Rapid F508del and F508C assay using fluorescent hybridization probes

Amplification and fluorescent genotyping of the cystic fibrosis F508del locus was achieved from human genomic DNA in less than 30 min. The hybridization of adjacent fluorescent probes at the mutation site was monitored by resonance energy transfer between fluorescein and Cy5 during heating or cooling. Characteristic curves were obtained for each genotype; the first derivative of these fluorescent curves has a maximum at an apparent hybridization temperature (Tm) that is specific for each probe/allele duplex. The direction and rate of temperature change determines the difference between the apparent Tm and the true equilibrium Tm. One hundred and five sample were genotyped for the F508del cystic fibrosis mutation by heating and cooling curve profiles. These genotypes were validated by allele-specific amplification. Two fluorescein hybridization probes were designed to match the wild-type sequence perfectly from either codons 502 to 513 or from 504 to 511 on the cystic fibrosis transconductance regulator gene of chromosome 7. While genotyping for the F508del, an allele with the F508C base change was detected. For both F508del and F508C variants, the Tm shift from wild type was greater with a 24-mer probe than with a 35-mer probe. Fluorescent monitoring of hybridization probes is a versatile technique that can detect unexpected sequence alterations.     

Ancient DNA analysis of the delta F508 mutation

When working with highly degraded DNA, validating the results of a slightly polymorphic system always complicates the analysis because of the difficulties in recognizing contamination and artifacts. Recognition can be greatly simplified by employing a multiplex reaction that coamplifies the fragments together with several highly polymorphic markers, for instance, short tandem repeats. In this work, we successfully included newly designed oligonucleotide primers for the detection of delta F508, the most frequent mutation causing cystic fibrosis, in the commercial AmpFlSTR Profiler Plus PCR Amplification Kit (PE Applied Biosystems). This coamplification enabled us to test the hypothesis of a heterozygote advantage associated with cystic fibrosis-specifically, higher resistance to toxin-mediated diarrheas--in a Sicilian skeletal sample of individuals who died in a cholera epidemic in 1837. The proposed method should also be suitable for the genetic characterization of other slightly polymorphic loci tested on human and animal ancient DNA; it should permit simple authentication of results by comparing the fingerprints obtained from independent amplifications repeated several times.     

The cystic fibrosis ΔF508 mutation in the French population

Summary French families (n = 129) with at least one cystic fibrosis (CF) affected child and 44 unrelated subjects from the general population were tested for the presence of the ΔF508 mutation by the polymerase chain reaction. The ΔF508/CF mutation ratio (CF: uncharacterised CF mutations) was tested in the CF families with and without meconium ileus. The association between ΔF508 and CF mutations and restriction fragment length polymorphism haplotypes (XV2c and KM19) has been estimated; these data suggest that the CF chromosomes include a panel of independent and probably different mutations.     

A mouse model for the cystic fibrosis delta F508 mutation.

Most cystic fibrosis (CF) patients produce a mutant form (delta F508) of the cystic fibrosis transmembrane conductance regulator (CFTR), which is not properly processed in normal cells but is active as a chloride channel in several experimental systems. We used a double homologous recombination ('Hit and Run') procedure to generate a mouse model for the delta F508 mutation. Targeted embryonic stem (ES) cells (Hit clones) were found; of these either 80 or 20% of the clones had lost the delta F508 mutation, depending on the distance between the linearization site in the targeting construct and the delta F508 mutation. Correctly targeted clones underwent a second selection step resulting in ES cell clones (Run clones) heterozygous for the delta F508 mutation with an efficiency of 2-7%. Chimeric mice were generated and offspring homozygous for the delta F508 mutation showed electrophysiological abnormalities in nasal epithelium, gallbladder and in the intestine, and histological abnormalities in the intestine, typical of CF. Our data suggest that the delta F508 mice have residual delta F508 CFTR activity which would explain the mild pathology of the delta F508 mice. The delta F508 mouse may provide a useful model for the study of the processing defect of delta F508 CFTR and for the development of novel therapeutic approaches based on circumvention of the processing block.     

