Mutations In Cystic Fibrosis Transmembrane Regulator Research Articles

Restoring airway epithelial homeostasis in Cystic Fibrosis

Cystic fibrosis (CF), the most common life-threatening genetic disorder in Caucasians, is caused by recessive mutations in the Cystic Fibrosis Transmembrane Regulator (CFTR) gene encoding a chloride ion channel. Aberrant function of CFTR involves mucus- and sweat-producing epithelia affecting multiple organs, including airways and lungs. This condition facilitates the colonization of fungi, bacteria, or viruses. Recurrent antibiotic administration is commonly used to treat pathogen infections leading to the insurgence of resistant bacteria and to a chronic inflammatory state that jeopardizes airway epithelium repair. The phenotype of patients carrying CFTR mutations does not always present a strict correlation with their genotype, suggesting that the disease may occur because of multiple additive effects. Among them, the frequent microbiota dysbiosis observed in patients affected by CF, might be one cause of the discrepancy observed in their genotype-phenotype correlation. Interestingly, the abnormal polarity of the CF airway epithelium has been observed also under non-infectious and non-inflammatory conditions, suggesting that CFTR dysfunction “per se” perturbs epithelial homeostasis. New pathogen- or host-directed strategies are thus needed to counteract bacterial infections and restore epithelial homeostasis in individuals with CF. In this review, we summarized alternative cutting-edge approaches to high-efficiency modulator therapy that might be promising for these patients.

The NSAID glafenine rescues class 2 CFTR mutants via cyclooxygenase 2 inhibition of the arachidonic acid pathway

Most cases of cystic fibrosis (CF) are caused by class 2 mutations in the cystic fibrosis transmembrane regulator (CFTR). These proteins preserve some channel function but are retained in the endoplasmic reticulum (ER). Partial rescue of the most common CFTR class 2 mutant, F508del-CFTR, has been achieved through the development of pharmacological chaperones (Tezacaftor and Elexacaftor) that bind CFTR directly. However, it is not clear whether these drugs will rescue all class 2 CFTR mutants to a medically relevant level. We have previously shown that the nonsteroidal anti-inflammatory drug (NSAID) ibuprofen can correct F508del-CFTR trafficking. Here, we utilized RNAi and pharmacological inhibitors to determine the mechanism of action of the NSAID glafenine. Using cellular thermal stability assays (CETSAs), we show that it is a proteostasis modulator. Using medicinal chemistry, we identified a derivative with a fourfold increase in CFTR corrector potency. Furthermore, we show that these novel arachidonic acid pathway inhibitors can rescue difficult-to-correct class 2 mutants, such as G85E-CFTR > 13%, that of non-CF cells in well-differentiated HBE cells. Thus, the results suggest that targeting the arachidonic acid pathway may be a profitable way of developing correctors of certain previously hard-to-correct class 2 CFTR mutations.

New drugs in cystic fibrosis: what has changed in the last decade?

Cystic fibrosis (CF), a life-limiting chronic disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, affects more than 90,000 people worldwide. Until recently, the only available treatments were directed to symptom control, but they failed to change the course of the disease. New drugs developed in the last decade have the potential to change the expression, function, and stability of CFTR protein, targeting the basic molecular defect. The authors seek to provide an update on the new drugs, with a special focus on the most promising clinical trials that have been carried out to date. These newly approved drugs that target specific CFTR mutations are mainly divided into two main groups of CFTR modulators: potentiators and correctors. New therapies have opened the door for potentially disease-modifying, personalized treatments for patients with CF.

The prevalence of CFTR mutations in patients with chronic rhinosinusitis: A systematic review and meta-analysis.

