BackgroundCystic 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 FindingsTo 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/SignificanceDetermination 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.

Altogether, these results indicate that R560S is a class II mutation. However, unlike F508del, it cannot be rescued by any of the CFTR modulators tested.

A major challenge in cystic fibrosis (CF) research is applying mutation‐specific therapy to individual patients with diverse and rare CF transmembrane conductance regulator (CFTR) genotypes. Read‐through agents are currently the most promising approach for Class I mutations that introduce premature termination codons (PTCs) into CFTR mRNA. However, variations in degradation of PTC containing transcripts by nonsense mediated decay (NMD) might lower read‐through efficacy. Allele specific quantitative real time (qRT)‐PCR was used to measure variations in CFTR mRNA abundance for several PTC mutations in respiratory cells and intestinal organoids. The majority of PTC mutations were associated with reduced levels of relative mRNA transcript abundance (∼33% and 26% of total CFTR mRNA in respiratory cells and intestinal organoids, respectively, compared to >50% for non‐PTC causing mutations). These levels were generally not affected by PTC mutation type or position, but there could be twofold variations between individuals bearing the same genotype. Most PTC mutations in CFTR are subject to similar levels of NMD, which reduce but do not abolish PTC bearing mRNAs. Measurement of individual NMD levels in intestinal organoids and HNE cells might, therefore, be useful in predicting efficacy of PTC read‐through in the context of personalized CFTR modulator therapy.

Newborn screening (NBS) for cystic fibrosis (CF) has been shown to be advantageous for children with CF, and has thus been included in most NBS programs using various algorithms. With this study, we intend to establish the most appropriate algorithm for CF-NBS in the Portuguese population, to determine the incidence, and to contribute to elucidating the genetic epidemiology of CF in Portugal. This was a nationwide three-year pilot study including 255,000 newborns (NB) that were also screened for congenital hypothyroidism (CH) and 24 other metabolic disorders included in the Portuguese screening program. Most samples were collected in local health centers spread all over the country, between the 3rd and 6th days of life. The algorithm tested includes immunoreactive trypsinogen (IRT) determination, pancreatitis associated protein (PAP) as a second tier, and genetic study for cases referred to specialized clinical centers. Thirty-four CF cases were confirmed positive, thus indicating an incidence of 1:7500 NB. The p.F508del mutation was found in 79% of the alleles. According to the results presented here, CF-NBS is recommended to be included in the Portuguese NBS panel with a small adjustment regarding the PAP cutoff , which we expect to contribute to the improvement of the CF-NBS performance. According to our results, this algorithm is a valuable alternative for CF-NBS in populations with stringent rules for genetic studies.

The Cystic Fibrosis p.Ile1234Val missense mutation actually creates a new dual splicing site possibly used either as a new acceptor or donor. Here, we aimed to test the accuracy of in silico predictions by comparing them with in vitro and ex vivo functional analyses of this mutation for an accurate CF diagnosis/prognosis. To this end, we applied a new in vitro strategy using a CFTR mini-gene which includes the complete CFTR coding sequence plus intron 22 (short version) which allows the assessment of alternatively spliced mRNA levels as well as the properties of the resulting abnormal CFTR protein regarding processing, intracellular localization and function. Our data demonstrate that p.Ile1234Val leads to usage of the alternative splicing donor (but not acceptor) resulting in alternative CFTR transcripts lacking 18 nts of exon 22 which produce a truncated CFTR protein with residual Cl- channel function. These results recapitulate data from native tissues of a CF patient. In conclusion, the existing in silico prediction models have limited application and ex vivo functional assessment of mutation effects should be made. Alternatively the in vitro strategy adopted here can be applied to assess the disease liability of mutations for an accurate CF diagnosis/prognosis.

