Cystic Fibrosis Transmembrane Conductance Regulator Mutations Research Articles

VX-659–Tezacaftor–Ivacaftor in Patients with Cystic Fibrosis and One or Two Phe508del Alleles

BackgroundThe next-generation cystic fibrosis transmembrane conductance regulator (CFTR) corrector VX-659, in triple combination with tezacaftor and ivacaftor (VX-659–tezacaftor–ivacaftor), was developed to restore the function of Phe508del CFTR protein in patients with cystic fibrosis.MethodsWe evaluated the effects of VX-659–tezacaftor–ivacaftor on the processing, trafficking, and function of Phe508del CFTR protein using human bronchial epithelial cells. A range of oral VX-659–tezacaftor–ivacaftor doses in triple combination were then evaluated in randomized, controlled, double-blind, multicenter trials involving patients with cystic fibrosis who were heterozygous for the Phe508del CFTR mutation and a minimal-function CFTR mutation (Phe508del–MF genotypes) or homozygous for the Phe508del CFTR mutation (Phe508del–Phe508del genotype). The primary end points were safety and the absolute change from baseline in the percentage of predicted forced expiratory volume in 1 second (FEV1).ResultsVX-659–tezacaftor–ivacaftor significantly improved the processing and trafficking of Phe508del CFTR protein as well as chloride transport in vitro. In patients, VX-659–tezacaftor–ivacaftor had an acceptable safety and side-effect profile. Most adverse events were mild or moderate. VX-659–tezacaftor–ivacaftor resulted in significant mean increases in the percentage of predicted FEV1 through day 29 (P<0.001) of up to 13.3 points in patients with Phe508del–MF genotypes; in patients with the Phe508del–Phe508del genotype already receiving tezacaftor–ivacaftor, adding VX-659 resulted in a further 9.7-point increase in the percentage of predicted FEV1. The sweat chloride concentrations and scores on the respiratory domain of the Cystic Fibrosis Questionnaire–Revised improved in both patient populations.ConclusionsRobust in vitro activity of VX-659–tezacaftor–ivacaftor targeting Phe508del CFTR protein translated into improvements for patients with Phe508del–MF or Phe508del–Phe508del genotypes. VX-659 triple-combination regimens have the potential to treat the underlying cause of disease in approximately 90% of patients with cystic fibrosis. (Funded by Vertex Pharmaceuticals; VX16-659-101 and VX16-659-001 ClinicalTrials.gov numbers, NCT03224351 and NCT03029455.)

VX-445–Tezacaftor–Ivacaftor in Patients with Cystic Fibrosis and One or Two Phe508del Alleles

BackgroundVX-445 is a next-generation cystic fibrosis transmembrane conductance regulator (CFTR) corrector designed to restore Phe508del CFTR protein function in patients with cystic fibrosis when administered with tezacaftor and ivacaftor (VX-445–tezacaftor–ivacaftor).MethodsWe evaluated the effects of VX-445–tezacaftor–ivacaftor on Phe508del CFTR protein processing, trafficking, and chloride transport in human bronchial epithelial cells. On the basis of in vitro activity, a randomized, placebo-controlled, double-blind, dose-ranging, phase 2 trial was conducted to evaluate oral VX-445–tezacaftor–ivacaftor in patients heterozygous for the Phe508del CFTR mutation and a minimal-function mutation (Phe508del–MF) and in patients homozygous for the Phe508del CFTR mutation (Phe508del–Phe508del) after tezacaftor–ivacaftor run-in. Primary end points were safety and absolute change in percentage of predicted forced expiratory volume in 1 second (FEV1) from baseline.ResultsIn vitro, VX-445–tezacaftor–ivacaftor significantly improved Phe508del CFTR protein processing, trafficking, and chloride transport to a greater extent than any two of these agents in dual combination. In patients with cystic fibrosis, VX-445–tezacaftor–ivacaftor had an acceptable safety and side-effect profile. Most adverse events were mild or moderate. The treatment also resulted in an increased percentage of predicted FEV1 of up to 13.8 points in the Phe508del–MF group (P<0.001). In patients in the Phe508del–Phe508del group, who were already receiving tezacaftor–ivacaftor, the addition of VX-445 resulted in an 11.0-point increase in the percentage of predicted FEV1 (P<0.001). In both groups, there was a decrease in sweat chloride concentrations and improvement in the respiratory domain score on the Cystic Fibrosis Questionnaire–Revised.ConclusionsThe use of VX-445–tezacaftor–ivacaftor to target Phe508del CFTR protein resulted in increased CFTR function in vitro and translated to improvements in patients with cystic fibrosis with one or two Phe508del alleles. This approach has the potential to treat the underlying cause of cystic fibrosis in approximately 90% of patients. (Funded by Vertex Pharmaceuticals; VX16-445-001 ClinicalTrials.gov number, NCT03227471; and EudraCT number, 2017-000797-11.)