Undetected cystic fibrosis transmembrane regulator (CFTR) mutations may predispose individuals to develop CRS independent of formal CF diagnosis. The objective of this study was to determine the prevalence of CFTR mutations among individuals with CRS. A systematic search following PRISMA guidelines was performed. A meta-analysis was performed to calculate pooled estimates for the prevalence of any CFTR mutation and for the DF508mutation. The systematic search included all studies identifying adults diagnosed with CRS, with no limitation to region or publication date. Studies had to identify a sample of patients previously diagnosed with CRS but not with CF and reporting testing for the prevalence of CF or the CFTR gene mutation. Prevalence of CFTR mutations among the general CRS population, with subgroup analysis of individuals with the dF508mutation. The 6 included studies represented five countries: the United States, the UK, France, Poland and Finland. The pooled prevalence of CFTR mutations of any kind in CRS subjects without CF was 5.65% (RE 95% CI 2.99 - 10.41). The overall prevalence for the dF508mutation was 4.22% (RE 95% CI 1.71 - 10.07). These estimates were significantly higher than the baseline estimated prevalence of CFTR carrier status of 3%-4% in the general population. However, the clinical relevance of the presence of CFTR mutations in CRS patients who have not been diagnosed with CF is currently unclear. Future studies should include sweat chloride testing as a measure of CFTR function.

Abstract P402: Caveolin-1 Upregulation Leads To Decreased Erg And Stress-induced Ventricular Tachycardia In Cystic Fibrosis Hearts

Background: Cardiac manifestations of cystic fibrosis are classically accepted as cor pulmonale, yet emerging evidence supports that cardiac abnormalities can develop early when respiratory function is preserved. Myocardial cystic fibrosis transmembrane regulator (CFTR) channels, inactive in normal conditions, are activated in response to catecholamine stress. Methods and Results: In multiple mouse models of homozygous CFTR mutations (S489X or F508d), we demonstrate increased susceptibility to ventricular tachycardia (VT) in response to epinephrine, and this is associated with prolongation of QTc intervals (repolarization). Despite having normal left ventricular ejection fraction and histology, CF S489X mut/mut hearts showed 460 proteins significantly altered compared with WT hearts by tandem mass tag (TMT) proteomics. The Ingenuity Pathway Analysis of proteomics data revealed mitochondrial dysfunction and increased caveolin-1 signaling among the top pathways affected in CF S489X mut/mut hearts. In mouse hearts and human induced pluripotent stem cells derived cardiomyocytes (iPS-CM) with CFTR mutations, we observed increased ER stress and caveolin-1 (cav-1). These changes are concomitant with reduced levels of the ERG channel, a K+ channel contributing to phase 3 repolarization, mutations of which cause long QT syndrome II and VT. Patch clamp analysis showed decreased phase 2-3 repolarization current (including hERG current) in CF F508d iPS-CM compared with control iPS-CM, consistent with the findings of decreased membranous hERG and elevated Cav-1. Suppression of Cav-1 successfully restores membranous hERG in two separate lines of mutant iPS-CM generated from CF patients with F508d mutation. In F508d mut/mut mice, suppression of cardiac Cav-1 upregulation by reducing ER stress using TUDCA or crossing with heterozygous Cav-1 +/- abolished the stress-induced VT. Our analysis of a large-scale real-world clinical database from TriNetX, including 4520 CF men, 3510 CF women and the same number of well-matched controls demonstrated novel findings of increased risk for QT prolongation and VT in CF patients. Retrospective analysis of 126 CF patients with available EKG in our hospital revealed significant QTc prolongation in CF patients compared with matched controls, for both males (CF 447.8±4.5 vs cont. 429.2±2.5) and females (CF 454.0±3.5 vs cont.437.5±2.4). The QTc interval is not affected by lung function (%predicted FEV1) and is an independent predictor of survival in CF patients. Conclusions: In mice and iPS-CM models of CF, we demonstrated impaired repolarization and increased susceptibility to stress-induced VT. This is mediated by ER-stress-induced upregulation of Cav-1, which leads to internalization of ERG and subsequent decrease in repolarization current.

