ABSTRACT
RATIONALE: Cystic fibrosis (CF) is a monogenic life-shortening disease associated with highly variable individual disease progression which is difficult to predict. Here we assessed the association of forskolin-induced swelling (FIS) of patient-derived organoids with long-term CF disease progression in multiple organs and compared FIS with the golden standard biomarker sweat chloride concentration (SCC). METHODS: We retrieved 9-year longitudinal clinical data from the Dutch CF Registry of 173 people with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Individual CFTR function was defined by FIS, measured as the relative size increase of intestinal organoids after stimulation with 0.8â µM forskolin, quantified as area under the curve (AUC). We used linear mixed-effect models and multivariable logistic regression to estimate the association of FIS with long-term forced expiratory volume in 1â s % predicted (FEV1pp) decline and development of pancreatic insufficiency, CF-related liver disease and diabetes. Within these models, FIS was compared with SCC. RESULTS: FIS was strongly associated with longitudinal changes of lung function, with an estimated difference in annual FEV1pp decline of 0.32% (95% CI 0.11-0.54%; p=0.004) per 1000-point change in AUC. Moreover, increasing FIS levels were associated with lower odds of developing pancreatic insufficiency (adjusted OR 0.18, 95% CI 0.07-0.46; p<0.001), CF-related liver disease (adjusted OR 0.18, 95% CI 0.06-0.54; p=0.002) and diabetes (adjusted OR 0.34, 95% CI 0.12-0.97; p=0.044). These associations were absent for SCC. CONCLUSION: This study exemplifies the prognostic value of a patient-derived organoid-based biomarker within a clinical setting, which is especially important for people carrying rare CFTR mutations with unclear clinical consequences.
Subject(s)
Cystic Fibrosis , Exocrine Pancreatic Insufficiency , Biomarkers , Colforsin/pharmacology , Cystic Fibrosis/complications , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Disease Progression , Exocrine Pancreatic Insufficiency/complications , Humans , Mutation , OrganoidsABSTRACT
During pregnancy, cell-free DNA (cfDNA) in maternal blood encompasses a small percentage of cell-free fetal DNA (cffDNA), an easily accessible source for determination of fetal disease status in risk families through non-invasive procedures. In case of monogenic heritable disease, background maternal cfDNA prohibits direct observation of the maternally inherited allele. Non-invasive prenatal diagnostics (NIPD) of monogenic diseases therefore relies on parental haplotyping and statistical assessment of inherited alleles from cffDNA, techniques currently unavailable for routine clinical practice. Here, we present monogenic NIPD (MG-NIPD), which requires a blood sample from both parents, for targeted locus amplification (TLA)-based phasing of heterozygous variants selectively at a gene of interest. Capture probes-based targeted sequencing of cfDNA from the pregnant mother and a tailored statistical analysis enables predicting fetal gene inheritance. MG-NIPD was validated for 18 pregnancies, focusing on CFTR, CYP21A2, and HBB. In all cases we could predict the inherited alleles with >98% confidence, even at relatively early stages (8 weeks) of pregnancy. This prediction and the accuracy of parental haplotyping was confirmed by sequencing of fetal material obtained by parallel invasive procedures. MG-NIPD is a robust method that requires standard instrumentation and can be implemented in any clinic to provide families carrying a severe monogenic disease with a prenatal diagnostic test based on a simple blood draw.
