Pascale Fanen

Molecular characterization of cystic fibrosis: 16 novel mutations identified by analysis of the whole cystic fibrosis conductance transmembrane regulator (CFTR) coding regions and splice site junctions

The spectrum of cystic fibrosis (CF) mutations was determined in 105 patients by using denaturing gradient gel electrophoresis to screen the entire coding regions and adjacent cystic fibrosis transmembrane conductance regulator (CFTR) gene sequences. The nucleotide substitutions detected included 16 novel mutations, 11 previously described defects, and 11 nucleotide sequence polymorphisms. Among the novel mutations, 6 were of the missense type, 4 were nonsense mutations, 4 were frameshift defects, and 2 affected mRNA splicing. The mutations involved all the CFTR domains, including the R domain. Of the 61 non-delta F508 CF chromosomes studied, mutations were found on 36 (59%), raising the proportion of CF alleles characterized in our patient cohort to 88%. Given the efficacy of the screening method used, the remaining uncharacterized mutations probably lie in DNA sequences outside the regions studied, e.g., upstream-promoter sequences, the large introns, or putative regulatory regions. Our results further document the highly heterogeneous nature of CF mutations and provide the information required for DNA-based genetic testing.

Altered channel gating mechanism for CFTR inhibition by a high-affinity thiazolidinone blocker.

The thiazolidinone CFTRinh‐172 was identified recently as a potent and selective blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. Here, we characterized the CFTRinh‐172 inhibition mechanism by patch‐clamp and short‐circuit analysis using cells stably expressing wild‐type and mutant CFTRs. CFTRinh‐172 did not alter CFTR unitary conductance (8 pS), but reduced open probability by >90% with K i≈0.6 μM. This effect was due to increased mean channel closed time without changing mean channel open time. Short‐circuit current experiments indicated similar CFTRinh‐172 inhibitory potency (K i≈0.5 μM) for inhibition of Cl− current in wild‐type, G551D, and G1349D CFTR; however, K i was significantly reduced to 0.2 μM for ΔF508 CFTR. Our studies provide evidence for CFTR inhibition by CFTRinh‐172 by a mechanism involving altered CFTR gating.

Three point mutations in the cftr gene in french cystic fibrosis patients identification by denaturing gradient gel electrophoresis

SummaryThe cystic fibrosis (CF) gene was recently identified as a gene spanning 250 kilobases (kbp) and coding for a 1480 amino acid protein, cystic fibrosis transmembrane conductance regulator (CFTR). Approximately 70% of CF mutations involve a three-base-pair deletion in CFTR exon 10, resulting in the loss of a phenylalanine at position 508 in the gene product (ΔF508). In order to screen for other molecular defects, we have used a strategy based on denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified gene segments. This method, which permits rapid detection of any sequence change in a given DNA stretch, was used successfully to analyse 61 non-ΔF508 CF chromosomes from French CF patients. A study of CFTR exons 10, 11, 14a, 15 and 20 detected three mutations located in exons 14a, 15 and 20, along with several nucleotide sequence polymorphisms. These nucleotide changes were identified by direct sequencing of PCR fragments displaying altered electrophoretic behaviour, together with some of the polymorphisms and mutations previously characterized by others. The strategy presented here constitutes a valuable tool for the development of carrier testing for individuals or couples with a family history of cystic fibrosis, and will contribute to deciphering the functionally important regions of the CFTR gene.

Evidence for direct CFTR inhibition by CFTRinh-172 based on Arg347 mutagenesis

CFTR (cystic fibrosis transmembrane conductance regulator) is an epithelial Cl- channel inhibited with high affinity and selectivity by the thiazolidinone compound CFTR(inh)-172. In the present study, we provide evidence that CFTR(inh)-172 acts directly on the CFTR. We introduced mutations in amino acid residues of the sixth transmembrane helix of the CFTR protein, a domain that has an important role in the formation of the channel pore. Basic and hydrophilic amino acids at positions 334-352 were replaced with alanine residues and the sensitivity to CFTR(inh)-172 was assessed using functional assays. We found that an arginine-to-alanine change at position 347 reduced the inhibitory potency of CFTR(inh)-172 by 20-30-fold. Mutagenesis of Arg347 to other amino acids also decreased the inhibitory potency, with aspartate producing near total loss of CFTR(inh)-172 activity. The results of the present study provide evidence that CFTR(inh)-172 interacts directly with CFTR, and that Arg347 is important for the interaction.