Cross-linking of F508-CFTR promotes its trafficking to the plasma membrane

CFTR is a cAMP-activated chloride channel responsible for agonist stimulated chloride and fluid transport across epithelial surfaces.¹ Mutations in the CFTR gene lead to cystic fibrosis (CF) which affects the function of secretory organs like the intestine, the pancreas, the airways and the sweat glands. Most of the morbidity and mortality in CF has been linked to a decrease in airway function.² The ΔF508 mutation is the most common CF-related mutation in the Caucasian population and represents 90% of CF alleles. Homozygote carriers of this mutation present with a severe CF phenotype.³ The ΔF508 mutation causes misfolding of the nascent CFTR polypeptide, which leads to inefficient export from the endoplasmic reticulum (ER) and rapid degradation by the proteasome.⁴Key words: cystic fibrosis, endoplasmic reticulum, oligomer, processing mutation, curcuminGiven the frequency of the ΔF508 processing mutation and the severity of its corresponding phenotype, much research has focused on identifying compounds that restore the trafficking and function of this mutant at the plasma membrane. Several synthetic ‘correctors’ of ΔF508 mis-processing and ‘potentiators’ of mutant channel activity have been identified.^5,6 Natural compounds such as curcumin also have generated interest. Curcumin is an organic phenolic compound abundant in turmeric, an Indian spice extracted from the rhizome of Curcuma longa.⁷ Earlier studies performed using ΔF508/ΔF508 mouse models and human airway epithelial cell lines suggested that curcumin may act as a ΔF508-CFTR trafficking corrector.⁸ Also, we and others showed that curcumin stimulates CFTR channel activity in excised membrane patches.^9,10 This stimulation occurs in the absence of ATP binding, which is normally required for channel opening.¹⁰ Binding sites of correctors and potentiators within the CFTR polypeptide as well as the molecular mechanisms underlying the rescue of CFTR trafficking and function remain to be elucidated. In our attempt to understand how curcumin could circumvent the normally critical step of ATP binding to promote CFTR channel activity we investigated the effect of curcumin on CFTR conformation by using biochemical assays. We showed that curcumin caused dimerization of several CFTR channel constructs (including ΔF508-CFTR) in a dose- and time-dependent manner both in microsomes and within intact cells. This effect of curcumin on CFTR oligomerization is attributable to its reactive β-diketone groups, which may undergo an oxidation reaction with CFTR nucleophilic amino acid residues.¹¹ Importantly, CFTR channel activation by curcumin is unrelated to its cross-linking effect. We identified cyclic derivatives of curcumin that lack this cross-linking activity but still promote CFTR channel function.¹¹Here we examined the possibility that the cross-linking of ΔF508-CFTR channels by curcumin promotes the delivery of this ER processing mutant to the cell surface. We were motivated to test this possibility for three reasons: (i) our previous evidence that curcumin-induced dimers of wild-type CFTR polypeptides were detected at the cell surface where they remained over an hour after the removal of curcumin;¹¹ (ii) the very efficient cross-linking of the immature (ER) forms of wild-type CFTR and the ΔF508-CFTR mutant that we observed earlier¹¹ and (iii) prior evidence from our group that the ER export and cell surface delivery of ΔF508-CFTR polypeptides could be promoted by the co-expression of this mutant with certain CFTR fragments (trans-complementation).¹² The latter result might be due to the existence of ER retention ‘signals’ that are exposed on the ΔF508-CFTR polypeptide but become buried by interacting (complementing) fragments.Figure 1 provides evidence that ΔF508-CFTR oligomers that form in response to curcumin treatment do indeed appear at the surfaces of cultured airway epithelial cells (CF bronchial epithelial (CFBE) cells stably transfected with this CFTR mutant). Surface biotinylation assays were performed to detect the appearance of ΔF508-CFTR polypeptides at the cell surface. MESNA, a cell impermeant reducing agent that cleaves the biotin label, was used to verify the surface accessibility of the labeled ΔF508-CFTR polypeptides. ΔF508-CFTR polypeptides were precipititated with streptavidinagarose (surface pool) or with a CFTR monoclonal antibody (total pool). In the absence of curcumin treatment the great majority of the ΔF508-CFTR protein existed as the ER form (monomeric band B), as previously observed by many investigators (Fig. 1, lane 5). No band B was detected in the surface pool before or after curcumin treatment (Fig. 1, lanes 1, 2). As we reported earlier, treatment of the cells with 50 µM curcumin for 15 mins at 37°C cross-linked nearly all of the ΔF508-CFTR polypeptides into higher order complexes (e.g., dimers, termed band D here; lanes 6–8 in Fig. 1). Interestingly, these higher order forms of ΔF508-CFTR were readily apparent in the surface pool (Fig. 1, lane 2).Open in a separate windowFigure 1ΔF508-CFTR