Comprehensive Assessment of CFTR Modulators' Therapeutic Efficiency for N1303K Variant.

p.Asn1303Lys (N1303K) is a common missense variant of the CFTR gene, causing cystic fibrosis (CF). In this study, we initially evaluated the influence of CFTR modulators on the restoration of N1303K-CFTR function using intestinal organoids derived from four CF patients expressing the N1303K variant. The forskolin-induced swelling assay in organoids offered valuable insights about the beneficial effects of VX-770 + VX-661 + VX-445 (Elexacaftor + Tezacaftor + Ivacaftor, ETI) on N1303K-CFTR function restoration and about discouraging the prescription of VX-770 + VX-809 (Ivacaftor + Lumacaftor) or VX-770 + VX-661 (Ivacaftor + Tezacaftor) therapy for N1303K/class I patients. Then, a comprehensive assessment was conducted on an example of one patient with the N1303K/class I genotype to examine the ETI effect on the restoration of N1303K-CFTR function using in vitro the patient's intestinal organoids, ex vivo the intestinal current measurements (ICM) method and assessment of the clinical status before and after targeted therapy. All obtained results are consistent with each other and have proven the effectiveness of ETI for the N1303K variant. ETI produced a significant positive effect on forskolin-induced swelling in N1303K/class I organoids indicating functional improvement of the CFTR protein; ICM demonstrated that ETI therapy restored CFTR function in the intestinal epithelium after three months of treatment, and the patient improved his clinical status and lung function, increased his body mass index (BMI) and reduced the lung pathogenic flora diversity, surprisingly without improving the sweat test results.

Clinical and Functional Characteristics of the E92K CFTR Gene Variant in the Russian and Turkish Population of People with Cystic Fibrosis.

The pathogenic variant E92K (c.274G > A) of the CFTR gene is rare in America and Europe, but it is common for people with cystic fibrosis from Russia and Turkey. We studied the effect of the E92K genetic variant on the CFTR function. The function of the CFTR channel was studied using the intestinal current measurements (ICM) method. The effects of CFTR modulators on the restoration of the CFTR function were studied in the model of intestinal organoids. To assess the effect of E92K on pre-mRNA splicing, the RT-PCR products obtained from patients' intestinal organoid cultures were analyzed. Patients with the genetic variant E92K are characterized by an older age of diagnosis compared to homozygotes F508del and a high frequency of pancreatic sufficiency. The results of the sweat test and the ICM method showed partial preservation of the function of the CFTR channel. Functional analysis of CFTR gene expression revealed a weak effect of the E92K variant on mRNA-CFTR splicing. Lumacaftor (VX-809) has been shown to restore CFTR function in an intestinal organoid model, which allows us to consider the E92K variant as a promising target for therapy with CFTR correctors.

Exhaled breath analysis in adult patients with cystic fibrosis by real-time proton mass spectrometry.

BACKGROUND: Proton-transfer reaction time-of-flight mass spectrometry (PTR-TOF-MS) is a promising tool for a rapid online determination of exhaled volatile organic compounds (eVOCs) profiles in patients with cystic fibrosis (CF). OBJECTIVE: To detect VOC breath signatures specific to adult patients with CF compared with controls using PTR-TOF-MS. METHODS: 102 CF patients (54 M/48, mean age 25.6 ± 7.8 yrs) and 97 healthy controls (56 M/41F, mean age 25.8 ± 6.0 yrs) were examined. Samples from normal quiet breathing and forced expiratory maneuvers were analyzed with PTR-TOF-MS (Ionicon, Austria) to obtain VOC profiles listed as ions at various mass-to-charge ratios (m/z). RESULTS: PTR-TOF-MS analysis was able to detect 167 features in exhaled breath from CF patients and healthy controls. According to cluster analysis and LASSO regression, patients with CF and controls were separated. The most significant VOCs for CF were indole, phenol, dimethyl sulfide, and not indicated: m/z = 297.0720 ([C12H13N2O7 and C17H13O5]H + ), m/z = 281.0534 ([C19H7NO2, C12H11NO7 and C16H9O5]H + ) during five-fold cross-validation both in forced expiratory maneuver and in normal quiet breathing. CONCLUSION: PTR-TOF-MS is a promising method for determining the molecular composition of exhaled air specific to CF.

Identification of Exhaled Metabolites Correlated with Respiratory Function and Clinical Features in Adult Patients with Cystic Fibrosis by Real-Time Proton Mass Spectrometry.

