Genetic modification of cystic fibrosis with ΔF508 mutation of CFTR gene using the CRISPR system in peripheral blood mononuclear cells.

OBJECTIVES: Cystic fibrosis (CF) is an inherited autosomal recessive disease that is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The present study aimed to investigate the genetic modification of CF with ΔF508 mutation of the CFTR gene using CRISPR in peripheral blood mononuclear cells (PBMCs). MATERIALS AND METHODS: Two single guide RNAs were designed to target sequences in the CFTR gene. The transfection efficiency of PBMC cells was examined through evaluation of green fluorescent protein (GFP) expression using fluorescent microscopy. Moreover, a sgRNA-Cas9 plasmid was tested to target the CFTR gene. The ΔF508 gene modification was evaluated and confirmed by PCR and Sanger sequencing methods. RESULTS: Our results indicate the feasibility of site-specific gene targeting with the CRISPR/Cas9 system. 33% of the samples were corrected using CRISPR in mutant locus and confirmed by sequence blast at NCBI databases and primers outside the arm locus. CRISPR/Cas9 approach represents an efficient tool to repair the ΔF508 mutation of the CFTR gene in PBMC Cells. CONCLUSION: Therefore, the CRISPR system can be highly efficient and specific and provides a powerful approach for genetic engineering of cells and model animals. Generally, the proposed method opens new insights into the treatment of human diseases.

Plasma Levels of the Bioactive Sphingolipid Metabolite S1P in Adult Cystic Fibrosis Patients: Potential Target for Immunonutrition?

Recent research has linked sphingolipid (SL) metabolism with cystic fibrosis transmembrane conductance regulator (CFTR) activity, affecting bioactive lipid mediator sphingosine-1-phosphate (S1P). We hypothesize that loss of CFTR function in cystic fibrosis (CF) patients influenced plasma S1P levels. Total and unbound plasma S1P levels were measured in 20 lung-transplanted adult CF patients and 20 healthy controls by mass spectrometry and enzyme-linked immunosorbent assay (ELISA). S1P levels were correlated with CFTR genotype, routine laboratory parameters, lung function and pathogen colonization, and clinical symptoms. Compared to controls, CF patients showed lower unbound plasma S1P, whereas total S1P levels did not differ. A positive correlation of total and unbound S1P levels was found in healthy controls, but not in CF patients. Higher unbound S1P levels were measured in ΔF508-homozygous compared to ΔF508-heterozygous CF patients (p = 0.038), accompanied by higher levels of HDL in ΔF508-heterozygous patients. Gastrointestinal symptoms were more common in ΔF508 heterozygotes compared to ΔF508 homozygotes. This is the first clinical study linking plasma S1P levels with CFTR function and clinical presentation in adult CF patients. Given the emerging role of immunonutrition in CF, our study might pave the way for using S1P as a novel biomarker and nutritional target in CF.

A defective flexible loop contributes to the processing and gating defects of the predominant cystic fibrosis-causing mutation.

People with the genetic disease cystic fibrosis (CF) often carry a deletion mutation ΔF508 on the gene encoding the CF transmembrane conductance regulator (CFTR) Cl- channel. This mutation greatly reduces the CFTR maturation process and slows the channel opening rate. Here, we investigate whether residues near F508 contribute to these defects in ΔF508-CFTR. Most deletion mutations, but not alanine substitutions, of individual residues from positions 503 to 513 impaired CFTR maturation. Interestingly, only protein processing of ΔY512-CFTR, like that of ΔF508-CFTR, was greatly improved by low-temperature culture at 27°C or small-molecule corrector C18. The 2 mutant Cl- channels were equally slow to open, suggesting that they may share common structural flaws. Studies on the H3-H4 loop that links residues F508 and Y512 demonstrate that G509A/V510G mutations, moving G509 1 position backward in the loop, markedly enhanced ΔF508-CFTR maturation and opening rate while promoting protein stability and persistence of the H3 helix in ΔF508 nucleotide-binding domain 1. Moreover, V510A/S511A mutations noticeably increased ΔY512-CFTR maturation at 27°C and its opening rate. Thus, loop abnormalities may contribute to ΔF508- and ΔY512-CFTR defects. Importantly, correcting defects from G509 displacement in ΔF508-CFTR may offer a new avenue for drug discovery and CF treatments.-Chen, X., Zhu, S., Zhenin, M., Xu, W., Bose, S. J., Wong, M. P.-F., Leung, G. P. H., Senderowitz, H., Chen, J.-H. A defective flexible loop contributes to the processing and gating defects of the predominant cystic fibrosis-causing mutation.