Subject(s)
Adrenal Hyperplasia, Congenital/diagnosis , Biomarkers/blood , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/diagnosis , Polymorphism, Single Nucleotide , Prenatal Diagnosis/methods , Steroid 21-Hydroxylase/genetics , Adrenal Hyperplasia, Congenital/blood , Adrenal Hyperplasia, Congenital/genetics , Cells, Cultured , Cystic Fibrosis/blood , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/blood , DNA/blood , DNA/genetics , Female , Haplotypes , Humans , Pregnancy , Steroid 21-Hydroxylase/bloodABSTRACT
Forskolin-induced swelling (FIS) of intestinal organoids from individuals with cystic fibrosis (CF) measures function of the cystic fibrosis transmembrane conductance regulator (CFTR), the protein mutated in CF.We investigated whether FIS corresponds with clinical outcome parameters and biomarkers of CFTR function in 34 infants diagnosed with CF. Relationships with FIS were studied for indicators of pulmonary and gastrointestinal disease.Children with low FIS had higher levels of immunoreactive trypsinogen (p=0.030) and pancreatitis-associated protein (p=0.039), more often had pancreatic insufficiency (p<0.001), had more abnormalities on chest computed tomography (p=0.049), and had lower z-scores for maximal expiratory flow at functional residual capacity (p=0.033) when compared to children with high FIS values. FIS significantly correlated with sweat chloride concentration (SCC) and intestinal current measurement (ICM) (r=â-0.82 and r=0.70, respectively; both p<0.001). Individual assessment of SCC, ICM and FIS suggested that FIS can help to classify individual disease severity.Thus, stratification by FIS identified subgroups that differed in pulmonary and gastrointestinal outcome parameters. FIS of intestinal organoids correlated well with established CFTR-dependent biomarkers such as SCC and ICM, and performed adequately at group and individual level in this proof-of-concept study.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/diagnosis , Exocrine Pancreatic Insufficiency/diagnosis , Organoids/pathology , Biomarkers/metabolism , Chlorides/metabolism , Cystic Fibrosis/complications , Female , Humans , Infant , Ion Transport , Linear Models , Male , Proof of Concept Study , Severity of Illness IndexABSTRACT
Small-molecule therapies that restore defects in cystic fibrosis transmembrane conductance regulator (CFTR) gating (potentiators) or trafficking (correctors) are being developed for cystic fibrosis (CF) in a mutation-specific fashion. Options for pharmacological correction of CFTR-p.Phe508del (F508del) are being extensively studied but correction of other trafficking mutants that may also benefit from corrector treatment remains largely unknown.We studied correction of the folding mutants CFTR-p.Phe508del, -p.Ala455Glu (A455E) and -p.Asn1303Lys (N1303K) by VX-809 and 18 other correctors (C1-C18) using a functional CFTR assay in human intestinal CF organoids.Function of both CFTR-p.Phe508del and -p.Ala455Glu was enhanced by a variety of correctors but no residual or corrector-induced activity was associated with CFTR-p.Asn1303Lys. Importantly, VX-809-induced correction was most dominant for CFTR-p.Phe508del, while correction of CFTR-p.Ala455Glu was highest by a subgroup of compounds called bithiazoles (C4, C13, C14 and C17) and C5.These data support the development of mutation-specific correctors for optimal treatment of different CFTR trafficking mutants, and identify C5 and bithiazoles as the most promising compounds for correction of CFTR-p.Ala455Glu.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/drug therapy , Cystic Fibrosis/genetics , Mutation , Aminopyridines/pharmacology , Benzodioxoles/pharmacology , Biopsy , Genotype , Homozygote , Humans , Organoids , Protein Folding , Protein Transport , Rectum/pathology , Thiazoles/chemistry , Treatment OutcomeABSTRACT
We hypothesized that people with cystic fibrosis (CF) who express CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations associated with residual function may benefit from G-protein coupled receptor (GPCR)-targeting drugs that can activate and enhance CFTR function.We used intestinal organoids to screen a GPCR-modulating compound library and identified ß2-adrenergic receptor agonists as the most potent inducers of CFTR function.ß2-Agonist-induced organoid swelling correlated with the CFTR genotype, and could be induced in homozygous CFTR-F508del organoids and highly differentiated primary CF airway epithelial cells after rescue of CFTR trafficking by small molecules. The in vivo response to treatment with an oral or inhaled ß2-agonist (salbutamol) in CF patients with residual CFTR function was evaluated in a pilot study. 10 subjects with a R117H or A455E mutation were included and showed changes in the nasal potential difference measurement after treatment with oral salbutamol, including a significant improvement of the baseline potential difference of the nasal mucosa (+6.35â mV, p<0.05), suggesting that this treatment might be effective in vivo Furthermore, plasma that was collected after oral salbutamol treatment induced CFTR activation when administered ex vivo to organoids.This proof-of-concept study suggests that organoids can be used to identify drugs that activate CFTR function in vivo and to select route of administration.
