Airway basal cells from human-induced pluripotent stem cells: a new frontier in cystic fibrosis research

Human-induced airway basal cells (hiBCs) derived from human-induced pluripotent stem cells (hiPSCs) offer a promising cell model for studying lung diseases, regenerative medicine, and developing new gene therapy methods. We analyzed existing differentiation protocols and proposed our own protocol for obtaining hiBCs, which involves step-by-step differentiation of hiPSCs into definitive endoderm, anterior foregut endoderm, NKX2.1+ lung progenitors, and cultivation on basal cell medium with subsequent cell sorting using the surface marker CD271 (NGFR). We derived hiBCs from two healthy cell lines and three cell lines with cystic fibrosis (CF). The obtained hiBCs, expressing basal cell markers (NGFR, KRT5, and TP63), could differentiate into lung organoids (LOs). We demonstrated that LOs derived from hiBCs can assess cystic fibrosis transmembrane conductance regulator (CFTR) channel function using the forskolin-induced swelling (FIS) assay. We also carried out non-viral (electroporation) and viral (recombinant adeno-associated virus (rAAV)) serotypes 6 and 9 and recombinant adenovirus (rAdV) serotype 5 transgene delivery to hiBCs and showed that rAAV serotype 6 is most effective against hiBCs, potentially applicable for gene therapy research

THE CONING AND MOLECULAR-GENETIC INVESTIGATION OF GENE BRPB FUNCTIONAL ROLE IN THE MODEL STRAIN STREPTOMYCES LIVIDANS TK 64 AND THE PRODUCER OF BIALOPHOS S.HYDROSCOPICUS ATCC21705

The odject of investigation: the gene of the positive regulation of genes clusters of the brpB bialaphos antibiotikherbicide biosynthesis from strain S.hydroscopicus. Offered has been the diagram of the transcription initiation of the bialaphos genes cluster biosynthesis with the participation of the product of the brpB cloned gene in strain of the S.hydroscopicus producer. Demonstrated has been the possibility of the genetic expression of structures of plasmides and phages Streptomyces in strain Phodocorcus rhodochrous M8. Gene brpB, taking part in the regulation of genes cluster of the bialaphos biosynthesis has been cloned, the gene properties have been studied. The test-system intended for the identification of matters, possessing the antiglutaminesynthetase activity, has been developed. Adapted have been the methods of the between-kin conjugation, intended for the introduction of the recombinant DNA into the industrial strains of the actinomycetesAvailable from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio

CTNS mRNA molecular analysis revealed a novel mutation in a child with infantile nephropathic cystinosis: a case report

BACKGROUND: Cystinosis is an autosomal recessive lysosomal storage disorder characterized by accumulation of cystine in lysosomes throughout the body. Cystinosis is caused by mutations in the CTNS gene that encodes the lysosomal cystine carrier protein cystinosin. CTNS mutations result in either complete absence or reduced cystine transporting function of the protein. The diagnosis of nephropathic cystinosis is generally based on measuring leukocyte cystine level, demonstration of corneal cystine crystals by the slit lamp examination and confirmed by genetic analysis of the CTNS gene. CASE PRESENTATION: A boy born to consanguineous Caucasian parents had the characteristic clinical features of the infantile nephropathic cystinosis including renal Fanconi syndrome (polydipsia/polyuria, metabolic acidosis, hypokalemia, hypophosphatemia, low molecular weight proteinuria, glycosuria, cystine crystals in the cornea) and elevated WBC cystine levels. Initially we performed RFLP analysis of the common in the Northern European population 57-kb deletion of proband's DNA, then a direct Sanger sequencing which revealed no mutations in the coding part of the CTNS gene. To confirm the diagnosis we performed RT-PCR analysis of total RNA obtained from patient-derived fibroblasts in combination with cDNA sequencing. This revealed the skipping of exon 4 and exon 5 in the CTNS in our patient. Therefore, we detected a novel 9-kb homozygous deletion in the CTNS gene at genomic DNA level, spanning region from intron 3 to intron 5. In order to identify the inheritance pattern of the deletion we analyzed DNA of proband's mother and father. Both parents were found to be heterozygous carriers of the CTNS mutation. CONCLUSIONS: Analysis of CTNS gene transcript allowed to identify a large homozygous deletion in the patient with infantile nephropathic cystinosis. Mutational detection at RNA level may be an efficient tool to establish the genetic defect in some cystinosis patients.status: publishe

Previously Undescribed Gross HACE1 Deletions as a Cause of Autosomal Recessive Spastic Paraplegia

Spastic paraplegia and psychomotor retardation with or without seizures (SPPRS, OMIM 616756) is a rare genetic disease caused by biallelic pathogenic variants in the HACE1 gene. Originally, these mutations have been reported to be implicated in tumor predisposition. Nonetheless, via whole exome sequencing in 2015, HACE1 mutations were suggested to be the cause of a new autosomal recessive neurodevelopmental disorder, which is characterized by spasticity, muscular hypotonia, and intellectual disability. To date, 14 HACE1 pathogenic variants have been described; these variants have a loss-of-function effect that leads to clinical presentations with variable severities. However, gross deletions in the HACE1 gene have not yet been mentioned as a cause of spastic paraplegia. Here, we report a clinical case involving a 2-year-old male presenting with spasticity, mainly affecting the lower limbs, and developmental delay. Exome sequencing, chromosomal microarray analysis, and mRNA analysis were used to identify the causative gene. We revealed that the clinical findings were due to previously undescribed HACE1 biallelic deletions. We identified the deletion of exon 7: c.(534+1_535-1)_(617+1_618-1)del (NM_020771.4) and the gross deletion in the 6q16.3 locus, which affected the entire HACE1 gene: g.105018931_105337494del, (GRCh37). A comprehensive diagnostic approach for the patients with originally homozygous mutations in HACE1 is required since false homozygosity results are possible. More than 80% of the described mutations were reported to be homozygous. Initial hemizygosity is hard to detect by quantitative methods, and this may challenge molecular diagnostic identification in patients with spastic paraplegia