Identificación de la mutación ΔF508 en afectados con íleo meconial

Objetivo: Identificar la mutación ΔF508 en pacientes con íleo meconial. Ambiente: En el Instituto de Investigaciones Genéticas de la Facultad de Medicina de la Universidad del Zulia. Maracaibo. Métodos: Se estudiaron diez pacientes con ileo meconial. La detección de la mutación ΔF508 se realizó a partir de amplificación por reacción en cadena de la polimerasa de un segmento del gen de fibrosis quística de 98 pares de bases que contiene el codón que codifica a la fenilalanina en la posición 508 y el cual está ausente en los que tienen la mutación. Resultados: Se detectó la mutación ΔF508 en ambos alelos del gen de la fibrosis quística en tres pacientes, en un solo alelo en cinco y en dos no se identificó el alelo ΔF508 en su patrón molecular. Conclusión: El íleo meconial fue el marcador que sugirió el diagnóstico de fibrosis quística y permitió el asesoramiento genético de las familias al confirmar la presencia de la mutación ΔF508.

Objective: To perform ΔF508 mutation in patients with meconium ileus. Setting: In the Genetic Research Institute of the Faculty of Medicine. University of Zulia. Maracaibo. Methods: We studied 10 patients with meconium ileus. Detection of the mutation was performed from the amplification of a 98 pair of bases cystic fibrosis gene segment which contains the codon that encodes fenilalanine in the 508 position by polymerase chain reaction. This amplified product is absent in those who have the mutation. Results: The ΔF508 mutation was detected in both alleles of the cystic fibrosis gene in 3 patients, 5 were heterozygous for this mutation and in two patients were undetectable. Conclusion: Meconium ileus was the marker that suggested the diagnosis of cystic fibrosis and allowed the genetic counseling in this family to confirm the presence of the ΔF508 mutation.

Frequency of Genotype With ΔF508 Mutation in CFTR Gene Among Iranian Cystic Fibrosis Patients With Pancreatic Insufficiency.

BACKGROUND: Cystic fibrosis (CF) is the most prevalent lethal autosomal recessive disease with a broad spectrum of phenotypes. Mutation of ΔF508 in the CFTR gene is the most important and lethal mutation in CF, which contains 70% of all predisposing mutations for CF worldwide. OBJECTIVES: Determining frequency of genotypes with ΔF508 mutation in CFTR gene, and evaluation of correlation between genotype and phenotype of Iranian patients with CF. PATIENTS AND METHODS: Thirty six patients were included in this cross sectional study. ΔF508 mutations in both alleles of the CFTR gene were checked. RESULTS: Among 36 pediatric patients, ΔF508 mutation was detected in 9 (25%) patients; 2 patients were heterozygous, and 7 patients homozygous for this mutation. From overall 72 tracked alleles, 11 (15.2%) were found to have ΔF508 mutations. Differences in prevalence of dyspnea and bronchiectasis were significant in homozygote group, compared with non-mutated group for ΔF508. CONCLUSIONS: It seems that more ΔF508 mutated alleles lead to more severe symptoms of CF.

Profile of cystic fibrosis in a single referral center in Egypt.