Subject(s)
Adrenergic beta-2 Receptor Agonists/pharmacology , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/drug therapy , Cystic Fibrosis/metabolism , Administration, Oral , Albuterol/administration & dosage , Biological Assay , Bronchi/pathology , Cell Line , Cells, Cultured , Chlorides/chemistry , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Drug Evaluation, Preclinical , Epithelial Cells/metabolism , Epithelium/metabolism , Humans , Mutation , Nasal Mucosa/drug effects , Nasal Mucosa/metabolism , Organoids , Pilot Projects , Respiratory System/metabolism , Signal TransductionSubject(s)
Aminophenols/therapeutic use , Aminopyridines/therapeutic use , Benzodioxoles/therapeutic use , Cystic Fibrosis/drug therapy , Organoids/drug effects , Quinolones/therapeutic use , Rectum/drug effects , Adolescent , Adult , Aminophenols/pharmacology , Aminopyridines/pharmacology , Benzodioxoles/pharmacology , Child , Chlorides/analysis , Colforsin , Cystic Fibrosis/genetics , Cystic Fibrosis/physiopathology , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Drug Combinations , Edema , Female , Forced Expiratory Volume , Homozygote , Humans , In Vitro Techniques , Male , Membrane Potentials/physiology , Middle Aged , Nasal Mucosa/physiopathology , Precision Medicine , Prospective Studies , Quinolones/pharmacology , Sweat/chemistry , Treatment Outcome , Vital Capacity , Young AdultABSTRACT
Background: Cystic fibrosis (CF) is a rare hereditary disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Recent therapies enable effective restoration of CFTR function of the most common F508del CFTR mutation. This shifts the unmet clinical need towards people with rare CFTR mutations such as nonsense mutations, of which G542X and W1282X are most prevalent. CFTR function measurements in patient-derived cell-based assays played a critical role in preclinical drug development for CF and may play an important role to identify new drugs for people with rare CFTR mutations. Methods: Here, we miniaturised the previously described forskolin-induced swelling (FIS) assay in intestinal organoids from a 96-well to a 384-well plate screening format. Using this novel assay, we tested CFTR increasing potential of a 1400-compound Food and Drug Administration (FDA)-approved drug library in organoids from donors with W1282X/W1282X CFTR nonsense mutations. Results: The 384-well FIS assay demonstrated uniformity and robustness based on coefficient of variation and Z'-factor calculations. In the primary screen, CFTR induction was limited overall, yet interestingly, the top five compound combinations that increased CFTR function all contained at least one statin. In the secondary screen, we indeed verified that four out of the five statins (mevastatin, lovastatin, simvastatin and fluvastatin) increased CFTR function when combined with CFTR modulators. Statin-induced CFTR rescue was concentration-dependent and W1282X-specific. Conclusions: Future studies should focus on elucidating genotype specificity and mode-of-action of statins in more detail. This study exemplifies proof of principle of large-scale compound screening in a functional assay using patient-derived organoids.
ABSTRACT
The nasal and bronchial epithelium are unified parts of the respiratory tract that are affected in the monogenic disorder cystic fibrosis (CF). Recent studies have uncovered that nasal and bronchial tissues exhibit intrinsic variability, including differences in mucociliary cell composition and expression of unique transcriptional regulatory proteins which relate to germ layer origin. In the present study, we explored whether intrinsic differences between nasal and bronchial epithelial cells persist in cell cultures and affect epithelial cell functioning in CF. Comparison of air-liquid interface (ALI) differentiated epithelial cells from subjects with CF revealed distinct mucociliary differentiation states of nasal and bronchial cultures. Moreover, using RNA sequencing we identified cell type-specific signature transcription factors in differentiated nasal and bronchial epithelial cells, some of which were already poised for expression in basal progenitor cells as evidenced by ATAC sequencing. Analysis of differentiated nasal and bronchial epithelial 3D organoids revealed distinct capacities for fluid secretion, which was linked to differences in ciliated cell differentiation. In conclusion, we show that unique phenotypical and functional features of nasal and bronchial epithelial cells persist in cell culture models, which can be further used to investigate the effects of tissue-specific features on upper and lower respiratory disease development in CF.