oligomers detected at the surfaces of airway epithelial cells after curcumin treatment. ΔF508-CFTR expressing CFBE cells were treated with curcumin (50 µM) for 15 min at 37°C. Cell surface proteins were then biotinylated (Sulfo-NHS-SS-Biotin, 1 mg/ml) for 30 min at 4°C followed by cell lysis with 1% Triton X-100. Surface proteins were isolated by streptavidin pulldown and ΔF508-CFTR was isolated from the total cell protein pool by immunoprecipitation with an anti-CFTR C-terminus antibody (clone 24-1, R&D systems). After SDS-PAGE the ΔF508-CFTR signal was detected by immunoblotting using the 24-1 antibody described above. (SP: streptavidin pulldown; IP: immunoprecipitation). As an additional control curcumin-treated cells were treated with the cell impermeant MESNA after biotinylation to strip the biotin off the cell surface proteins with which it had reacted.CFTR oligomers also can be generated by standard chemical cross-linkers such as DSS, as previously reported by others and confirmed by us.¹³ Figure 2 shows that oligomers of ΔF508-CFTR that are induced by DSS treatment also appear in the surface pool. These experiments were performed using transiently transfected HEK-293T cells with 30 µM curcumin as a positive control. Quantitative densitometry results are shown in Figure 3. By titrating the DSS concentration we observed a dose-dependent disappearance of the monomeric band B form, a corresponding increase in the band D (dimer) pool and the appearance of higher order oligomers (band E) which prevailed at higher DSS concentrations (see total cell pool data in right-hand). A small amount of the band D form was detected in the absence of DSS or curcumin treatment, which might represent some spontaneous cross-linking of ΔF508-CFTR polypeptides under these conditions. The DSS and curcumin-induced ΔF508-CFTR oligomers were readily detected in the surface pool. The densitometry analysis revealed that 20 ± 5% and 33 ± 19% of the total oligomer pool (combined bands D and E) was found in the surface pool after treatment with 0.1 mM DSS (n = 3) or 30 µM curcumin (n = 3), respectively, which corresponded to a 17 ± 7 and 26 ± 20 fold increase compared to the control condition (i.e., no DSS or no curcumin).Open in a separate windowFigure 2ΔF508-CFTR oligomers detected at the surfaces of HEK cells after DSS or curcumin treatment. ΔF508-CFTR expressing HEK cells were treated with the indicated concentrations of DSS or with 30 µM curcumin (*) for 15 min at 37°C. Cell surface proteins were then biotinylated and isolated by streptavidin pulldown as described above. ΔF508-CFTR was immunoprecipitated from the total cell protein pool with the 24-1 antibody and detected by immunoblotting as before (SP: streptavidin pulldown; IP: immunoprecipitation). Band B corresponds to ΔF508 monomer (ER form). Band D corresponds to ΔF508 dimer. Band E corresponds to a higher degree of ΔF508 oligomerization. Each panel corresponds to a different exposure of the same blot.Open in a separate windowFigure 3Dose-dependent expression of ΔF508-CFTR oligomers at the surfaces of HEK cells after DSS treatment. CFTR signals detected by the 24-1 antibody from three different experiments as the one described in Figure 2 were analyzed using the ImageJ software (from the National Institute of Health). (A) band B signal intensity is plotted as a function of the DSS concentrations. Signals analyzed correspond to ΔF508-CFTR band B immunoprecipitated by the 24-1 antibody. (B) band D plus band E signal intensities are plotted as a function of the DSS concentration. Signals analyzed correspond to the sum of ΔF508-CFTR band D and band E immunoprecipitated by the 24-1 antibody. (C) band D plus band E signal intensities at the cell surface are plotted as a function of the DSS concentration. Signals analyzed correspond to the sum of ΔF508-CFTR band D and band E isolated from the surfaces of ΔF508-CFTR expressing HEK cells by biotinylation and streptavidin pulldown. (D) the ratio between the amount of band E and D at the surfaces of ΔF508-CFTR expressing HEK cells is plotted as a function of the DSS concentration. Error bars are SEMs.Altogether these data indicate that the cross-linking of ΔF508-CFTR band B into oligomers by curcumin or DSS allows ΔF508-CFTR to traffic to the cell surface. This effect might be caused by the burial of ER retention motifs within the oligomer, which also could explain our previous trans-complementation results in which we observed that certain CFTR fragments promote the cell surface delivery of this processing mutant.¹² Although non-specific protein cross-linkers like DSS would not be therapeutically beneficial, more specific CFTR cross-linkers (perhaps curcumin?) may be worth considering for treating CF disease linked to ER processing mutations in CFTR. In this regard, we note that cross-linked CFTR polypeptides appear to retain chloride channel activity. Namely, in our prior excised patch clamp studies we observed stable CFTR channel activity when these patches were exposed to curcumin at doses and times that promote robust cross-linking of CFTR polypeptides.^10,11     