A review of inflammatory mechanism in airway diseases.

Inflammation in the lung is the body's natural response to injury. It acts to remove harmful stimuli such as pathogens, irritants, and damaged cells and initiate the healing process. Acute and chronic pulmonary inflammation are seen in different respiratory diseases such as; acute respiratory distress syndrome, chronic obstructive pulmonary disease (COPD), asthma, and cystic fibrosis (CF). In this review, we found that inflammatory response in COPD is determined by the activation of epithelial cells and macrophages in the respiratory tract. Epithelial cells and macrophages discharge transforming growth factor-β (TGF-β), which trigger fibroblast proliferation and tissue remodeling. Asthma leads to airway hyper-responsiveness, obstruction, mucus hyper-production, and airway-wall remodeling. Cytokines, allergens, chemokines, and infectious agents are the main stimuli that activate signaling pathways in epithelial cells in asthma. Mutation of the CF transmembrane conductance regulator (CFTR) gene results in CF. Mutations in CFTR influence the lung epithelial innate immune function that leads to exaggerated and ineffective airway inflammation that fails to abolish pulmonary pathogens. We present mechanistic computational models (based on ordinary differential equations, partial differential equations and agent-based models) that have been applied in studying the complex physiological and pathological mechanisms of chronic inflammation in different airway diseases. The scope of the present review is to explore the inflammatory mechanism in airway diseases and highlight the influence of aging on airways' inflammation mechanism. The main goal of this review is to encourage research collaborations between experimentalist and modelers to promote our understanding of the physiological and pathological mechanisms that control inflammation in different airway diseases.

Emerging microRNA Therapeutic Approaches for Cystic Fibrosis.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and remains the most common life-shortening diseases affecting the exocrine organs. The absence of this channel results in an imbalance of ion concentrations across the cell membrane and results in more abnormal secretion and mucus plugging in the gastrointestinal tract and in the lungs of CF patients. The direct introduction of fully functional CFTR by gene therapy has long been pursued as a therapeutical option to restore CFTR function independent of the specific CFTR mutation, but the different clinical trials failed to propose persuasive evidence of this strategy. The last ten years has led to the development of new pharmacotherapies which can activate CFTR function in a mutation-specific manner. Although approximately 2,000 different disease-associated mutations have been identified, a single codon deletion, F508del, is by far the most common and is present on at least one allele in approximately 70% of the patients in CF populations. This strategy is limited by chemistry, the knowledge on CFTR and the heterogenicity of the patients. New research efforts in CF aim to develop other therapeutical approaches to combine different strategies. Targeting RNA appears as a new and an important opportunity to modulate dysregulated biological processes. Abnormal miRNA activity has been linked to numerous diseases, and over the last decade, the critical role of miRNA in regulating biological processes has fostered interest in how miRNA binds to and interacts explicitly with the target protein. Herein, this review describes the different strategies to identify dysregulated miRNA opens up a new concept and new opportunities to correct CFTR deficiency. This review describes therapeutic applications of antisense techniques currently under investigation in CF.