Background: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the dysfunctional Cl^- channel in Cystic Fibrosis, undergoes complex biosynthesis at the endoplasmic reticulum involving several molecular chaperones including Hsp70 and many co-chaperones. Bcl-2- associated athanogenes (BAGs) constitute a protein family sharing an Hsc70-binding domain. BAG-1 possesses an ubiquitin-like domain (Ub-LD) responsible for proteasomal association and for promoting substrate release from Hsc70/Hsp70 in vitro by accelerating the chaperone ATP/ADP exchange rate. Methods: Herein, we studied the in vivo effect of BAG-1 on the turnover and processing of wild type (wt)- and F508del-CFTR, the most frequent mutation in CF patients. Results: Results show that BAG-1 associates with both wt- and F508del-CFTR (in higher yields with the latter) through its Ub-LD and independently of Hsc70. Moreover, the immature form of F508del-CFTR (but not of wt-CFTR) is stabilized by BAG-1 overexpression, albeit in a cell-type specific way, without detectable maturation. Data also show that BAG-1 and the proteasome inhibitor ALLN are not additive on stabilizing F508del-CFTR and this effect depends on BAG-1 Ub-LD. Moreover, under BAG-1 overexpression, a reduction in ubiquitinylated-CFTR occurs suggesting that BAG-1 competes with Ub. Conclusion: Overall, data are compatible with a mechanism in which BAG-1 stabilizes F508del-CFTR by direct binding, probably competing out ubiquitin to partially avoid its proteasomal degradation.

Even with advent of next generation sequencing complete sequencing of large disease-associated genes and intronic regions is economically not feasible. This is the case of cystic fibrosis transmembrane conductance regulator (CFTR), the gene responsible for cystic fibrosis (CF). Yet, to confirm a CF diagnosis, proof of CFTR dysfunction needs to be obtained, namely by the identification of two disease-causing mutations. Moreover, with the advent of mutation-based therapies, genotyping is an essential tool for CF disease management. There is, however, still an unmet need to genotype CF patients by fast, comprehensive and cost-effective approaches, especially in populations with high genetic heterogeneity (and low p.F508del incidence), where CF is now emerging with new diagnosis dilemmas (Brazil, Asia, etc). Herein, we report an innovative mRNA-based approach to identify CFTR mutations in the complete coding and intronic regions. We applied this protocol to genotype individuals with a suspicion of CF and only one or no CFTR mutations identified by routine methods. It successfully detected multiple intronic mutations unlikely to be detected by CFTR exon sequencing. We conclude that this is a rapid, robust and inexpensive method to detect any CFTR coding/intronic mutation (including rare ones) that can be easily used either as primary approach or after routine DNA analysis.

Cystic Fibrosis (CF) is caused by mutations in the CF Transmembrane conductance Regulator (CFTR), the only ATP-binding cassette (ABC) transporter functioning as a channel. Unique to CFTR is a regulatory domain which includes a highly conformationally dynamic region—the regulatory extension (RE). The first nucleotide-binding domain of CFTR contains another dynamic region—regulatory insertion (RI). Removal of RI rescues the trafficking defect of CFTR with F508del, the most common CF-causing mutation. Here we aimed to assess the impact of RE removal (with/without RI or genetic revertants) on F508del-CFTR trafficking and how CFTR modulator drugs VX-809/lumacaftor and VX-770/ivacaftor rescue these variants. We generated cell lines expressing ΔRE and ΔRI CFTR (with/without genetic revertants) and assessed CFTR expression, stability, plasma membrane levels, and channel activity. Our data demonstrated that ΔRI significantly enhanced rescue of F508del-CFTR by VX-809. While the presence of the RI seems to be precluding full rescue of F508del-CFTR processing by VX-809, this region appears essential to rescue its function by VX-770, suggesting some contradictory role in rescue of F508del-CFTR by these two modulators. This negative impact of RI removal on VX-770-stimulated currents on F508del-CFTR can be compensated by deletion of the RE which also leads to the stabilization of this mutant. Despite both regions being conformationally dynamic, RI precludes F508del-CFTR processing while RE affects mostly its stability and channel opening.