Research Letters

Pulmonary disease severity in men with ΔF508 cystic fibrosis and residual chloride secretion

CFTR function measurements in intestinal organoids may help to better characterise individual disease expression in F508del homozygous people. Our objective was to study correlations between CFTR function as measured with forskolin-induced swelling in rectal organoids with clinical parameters in adult patients with homozygous F508del mutations.

Multicentre observational study. Thirty-four adults underwent rectal biopsy, pulmonary function tests (FEV₁ and FVC), chest X-ray and chest CT. Body-mass index (BMI) was assessed at study visit and exacerbation rate was determined during five years prior to study visit. Organoids were cultured and measured after stimulation with 5 µm forskolin for three hours to quantitate CFTR residual function.

FIS was positively correlated with FEV₁ (r = 0.36, 95% CI 0.02–0.62, p = 0.04) and BMI (r = 0.42, 95% CI 0.09–0.66, p = 0.015). FIS was negatively correlated with PRAGMA-CF CT score for% of disease (r = −0.37, 95% CI -0.62- -0.03, p = 0.049). We found no significant correlation between FIS and chest radiography score for CF (r = −0.16, 95% CI -0.48–0.20, p = 0.44). We observed a trend between higher FIS and a lower mean number of exacerbations over the last 5 years of observation, but this was not statistically significant (Poisson regression, p = 0.089).

FIS of intestinal organoids varied between subjects with homozygous F508del and correlated with pulmonary and nutritional parameters. These findings suggest that differences at low CFTR residual function may contribute to clinical heterogeneity in F508del homozygous patients and small changes in CFTR residual function might impact long-term disease expression.

Variation in cystic fibrosis (CF) phenotypes, including lung disease severity, age of onset of persistent Pseudomonas aeruginosa (P. aeruginosa) lung infection, and presence of meconium ileus (MI), has been partially explained by genome-wide association studies (GWASs). It is not expected that GWASs alone are sufficiently powered to uncover all heritable traits associated with CF phenotypic diversity. Therefore, we utilized gene expression association from lymphoblastoid cells lines from 754 p.Phe508del CF-affected homozygous individuals to identify genes and pathways. LPAR6, a G protein coupled receptor, associated with lung disease severity (false discovery rate q value = 0.0006). Additional pathway analyses, utilizing a stringent permutation-based approach, identified unique signals for all three phenotypes. Pathways associated with lung disease severity were annotated in three broad categories: (1) endomembrane function, containing p.Phe508del processing genes, providing evidence of the importance of p.Phe508del processing to explain lung phenotype variation; (2) HLA class I genes, extending previous GWAS findings in the HLA region; and (3) endoplasmic reticulum stress response genes. Expression pathways associated with lung disease were concordant for some endosome and HLA pathways, with pathways identified using GWAS associations from 1,978 CF-affected individuals. Pathways associated with age of onset of persistent P. aeruginosa infection were enriched for HLA class II genes, and those associated with MI were related to oxidative phosphorylation. Formal testing demonstrated that genes showing differential expression associated with lung disease severity were enriched for heritable genetic variation and expression quantitative traits. Gene expression provided a powerful tool to identify unrecognized heritable variation, complementing ongoing GWASs in this rare disease.