Molecular and Functional Basis of Cystic Fibrosis in Indian Patients: Genetic, Diagnostic and Therapeutic Implications

Cystic fibrosis (CF, MIM#219700) is a common autosomal recessive disorder among Caucasians, which was considered as rare disease for Indian population. CF is caused due to presence of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In this study, we established a spectrum of mutations from both classical CF as well as from infertile male patients with congenital absence of vas deferens (CAVD). In Indian classical CF patients, we reported 14 previously known and eight novel mutations, viz. 3986-3987 delC, 876-6 del4, 1792 InsA, L69H, S158N, Q493L, 1530L and E1329Q. The frequency of delta 508 was found to be 27%. Absolute linkage between delta 508 and KM19-GATT TUB9-M470V-T854T haplotype predicts a relatively recent appearance of delta 508 mutations in Indian population. The CFTR gene analysis in CAVD infertile males documented 13 different CFTR gene mutations and 1 intronic variant that led to aberrant splicing. P.Phe 508 del (n= 16) and p.Arg 117 His (n=4) were among the common severe forms of CFTR mutations identified. The IVS-8-T5 allele (mild form of mutations) was formed with an allele frequency of 28.3%. Eight novel mutations were also found in the CFTR gene from our patient cohort. We also investigated whether genetic modifiers, viz. transforming growth factor (TGF-β) and endothelial receptor type A (EDNRA) of CF lung disease also predispose to CAVD in association with CFTR mutations, which were associated with the CAVD phenotype. Functional characterization of identified 11 novel CFTR gene mutations disclosed that a significant reduction in channel activity for L69H and S549N mutants in CFTR expressing cells was observed whereas impaired CFTR protein maturation was noticed only in L69H substitute CFTR. CFTR correctors (VX809) rescued the defect due to L69H mutation, which is evidenced from detection of C band in L69H mutant expressing cells pre-treated with VX809. The chloride channel activity in S549N and L69H mutant CFTR was also restored in presence of CFTR potentiators VX770. Above findings confirms heterogeneity of CFTR mutations in Indian classical and non-classical CF patients. They may help in developing a strategy to develop counseling and therapeutic approach for CF patients in India.

Cost-effectiveness analysis of lumacaftor and ivacaftor combination for the treatment of patients with cystic fibrosis in the United States

BackgroundLumacaftor/ivacaftor was approved by the Food and Drug Administration (FDA) as a combination treatment for Cystic Fibrosis (CF) patients who are homozygous for the F508del mutation. The objective of this study was to assess the cost-effectiveness of lumacaftor/ivacaftor combination for the treatment of CF homozygous for F508del CF Transmembrane Conductance Regulator (CFTR) mutation.MethodsA Markov-state transition model following a cohort of 12 year-old CF patients homozygous for F508del CFTR mutation in the United States (US) over two, four, six, eight and ten years from a payer’s perspective was developed using TreeAge Pro 2016. Markov states included: mild (percentage of predicted forced expiratory volume in 1 s or FEV1 > 70%), moderate (FEV1 40–70%), severe (FEV1 < 40%) disease, post-transplant, and death. Pulmonary exacerbation and lung transplant were included as transition states. All the input parameters were estimated from the literature. A 1-year cycle length and 3% discount rate were applied. To assess uncertainty in long-term treatment effects, several scenarios were modelled: 100% long-term effectiveness (base-case), defined as improvement in FEV1 in the first year followed by no annual FEV1 decline and a constant reduction in pulmonary exacerbations throughout, 75%, 50%, 25% and 0% (worst case) long-term effectiveness, where treatment effects were intermediate from the second year of treatment until the end of the time horizon. Other scenarios included changing the starting age of the cohort to 6 and 25 years. Primary outcome included incremental cost-effectiveness ratio (ICER) in terms of cost per quality adjusted life year (QALY) gained. One-way and probabilistic sensitivity analyses were performed to determine uncertainty.ResultsUnder the base-case, Lumacaftor/ivacaftor resulted in higher QALYs (7.29 vs 6.84) but at a very high cost ($1,778,920.88) compared to usual care ($116,155.76) over a 10-year period. The ICER for base-case and worst-case scenarios were $3,655,352 / QALY, and $8,480,265/QALY gained, respectively. In the base-case, lumacaftor/ivacaftor was cost-effective at a threshold of $150,000/QALY-gained when annual drug costs were lower than $4153. The results were not substantially affected by the sensitivity analyses.ConclusionsThe intervention produces large QALY gains but at an extremely high cost, resulting in an ICER that would not typically be covered by any insurer. Lumacaftor/ivacaftor’s status as an orphan drug complicates coverage decisions.

Correctors (specific therapies for class II CFTR mutations) for cystic fibrosis.