Correction of G551D-CFTR transport defect in epithelial monolayers by genistein but not by CPX or MPB-07

This study compares the effect of three chemically unrelated cystic fibrosis transmembrane conductance regulator (CFTR) activators on epithelial cell monolayers expressing the G551D‐CFTR mutant. We measured Cl− transport as the amplitude of short‐circuit current in response to the membrane permeable cAMP analogue 8‐(4‐chlorophenylthio)adenosine‐3′‐5′‐cyclic monophosphate (CPT‐cAMP) alone or in combination with a CFTR opener. The correction of G551D‐CFTR defect was quantified by comparison with maximal activity elicited in cells expressing wild type CFTR. To this end we used Fisher rat thyroid (FRT) cells transfected with wild type or G551D CFTR, and primary cultures of human nasal epithelial cells. In both types of epithelia, cAMP caused activation of Cl− transport that was inhibited by glibenclamide and not by 4,4′‐diisothiocyanato‐stilbene‐2,2′‐disulfonic acid. After normalising for CFTR expression, the response of FRT‐G551D epithelia was 1% that of wild type monolayers. Addition of genistein (10–200 μM), but not of 8‐cyclopentyl‐1,3‐dipropylxanthine (CPX, 1–100 μM) or of the benzo[c]quinolizinium MPB‐07 (10–200 μM) to FRT‐G551D epithelia pre‐treated with cAMP, stimulated a sustained current that at maximal genistein concentration corresponded to 30% of the response of wild type epithelia. The genistein dose‐response curve was bell‐shaped due to inhibitory activity at the highest concentrations. The dose‐dependence in G551D cells was shifted with respect to wild type CFTR so that higher genistein concentrations were required to observe activation and inhibition, respectively. On human nasal epithelia the correction of G551D‐CFTR defective conductance obtained with genistein was 20% that of wild type. The impressive effect of genistein suggests that it might correct the Cl− transport defect on G551D patients.

Genetics of cystic fibrosis: CFTR mutation classifications toward genotype-based CF therapies.

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial anion channel. Since the identification of the disease in 1938 and up until 2012, CF patients have been treated exclusively with medications aimed at bettering their respiratory, digestive, inflammatory and infectious symptoms. The identification of the CFTR gene in 1989 gave hopes of rapidly finding a cure for the disease, for which over 1950 mutations have been identified. Since 2012, recent approaches have enabled the identification of small molecules targeting either the CFTR protein directly or its key processing steps, giving rise to novel promising therapeutic tools. This review presents the current CFTR mutation classifications according to their clinical consequences and to their effect on the structure and function of the CFTR channel. How these classifications are essential in the establishment of mutation-targeted therapeutic strategies is then discussed. The future of CFTR-targeted treatment lies in combinatory therapies that will enable CF patients to receive a customized treatment.

Two Mild Cystic Fibrosis-associated Mutations Result in Severe Cystic Fibrosis When Combined in Cis and Reveal a Residue Important for Cystic Fibrosis Transmembrane Conductance Regulator Processing and Function