Subject(s)
Cystic Fibrosis , Humans , Cystic Fibrosis/genetics , Cystic Fibrosis/metabolism , Cells, Cultured , Respiratory Mucosa/metabolism , Nose , Epithelial Cells/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolismABSTRACT
Metazoan genomes contain thousands of sequence tracts that match the guanine-quadruplex (G4) DNA signature G(3)N(x)G(3)N(x)G(3)N(x)G(3), a motif that is intrinsically mutagenic, probably because it can form secondary structures during DNA replication. Here we show how and to what extent this feature can be used to generate deletion alleles of many Caenorhabditis elegans genes.
Subject(s)
Caenorhabditis elegans/genetics , DNA/genetics , G-Quadruplexes , Gene Deletion , Mutagenesis , Alleles , Animals , DNA/chemistryABSTRACT
Cystic fibrosis is caused by genetic defects that impair the CFTR channel in airway epithelial cells. These defects may be overcome by specific CFTR modulating drugs, for which the efficacy can be predicted in a personalized manner using 3D nasal-brushing-derived airway organoids in a forskolin-induced swelling assay. Despite of this, previously described CFTR function assays in 3D airway organoids were not fully optimal, because of inefficient organoid differentiation and limited scalability. In this report, we therefore describe an alternative method of culturing nasal-brushing-derived airway organoids, which are created from an equally differentiated airway epithelial monolayer of a 2D air-liquid interface culture. In addition, we have defined organoid culture conditions, with the growth factor/cytokine combination neuregulin-1<i>ß</i> and interleukin-1<i>ß</i>, which enabled consistent detection of CFTR modulator responses in nasal-airway organoid cultures from subjects with cystic fibrosis.
Subject(s)
Cystic Fibrosis , Cells, Cultured , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Epithelial Cells , Humans , OrganoidsABSTRACT
To safeguard genetic integrity, cells have evolved an accurate but not failsafe mechanism of DNA replication. Not all DNA sequences tolerate DNA replication equally well [1]. Also, genomic regions that impose structural barriers to the DNA replication fork are a potential source of genetic instability [2, 3]. Here, we demonstrate that G4 DNA-a sequence motif that folds into quadruplex structures in vitro [4, 5]-is highly mutagenic in vivo and is removed from genomes that lack dog-1, the C. elegans ortholog of mammalian FANCJ [6, 7], which is mutated in Fanconi anemia patients [8-11]. We show that sequences that match the G4 DNA signature G3-5N1-3G3-5N1-3G3-5N1-3G3-5 are deleted in germ and somatic tissues of dog-1 animals. Unbiased aCGH analyses of dog-1 genomes that were allowed to accumulate mutations in >100 replication cycles indicate that deletions are found exclusively at G4 DNA; deletion frequencies can reach 4% per site per animal generation. We found that deletion sizes fall short of Okazaki fragment lengths [12], and no significant microhomology was observed at deletion junctions. The existence of 376,000 potentially mutagenic G4 DNA sites in the human genome could have major implications for the etiology of hereditary FancJ and nonhereditary cancers.