Cystic fibrosis being a polyendocrine disease (Review)

Ген CFTR (cystic fibrosis transmembrane regulator) кодирует синтез одноименного белка, который функционирует как прямой активатор анионного транспорта. Cl– является наиболее распространенным анионом, в качестве антагониста катионов натрия и калия обеспечивает электронейтральность клеточных мембран в покое, вместе с катионами служит важным осмолитом и формирует поток воды через клеточные мембраны для трансэпителиальной секреции. Железистые клетки при муковисцидозе (МВ) задерживают Cl– и Na+, а выделяемый ими секрет отличается чрезмерной вязкостью. Субнормальная активность CFTR приводит к застою мукоцилиарного клиренса, торможению кишечного транспорта.Помимо экзокринных нарушений, мутации CFTR ассоциируются с уменьшением объема, массы, усилением апоптоза β-клеток поджелудочной железы, значительным подавлением экзоцитоза инсулина в ответ на стимуляцию глюкозой и глюкагоноподобным пептидом-1, гиперглюкагонемией на фоне дефекта подавления инсулином функции α-клеток, но снижением максимальной емкости α-клеток.Дефицит и прогрессирующее снижение минеральной плотности костной ткани являются ожидаемыми вторичными проявлениями МВ ввиду панкреатической экзокринной недостаточности с мальабсорбцией питательных веществ и жирорастворимых витаминов. Но у пациентов с мутацией F508del установлены значимое уменьшение синтеза в остеобластической формации OPG, COX-2, PGE2, повышение активности антианаболического NF-kB. Речь идет о дефекте канонического сигнального пути (Wnt/β-катенин), который регулирует экспрессию генов-активаторов остеобластогенеза, разобщении этапности физиологического костного ремоделирования.Помимо врожденной двусторонней или односторонней аплазии семявыносящих протоков, увеличение частоты мутаций CFTR также обнаруживается при необструктивной азооспермии, олиго-, астено- и тератоспермии. CFTR участвует во входе HCO3– в клетки Сертоли для запуска cAMP-зависимой транскрипции, и его дефекты приводят к подавлению ФСГ-зависимой экспрессии генов сперматогенеза, потере последовательности в каскаде Wnt, разрушению зависимого от PGE2 трансэпителиального взаимодействия и, как следствие, гематотестикулярного барьера.МВ характеризуется, наряду с классическими признаками, эндокринной дисфункцией поджелудочной железы, остеопорозом с подавлением остеобластогенеза, дефектом сперматогенеза.

Cellular Interactions between Human Airway Epithelial Cells and Cystic Fibrosis Macrophages result in Elevated Inflammation and Impaired Multiciliogenesis