Background Cystic fibrosis (CF) is a common life‐shortening condition caused by mutation in the gene that codes for that codes for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which functions as a salt transporter. F508del, the most common CFTR mutation that causes CF, is found in up to 80% to 90% of people with CF. In people with this mutation, a full length of protein is transcribed, but recognised as misfolded by the cell and degraded before reaching the cell membrane, where it needs to be positioned to effect transepithelial salt transport. This severe mutation is associated with no meaningful CFTR function. A corrective therapy for this mutation could positively impact on an important proportion of the CF population.

Brushed nasal epithelial cells are a surrogate for bronchial epithelial CFTR studies.

Recent advances in the management of cystic fibrosis (CF) target underlying defects in the CF transmembrane conductance regulator (CFTR) protein, but efficacy analyses remain limited to specific genotype-based subgroups. Patient-derived model systems may therefore aid in expanding access to these drugs. Brushed human nasal epithelial cells (HNEs) are an attractive tissue source, but it remains unclear how faithfully they recapitulate human bronchial epithelial cell (HBE) CFTR activity. We examined this gap using paired, brushed HNE/HBE samples from pediatric CF subjects with a wide variety of CFTR mutations cultured at the air-liquid interface. Growth and structural characteristics for the two cell types were similar, including differentiation into mature respiratory epithelia. In electrophysiologic analysis, no correlation was identified between nasal and bronchial cultures in baseline resistance or epithelial sodium channel (ENaC) activity. Conversely, robust correlation was demonstrated between nasal and bronchial cultures in both stimulated and inhibited CFTR activity. There was close correlation in modulator-induced change in CFTR activity, and CFTR activity in both cell types correlated with in vivo sweat chloride measurements. These data confirm that brushed HNE cell cultures recapitulate the functional CFTR characteristics of HBEs with fidelity and are therefore an appropriate noninvasive HBE surrogate for individualized CFTR analysis.

Synthesis and characterization of a photoaffinity labelling probe based on the structure of the cystic fibrosis drug ivacaftor

Cystic Fibrosis (CF) is a genetic disorder caused by loss-of-function mutations to the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Ivacaftor (1) was the first therapeutic approved for the treatment of CF that is able to restore gating activity to certain CFTR variants although the mechanism of action is poorly understood. Herein we describe the synthesis of a photoaffinity labelling (PAL) probe (2) based on the structure of ivacaftor incorporating a photoreactive diazirine moiety for use in labelling studies designed to identify the binding site for ivacaftor on mutant CFTR. The PAL probe 2 retained potentiation activity, with a potency similar to 1, using a Fluorescent Imaging Plate Reader (FLIPR®) assay measuring ion conductance potentiation of wild type (Wt)-CFTR. Photolabelling experiments with human serum albumin (HSA) as a model protein have shown that probe 2 can label HSA in a manner consistent with observed and predicted binding.

Primary sclerosing cholangitis is associated with abnormalities in CFTR

BackgroundThe etiology of primary sclerosing cholangitis (PSC) is unknown. PSC and Cystic Fibrosis related liver disease have common features: chronic inflammation, biliary damage and similar cholangiographic findings. It is unknown whether or not PSC is related to cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. We hypothesize that a sub-group of PSC patients may be a “single-organ” presentation of CF. MethodsPatients with PSC underwent nasal potential difference (NPD) measurement, sweat chloride measurement and complete CFTR sequencing by new generation sequencing. Results6/32 patients aged 46 ± 13 yrs. had CFTR causing mutations on one allele and 19 had CFTR polymorphisms; 6/23 tested had abnormal and 21 had intermediate sweat tests; 4/32 patients had abnormal NPD. One patient had chronic pancreatitis and was infertile. Conclusions19% of PSC patients had features of CFTR related disorder, 19% carry CFTR mutations and 50% had CFTR polymorphisms. In some patients, PSC may be a single organ presentation of CF or a CFTR-related disorder.

Long-term culture and cloning of primary human bronchial basal cells that maintain multipotent differentiation capacity and CFTR channel function.