Subject(s)
Basic-Leucine Zipper Transcription Factors/genetics , Caenorhabditis elegans/genetics , Fanconi Anemia Complementation Group Proteins/genetics , G-Quadruplexes , Genomic Instability , Mutation , Animals , Base Sequence , Caenorhabditis elegans Proteins/genetics , DNA Helicases/genetics , DNA Replication , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Sequence Analysis, DNA , Sequence DeletionABSTRACT
INTRODUCTION: Variability in disease severity and CFTR modulator responses exists between patients with identical CFTR genotypes. Here, we characterized transcription, translation and function of R117H-CFTR using intestinal organoids and correlated them with in vitro responses to ivacaftor (VX-770). METHODS: Organoids were generated from individuals possessing at least one R117H-CFTR allele. The forskolin-induced swelling (FIS) assay was used to measure CFTR function and response to VX-770 treatment. R117H-CFTR protein and mRNA expression levels were determined in parallel and Pearson's correlation coefficients were assessed. RESULTS: Variability in R117H-CFTR FIS responses was observed and correlated significantly with mRNA and protein expression. Response to VX-770 treatment in organoids correlated with mRNA and protein expression as well. CONCLUSIONS: Our results indicate that gene expression, protein expression and CFTR function are strongly correlated in organoids from people with CFTR-R117H-7T/9T, which may suggest that CFTR gene expression may have consequences for CF diagnosis, prognosis and therapeutic benefit.
Subject(s)
Aminophenols/pharmacology , Chloride Channel Agonists/pharmacology , Colon/drug effects , Cystic Fibrosis Transmembrane Conductance Regulator/drug effects , Cystic Fibrosis/metabolism , Organoids/drug effects , Quinolones/pharmacology , Cell Culture Techniques , Colon/metabolism , Cystic Fibrosis/drug therapy , Cystic Fibrosis/pathology , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Humans , Organoids/metabolism , RNA, Messenger/metabolismABSTRACT
This protocol describes the isolation, handling, culture of, and experiments with human colon stem cell organoids in the context of cystic fibrosis (CF). In human colon organoids, the function of cystic fibrosis transmembrane conductance regulator (CFTR) protein and its rescue by CFTR modulators can be quantified using the forskolin-induced swelling assay. Implementation procedures and validation experiments are described for six CF human colon organoid lines, and representative CFTR genotypes are tested for basal CFTR function and response to CFTR-modulating drugs. For complete details on the use and execution of this protocol, please refer to Dekkers et al (2016) and Berkers and van Mourik (2019).
Subject(s)
Biological Assay/methods , Colforsin/pharmacology , Colon , Cystic Fibrosis/metabolism , Organoids , Cells, Cultured , Colon/drug effects , Colon/metabolism , Edema , Humans , Organoids/drug effects , Organoids/metabolism , Reproducibility of ResultsABSTRACT
BACKGROUND: The natural food supplements curcumin and genistein, and the drug ivacaftor were found effective as CFTR potentiators in the organoids of individuals carrying a S1251N gating mutation, possibly in a synergistic fashion. Based on these in vitro findings, we evaluated the clinical efficacy of a treatment with curcumin, genistein and ivacaftor, in different combinations. METHODS: In three multi-center trials people with CF carrying the S1251N mutation were treated for 8 weeks with curcumin+genistein, ivacaftor and ivacaftor+genistein. We evaluated change in lung function, sweat chloride concentration, CFQ-r, BMI and fecal elastase to determine the clinical effect. We evaluated the pharmacokinetic properties of the compounds by evaluating the concentration in plasma collected after treatment and the effect of the same plasma on the intestinal organoids. RESULTS: A clear clinical effect of treatment with ivacaftor was observed, evidenced by a significant improvement in clinical parameters. In contrast we observed no clear clinical effect of curcumin and/or genistein, except for a small but significant reduction in sweat chloride and airway resistance. Plasma concentrations of the food supplements were low, as was the response of the organoids to this plasma. CONCLUSIONS: We observed a clear clinical effect of treatment with ivacaftor, which is in line with the high responsiveness of the intestinal organoids to this drug. No clear clinical effect was observed of the treatment with curcumin and/or genistein, the low plasma concentration of these compounds emphasizes that pharmacokinetic properties of a compound have to be considered when in vitro experiments are performed.