ΔF508 deletion in cystic fibrosis in Italian families

Summary In 20 Italian families with cystic fibrosis (CF), restriction fragment length polymorphisms were detected by five linked markers; a strong linkage disequilibrium is observed between the haplotype B (alleles 2/1 with respect to KM19/XV2c) and CF. The frequency of the ΔF508 deletion in CF chromosomes of this sample is 50%. A significant correlation is found between the absence of the ΔF508 mutation and pancreatic sufficiency.     

Activation of Delta F508 CFTR in an epithelial monolayer

The F508 mutation leads to retention of cystic fibrosistransmembrane conductance regulator (CFTR) in the endoplasmic reticulum and rapid degradation by the proteasome and other proteolytic systems.In stably transfected LLC-PK₁(porcine kidney) epithelial cells, F508 CFTR conforms to thisparadigm and is not present at the plasma membrane. WhenLLC-PK₁ cells or human nasal polyp cells derived from a F508 homozygous patient are grown on plastic dishes and treated with an epithelial differentiating agent (DMSO, 2%for 4 days) or when LLC-PK₁ cellsare grown as polarized monolayers on permeable supports, plasmamembrane F508 CFTR is significantly increased. Moreover, whenconfluent LLC-PK₁ cells expressingF508 CFTR were treated with DMSO and mounted in an Ussing chamber, afurther increase in cAMP-activated short-circuit current (i.e., ~7µA/cm²;P < 0.00025 compared with untreatedcontrols) was observed. No plasma membrane CFTR was detected after DMSOtreatment in nonepithelial cells (mouse L cells) expressing F508CFTR. The experiments describe a way to augment F508 CFTR maturationin epithelial cells that appears to act through a novel mechanism andallows insertion of functional F508 CFTR in the plasma membranes oftransporting cell monolayers. The results raise the possibility thatincreased epithelial differentiation might increase the delivery ofF508 CFTR from the endoplasmic reticulum to the Golgi, where theF508 protein is shielded from degradative pathways such as theproteasome and allowed to mature.

     

Calumenin contributes to ER-Ca2+ homeostasis in bronchial epithelial cells expressing WT and F508del mutated CFTR and to F508del-CFTR retention

Cystic Fibrosis (CF) is the most frequent fatal genetic disease in Caucasian populations. Mutations in the chloride channel CF Transmembrane Conductance Regulator (CFTR) gene are responsible for functional defects of the protein and multiple associated dysregulations. The most common mutation in patients with CF, F508del-CFTR, causes defective CFTR protein folding. Thus minimal levels of the receptor are expressed at the cell surface as the mutated CFTR is retained in the endoplasmic reticulum (ER) where it correlates with defective calcium (Ca²⁺) homeostasis. In this study, we discovered that the Ca²⁺ binding protein Calumenin (CALU) is a key regulator in the maintenance of ER-Ca²⁺ calcium homeostasis in both wild type and F508del-CFTR expressing cells. Calumenin modulates SERCA pump activity without drastically affecting ER-Ca²⁺ concentration. In addition, reducing Calumenin expression in CF cells results in a partial restoration of CFTR activity, highlighting a potential function of Calumenin in CFTR maturation. These findings demonstrate a pivotal role for Calumenin in CF cells, providing insights into how modulation of Calumenin expression or activity may be used as a potential therapeutic tool to correct defects in F508del-CFTR.     

Extent of rescue of F508del-CFTR function by VX-809 and VX-770 in human nasal epithelial cells correlates with SNP rs7512462 in SLC26A9 gene in F508del/F508del Cystic Fibrosis patients

BackgroundWe analyzed the CFTR response to VX-809/VX-770 drugs in conditionally reprogrammed cells (CRC) of human nasal epithelium (HNE) from F508del/F508del patients based on SNP rs7512462 in the Solute Carrier Family 26, Member 9 (SLC26A9; MIM: 608481) gene.MethodsThe I_sc-eq measurements of primary nasal epithelial cells from F508del/F508del patients (n = 12) for CFTR function were performed in micro Ussing chambers and compared with non-CF controls (n = 2). Data were analyzed according to the rs7512462 genotype which were determined by real-time PCR.ResultsThe CRC-HNE cells from F508del/F508del patients evidenced high variability in the basal levels of CFTR function. Also, the rs7512462*C allele showed an increased basal CFTR function and higher responses to VX-809 + VX-770. The rs7512462*CC + CT genotypes together evidenced CFTR function levels of 14.89% relatively to wt/wt (rs7512462*CT alone-15.29%) i.e., almost double of rs7512462*TT (7.13%). Furthermore, sweat [Cl⁻] and body mass index of patients also evidenced an association with the rs7512462 genotype.ConclusionThe CFTR function can be performed in F508del/F508del patient-derived CRC-HNEs and its function and responses to VX-809 + VX-770 combination as well as clinical data, are all associated with the rs7512462 variant, which partially sheds light on the generally inter-individual phenotypic variability and in personalized responses to CFTR modulator drugs.     