Introduction Patients with chronic lung diseases, such as cystic fibrosis (CF), are often characterized with chronic inflammation and impaired bacterial clearance. This suggests that their respiratory innate immune response is inadequate. Macrophages are the sentinel cells of the respiratory system acting as the first line of defense against invading pathogens, orchestrating wound healing, and maintaining tissue homeostasis. The phenotype and function of macrophages are governed by signals they receive from their microenvironment. Despite this they are often studied in isolation in vitro. We hypothesized that changes in the innate immune response in CF result from pathological signaling between macrophages and airway epithelium. Methods Primary lung macrophages, isolated from lung explants from CF patient and patients with no prior evidence of chronic lung disease, were co-cultured with human bronchial epithelial cell lines with or without mutations in the cystic fibrosis transmembrane regulator (CFTR), NuLi (control) and CuFi (CF). Cell lines were used for the initial experiments to reduce patient to patient variability due to factors such as treatment and bacterial colonization, allowing to focus on the specific role of CFTR mutations. A co-culture model at the air-liquid interface was developed to determine whether epithelial interactions with CFTR-mutant macrophages influenced basal stem cell differentiation. Tumor necrosis factor alpha (TNFα) and interleukins 6 and 8 (IL-6/8) were profiled by stimulating cultures with lipopolysaccharide (LPS) for 24 hours and analyzing the culture supernatants. Results Substantial differences in theinflammatory profile were observed in the presence of CFTR mutations in the co-culture model. A 2-fold increase in TNFα after LPS, was observed comparing CF to WT macrophage monocultures; this suggests they may maintain a hyperinflammatory physiological memory of the CF lung. The presence of epithelial cells suppresses TNFα release when compared to monoculture controls. In contrast, IL-6 is increased in the co-cultures. Additionally, CFTR mutant epithelial cells significantly increase IL-8 (3-fold increase, P<0.05), even in the absence of LPS, this response further exacerbated after LPS stimulation (1.5-fold increase, P<0.05), compared to WT controls. Finally, our data supports a role for macrophages in stimulating ciliogenesis in WT epithelium which is suppressed by the presence of CFTR-mutant macrophages (P<0.05). Conclusion This study highlights the importance of studying macrophages in a physiological microenvironment and supports a pathogenic role for CFTR-mutant epithelium in stimulating lung macrophages to a hyper-inflammatory phenotype. In addition, CF macrophages lose a capacity to promote efficient epithelial regeneration and homeostasis. This highlights the importance of understanding the cellular mechanisms regulated by macrophage-epithelial interactions, regulated by CFTR, to identify new therapeutic targets in CF.

Муковисцидоз как полиэндокринное заболевание (обзор литературы)

The cystic fibrosis transmembrane regulator ( CFTR ) gene encodes the synthesis of a protein of the same name, which functions as a direct activator of anionic transport. Chloride is the most abundant anion; as an antagonist of Na + and K + , it provides electroneutrality of cell membranes at rest; together with cations, it serves as an important osmolyte and forms water flow across cell membranes for transepithelial secretion. Glandular cells in CF trap Cl – and Na + , and the prodused secretion is excessively viscous. Subnormal CFTR activity leads to stagnation of mucociliary clearance, inhibition of intestinal transport. In addition to exocrine disorders, CFTR mutations are associated with a decrease in volume, mass, increased apoptosis of β-cells of the pancreas, a significant suppression of insulin exocytosis in response to stimulation with glucose and glucagon-like peptide-1, hyperglucagonemia against the background of a defect in the suppression of α-cell function by insulin, but a decrease in maximum capacity α-cells. Deficiency and progressive decline in bone mineral density is an expected secondary manifestation of CF due to pancreatic exocrine insufficiency with malabsorption of nutrients and fat-soluble vitamins. However, in patients with the F508del mutation, a significant decrease in the synthesis of OPG, COX-2, PGE2 in the osteoblastic formation, and an increase in the activity of the antianabolic NF-kB were found. We are talking about a defect in the canonical signaling pathway (Wnt/β-catenin), which regulates the expression of genes-activators of osteoblastogenesis, dissociation of the stages of physiological bone remodeling. In addition to congenital bilateral or unilateral aplasia of the vas deferens, an increase in the frequency of CFTR mutations is also found in non-obstructive azoospermia, oligo-, astheno- and teratospermia. CFTR is involved in the entry of HCO 3 – into Sertoli cells to trigger cAMP-dependent transcription and its defects lead to suppression of FSH-dependent gene expression of spermatogenesis, loss of sequence in the Wnt cascade, destruction of the PGE2-dependent transepithelial interaction and, as a consequence, the blood-testicular barrier. CF is characterized, along with classical signs, by endocrine dysfunction of the pancreas, osteoporosis with suppression of osteoblastogenesis, and a defect in spermatogenesis.