The number of complex cystic fibrosis transmembrane conductance regulator (CFTR) genotypes identified as having double-mutant alleles with two mutations inheritedin cis has been growing. We investigated the structure-function relationships of a severe cystic fibrosis (CF)-associated double mutant (R347H-D979A) to evaluate the contribution of each mild mutation to the phenotype. CFTRmutants expressed in HeLa cells were analyzed for protein biosynthesis and Cl− channel activity. Our data show that R347H is associated with mild defective Cl− channel activity and that the D979A defect leads to misprocessing. The mutant R347H-D979A combines both defects for a dramatic decrease in Cl−current. To decipher the molecular mechanism of this phenotype, single and double mutants with different charge combinations at residues 347 and 979 were constructed as charged residues were involved in this complex genotype. These studies revealed that residue 979, located in the third cytoplasmic loop, is critical for CFTR processing and Cl− channel activity highlighting the role of charged residues. These results have also important implications for CF, as they show that two mutations in cis can act in concert to alter dramatically CFTR function contributing to the wide phenotypic variability of CF disease.

Alternative Splicing at a NAGNAG Acceptor Site as a Novel Phenotype Modifier

Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of the mild to asymptomatic phenotype observed in cystic fibrosis patients homozygous for the E831X mutation (2623G>T) in the CFTR gene. Analyses performed on nasal epithelial cell mRNA detected three distinct isoforms, a considerably more complex situation than expected for a single nucleotide substitution. Structure-function studies and in silico analyses provided the first experimental evidence of an indel of a stop codon by alternative splicing at a NAGNAG acceptor site. In addition to contributing to proteome plasticity, alternative splicing at a NAGNAG tandem site can thus remove a disease-causing UAG stop codon. This molecular study reveals a naturally occurring mechanism where the effect of either modifier genes or epigenetic factors could be suspected. This finding is of importance for genetic counseling as well as for deciding appropriate therapeutic strategies.

COMMD1-Mediated Ubiquitination Regulates CFTR Trafficking

The CFTR (cystic fibrosis transmembrane conductance regulator) protein is a large polytopic protein whose biogenesis is inefficient. To better understand the regulation of CFTR processing and trafficking, we conducted a genetic screen that identified COMMD1 as a new CFTR partner. COMMD1 is a protein associated with multiple cellular pathways, including the regulation of hepatic copper excretion, sodium uptake through interaction with ENaC (epithelial sodium channel) and NF-kappaB signaling. In this study, we show that COMMD1 interacts with CFTR in cells expressing both proteins endogenously. This interaction promotes CFTR cell surface expression as assessed by biotinylation experiments in heterologously expressing cells through regulation of CFTR ubiquitination. In summary, our data demonstrate that CFTR is protected from ubiquitination by COMMD1, which sustains CFTR expression at the plasma membrane. Thus, increasing COMMD1 expression may provide an approach to simultaneously inhibit ENaC absorption and enhance CFTR trafficking, two major issues in cystic fibrosis.

Combined pulmonary fibrosis and emphysema syndrome associated with ABCA3 mutations

To the Editor: Herein, we present the first report of combined pulmonary fibrosis and emphysema (CPFE) in an adult patient who was compound heterozygous for mutations of the ATP-binding cassette subfamily A member 3 gene ( ABCA3 , MIM 601615). A 41-year-old nonsmoking male presented with dyspnoea on mild exertion. The patient’s medical history indicated neonatal respiratory distress, gastro-oesophageal reflux and pneumonia 8 years previously that resolved with antibiotics. His physical examination revealed a mild pectus excavatum, finger clubbing and bilateral basal crackles. High-resolution computed tomography (HRCT) of the chest showed voluminous emphysema in the upper zones of the lungs associated with honeycomb fibrosis and ground-glass opacity in lower lobes, predominating in left lung (fig. 1). The bronchoalveolar lavage differential cell count was 67% macrophages, 22% neutrophils and 8% lymphocytes. Pulmonary function tests showed: total lung capacity of 75%, vital capacity (VC) of 50%, residual volume of 134%; forced expiratory volume in 1 s (FEV1) of 49%, diffusing capacity of the lung for carbon monoxide of 38% predicted, FEV1/VC of 74%, and arterial oxygen tension at room air was 96 mmHg. During a 6-min walk test the peripheral oxygen saturation decreased from 96% at rest to 90% after 630 m (80% of predicted value). A lung biopsy was not performed. …