While primary cystic fibrosis (CF) and non-CF human bronchial epithelial basal cells (HBECs) accurately represent in vivo phenotypes, one barrier to their wider use has been a limited ability to clone and expand cells in sufficient numbers to produce rare genotypes using genome-editing tools. Recently, conditional reprogramming of cells (CRC) with a Rho-associated protein kinase (ROCK) inhibitor and culture on an irradiated fibroblast feeder layer resulted in extension of the life span of HBECs, but differentiation capacity and CF transmembrane conductance regulator (CFTR) function decreased as a function of passage. This report details modifications to the standard HBEC CRC protocol (Mod CRC), including the use of bronchial epithelial cell growth medium, instead of F medium, and 2% O2, instead of 21% O2, that extend HBEC life span while preserving multipotent differentiation capacity and CFTR function. Critically, Mod CRC conditions support clonal growth of primary HBECs from a single cell, and the resulting clonal HBEC population maintains multipotent differentiation capacity, including CFTR function, permitting gene editing of these cells. As a proof-of-concept, CRISPR/Cas9 genome editing and cloning were used to introduce insertions/deletions in CFTR exon 11. Mod CRC conditions overcome many barriers to the expanded use of HBECs for basic research and drug screens. Importantly, Mod CRC conditions support the creation of isogenic cell lines in which CFTR is mutant or wild-type in the same genetic background with no history of CF to enable determination of the primary defects of mutant CFTR.

Effectiveness of ivacaftor in cystic fibrosis patients with non-G551D gating mutations

BackgroundThe cystic fibrosis transmembrane conductance regulator (CFTR) potentiator ivacaftor is approved for patients with CF with gating and residual function CFTR mutations. We report the results of an observational study investigating its effects in CF patients with non-G551D gating mutations. MethodsPatients with non-G551D gating mutations were recruited to an open-label study evaluating ivacaftor. Primary outcomes included: lung function, sweat chloride, weight gain, and quality of life scores. ResultsTwenty-one subjects were enrolled and completed 6 months follow-up on ivacaftor; mean age was 25.6 years with 52% <18. Baseline ppFEV1 was 68% and mean sweat chloride 89.6 mEq/L. Participants experienced significant improvements in ppFEV1 (mean absolute increase of 10.9% 95% CI = [2.6,19.3], p = 0.0134), sweat chloride (−48.6 95% CI = [−67.4,-29.9], p < 0.0001), and weight (5.1 kg, 95% CI = [2.8, 7.3], p = 0.0002). ConclusionsPatients with non-G551D gating mutations experienced improved lung function, nutritional status, and quality of life. This study supports ongoing use of ivacaftor for patients with these mutations.

Innovative Therapeutic Strategies for Cystic Fibrosis: Moving Forward to CRISPR Technique.

One of the most revolutionary technologies in recent years in the field of molecular biology is CRISPR-Cas9. CRISPR technology is a promising tool for gene editing that provides researchers the opportunity to easily alter DNA sequences and modify gene function. Its many potential applications include correcting genetic defects, treating and preventing the spread of diseases. Cystic fibrosis (CF) is one of the most common lethal genetic diseases caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Although CF is an old acquaintance, there is still no effective/resolutive cure. Life expectancy has improved thanks to the combination of various treatments, but it is generally below average. Recently, a significant number of additional key medications have become licensed in Europe for the CF treatment including CFTR modulators. But innovative genomically-guided therapies have begun for CF and it is predictable that this will lead to rapid improvements in CF clinical disease and survival in the next decades. In this way, CRISPR-Cas9 approach may represent a valid tool to repair the CFTR mutation and hopeful results were obtained in tissue and animal models of CF disease.

Cystic fibrosis-related diabetes is caused by islet loss and inflammation.

Cystic fibrosis-related (CF-related) diabetes (CFRD) is an increasingly common and devastating comorbidity of CF, affecting approximately 35% of adults with CF. However, the underlying causes of CFRD are unclear. Here, we examined cystic fibrosis transmembrane conductance regulator (CFTR) islet expression and whether the CFTR participates in islet endocrine cell function using murine models of β cell CFTR deletion and normal and CF human pancreas and islets. Specific deletion of CFTR from murine β cells did not affect β cell function. In human islets, CFTR mRNA was minimally expressed, and CFTR protein and electrical activity were not detected. Isolated CF/CFRD islets demonstrated appropriate insulin and glucagon secretion, with few changes in key islet-regulatory transcripts. Furthermore, approximately 65% of β cell area was lost in CF donors, compounded by pancreatic remodeling and immune infiltration of the islet. These results indicate that CFRD is caused by β cell loss and intraislet inflammation in the setting of a complex pleiotropic disease and not by intrinsic islet dysfunction from CFTR mutation.