Post-translational disruption of the delta F508 cystic fibrosis transmembrane conductance regulator (CFTR)-molecular chaperone complex with geldanamycin stabilizes delta F508 CFTR in the rabbit reticulocyte lysate

The DeltaF508 mutation of cystic fibrosis transmembrane conductance regulator (CFTR) is a trafficking mutant, which is retained and degraded in the endoplasmic reticulum by the ubiquitin-proteasome pathway. The mutant protein fails to reach a completely folded conformation that is no longer a substrate for ubiquitination ("stable B"). Wild type protein reaches this state with 25% efficiency. In this study the rabbit reticulocyte lysate with added microsomal membranes has been used to reproduce the post-translational events in the folding of wild type and DeltaF508 CFTR. In this system wild type CFTR does not reach the stable B form if the post-translational temperature is 37 degrees C, whereas at 30 degrees C the behavior of both wild type and mutant proteins mimics that observed in the cell. Geldanamycin stabilizes DeltaF508 CFTR with respect to ubiquitination only when added post-translationally. The interaction of wild type and mutant CFTR with the molecular chaperones heat shock cognate 70 (hsc70) and heat shock protein 90 (hsp90) has been assessed. Release of wild type protein from hsc70 coincides with the cessation of ubiquitination and formation of stable B. Geldanamycin immediately prevents the binding of hsp90 to DeltaF508 CFTR, and after a delay releases it from hsc70. Release of mutant protein from hsc70 also coincides with the formation of stable B DeltaF508 CFTR.     

Role for phospholipid interactions in the trafficking defect of Delta F508-CFTR

Cystic fibrosis commonly occurs as a consequence of the DeltaF508 mutation in the first nucleotide binding fold domain (NBF-1) of CFTR. The mutation causes retention of the mutant CFTR molecule in the endoplasmic reticulum, and this aberrant trafficking event is believed to be due to defective interactions between the mutant NBF-1 domain and other cellular factors in the endoplasmic reticulum. Since the NBF-1 domain has been shown to interact with membranes, we wanted to investigate whether NBF-1 and CFTR interactions with specific phospholipid chaperones might play a role in trafficking. We have found that the recombinant wild-type NBF-1 interacts selectively with phosphatidylserine (PS) rather than phosphatidylcholine (PC). By contrast, NBF-1 carrying the DeltaF508 mutation loses the ability to discriminate between these two phospholipids. In cells expressing DeltaF508-CFTR, replacement of PC by noncharged analogues results in an absolute increase in CFTR expression. In addition, we detected progressive expression of higher molecular weight CFTR forms. Thus, phospholipid chaperones may be important for CFTR trafficking, and contribute to the pathology of cystic fibrosis.     

Cross-linking of ΔF508-CFTR promotes its trafficking to the plasma membrane

CFTR is a cAMP-activated chloride channel responsible for agonist stimulated chloride and fluid transport across epithelial surfaces.¹ Mutations in the CFTR gene lead to cystic fibrosis (CF) which affects the function of secretory organs like the intestine, the pancreas, the airways and the sweat glands. Most of the morbidity and mortality in CF has been linked to a decrease in airway function.² The ΔF508 mutation is the most common CF-related mutation in the Caucasian population and represents 90% of CF alleles. Homozygote carriers of this mutation present with a severe CF phenotype.³ The ΔF508 mutation causes misfolding of the nascent CFTR polypeptide, which leads to inefficient export from the endoplasmic reticulum (ER) and rapid degradation by the proteasome.⁴     

First analysis of the F508 deletion in cystic fibrosis patients from the GDR

Summary Cystic fibrosis (CF) patients (n = 157) from the GDR were analysed for the occurrence of the recently discovered 3bp deletion causing CF. About 50% of all investigated patients were homozygotes and about 30% heterozygotes for this deletion. Of the analysed CF chromosomes from these patients, 62% carry the deletion, which is in strong linkage disequilibrium with the KM19 restriction fragment length polymorphism allele 2 and the 1/2 XV2c/KM19 haplotype.