Cystic fibrosis (CF) is a hereditary disease characterized by the progression of respiratory disorders, especially in adult patients. The purpose of the study was to identify volatile organic compounds (VOCs) as predictors of respiratory dysfunction, chronic respiratory infections of Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia, and VOCs associated with severe genotype and highly effective modulator treatment (HEMT). Exhaled breath samples from 102 adults with CF were analyzed using PTR-TOF-MS, obtained during a forced expiratory maneuver and normal quiet breathing. Using cross-validation and building gradient boosting classifiers (XGBoost), the importance of VOCs for functional and clinical outcomes was determined. The presence of the previously identified VOCs indole, phenol, and dimethyl sulfide were metabolic outcomes associated with impaired respiratory function. New VOCs associated with respiratory disorders were methyl acetate, carbamic acid, 1,3-Pentadiene, and 2,3-dimethyl-2-butene; VOCs associated with the above mentioned respiratory pathogens were non-differentiable nitrogen-containing organic compounds m/z = 47.041 (CH5NO)+ and m/z = 44.044 (C2H5NH+), hydrocarbons (cyclopropane, propene) and methanethiol; and VOCs associated with severe CFTR genotype were non-differentiable VOC m/z = 281.053. No significant features associated with the use of HEMT were identified. Early non-invasive determination of VOCs as biomarkers of the severity of CF and specific pathogenic respiratory flora could make it possible to prescribe adequate therapy and assess the prognosis of the disease. However, further larger standardized studies are needed for clinical use.

Comprehensive semen examination in patients with pancreatic-sufficient and pancreatic-insufficient cystic fibrosis.

We examined a cohort of 93 cystic fibrosis (CF) male patients who were pancreatic-sufficient (PS-CF; n=40) or pancreatic-insufficient (PI-CF; n = 53). Complex semen examination was performed, including standard semen analysis, quantitative karyological analysis (QKA) of immature germ cells (IGCs), transmission electronic microscopy (TEM), biochemical analysis, and sperm DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling (TUNEL) assay. Azoospermia was diagnosed in 83 (89.2%) patients. The other 10 (10.8%) patients were found to be nonazoospermic and showed various spermatological diagnoses (asthenozoospermia, n = 2; asthenoteratozoospermia, n = 3; oligoasthenozoospermia, n = 1; oligoasthenoteratozoospermia, n = 3; and normozoospermia, n = 1) with no specific morphological abnormalities. Oligospermia was detected in 89.2% azoospermic and 30.0% nonazoospermic patients. Low seminal pH (<7.0) was found in 74 (89.2%) of 83 azoospermic patients. Moderate leukocytospermia (2.0 × 10 6 -2.2 × 10 6 ml -1 ) was revealed in 2.4% azoospermic and 40.0% nonazoospermic semen samples. The signs of partial meiotic arrest at prophase I were found in 4 of 6 nonazoospermic patients examined by QKA of IGCs. The content of fructose and citrate was low in oligospermic and normal in nonoligospermic semen samples. An increased percentage (>30%) of spermatozoa with noncondensed ("immature") chromatin was revealed in 2 of 6 nonazoospermic semen samples analyzed by TEM.

Clinical Presentation of the c.3844T>C (p.Trp1282Arg, W1282R) Variant in Russian Cystic Fibrosis Patients.

The goal was to study the phenotypic manifestations of c.3844T>C (p.Trp1282Arg, W1282R) variant, a CF-causing mutation, in patients from the Russian Federation. Clinical manifestations and complications (the age at CF diagnosis, sweat test, pancreatic status, lung function, microbial infection, body mass index (BMI), the presence of meconium ileus (MI), diabetes, and severe liver disease) were compared in four groups: group 1-patients carrying c.3844T>C and severe class I or II variant in trans; group 2-3849+10kbC>T/F508del patients; group 3-F508del/F508del patients; and group 4-patients with W1282R and "mild" variant in trans. Based on the analyses, W1282R with class I or II variant in trans appears to cause at least as severe CF symptoms as F508del homozygotes as reflected in the early age of diagnosis, high sweat chloride concentration, insufficient pancreatic function, and low lung function, in contrast to 3849+10kbC-T/F508del compound heterozygotes having milder clinical phenotypes. The W1282R pathogenic variant is seemed to lead to severe disease phenotype with pancreatic insufficiency similarly to the F508del homozygous genotype.

Targeted sequencing reveals complex, phenotype-correlated genotypes in cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) is one of the most common life-threatening genetic disorders. Around 2000 variants in the CFTR gene have been identified, with some proportion known to be pathogenic and 300 disease-causing mutations have been characterized in detail by CFTR2 database, which complicates its analysis with conventional methods. METHODS: We conducted next-generation sequencing (NGS) in a cohort of 89 adult patients negative for p.Phe508del homozygosity. Complete clinical and demographic information were available for 84 patients. RESULTS: By combining MLPA with NGS, we identified disease-causing alleles in all the CF patients. Importantly, in 10% of cases, standard bioinformatics pipelines were inefficient in identifying causative mutations. Class IV-V mutations were observed in 38 (45%) cases, predominantly ones with pancreatic sufficient CF disease; rest of the patients had Class I-III mutations. Diabetes was seen only in patients homozygous for class I-III mutations. We found that 12% of the patients were heterozygous for more than two pathogenic CFTR mutations. Two patients were observed with p.[Arg1070Gln, Ser466*] complex allele which was associated with milder pulmonary obstructions (FVC 107 and 109% versus 67%, CI 95%: 63-72%; FEV 90 and 111% versus 47%, CI 95%: 37-48%). For the first time p.[Phe508del, Leu467Phe] complex allele was reported, observed in four patients (5%). CONCLUSION: NGS can be a more information-gaining technology compared to standard methods. Combined with its equivalent diagnostic performance, it can therefore be implemented in the clinical practice, although careful validation is still required.