteins play a key role in the apical polarization of ion channels in epithelial cells. J. Clin. Invest. 104:1353–1361 (1999). PMID:10562297

Screening for five mutations detects 97% of cystic fibrosis (CF) chromosomes and predicts a carrier frequency of 1:29 in the Jewish Ashkenazi population

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abeliovich, D.; Lavon, I.P.; Lerer, I.

1992-11-01

To determine the distribution and frequency of cystic fibrosis (CF) mutations in the Israeli population, the authors have screened 96 patients for 11 relatively common mutations. Five mutations - [Delta]F508, G542X, W1282X, N1303K, and 3849 + 10kb C[yields]T-were found to account for 97% of the CF alleles in the Ashkenazi Jews. In contrast, of the 11 mutations tested, only [Delta]F508 was detected in Jewish patients of Sephardic or Oriental origin, accounting for 43% of the CF alleles. Four mutations - [Delta]F508, G542X, W1282X, and N1303K- accounted for 55% of the CF alleles in Arab patients. In a pilot screening study,more » a random sample of 424 Ashkenazi individuals was analyzed for three mutations - [Delta]F508, W128X, and G542X. Thirteen individuals were detected as heterozygotes (six for [Delta]F508 and seven for W1282X), predicting a heterozygote frequency of 1:29. This is similar to the frequency of carriers in the Caucasian population of northern European ancestry. On the basis of these data, the Ashkenazi populations is considered to be a candidate for CF heterozygote screening. 32 refs., 2 tabs.« less

Comprehensive CFTR gene analysis of the French cystic fibrosis screened newborn cohort: implications for diagnosis, genetic counseling, and mutation-specific therapy.

PubMed

Audrézet, Marie Pierre; Munck, Anne; Scotet, Virginie; Claustres, Mireille; Roussey, Michel; Delmas, Dominique; Férec, Claude; Desgeorges, Marie

2015-02-01

Newborn screening (NBS) for cystic fibrosis (CF) was implemented throughout France in 2002. It involves a four-tiered procedure: immunoreactive trypsin (IRT)/DNA/IRT/sweat test [corrected] was implemented throughout France in 2002. The aim of this study was to assess the performance of molecular CFTR gene analysis from the French NBS cohort, to evaluate CF incidence, mutation detection rate, and allelic heterogeneity. During the 8-year period, 5,947,148 newborns were screened for cystic fibrosis. The data were collected by the Association Française pour le Dépistage et la Prévention des Handicaps de l'Enfant. The mutations identified were classified into four groups based on their potential for causing disease, and a diagnostic algorithm was proposed. Combining the genetic and sweat test results, 1,160 neonates were diagnosed as having cystic fibrosis. The corresponding incidence, including both the meconium ileus (MI) and false-negative cases, was calculated at 1 in 4,726 live births. The CF30 kit, completed with a comprehensive CFTR gene analysis, provides an excellent detection rate of 99.77% for the mutated alleles, enabling the identification of a complete genotype in 99.55% of affected neonates. With more than 200 different mutations characterized, we confirmed the French allelic heterogeneity. The very good sensitivity, specificity, and positive predictive value obtained suggest that the four-tiered IRT/DNA/IRT/sweat test procedure may provide an effective strategy for newborn screening for cystic fibrosis.

CFTR Modulators for the Treatment of Cystic Fibrosis.

PubMed

Pettit, Rebecca S; Fellner, Chris

2014-07-01

Defects in a single gene lead to the defective proteins that cause cystic fibrosis, making the disease an ideal candidate for mutation-targeted therapy. Although ivacaftor is currently the only FDA-approved CFTR modifier, others are in development.

A CFTR potentiator in patients with cystic fibrosis and the G551D mutation.

PubMed

Ramsey, Bonnie W; Davies, Jane; McElvaney, N Gerard; Tullis, Elizabeth; Bell, Scott C; Dřevínek, Pavel; Griese, Matthias; McKone, Edward F; Wainwright, Claire E; Konstan, Michael W; Moss, Richard; Ratjen, Felix; Sermet-Gaudelus, Isabelle; Rowe, Steven M; Dong, Qunming; Rodriguez, Sally; Yen, Karl; Ordoñez, Claudia; Elborn, J Stuart

2011-11-03

Increasing the activity of defective cystic fibrosis transmembrane conductance regulator (CFTR) protein is a potential treatment for cystic fibrosis. We conducted a randomized, double-blind, placebo-controlled trial to evaluate ivacaftor (VX-770), a CFTR potentiator, in subjects 12 years of age or older with cystic fibrosis and at least one G551D-CFTR mutation. Subjects were randomly assigned to receive 150 mg of ivacaftor every 12 hours (84 subjects, of whom 83 received at least one dose) or placebo (83, of whom 78 received at least one dose) for 48 weeks. The primary end point was the estimated mean change from baseline through week 24 in the percent of predicted forced expiratory volume in 1 second (FEV(1)). The change from baseline through week 24 in the percent of predicted FEV(1) was greater by 10.6 percentage points in the ivacaftor group than in the placebo group (P<0.001). Effects on pulmonary function were noted by 2 weeks, and a significant treatment effect was maintained through week 48. Subjects receiving ivacaftor were 55% less likely to have a pulmonary exacerbation than were patients receiving placebo, through week 48 (P<0.001). In addition, through week 48, subjects in the ivacaftor group scored 8.6 points higher than did subjects in the placebo group on the respiratory-symptoms domain of the Cystic Fibrosis Questionnaire-revised instrument (a 100-point scale, with higher numbers indicating a lower effect of symptoms on the patient's quality of life) (P<0.001). By 48 weeks, patients treated with ivacaftor had gained, on average, 2.7 kg more weight than had patients receiving placebo (P<0.001). The change from baseline through week 48 in the concentration of sweat chloride, a measure of CFTR activity, with ivacaftor as compared with placebo was -48.1 mmol per liter (P<0.001). The incidence of adverse events was similar with ivacaftor and placebo, with a lower proportion of serious adverse events with ivacaftor than with placebo (24% vs. 42

Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle.

