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Background: Cystic Fibrosis (CF) is caused by ~1,900 mutations in the CF transmembrane conductance regulator (CFTR) gene encoding for a cAMP-regulated chloride (Cl-) channel expressed in several epithelia. Clinical features are dominated by respiratory symptoms, but there is variable organ involvement thus causing diagnostic dilemmas, especially for non-classic cases. Methodology/Principal Findings: To further establish measurement of CFTR function as a sensitive and robust biomarker for diagnosis and prognosis of CF, we herein assessed cholinergic and cAMP-CFTR-mediated Cl- secretion in 524 freshly excised rectal biopsies from 118 individuals, including patients with confirmed CF clinical diagnosis (n = 51), individuals with clinical CF suspicion (n = 49) and age-matched non-CF controls (n = 18). Conclusive measurements were obtained for 96% of cases. Patients with "Classic CF", presenting earlier onset of symptoms, pancreatic insufficiency, severe lung disease and low Shwachman-Kulczycki scores were found to lack CFTR-mediated Cl- secretion (<5%). Individuals with milder CF disease presented residual CFTR-mediated Cl- secretion (10-57%) and non-CF controls show CFTR-mediated Cl- secretion ≥30-35% and data evidenced good correlations with various clinical parameters. Finally, comparison of these values with those in "CF suspicion" individuals allowed to confirm CF in 16/49 individuals (33%) and exclude it in 28/49 (57%). Statistical discriminant analyses showed that colonic measurements of CFTR-mediated Cl- secretion are the best discriminator among Classic/Non-Classic CF and non-CF groups. Conclusions/Significance: Determination of CFTR-mediated Cl- secretion in rectal biopsies is demonstrated here to be a sensitive, reproducible and robust predictive biomarker for the diagnosis and prognosis of CF. The method also has very high potential for (pre-)clinical trials of CFTR-modulator therapies. [ABSTRACT FROM AUTHOR]

Synonymous single nucleotide polymorphisms (sSNPs), which change a nucleotide, but not the encoded amino acid, are perceived as neutral to protein function and thus, classified as benign. We report a patient who was diagnosed with cystic fibrosis (CF) at an advanced age and presented very mild CF symptoms. The sequencing of the whole cystic fibrosis transmembrane conductance regulator (CFTR) gene locus revealed that the patient lacks known CF-causing mutations. We found a homozygous sSNP (c.1584G>A) at the end of exon 11 in the CFTR gene. Using sensitive molecular methods, we report that the c.1584G>A sSNP causes cognate exon skipping and retention of a sequence from the downstream intron, both of which, however, occur at a relatively low frequency. In addition, we found two other sSNPs (c.2562T>G (p.Thr854=) and c.4389G>A (p.Gln1463=)), for which the patient is also homozygous. These two sSNPs stabilize the CFTR protein expression, compensating, at least in part, for the c.1584G>A-triggered inefficient splicing. Our data highlight the importance of considering sSNPs when assessing the effect(s) of complex CFTR alleles. sSNPs may epistatically modulate mRNA and protein expression levels and consequently influence disease phenotype and progression. [ABSTRACT FROM AUTHOR]

pwCF carrying the Q359K/T360K variant may have significant clinical benefit from treatment with ETI that may exceed improvements observed with TI treatment. These data support routine clinical use of ETI in this rare patient group. https://bit.ly/45DjFw9., Competing Interests: Conflict of interest: K. Yaacoby-Bianu declares grants and support for attending meetings, receiving fees from Sanofi outside the submitted work. Conflict of interest: D. Prais declares grants, consulting fees and payment for lectures from Vertex, outside the submitted work. Conflict of interest: R. Cohen declares payment for lectures from GSK, and support for attending meetings, receiving fees from Boehringer Ingelheim, AstraZeneca and Kamada, outside the submitted work. Conflict of interest: M. Shteinberg declares grants from GSK, Trudell and the Tel Aviv League for Lung Disease during the conduct of the study; consulting fees from AstraZeneca, Boehringer Ingelheim, Dexcel, Kamada, Synchrony Medical, Trumed, Vertex and Zambon; lecture fees from AstraZeneca, Boehringer Ingelheim, GSK, Kamada, Sanofi and Insmed; support for attending meetings from AstraZeneca, Boehringer Ingelheim, Kamada, Rafa and GSK; participation on advisory boards for Bonus Biotherapeutics, Boehringer Ingelheim and AstraZeneca; board fees from the American Journal of Respiratory and Critical Care Medicine; and receipt of equipment from Trudell, outside the submitted work. Conflict of interest: M. Heching, M.R. Kramer, E. Shmueli, F. Vermeulen, A.S. Ramalho and G. Livnat declare that they have no conflicts of interest., (Copyright ©The authors 2025.)