Subject(s)
Aminophenols/pharmacokinetics , Chloride Channel Agonists/pharmacokinetics , Curcumin/pharmacokinetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/drug therapy , Genistein/pharmacokinetics , Quinolones/pharmacokinetics , Adolescent , Adult , Child , Cystic Fibrosis/genetics , Female , Humans , Male , Organoids/drug effectsABSTRACT
BACKGROUND: 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. METHODS: 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⯵m forskolin for three hours to quantitate CFTR residual function. FINDINGS: FIS was positively correlated with FEV1 (râ¯=â¯0.36, 95% CI 0.02-0.62, pâ¯=â¯0.04) and BMI (râ¯=â¯0.42, 95% CI 0.09-0.66, pâ¯=â¯0.015). FIS was negatively correlated with PRAGMA-CF CT score for% of disease (r = -0.37, 95% CI -0.62- -0.03, pâ¯=â¯0.049). We found no significant correlation between FIS and chest radiography score for CF (r = -0.16, 95% CI -0.48-0.20, pâ¯=â¯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, pâ¯=â¯0.089). INTERPRETATION: 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.
Subject(s)
Colforsin/pharmacology , Cystic Fibrosis Transmembrane Conductance Regulator , Cystic Fibrosis , Organoids , Rectum , Adjuvants, Immunologic/pharmacology , Adult , Biopsy/methods , Correlation of Data , Cystic Fibrosis/diagnosis , Cystic Fibrosis/genetics , Cystic Fibrosis/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Female , Humans , Male , Mutation , Nutritional Status , Organoids/drug effects , Organoids/metabolism , Organoids/pathology , Rectum/metabolism , Rectum/pathology , Respiratory Function Tests , Severity of Illness IndexABSTRACT
Adenine base editing (ABE) enables enzymatic conversion from A-T into G-C base pairs. ABE holds promise for clinical application, as it does not depend on the introduction of double-strand breaks, contrary to conventional CRISPR/Cas9-mediated genome engineering. Here, we describe a cystic fibrosis (CF) intestinal organoid biobank, representing 664 patients, of which ~20% can theoretically be repaired by ABE. We apply SpCas9-ABE (PAM recognition sequence: NGG) and xCas9-ABE (PAM recognition sequence: NGN) on four selected CF organoid samples. Genetic and functional repair was obtained in all four cases, while whole-genome sequencing (WGS) of corrected lines of two patients did not detect off-target mutations. These observations exemplify the value of large, patient-derived organoid biobanks representing hereditary disease and indicate that ABE may be safely applied in human cells.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Cystic Fibrosis , Adenine , Biological Specimen Banks , CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems/genetics , Codon, Nonsense , Cystic Fibrosis/genetics , Gene Editing , Humans , Organoids/metabolismABSTRACT
Cystic fibrosis is caused by mutations in the CFTR gene, which are subdivided into six classes. Mutants of classes III and IV reach the cell surface but have limited function. Most class-III and class-IV mutants respond well to the recently approved potentiator VX-770, which opens the channel. We here revisited function and folding of some class-IV mutants and discovered that R347P is the only one that leads to major defects in folding. By this criterion and by its functional response to corrector drug VX-809, R347P qualifies also as a class-II mutation. Other class-IV mutants folded like wild-type CFTR and responded similarly to VX-809, demonstrating how function and folding are connected. Studies on both types of defects complement each other in understanding how compounds improve mutant CFTR function. This provides an attractive unbiased approach for characterizing mode of action of novel therapeutic compounds and helps address which drugs are efficacious for each cystic fibrosis disease variant.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/genetics , Cystic Fibrosis/pathology , Protein Folding/drug effects , Alleles , Aminophenols/pharmacology , Aminopyridines/pharmacology , Benzodioxoles/pharmacology , Biopsy , Cystic Fibrosis Transmembrane Conductance Regulator/classification , Genotype , HEK293 Cells , Humans , Mutation , Organoids/drug effects , Protein Structure, Tertiary/drug effects , Quinolones/pharmacology , Rectum/pathology , TransfectionABSTRACT
In vitro drug tests using patient-derived stem cell cultures offer opportunities to individually select efficacious treatments. Here, we provide a study that demonstrates that in vitro drug responses in rectal organoids from individual patients with cystic fibrosis (CF) correlate with changes in two in vivo therapeutic endpoints. We measured individual in vitro efficaciousness using a functional assay in rectum-derived organoids based on forskolin-induced swelling and studied the correlation with in vivo effects. The in vitro organoid responses correlated with both change in pulmonary response and change in sweat chloride concentration. Receiver operating characteristic curves indicated good-to-excellent accuracy of the organoid-based test for defining clinical responses. This study indicates that an in vitro assay using stem cell cultures can prospectively select efficacious treatments for patients and suggests that biobanked stem cell resources can be used to tailor individual treatments in a cost-effective and patient-friendly manner.