Contribution of common CFTR variants (M470V, T854, and Q1463) to cystic fibrosis in Tunisia: haplotype analysis.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane regulator (CFTR) protein, a chloride channel located in the epithelial cell membrane. Over than 2,000CFTR mutations have been identified, which contribute to the variety of clinical phenotypes of CF. We performed a case-control study to determine p.Met470Val (M470V), p.Thr854= (T854) and p.Gln1463= (Q1463) polymorphisms frequencies in CF patients and healthy controls and to elaborate haplotype based on these SNPs. The genotyping of M470V (exon 10), T854 (exon 14a), and Q1463 (exon 24) variants were identified using polymorphism restriction fragment length polymorphism (RFLP). Statistical difference was noted in the genotype distribution of two markers, M470V and T854, between CF and control groups. However, the Q1463 polymorphism is not identified in two studied groups. Three haplotypes were found in CF patients and controls. An exclusive association between the ancestral haplotype 1-1-2 and p.Phe508del (F508del) mutation was shown. In Tunisia, this is the first work to be interested in the analysis of M470V, T854 and Q1463 polymorphisms and haplotypes associated with the most common mutation, F508del, in the Tunisian population and worldwide.

MP38-02 CFTR GENE MUTATIONS IN SAUDI INFERTILE MEN WITH AZOOSPERMIA OR OLIGOASTHENOTERATOZOOSPERMIA

INTRODUCTION AND OBJECTIVE: Some mutations of cystic fibrosis transmembrane regulator (CFTR) gene may result in impaired spermatogenesis or cause a congenital absence of vas deferens which is manifested as isolated male infertility. The aim of this study is to examine the frequency and analyse the spectrum of CFTR gene variations in Saudi men who presented with idiopathic primary infertility. METHODS: Design and Setting: Prospective observational study conducted in King Faisal Specialist Hospital and Research Centre (Gen. Org), Jeddah, Saudi Arabia.Population: An unselected group of 50 Saudi men with idiopathic primary infertility who had either an Oligoasthenoteratozoospermia or azoospermia and presented to the andrology clinic between January 2017 and January 2019.Intervention: Genomic DNA extracted from a peripheral blood sample of each subject by a polymerase chain reaction and direct sequencing to identify all the variants of the entire coding regions and boundaries of the 27 exons of the CFTR gene. RESULTS: This study identified 10 CFTR gene mutations and polymorphisms in 7 (14%) subjects (100 chromosomes). Six subjects (12%) had two or more variants. The detected variants andpolymorphisms were: c.1408G>A, c.4389G>A, c.2562T>G, c.869+11C>T, c.2909-92G>A, c.3469-65C>A, c.1210-6delT, c.1210-6T>A, c.2988+1G>A, and c.1210-13GT>TG. CONCLUSIONS: We have shown that 14% of the study’s subjects had one or more CFTR mutation(s) and were compound in most affected patients. It seems that this frequency is higher than that in the general population. The spectrum of CFTR gene mutations in these subjects is similar to that reported worldwide.Source of Funding: King Faisal Specialist Hospital and Research Center.

Homozygous 5T alleles, clinical presentation and genetic analysis within a family with congenital bilateral absence of the vas deferens

Objective: To study the cystic fibrosis transmembrane regulator(CFTR) genotypes and genetic characteristics of a Chinese family with Congenital bilateral absence of vas deferens(CBAVD). Methods: Two 33/29-years-old brothers presented with CBAVD-caused obstructive azoospermia were diagnosed on the basis of scrotal palpation, analysis of semen and ultrasound tests. We extracted their genomic DNA as well as their healthy parents' from the peripheral blood leukocytes. To identify CFTR mutations, each of the 27 exons of the CFTR gene and their flanking splice sites sequences were amplified by polymerase chain reaction(PCR) and subsequently studied with Sanger sequencing. Mutations/variations were identified and compared with the control sequence searched in the NCBI database. Results: Homozygous 5T mutation at the splicing site ahead of exon 10 of the CFTR gene was identified in both brothers in association with 13TG and 12TG alleles(13TG-5T/12TG-5T), one of those was inherited from the mother(13TG-5T/11TG-7T), the other was from the father(12TG-5T/12TG-7T). All of the results above had been excluded the presence of other mutations. Genetic study of this family supports that homozygous 5T mutation is associated with CBAVD. Individuals with homozygous 5T alleles are 20 times more possible to transmit this deleterious variant to the next generation than general population. Conclusions: This family we analysed agrees with the previous conclusion that 5T allele is a deleterious and heritable mutation which could cause CBAVD. Considering better genetic counseling, CFTR gene detection and Preimplantation genetic diagnosis(PGD) are suggested for CBAVD couples who seek for reproductive assistance.