A transcription factor network represses CFTR gene expression in airway epithelial cells.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the inherited disorder cystic fibrosis (CF). Lung disease is the major cause of CF morbidity, though CFTR expression levels are substantially lower in the airway epithelium than in pancreatic duct and intestinal epithelia, which also show compromised function in CF. Recently developed small molecule therapeutics for CF are highly successful for one specific CFTR mutation and have a positive impact on others. However, the low abundance of CFTR transcripts in the airway limits the opportunity for drugs to correct the defective substrate. Elucidation of the transcriptional mechanisms for the CFTR locus has largely focused on intragenic and intergenic tissue-specific enhancers and their activating trans-factors. Here, we investigate whether the low CFTR levels in the airway epithelium result from the recruitment of repressive proteins directly to the locus. Using an siRNA screen to deplete ∼1500 transcription factors (TFs) and associated regulatory proteins in Calu-3 lung epithelial cells, we identified nearly 40 factors that upon depletion elevated CFTR mRNA levels more than 2-fold. A subset of these TFs was validated in primary human bronchial epithelial cells. Among the strongest repressors of airway expression of CFTR were Krüppel-like factor 5 and Ets homologous factor, both of which have pivotal roles in the airway epithelium. Depletion of these factors, which are both recruited to an airway-selective cis-regulatory element at -35 kb from the CFTR promoter, improved CFTR production and function, thus defining novel therapeutic targets for enhancement of CFTR.

Structure of Transmembrane Helix 8 and Possible Membrane Defects in CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that regulates the flow of anions across epithelia. Mutations in CFTR cause cystic fibrosis. CFTR belongs to the ATP-binding cassette transporter superfamily, and gating is controlled by phosphorylation and ATP binding and hydrolysis. Recently obtained ATP-free and ATP-bound structures of zebrafish CFTR revealed an unwound segment of transmembrane helix (TM) 8, which appears to be a unique feature of CFTR not present in other ATP-binding cassette transporter structures. Here, using μs-long molecular dynamics simulations, we investigate the interactions formed by this TM8 segment with nearby helices in both ATP-free and ATP-bound states. We highlight ATP-dependent interactions as well as the structural role of TM8 in maintaining the functional architecture of the pore via interactions common to both the ATP-bound and ATP-free state. The results of the molecular dynamics simulations are discussed in the context of the gating mechanism of CFTR.

Clinical characterization and diagnosis of cystic fibrosis through exome sequencing in Chinese infants with Bartter-syndrome-like hypokalemia alkalosis

Cystic fibrosis (CF) is a fatal autosomal-recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. CF is characterized by recurrent pulmonary infection with obstructive pulmonary disease. CF is common in the Caucasian population but is rare in the Chinese population. The symptoms of early-stage CF are often untypical and may sometimes manifest as Bartter syndrome (BS)-like hypokalemic alkalosis. Therefore, the ability of doctors to differentiate CF from BS-like hypokalemic alkalosis in Chinese infants is a great challenge in the timely and accurate diagnosis of CF. In China, sporadic CF has not been diagnosed in children younger than three years of age to date. Three infants, who were initially admitted to our hospital over the period of June 2013 to September 2014 with BS-like hypokalemic alkalosis, were diagnosed with CF through exome sequencing and sweat chloride measurement. The compound heterozygous mutations of the CFTR gene were detected in two infants, and a homozygous missense mutation was found in one infant. Among the six identified mutations, two are novel point mutations (c.1526G > C and c.3062C > T) that are possibly pathogenic. The three infants are the youngest Chinese patients to have been diagnosed with sporadic CF at a very early stage. Follow-up examination showed that all of the cases remained symptom-free after early intervention, indicating the potential benefit of very early diagnosis and timely intervention in children with CF. Our results demonstrate the necessity of distinguishing CF from BS in Chinese infants with hypokalemic alkalosis and the significant diagnostic value of powerful exome sequencing for rare genetic diseases. Furthermore, our findings expand the CFTR mutation spectrum associated with CF.