The most common mutation in cystic fibrosis (CF) is a deletion of Phe at position 508 (ΔF508-CFTR). ΔF508-CFTR is a trafficking mutant that is retained in the ER, unable to reach the plasma membrane. To identify compounds and drugs that rescue this trafficking defect, we screened a kinase inhibitor library enriched for small molecules already in the clinic or in clinical trials for the treatment of cancer and inflammation, using our recently developed high-content screen technology (Trzcinska-Daneluti et al. Mol. Cell. Proteomics 8:780, 2009). The top hits of the screen were further validated by (1) biochemical analysis to demonstrate the presence of mature (Band C) ΔF508-CFTR, (2) flow cytometry to reveal the presence of ΔF508-CFTR at the cell surface, (3) short-circuit current (Isc) analysis in Ussing chambers to show restoration of function of the rescued ΔF508-CFTR in epithelial MDCK cells stably expressing this mutant (including EC₅₀ determinations), and importantly (4) Isc analysis of Human Bronchial Epithelial (HBE) cells harvested from homozygote ΔF508-CFTR transplant patients. Interestingly, several inhibitors of receptor Tyr kinases (RTKs), such as SU5402 and SU6668 (which target FGFRs, VEGFR, and PDGFR) exhibited strong rescue of ΔF508-CFTR, as did several inhibitors of the Ras/Raf/MEK/ERK or p38 pathways (e.g. (5Z)-7-oxozeaenol). Prominent rescue was also observed by inhibitors of GSK-3β (e.g. GSK-3β Inhibitor II and Kenpaullone). These results identify several kinase inhibitors that can rescue ΔF508-CFTR to various degrees, and suggest that use of compounds or drugs already in the clinic or in clinical trials for other diseases can expedite delivery of treatment for CF patients.

In the majority of cases, there is no difficulty in diagnosing Cystic Fibrosis (CF). However, there may be wide variation in signs and symptoms between individuals which encourage the scientific community to constantly improve the diagnostic tests available and develop better methods to come to a final diagnosis in patients with milder phenotypes. This paper is the result of discussions held at meetings of the European Cystic Fibrosis Society Diagnostic Network supported by EuroCareCF. CFTR bioassays in the nasal epithelium (nasal potential difference measurements) and the rectal mucosa (intestinal current measurements) are discussed in detail including efforts to standardize the techniques across Europe. New approaches to evaluate the sweat gland, future of genetic testing and methods on the horizon like CFTR expression in human leucocytes and erythrocytes are discussed briefly.

Cystic Fibrosis is caused by mutations in CFTR, with a deletion of a phenylalanine at position 508 (F508del-CFTR) representing the most common mutation. The F508del-CFTR protein exhibits a trafficking defect and is retained in the endoplasmic reticulum. Here we describe the development of a high-content screen based on a functional assay to identify proteins that correct the F508del-CFTR defect. Using a HEK293 MSR GripTite cell line that stably expresses F508del-CFTR, we individually co-expressed ∼450 unique proteins fused to the Cl⁻-sensitive YFP(H148Q/I152L) mutant. We then tested correction of F508del-CFTR function by the CI⁻/l⁻ exchange method following stimulation with forskolin/IBMX/genistein, using quantitative recordings in multiple individual cells with a high-content (high-throughput) Cellomics KSR imaging system. Using this approach, we identified several known and novel proteins that corrected F508del-CFTR function, including STAT1, Endothelin 1, HspA4, SAPK substrate protein 1, AP2M1, LGALS3/galectin-3, Trk-fused gene, Caveolin 2, PAP/REG3α, and others. The ability of these correctors to rescue F508del-CFTR trafficking was then validated by demonstrating their enhancement of maturation (appearance of band C) and by cell surface expression of F508del-CFTR bearing HA tag at the ectodomain using confocal microscopy and flow cytometry. These data demonstrate the utility of high-content analyses for identifying proteins that correct mutant CFTR and discover new proteins that stimulate this correction. This assay can also be utilized for RNAi screens to identify inhibitory proteins that block correction of F508del-CFTR, small molecule, and peptide screens.

Abnormal airway ion transport is a feature of cystic fibrosis. The aim of this study was to investigate whether distinct components of ion transport are associated with the clinical expression and severity of the disease.

Univariate and multivariate analyses were used to study interaction effects between nasal potential difference parameters and clinical status, recorded at stable conditions, in 75 F508del homozygous young adults.

All patients demonstrated increased sodium and reduced chloride conductances. Less sodium transport abnormalities were related to better respiratory function and nutrition. Presentation with digestive symptoms at diagnosis was associated with lower chloride conductance. With an accuracy of 85% good nutritional status was linked to more preserved lung function, increasing age and more preserved chloride conductance.

Ion transport abnormalities have distinct clinical outcomes. Sodium conductance relates to respiratory function and nutrition; chloride conductance to nutrition and presentation with digestive symptoms at diagnosis.