Assessment of p.Phe508del-CFTR functional restoration in pediatric primary cystic fibrosis airway epithelial cells.

BackgroundMutations in the cystic fibrosis transmembrane regulator (CFTR) gene can reduce function of the CFTR ion channel activity and impair cellular chloride secretion. The gold standard method to assess CFTR function of ion transport using the Ussing chamber requires a high number of airway epithelial cells grown at air-liquid interface, limiting the application of this method for high throughput screening of potential therapeutic compounds in primary airway epithelial cells (pAECs) featuring less common CFTR mutations. This study assessed an alternative approach, using a small scale halide assay that can be adapted for a personalized high throughput setting to analyze CFTR function of pAEC.MethodsPediatric pAECs derived from children with CF (pAECCF) were established and expanded as monolayer cultures, before seeding into 96-well plates for the halide assay. Cells were then transduced with an adenoviral construct containing yellow fluorescent protein (eYFP) reporter gene, alone or in combination with either wild-type CFTR (WT-CFTR) or p.Phe508del CFTR. Four days post transduction, cells were stimulated with forskolin and genistein, and assessed for quenching of the eYFP signal following injection of iodide solution into the assay media.ResultsData showed that pAECCF can express eYFP at high efficiency following transduction with the eYFP construct. The halide assay was able to discriminate functional restoration of CFTR in pAECCF treated with either WT-CFTR construct or the positive controls syntaxin 8 and B-cell receptor-associated protein 31 shRNAs.SignificanceThe current study demonstrates that the halide assay can be adapted for pediatric pAECCF to evaluate restoration of CFTR function. With the ongoing development of small molecules to modulate the folding and/or activity of various mutated CFTR proteins, this halide assay presents a small-scale personalized screening platform that could assess therapeutic potential of molecules across a broad range of CFTR mutations.

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

IntroductionNearly 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. ObjectiveTo 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. MethodsGATT 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. ResultsNine 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. ConclusionCFTR 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.

F508del CFTR gene mutation in patients with allergic bronchopulmonary aspergillosis

ABSTRACTObjective: The F508del mutation occurs in approximately 3.5% of Caucasian population of Northern Europe. Heterozygotes have increased risk for asthma and reduced pulmonary function. Allergic bronchopulmonary aspergillosis (ABPA) is more common in patients with cystic fibrosis (CF). We aimed to establish the frequency of F508del mutation in adult patients with ABPA. Methods: A retrospective matched case-control study of CF genotyped patients with ABPA seen at the National Aspergillosis Centre was undertaken. Key data were collected retrospectively from medical records, including respiratory comorbidities, total IgE, Aspergillus IgG and IgE, and immunoglobulins. Cystic fibrosis transmembrane regulator (CFTR) gene mutation analysis included multiplex PCR and sequencing. Results: From a cohort of 189 ABPA patients, 156 were screened for common mutations and variants in the CFTR gene. Eighteen were heterozygous for at least one CFTR mutation; 12 (7.7%) were heterozygous for the F508del, notably; 3 were heterozygous for the intron 8 5T variant; and 1 for an intronic variant of uncertain significance, c.3139 + 18C>T. Eight (67%) had asthma, 7 (58%) had CT-defined bronchiectasis, 4 (33%) hypergammaglobulinemia (>16 g/L), 3 (25%) sinusitis and 1 (8%) chronic pulmonary aspergillosis. Eight (67%) had elevated Aspergillus IgG antibodies (42–98 mg/L), and 8 (67%) had total IgE above 1,000 KIU/L. Two individuals heterozygous for the F508del mutation and the TG12T5 variant were diagnosed with CF, leading to a de novo CF discovery rate of 1.3%. Conclusions: In our ABPA patient cohort, the presence of the delta F508 mutation was higher than that seen in general population. Genetic counseling for CFTR genotyping might be appropriate for these patients.