Cystic fibrosis (CF) is a life-threatening disorder characterised by decreased pulmonary mucociliary and pathogen clearance, and an exaggerated inflammatory response leading to progressive lung damage. CF is caused by bi-allelic pathogenic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel. CFTR is expressed in endothelial cells (ECs) and EC dysfunction has been reported in CF patients, but a role for this ion channel in ECs regarding CF disease progression is poorly described.We used an unbiased RNA sequencing approach in complementary models of CFTR silencing and blockade (by the CFTR inhibitor CFTRinh-172) in human ECs to characterise the changes upon CFTR impairment. Key findings were further validated in vitro and in vivo in CFTR-knockout mice and ex vivo in CF patient-derived ECs.Both models of CFTR impairment revealed that EC proliferation, migration and autophagy were downregulated. Remarkably though, defective CFTR function led to EC activation and a persisting pro-inflammatory state of the endothelium with increased leukocyte adhesion. Further validation in CFTR-knockout mice revealed enhanced leukocyte extravasation in lung and liver parenchyma associated with increased levels of EC activation markers. In addition, CF patient-derived ECs displayed increased EC activation markers and leukocyte adhesion, which was partially rescued by the CFTR modulators VX-770 and VX-809.Our integrated analysis thus suggests that ECs are no innocent bystanders in CF pathology, but rather may contribute to the exaggerated inflammatory phenotype, raising the question of whether normalisation of vascular inflammation might be a novel therapeutic strategy to ameliorate the disease severity of CF., Competing Interests: Conflict of interest: M. Declercq has nothing to disclose. Conflict of interest: P. de Zeeuw has nothing to disclose. Conflict of interest: N.V. Conchinha has nothing to disclose. Conflict of interest: V. Geldhof has nothing to disclose. Conflict of interest: A.S. Ramalho has nothing to disclose. Conflict of interest: M. García-Caballero has nothing to disclose. Conflict of interest: K. Brepoels has nothing to disclose. Conflict of interest: M. Ensinck has nothing to disclose. Conflict of interest: M.S. Carlon has nothing to disclose. Conflict of interest: M.J. Bird has nothing to disclose. Conflict of interest: S. Vinckier has nothing to disclose. Conflict of interest: M. Proesmans has nothing to disclose. Conflict of interest: F. Vermeulen has nothing to disclose. Conflict of interest: L. Dupont has nothing to disclose. Conflict of interest: B. Ghesquière has nothing to disclose. Conflict of interest: M. Dewerchin has nothing to disclose. Conflict of interest: P. Carmeliet has nothing to disclose. Conflict of interest: D. Cassiman has nothing to disclose. Conflict of interest: L. Treps has nothing to disclose. Conflict of interest: G. Eelen has nothing to disclose. Conflict of interest: P. Witters has nothing to disclose., (Copyright ©ERS 2021.)