The classification of ATP-binding cassette subfamily A member 3 mutations using the cystic fibrosis transmembrane conductance regulator classification system.

ImportanceThe ATP‐binding cassette subfamily A member 3 (ABCA3) protein plays a vital role in surfactant homeostasis. Mutations in the ABCA3 gene lead to the development of interstitial lung disease. In the most severe manifestation, mutations can lead to a fatal respiratory distress syndrome in neonates. ABCA3 belongs to the same ATP‐binding cassette transporter superfamily as the cystic fibrosis transmembrane conductance regulator (CFTR), the gene that causes cystic fibrosis.ObjectiveTo classify ABCA3 mutations in a manner similar to CFTR mutations in order to take advantage of recent advances in therapeutics.MethodsSequence homology between the CFTR protein and the ABCA3 protein was established. The region of CFTR that is a target for the new potentiator class of drugs was of particular interest. We performed a literature search to obtain all published mutations that were thought to be disease causing. We classified these mutations using the established CFTR classification system. When possible, we drew on previous experimental classification of ABCA3 mutations.ResultsAlthough the proteins share the same overall structure, only a 19% identity was established between CFTR and ABCA3. The CFTR therapeutic target region has a 22% homology with the corresponding ABCA3 region. Totally 233 unique protein mutations were identified. All protein mutations were classified and mapped to a schematic diagram of the ABCA3 protein.InterpretationThis new classification system for ABCA3, based on CFTR classification, will likely aid further research of clinical outcomes and identification of mutation‐tailored therapeutics, with the aim for improving clinical care for patients with ABCA3 mutations.

Chronic β2AR stimulation limits CFTR activation in human airway epithelia.

Traditional pulmonary therapies for cystic fibrosis (CF) target the downstream effects of CF transmembrane conductance regulator (CFTR) dysfunction (the cause of CF). Use of one such therapy, β-adrenergic bronchodilators (such as albuterol), is nearly universal for airway clearance. Conversely, novel modulator therapies restore function to select mutant CFTR proteins, offering a disease-modifying treatment. Recent trials of modulators targeting F508del-CFTR, the most common CFTR mutation, suggest that chronic β-agonist use may undermine clinical modulator benefits. We therefore sought to understand the impact of chronic or excess β-agonist exposure on CFTR activation in human airway epithelium. The present studies demonstrate a greater than 60% reduction in both wild-type and modulator-corrected F508del-CFTR activation following chronic exposure to short- and long-acting β-agonists. This reduction was due to reduced cellular generation of cAMP downstream of the β-2 adrenergic receptor-G protein complex. Our results point towards a posttranscriptional reduction in adenylyl cyclase function as the mechanism of impaired CFTR activation produced by prolonged β-agonist exposure. β-Agonist-induced CFTR dysfunction was sufficient to abrogate VX809/VX770 modulation of F508del-CFTR in vitro. Understanding the clinical relevance of our observations is critical for CF patients using these drugs, and for investigators to inform future CFTR modulator drug trials.

CFTR mutation enhances Dishevelled degradation and results in impairment of Wnt-dependent hematopoiesis

Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF) with a multitude of clinical manifestations. Some CF patients develop clinically significant anemia, suggesting that CFTR may regulate hematopoiesis. Here, we report that cftr mutant zebrafish model exhibits primitive and definitive hematopoietic defects with impaired Wnt signaling. Cftr is found to interact, via its PDZ-binding domain (PDZBD), with Dishevelled (Dvl), a key component of Wnt signaling required for hematopoietic progenitor specification, thus protecting Dvl from Dapper1 (Dpr1)-induced lysosomal degradation. Defective hematopoiesis and impaired Wnt signaling in cftr mutant can be rescued by overexpression of wild-type or channel function-defective G551D mutant CFTR with an intact PDZBD, but not Cftr with mutations in the PDZBD. Analysis of human database (http://r2.amc.nl) shows that CFTR is positively correlated with DVL2 and Wnt-related hematopoietic factors in human blood system. The results reveal a previously unrecognized role of CFTR, which is independent of its channel function, in regulating DVL degradation and thus Wnt signaling required for hematopoiesis in both zebrafish and humans, providing an explanation for the anemic phenotype of CF patients.