It was generally believed that Cystic fibrosis (CF) is rare among Arabs; however, the few studies available from Egypt and other Arabic countries suggested the presence of many undiagnosed patients. The aim of the present study was to determine the frequency of CF patients out of the referred cases in a single referral hospital in Egypt. A total of 100 patients clinically suspected of having CF were recruited from the CF clinic of the Allergy and Pulmonology Unit, Children's Hospital, Cairo University, Egypt, throughout a 2 year period. Sweat chloride testing was done for all patients using the Wescor macroduct system for collection of sweat. Quantitative analysis for chloride was then done by the thiocyanate colorimetric method. Patients positive for sweat chloride (⩾60 mmol/L) were tested for the ΔF508 mutation using primer specific PCR for cystic fibrosis transmembrane conductance regulator (CFTR) gene. Thirty-six patients (36%) had a positive sweat chloride test. The main clinical presentations in patients were chronic cough in 32 (88.9%), failure to thrive in 27 (75%), steatorrhea in 24 (66.7%), and hepatobiliary involvement in 5 (13.9%). Positive consanguinity was reported in 50% of CF patients. Thirty-two patients were screened for ΔF508 mutation. Positive ΔF508 mutation was detected in 22 (68.8%) patients, 8 (25%) were homozygous, 14 (43.8%) were heterozygous, and 10 (31.3%) tested were negative. CF was diagnosed in more than third of patients suspected of having the disease on clinical grounds. This high frequency of CF among referred patients indicates that a high index of suspicion and an increasing availability of diagnostic tests lead to the identification of a higher number of affected individuals.

Tissue-specific control of CFTR endocytosis by Dab2: Cargo recruitment as a therapeutic target.

Clathrin-mediated endocytosis dynamically regulates cell membrane abundance of CFTR and plays an essential role in CFTR-dependent Cl(-) conductance in fluid-transporting epithelia. It requires two closely related, but distinct processes: assembly of the clathrin coat and recruitment of cargo proteins for endocytosis. The assembly polypeptide-2 complex (AP-2) is the prototypical endocytic adaptor responsible for optimal clathrin coat formation. Disabled-2 (Dab2) is a clathrin associated sorting protein (CLASP) that also mediates clathrin assembly and cargo selection. Both of these complexes have clearly been shown to play roles in CFTR endocytosis in cells that endogenously express the channel. However, their precise functions exhibit cell-specific differences. While Dab2 appears to play a central role in CFTR recruitment to the clathrin coat in airway epithelial cells, it does not play a direct role in CFTR endocytosis in intestinal epithelial cells. Here, we review our current understanding of the role of Dab2 in CFTR endocytosis in different tissues. Next, we present new data demonstrating the role of Dab2 in endocytosis of the most commonly mutated CFTR gene product, ∆F508-CFTR, in human airwy epithelial cells. Finally we discuss the potential therapeutic implications of targeting the functional interaction between ∆F508-CFTR and Dab2.

Acid and base secretion in freshly excised nasal tissue from cystic fibrosis patients with ΔF508 mutation.

BACKGROUND: Cystic fibrosis (CF) is caused by a misfunctional CF transmembrane conductance regulator (CFTR) protein, which is believed to contributes to the regulation of the airway surface liquid (ASL) pH. This study investigated acid and base secretion in freshly excised human nasal tissues from CF patients homozygous for the ΔF508 mutation. METHODS: Human nasal mucosa was collected during sinus surgery and investigated in Ussing chambers. Mucosal equilibrium pH values and rate of acid and base secretion were determined using the pH-stat technique. RESULTS: The equilibrium pH of nasal epithelia from ΔF508 CF patients with chronic rhinosinusitis (CRS) was pH = 7.08 ± 0.09 and was significantly lower compared to nasal epithelia from CRS patients without CF (pH = 7.33 ± 0.06) and normal subjects (pH = 7.34 ± 0.08, n = 6). The rate of base secretion in CF nasal tissues was 11.8 ± 2.4 nmol · min(−1) · cm(−2), which was significantly lower than normal (57.2 ± 9.2 nmol · min(−1) · cm(−2)). The HCO3(−) secretory rate was further increased by forskolin by 16.1% in normal, but not in CF tissues. CONCLUSION: Our data suggests that CF patients exhibited significantly lower base secretion by the nasal airway epithelium. It is possible that improper regulation of ASL pH in CF may negatively impact the innate host defense system.