Rationale: Given the vast number of cystic fibrosis transmembrane conductance regulator ( CFTR ) mutations, biomarkers predicting benefit from CFTR modulator therapies are needed for subjects with cystic fibrosis (CF)., Objectives: To study CFTR function in organoids of subjects with common and rare CFTR mutations and evaluate correlations between CFTR function and clinical data., Methods: Intestinal organoids were grown from rectal biopsies in a cohort of 97 subjects with CF. Residual CFTR function was measured by quantifying organoid swelling induced by forskolin and response to modulators by quantifying organoid swelling induced by CFTR correctors, potentiator and their combination. Organoid data were correlated with clinical data from the literature., Results: Across 28 genotypes, residual CFTR function correlated (r 2 =0.87) with sweat chloride values. When studying the same genotypes, CFTR function rescue by CFTR modulators in organoids correlated tightly with mean improvement in lung function (r 2 =0.90) and sweat chloride (r 2 =0.95) reported in clinical trials. We identified candidate genotypes for modulator therapy, such as E92K, Q237E, R334W and L159S. Based on organoid results, two subjects started modulator treatment: one homozygous for complex allele Q359K&#95;T360K, and the second with mutation E60K. Both subjects had major clinical benefit., Conclusions: Measurements of residual CFTR function and rescue of function by CFTR modulators in intestinal organoids correlate closely with clinical data. Our results for reference genotypes concur with previous results. CFTR function measured in organoids can be used to guide precision medicine in patients with CF, positioning organoids as a potential in vitro model to bring treatment to patients carrying rare CFTR mutations., Competing Interests: Conflict of interest: A.S. Ramalho has nothing to disclose. Conflict of interest: E. Fürstová has nothing to disclose. Conflict of interest: A.M. Vonk has nothing to disclose. Conflict of interest: M. Ferrante reports grants and personal fees from Amgen, grants, personal fees and non-financial support from Biogen, Janssen, Pfizer and Takeda, personal fees and non-financial support from Boehringer Ingelheim, MSD, Falk and Ferring, personal fees from Sandoz, Lamepro, and Mylan, outside the submitted work. Conflict of interest: C. Verfaillie has nothing to disclose. Conflict of interest: L. Dupont has nothing to disclose. Conflict of interest: M. Boon is a member of the European Reference Network for Rare Respiratory Diseases (ERN-LUNG) – Project ID number 739546. Conflict of interest: M. Proesmans has nothing to disclose. Conflict of interest: J.M. Beekman reports personal fees from various industries (Vertex, Proteostasis, Teva, others) for costs related to conference presentations, outside the submitted work; and has a patent WO2013093812A3 with royalties paid by Hubrecht Organoid Technology. Conflict of interest: I. Sarouk has nothing to disclose. Conflict of interest: C. Vazquez Cordero has nothing to disclose. Conflict of interest: F. Vermeulen has nothing to disclose. Conflict of interest: K. De Boeck has provided consultancy for Boehringer, Protalix, Raptor, Novabiotics, Eloxx and Chiesi, has been member of steering committees/advisory boards and been PI in studies for Vertex, has provided consultancy and been PI in studies for Galapagos, and has received speaker fees from Teva, outside the submitted work., (Copyright ©ERS 2021.)

Quantification and analysis of CFTR transcripts is of crucial importance not only for cystic fibrosis (CF) diagnosis and prognosis, but also in evaluating the efficiency of various therapeutic approaches to CF, including gene therapy. Reverse transcription (RT) followed by quantitative polymerase chain reaction (qPCR) is at present the most sensitive method for transcript abundance measurement. Classical RNA-based methods require significant expression levels in target samples for appropriate analysis, thus PCR-based methods have evolved towards reliable quantification. In this chapter we describe and discuss several protocols for the quantitative analysis of CFTR transcripts, including those variants that result from alternative splicing.

Cultured airway epithelial cells are widely used in cystic fibrosis (CF) research as in vitro models that mimic the in vivo manifestations of the disease and help to define a specific cellular phenotype. Recently, a number of in vitro studies have used an airway adenocarcinoma cell line, Calu-3 that expresses submucosal gland cell features and significant levels of the wild-type CFTR mRNA and protein. We further characterized previously described CF tracheobronchial gland cell lines, CFSMEo- and 6CFSMEo- and determined that these cell lines are compound heterozygotes for the F508del and Q2X mutations, produce vestigial amounts of CFTR mRNA, and do not express detectable CFTR protein. Electrophysiologically, both cell lines are characteristically CF in that they lack cAMP-induced Cl- currents. In this study the cell lines are evaluated in the context of their role as the CF correlate to the Calu-3 cells. Together these cell systems provide defined culture systems to study the biology and pathology of CF. These airway epithelial cell lines may also be a useful negative protein control for numerous studies involving gene therapy by cDNA complementation or gene targeting., (Copyright (c) 2005 S. Karger AG, Basel.)