Subject(s)
Cystic Fibrosis/therapy , Organoids/pathology , Rectum/pathology , Cystic Fibrosis/pathology , Female , Humans , MaleABSTRACT
BACKGROUND: New functional assays using primary human intestinal adult stem cell cultures can be valuable tools to study epithelial defects in human diseases such as cystic fibrosis. METHODS: CFTR-mediated ion transport was measured in rectal organoid-derived monolayers grown from subjects with various CFTR mutations and compared to donor-matched intestinal current measurements (ICM) in rectal biopsies and forskolin-induced swelling of rectal organoids. RESULTS: Rectal organoid-derived monolayers were generated within four days. Ion transport measurements of CFTR function using these monolayers correlated with ICM and organoid swelling (râ¯=â¯0.73 and 0.79 respectively). Culturing the monolayers under differentiation conditions enhanced the detection of mucus-secreting cells and was accompanied by reduced CFTR function. CONCLUSIONS: CFTR-dependent intestinal epithelial ion transport properties can be measured in rectal organoid-derived monolayers of subjects and correlate with donor-matched ICM and rectal organoid swelling.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator , Cystic Fibrosis , Epithelial Cells/metabolism , Ion Transport/physiology , Cells, Cultured , Cystic Fibrosis/genetics , Cystic Fibrosis/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Humans , Models, Biological , Mutation , Organoids/physiology , RectumABSTRACT
BACKGROUND: Germline chromothripsis causes complex genomic rearrangements that are likely to affect multiple genes and their regulatory contexts. The contribution of individual rearrangements and affected genes to the phenotypes of patients with complex germline genomic rearrangements is generally unknown. METHODS: To dissect the impact of germline chromothripsis in a relevant developmental context, we performed trio-based RNA expression analysis on blood cells, induced pluripotent stem cells (iPSCs), and iPSC-derived neuronal cells from a patient with de novo germline chromothripsis and both healthy parents. In addition, Hi-C and 4C-seq experiments were performed to determine the effects of the genomic rearrangements on transcription regulation of genes in the proximity of the breakpoint junctions. RESULTS: Sixty-seven genes are located within 1 Mb of the complex chromothripsis rearrangements involving 17 breakpoints on four chromosomes. We find that three of these genes (FOXP1, DPYD, and TWIST1) are both associated with developmental disorders and differentially expressed in the patient. Interestingly, the effect on TWIST1 expression was exclusively detectable in the patient's iPSC-derived neuronal cells, stressing the need for studying developmental disorders in the biologically relevant context. Chromosome conformation capture analyses show that TWIST1 lost genomic interactions with several enhancers due to the chromothripsis event, which likely led to deregulation of TWIST1 expression and contributed to the patient's craniosynostosis phenotype. CONCLUSIONS: We demonstrate that a combination of patient-derived iPSC differentiation and trio-based molecular profiling is a powerful approach to improve the interpretation of pathogenic complex genomic rearrangements. Here we have applied this approach to identify misexpression of TWIST1, FOXP1, and DPYD as key contributors to the complex congenital phenotype resulting from germline chromothripsis rearrangements.