An "ex vivo model" contributing to the diagnosis and evaluation of new drugs in cystic fibrosis.

SUMMARYCystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. About 2000 mutations have been described so far. We setup an ex vivo model of human nasal epithelial cells (HNECs) to study CF patients testing the effect of novel mutations and molecular therapies. We performed sampling (by brushing), followed by culture and analysis of HNECs using a series of molecular techniques. We performed 50 brushings from CF patients and controls. Using cultured cells, we: i) demonstrated the widely heterogeneous CFTR expression in patients and in controls; ii) defined the splicing effect of a CFTR mutation; iii) assessed the CFTR gating activity in patients bearing different mutations; iv) demonstrated that butyrate significantly enhances CFTR expression. Based on our data, we can conclude: 1) HNEC brushing is performed without anaesthesia and is well tolerated in all CF patients (children and adults); 2) HNECs can be preserved for up to 48 hours before culture allowings multicentre studies; 3) HNECs culture can be considered a suitable model to study the molecular effects of new CFTR gene mutations and/or uncertain meaning specific mutations of carriers; 4) an ex vivo model of HNECs may be used to evaluate, before human use, the effect of new drugs on patients’ cells bearing specific CFTR mutations; 5) the methodology is adequate for a quantitative measurement, by fluorescence, of the CFTR gating activity of the HNECs from patients with different genotypes identifying: a) CF patients bearing two severe mutations with an activity < 10% (compared to controls – 100%); b) CF patients bearing at least a mild mutation with an activity of 10-20%; c) CF carriers (heterozygous subjects) with an activity between 40-70%.

Changes in lipid raft proteome upon TNF-α stimulation of cystic fibrosis cells

We have previously shown (i) that the cystic fibrosis transmembrane regulator (CFTR) locates to lipid raft-like microdomains of epithelial cells upon TNF-α proinflammatory stimulation; and (ii) that TNF-α increases the membrane localization and the channel function of F508del-mutated CFTR. In the present work, we hypothesized that CFTR mutations modify the proteome of lipid rafts in the same proinflammatory conditions. We prepared lipid rafts from HeLa cells transfected with either wild-type or F508del-CFTR and incubated for 10min with 100U/mL of TNF-α. Proteins were extracted, trypsin digested, and peptides analyzed by high resolution MS. Proteins were quantified by a stable isotope labeling with amino acids in cell culture approach. Out of the 22 proteins differentially recruited in lipid rafts after proinflammatory exposure, 17 were increased in F508del cells with respect to wild-type, including two G-protein coupled receptors, three anion transporters, and one cell surface mucin. In both HeLa and bronchial epithelial cells we confirmed that G-protein coupled receptor 5A relocates to lipid rafts along with F508del-CFTR after TNF-α treatment. These results could enlighten the cross-talk between CFTR and TNF-α and its impact on the cell response to proinflammatory challenge. Biological significanceCFTR mutations are at the origin of cystic fibrosis. The latter disease is characterized, among other symptoms, by a defective management of infection and inflammation in the airways. Short exposure to the proinflammatory cytokine TNF-α targets mutated CFTR to the plasma membrane and increases its chloride channel activity. The results hereby presented show a substantial modification of the lipid raft proteome in the same conditions, and may enlighten the effect of this cytokine and the role of CFTR in the cell response to inflammation.