PubMed

Jih, Kang-Yang; Hwang, Tzyh-Chang

2013-03-12

Vx-770 (Ivacaftor), a Food and Drug Administration (FDA)-approved drug for clinical application to patients with cystic fibrosis (CF), shifts the paradigm from conventional symptomatic treatments to therapeutics directly tackling the root of the disease: functional defects of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel caused by pathogenic mutations. The underlying mechanism for the action of Vx-770 remains elusive partly because this compound not only increases the activity of wild-type (WT) channels whose gating is primarily controlled by ATP binding/hydrolysis, but also improves the function of G551D-CFTR, a disease-associated mutation that abolishes CFTR's responsiveness to ATP. Here we provide a unified theory to account for this dual effect of Vx-770. We found that Vx-770 enhances spontaneous, ATP-independent activity of WT-CFTR to a similar magnitude as its effects on G551D channels, a result essentially explaining Vx-770's effect on G551D-CFTR. Furthermore, Vx-770 increases the open time of WT-CFTR in an [ATP]-dependent manner. This distinct kinetic effect is accountable with a newly proposed CFTR gating model depicting an [ATP]-dependent "reentry" mechanism that allows CFTR shuffling among different open states by undergoing multiple rounds of ATP hydrolysis. We further examined the effect of Vx-770 on R352C-CFTR, a unique mutant that allows direct observation of hydrolysis-triggered gating events. Our data corroborate that Vx-770 increases the open time of WT-CFTR by stabilizing a posthydrolytic open state and thereby fosters decoupling between the gating cycle and ATP hydrolysis cycle. The current study also suggests that this unique mechanism of drug action can be further exploited to develop strategies that enhance the function of CFTR.

The hypertonic environment differentially regulates wild-type CFTR and TNR-CFTR chloride channels.

PubMed

Lassance-Soares, Roberta M; Cheng, Jie; Krasnov, Kristina; Cebotaru, Liudmila; Cutting, Garry R; Souza-Menezes, Jackson; Morales, Marcelo M; Guggino, William B

2010-01-01

This study tested the hypotheses that the hypertonic environment of the renal medulla regulates the expression of cystic fibrosis transmembrane conductance regulator protein (CFTR) and its natural splice variant, TNR-CFTR. To accomplish this, Madin-Darby canine kidney (MDCK) stable cell lines expressing TNR-CFTR or CFTR were used. The cells were treated with hypertonic medium made with either NaCl or urea or sucrose (480 mOsm/kg or 560 mOsm/kg) to mimic the tonicity of the renal medulla environment. Western blot data showed that CFTR and TNR-CFTR total cell protein is increased by hypertonic medium, but using the surface biotinylation technique, only CFTR was found to be increased in cell plasma membrane. Confocal microscopy showed TNR-CFTR localization primarily at the endoplasmic reticulum and plasma membrane. In conclusion, CFTR and TNR-CFTR have different patterns of distribution in MDCK cells and they are modulated by a hypertonic environment, suggesting their physiological importance in renal medulla. Copyright © 2010 S. Karger AG, Basel.

A novel fluorescent sensor for measurement of CFTR function by flow cytometry.

PubMed

Vijftigschild, Lodewijk A W; van der Ent, Cornelis K; Beekman, Jeffrey M

2013-06-01

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis. CFTR-dependent iodide transport measured by fluorescent quenching of ectopically expressed halide-sensitive yellow fluorescent protein (YFP) is widely being used to study CFTR function by microscopy or plate readers. Since YFP fluorescence in these systems is dependent on YFP expression levels and iodide concentration, differences in sensor expression level between experimental units are normalized at the start of each experiment. To allow accurate measurement of CFTR function by flow cytometry, we reasoned that co-expression of an iodide insensitive fluorescent protein would allow for normalization of sensor expression levels and more accurate quantification of CFTR function. Our data indicated that dsRed and mKate fluorescence are iodide insensitive, and we determined an optimal format for co-expression of these fluorescent proteins with halide-sensitive YFP. We showed using microscopy that ratiometric measurement (YFP/mKate) corrects for differences in sensor expression levels. Ratiometric measurements were essential to accurately measure CFTR function by flow cytometry that we here describe for the first time. Mixing of wild type or mutant CFTR expressing cells indicated that addition of approximately 10% of wild type CFTR expressing cells could be distinguished by ratiometric YFP quenching. Flow cytometric ratiometric YFP quenching also allowed us to study CFTR mutants associated with differential residual function upon ectopic expression. Compared with conventional plate-bound CFTR function assays, the flow cytometric approach described here can be used to study CFTR function in suspension cells. It may be further adapted to study CFTR function in heterologous cell populations using cell surface markers and selection of cells that display high CFTR function by cell sorting. Copyright © 2013 International Society for Advancement of Cytometry.

Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle