BACKGROUND:-Nasal Potential Difference (NPD) is an established diagnostic tool for CF. However, standardized values for very young patients are lacking. AIM:- To evaluate the feasibility of performing NPD testing in young children. METHODS:- We modified the standard NPD protocol for young children by shortening the infusion time per solution to 2 min. No sedation was required. RESULTS:- Fifty-five children aged 6 days to 3 years were enrolled from 2010 to 2024, with 50 completing the modified NPD protocol, 2 failing to tolerate the procedure, and 3 having inconclusive results. Fourty-eight results were normal, and 2 abnormal. In the normal group, the basal potential difference (PD) averaged -17 ± 6 mV, amiloride response was 7 ± 3 mV, and chloride-free + isoproterenol response was -9 ± 5 mV. The two abnormal results were observed in a 12-month-old with failure to thrive and a 6-day-old with meconium plug, presenting with basal PD of -30 mV and -28 mV respectively, with no cAMP response. CF was diagnosed in the 12-month-old based on clinical symptoms and sweat test of 70 meq/L. The 6-day-old, is being followed for mild asthma with normal repeat sweat testing. None of the children with normal NPD results were diagnosed with CF. CONCLUSION:-NPD testing is feasable in very young children. This protocol could be valuable for investigating cases of CF screened positive indeterminate diagnosis (CFSPID) or CF metabolic syndrome (CFMS), and for pre- and post-testing in clinical trials. Further validation studies are warranted.

This is the third paper in the series providing updated information and recommendations for people with cystic fibrosis transmembrane conductance regulator (CFTR)-related disorder (CFTR-RD). This paper covers the individual disorders, including the established conditions - congenital absence of the vas deferens (CAVD), diffuse bronchiectasis and chronic or acute recurrent pancreatitis - and also other conditions which might be considered a CFTR-RD, including allergic bronchopulmonary aspergillosis, chronic rhinosinusitis, primary sclerosing cholangitis and aquagenic wrinkling. The CFTR functional and genetic evidence in support of the condition being a CFTR-RD are discussed and guidance for reaching the diagnosis, including alternative conditions to consider and management recommendations, is provided. Gaps in our knowledge, particularly of the emerging conditions, and future areas of research, including the role of CFTR modulators, are highlighted.

ETI offers therapeutic opportunities for most CF patients but not for many with rare mutations. We established patient-derived organoids with the Q1100P mutation, not reported in CFTR2. We obtained biopsies from 3 patients heterozygous for Q1100P and a stop mutation (C225X, likely nonresponsive to ETI) or a missense (K163E). We investigated CFTR function after treatment with modulators. In Q1100P/C225X organoids, TEZ had no effect. CFTR function was partially restored by ELX and further enhanced in combination with TEZ, without benefit from IVA. The introduction of a proline (P) instead of the WT glutamine (Q) at residue 1100 is expected to disrupt the TM11 α-helix structure. A structure of CFTR in complex with ELX shows ELX binding to 2 residues flanking Q1100P. We suggest that ELX binding corrects the structural defect, leading to functional correction of CFTR. The additional benefit from TEZ is consistent with studies showing synergy between TEZ/ELX in improving CFTR-F508del folding and function. In Q1100P/K163E organoids, TEZ partially restored CFTR function, and ELX alone significantly restored function. The TEZ/ELX combination did not show an additive effect, neither did addition of IVA. In these organoids, function restoration might also arise from K163E, a missense mutation in a conserved lysine residue at the ICL1-NBD1 interphase, crucial for CFTR folding and corrected by TEZ. Further studies with organoids from a patient homozygous for K163E are ongoing and will shed light on the molecular basis of modulator response. Based on our results, treatment with ETI was approved by the health provider for 2 Q1100P/K163E patients. Improvement was immediate: FEV1 raised by 15% and 21%, accompanied by a decrease in LCI (5.6 & 5 points), and in sweat chloride (9 & 30 mmol/L). Together, we show how knowledge of the CFTR structure and modulator binding sites can guide in prioritizing the analysis of rare mutations to existing treatments in patient-derived organoids.

Background: Elexacaftor-tezacaftor-ivacaftor (ETI) offers therapeutic opportunities for most people with CF but not for many with rare mutations.Many Israeli's with CF carry rare mutations to which the response to ETI is not known.Knowledge of the CFTR structure and modulator binding sites can help predict the response and prioritize the order of the in vitro analysis in organoids from people with rare mutations.Furthermore, the response of rare mutations to currently available modulators can assist in defining specific regions important for CFTR maturation and function.We studied the effect of different modulator combinations on two ultrarare missense mutations, Q1100P and K163E, that are not reported in the CFTR2 database (https://cftr2.org/).The introduction of a proline (P) instead of the wt glutamine (Q) at residue 1100 is expected to disrupt the TM11 α-helix structure.A structure of CFTR in complex with elexacaftor shows elexacaftor binding to two residues flanking Q1100P; thus we suggest that elexacaftor binding has the potential to correct the structural defect, leading to functional correction of Q1100P.K163 participates in the intracellular loop 1-transmembrane domain 1 interface, which is crucial for CFTR folding.Mutations in this region are corrected by VX809.Thus, we hypothesized that the mutation could be corrected by VX661.Methods: We established patient-derived organoids harboring Q1100P and K163E.We obtained rectal biopsies and established organoid cultures from four patients: two heterozygous for Q1100P and K163E, one heterozygous for Q1100P and C225X, and one homozygous for the K163E mutation.We investigated CFTR function after treatment with different combinations of the modulators elexacaftor, tezacaftor, and ivacaftor using the forskolin-induced swelling assay.Results: In Q1100P/C225X organoids, tezacaftor had no effect.CFTR function was partially restored by elexacaftor and further enhanced in combination with tezacaftor, without benefit from ivacaftor.The additional benefit from tezacaftor is consistent with studies showing synergy between tezacaftor and elexacaftor in improving CFTR-F508del folding and function.In Q1100P/K163E organoids, tezacaftor partially restored CFTR function, and elexacaftor alone significantly restored function.The tezacaftor-elexacaftor combination did not show an additive effect, nor did addition of ivacaftor.In these organoids, function restoration might also arise from K163E.Further studies with organoids from a patient homozygous for K163E showed a similar response pattern to that of Q1100P/K163E organoids: partial restoration with tezacaftor alone and a maximal significant restoration with elexacaftor alone without additional benefit from tezacaftor or ivacaftor.Based on our results, treatment with ETI was approved by the health provider for the two Q1100P/K163E patients and the K163E/K163E patient.Improvement was immediate: FEV 1 increased by 15% to 24%, accompanied by a decrease in lung clearance index and sweat chloride.Conclusions: In vitro analysis of the response of rare mutations to currently available modulators is important for identifying people with potential to respond and benefit from modulator treatment.Our results suggest that knowledge of the CFTR structure and modulator binding sites can help with prioritizing patients likely to respond to treatment in the analysis of rare mutations in patient-derived organoids and with understanding regions important for CFTR domain interaction and function.There might be a ceiling effect in CFTR activity restoration, because the effect of the modulators on two alleles was similar to that of one allele.Not all CFTR mutations require treatment with all ETI components, and analysis of modulator combinations can minimize unnecessary drug exposure of patients.

The spectrum of disorders involving CFTR (cystic fibrosis transmembrane conductance regulator) dysfunction correlates with a continuous gradient of CFTR function defined by the combination of two allelic CFTR variants. CFTR-related disorders are clinical entities with features of cystic fibrosis (CF) and evidence for presence of CFTR dysfunction but not meeting criteria for diagnosis of CF. Individuals with CFTR-RDs demonstrate a wide range of CFTR activity and are still under-recognized or misclassified. The level of CFTR dysfunction may be measured in vivo (sweat testing, nasal potential difference measurements) and/or by ex vivo tests (intestinal current measurement), or indirectly indicated by CFTR variants, as alteration in sequence of the CFTR gene translates into CFTR dysfunction. CFTR bioassays can aid in the diagnosis of individuals with CF, but we lack parameters to differentiate CF from CFTR-RD. In the era of the CFTR modulators and their potential clinical benefit, it is of utmost importance to diagnose CFTR-RD as unambiguously as possible. We therefore propose the following to define compatible CFTR dysfunction in a person with a suspected diagnosis of CFTR-RD : (1) evidence of CFTR dysfunction in vivo or ex vivo in at least two different CFTR functional test types, or (2) One CFTR variant known to reduce CFTR function and evidence of CFTR dysfunction in vivo or ex vivo in at least two different CFTR functional test types, or (3) Two CFTR variants shown to reduce CFTR function, with at most one CF-causing variant.

More than five decades after the introduction of the quantitative pilocarpine iontophoresis technique, surveys still highlight inconsistencies in the performance and reporting of sweat tests in Europe. The sweat test remains key for the Cystic Fibrosis (CF) diagnostic pathway for all age groups, as it reflects the basic pathophysiological defect in the sweat gland. It is also critical following newborn screening as a confirmatory diagnostic step. Despite its importance, sweat test quality is variable whether performed in the laboratory or as a point of care test. The ECFS DNWG aims to improve sweat test performance, taking into account the barriers and issues identified in the European survey; the previous step in the ECFS sweat test project. This manuscript proposes a grading of sweat test guidance from "acceptable" to "optimal", aiming to pragmatically improve quality while taking into account local situations, especially in resource-limited settings. (c) 2022 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Background: Ataluren was developed for potential treatment of nonsense-mutation cystic fibrosis (CF). A previous phase 3 ataluren study failed to meet its primary efficacy endpoint, but post-hoc analyses suggested that aminoglycosides may have interfered with ataluren's action. Thus, this subsequent trial (NCT02139306) was designed to assess the efficacy and safety of ataluren in patients with nonsense-mutation CF not receiving aminoglycosides. Methods: Eligible subjects with nonsense-mutation CF (aged >= 6 years; percent predicted (pp) FEV1 >= 40 and <= 90) from 75 sites in 16 countries were randomly assigned in double-blinded fashion to receive oral ataluren or matching placebo thrice daily for 48 weeks. The primary endpoint was absolute change in average ppFEV(1) from baseline to the average of Weeks 40 and 48. Findings: 279 subjects were enrolled; 138 subjects in the ataluren arm and 136 in the placebo arm were evaluable for efficacy. Absolute ppFEV(1) change from baseline did not differ significantly between the ataluren and placebo groups at Week 40 (-0.8 vs -1.8) or Week 48 (-1.7 vs -2.4). Average ppFEV(1) treatment difference from baseline to Weeks 40 and 48 was 0.6 (95% CI -1.3, 2.5; p = 0.54). Pulmonary exacerbation rate per 48 weeks was not significantly different (ataluren 0.95 vs placebo 1.13; rate ratio p = 0.40). Safety was similar between groups. No life-threatening adverse events or deaths were reported. Interpretation: Neither ppFEV(1) change nor pulmonary exacerbation rate over 48 weeks were statistically different between ataluren and placebo groups. Development of a nonsense-mutation CF therapy remains elusive. (C) 2020 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

BackgroundThe Q359K/T360K mutation, described in Jewish CF patients of Georgian decent, is of questionable clinical significance.MethodsClinical records of patients with the Q359K/T360K mutation from three CF centers were studied for phenotypic expression and putative mechanism of dysfunction. Computer models of mutant CFTR were constructed.ResultsNine patients (4 homozygous) of Georgian Jewish origin were included. Age at diagnosis was 9.4 (0.25–38.2) years, median (range). Sweat chloride was 106 ± 13 meq/L, mean ± SD. Nasal Potential Difference performed in three, was abnormal. All had pulmonary symptoms since early childhood and bronchiectasis. Median FEV1 was 88 (40–121)%. Five had chronic mucoid P. aeruginosa. Homozygous patients were pancreatic insufficient. Enzyme supplementation was initiated at 3.8 (1–14.7) years, median (range). Structural models hint at possible interference of this mutation with transmembrane chloride transport.ConclusionIn our cohort, the Q359K/T360K mutation resulted in a severe CF phenotype, although with residual early CFTR function. The CFTR2 database should consider defining this mutation as CF-causing.

Background The Q359K/T360K mutation, described in Jewish CF patients of Georgian decent, is of questionable clinical significance. Methods Clinical records of patients with the Q359K/T360K mutation from three CF centers were studied for phenotypic expression and putative mechanism of dysfunction. Computer models of mutant CFTR were constructed. Results Nine patients (4 homozygous) of Georgian Jewish origin were included. Age at diagnosis was 9.4 (0.25–38.2) years, median (range). Sweat chloride was 106 ± 13 meq/L, mean ± SD. Nasal Potential Difference performed in three, was abnormal. All had pulmonary symptoms since early childhood and bronchiectasis. Median FEV1 was 88 (40–121)%. Five had chronic mucoid P. aeruginosa. Homozygous patients were pancreatic insufficient. Enzyme supplementation was initiated at 3.8 (1–14.7) years, median (range). Structural models hint at possible interference of this mutation with transmembrane chloride transport. Conclusion In our cohort, the Q359K/T360K mutation resulted in a severe CF phenotype, although with residual early CFTR function. The CFTR2 database should consider defining this mutation as CF-causing.

Evidence based guidelines exist for sweat testing, which remains a key component of a diagnosis of cystic fibrosis (CF), especially following newborn bloodspot screening (NBS). There are emerging challenges with respect to maintaining a valid sweat test service, notably a smaller number of sweat tests ordered in regions with established NBS programmes where Pediatricians refer less children for sweat testing, younger patients and equipment becoming obsolete. The ECFS Diagnostic Network Working Group has undertaken a comprehensive survey to better define sweat test practice across Europe. The survey was completed by 136 European respondents representing a CF center or laboratory providing a sweat test service (65% from regions with NBS for CF). There was considerable variance in practice, often not consistent with guidelines. In particular collection of sweat from two sites was rarely reported in European centres in contrast to US guidelines. There was a range of different references quoted for cut-off for both a positive and intermediate test. Most responses suggest cost is becoming an increasing issue and is not sufficiently reimbursed. This work will inform best practice guidelines and resources to sustain and improve sweat testing in Europe.