Consequences of cystic fibrosis transmembrane regulator mutations on inflammatory cells

Consequences of cystic fibrosis transmembrane regulator mutations on inflammatory cells

Correctors of mutant CFTR enhance subcortical cAMP-PKA signaling through modulating ezrin phosphorylation and cytoskeleton organization.

The most common mutation of the cystic fibrosis transmembrane regulator (CFTR) gene, F508del, produces a misfolded protein resulting in its defective trafficking to the cell surface and an impaired chloride secretion. Pharmacological treatments partially rescue F508del CFTR activity either directly by interacting with the mutant protein and/or indirectly by altering the cellular protein homeostasis. Here, we show that the phosphorylation of ezrin together with its binding to phosphatidylinositol-4,5-bisphosphate (PIP2) tethers the F508del CFTR to the actin cytoskeleton, stabilizing it on the apical membrane and rescuing the sub-membrane compartmentalization of cAMP and activated PKA. Both the small molecules trimethylangelicin (TMA) and VX-809, which act as 'correctors' for F508del CFTR by rescuing F508del-CFTR-dependent chloride secretion, also restore the apical expression of phosphorylated ezrin and actin organization and increase cAMP and activated PKA submembrane compartmentalization in both primary and secondary cystic fibrosis airway cells. Latrunculin B treatment or expression of the inactive ezrin mutant T567A reverse the TMA and VX-809-induced effects highlighting the role of corrector-dependent ezrin activation and actin re-organization in creating the conditions to generate a sub-cortical cAMP pool of adequate amplitude to activate the F508del-CFTR-dependent chloride secretion.

Cystic Fibrosis: A Novel Pharmacologic Approach to Cystic Fibrosis Transmembrane Regulator Modulation Therapy.

Therapy for cystic fibrosis (CF) has progressed during the past several decades. Much of this progress is because of advances in genetic testing to precisely identify the underlying cause of CF transmembrane regulator (CFTR) dysfunction. However, with more than 1900 mutations that can produce a faulty CFTR, the management of CF can remain a challenge. Several innovative drugs recently approved by the Food and Drug Administration, termed genetic modulators, target the underlying disease by modulating the CFTR defect. This review provides physicians with an established simple classification scheme to guide their use of these drugs. The treatment challenge of 1900 CFTR mutations has been simplified into 6 physiologic classes, each paired with an available therapy to offer patients the most functional improvement. Drug therapy monitoring, adverse effects, and indications for discontinuation must also be considered.

Spectrum of cystic fibrosis mutations in Syrian patients.

Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasians. However, it is considered to be rare in Arabs. Reports of the cystic fibrosis transmembrane regulator (CFTR) mutations from Syrians are limited. The main aim of this study was to identify the frequency of CFTR mutations in 25 CF patients. To the best of our knowledge, this is the first comprehensive report about CF in Syrian patients. The main clinical presentations were respiratory system symptoms (recurrent pneumonia and chronic cough) in 16 (64%) patients, failure to thrive in 15 (60%), GI system symptoms (diarrhea, steatorrhea) in 15 (60%) and nasal polyps in 1 (4%). A total of 36 known mutations in the CFTR gene were screened among 25 CF Syrian patients. However, 13 different CFTR mutations were identified. These mutations in order of frequency were: ΔF508 (18%), W1282X (12%), I148T (6%), CFTRdel 2.3 (6%), 2182AA→G (6%), G542X (6%), N1303K (6%), G551D (4%), G85E (4%), R117H (4%), G85E (4%), R347P (2%), M.2183AA>G (2%) and 3199del6 (2%). However, 22% of the total mutations could not be detected in this study. Moreover, 142 healthy Syrian individuals were tested for ΔF508 and G551D mutations in an attempt to determine the carrier rate in the Syrian population. These two mutations were not detected in this cohort of healthy Syrians. These results provide important tools for adapting a molecular diagnostic test and prenatal diagnosis for the Syrian population.