Airway and Lung Organoids from Human-Induced Pluripotent Stem Cells Can Be Used to Assess CFTR Conductance

Airway and lung organoids derived from human-induced pluripotent stem cells (hiPSCs) are current models for personalized drug screening, cell–cell interaction studies, and lung disease research. We analyzed the existing differentiation protocols and identified the optimal conditions for obtaining organoids. In this article, we describe a step-by-step protocol for differentiating hiPSCs into airway and lung organoids. We obtained airway and lung organoids from a healthy donor and from five donors with cystic fibrosis. Analysis of the cellular composition of airway and lung organoids showed that airway organoids contain proximal lung epithelial cells, while lung organoids contain both proximal and distal lung epithelial cells. Forskolin-induced swelling of organoids derived from a healthy donor showed that lung organoids, as well as airway organoids, contain functional epithelial cells and swell after 24 h exposure to forskolin, which makes it a suitable model for analyzing the cystic fibrosis transmembrane conductance regulator (CFTR) channel conductance in vitro. Thus, our results demonstrate the feasibility of generating and characterizing airway and lung organoids from hiPSCs, which can be used for a variety of future applications

A novel variant m.641A>T in the mitochondrial MT-TF gene is associated with epileptic encephalopathy in adolescent

We present a 14-year-old girl with loss of motor functions, tetraplegia, epilepsy and nystagmus, caused by a novel heteroplasmic m.641A>T transition in an evolutionary conserved region of mitochondrial genome, affecting the aminoacyl stem of mitochondrial tRNA-Phe. In silico prediction, respirometry, Western blot and enzymatic analyses in skin fibroblasts support the pathogenicity of the m.641A>T substitution. This is the 18th MT-TF point mutation associated with a mitochondrial disorder. The onset and the severity of the disease, however, is unique in this case and broadens the clinical picture related to mutations of mitochondrial tRNA-Phe

Airway and Lung Organoids from Human-Induced Pluripotent Stem Cells Can Be Used to Assess CFTR Conductance

Airway and lung organoids derived from human-induced pluripotent stem cells (hiPSCs) are current models for personalized drug screening, cell–cell interaction studies, and lung disease research. We analyzed the existing differentiation protocols and identified the optimal conditions for obtaining organoids. In this article, we describe a step-by-step protocol for differentiating hiPSCs into airway and lung organoids. We obtained airway and lung organoids from a healthy donor and from five donors with cystic fibrosis. Analysis of the cellular composition of airway and lung organoids showed that airway organoids contain proximal lung epithelial cells, while lung organoids contain both proximal and distal lung epithelial cells. Forskolin-induced swelling of organoids derived from a healthy donor showed that lung organoids, as well as airway organoids, contain functional epithelial cells and swell after 24 h exposure to forskolin, which makes it a suitable model for analyzing the cystic fibrosis transmembrane conductance regulator (CFTR) channel conductance in vitro. Thus, our results demonstrate the feasibility of generating and characterizing airway and lung organoids from hiPSCs, which can be used for a variety of future applications

P.F508del editing in cells from cystic fibrosis patients.

Development of genome editing methods created new opportunities for the development of etiology-based therapies of hereditary diseases. Here, we demonstrate that CRISPR/Cas9 can correct p.F508del mutation in the CFTR gene in the CFTE29o- cells and induced pluripotent stem cells (iPSCs) derived from patients with cystic fibrosis (CF). We used several combinations of Cas9, sgRNA and ssODN and measured editing efficiency in the endogenous CFTR gene and in the co-transfected plasmid containing the CFTR locus with the p.F508del mutation. The non-homologous end joining (NHEJ) frequency in the CFTR gene in the CFTE29o- cells varied from 1.25% to 2.54% of alleles. The best homology-directed repair (HDR) frequency in the endogenous CFTR locus was 1.42% of alleles. In iPSCs, the NHEJ frequency in the CFTR gene varied from 5.5% to 12.13% of alleles. The best HDR efficacy was 2.38% of alleles. Our results show that p.F508del mutation editing using CRISPR/Cas9 in CF patient-derived iPSCs is a relatively rare event and subsequent cell selection and cultivation should be carried out

The Genetic Basis of the First Patient with Wiedemann–Rautenstrauch Syndrome in the Russian Federation

Bi-allelic pathogenic variations within POLR3A have been associated with a spectrum of hereditary disorders. Among these, a less frequently observed condition is Wiedemann–Rautenstrauch syndrome (WRS), also known as neonatal progeroid syndrome. This syndrome typically manifests neonatally and is characterized by growth retardation, evident generalized lipodystrophy with distinctively localized fat accumulations, sparse scalp hair, and atypical facial features. Our objective was to elucidate the underlying molecular mechanisms of Wiedemann–Rautenstrauch syndrome (WRS). In this study, we present a clinical case of a 7-year-old female patient diagnosed with WRS. Utilizing whole-exome sequencing (WES), we identified a novel missense variant c.3677T>C (p.Leu1226Pro) in the POLR3A gene (NM_007055.4) alongside two cis intronic variants c.1909+22G>A and c.3337-11T>C. Via the analysis of mRNA derived from fibroblasts, we reconfirmed the splicing-affecting nature of the c.3337-11T>C variant. Furthermore, our investigation led to the reclassification of the c.3677T>C (p.Leu1226Pro) variant as a likely pathogenic variant. Therefore, this is the first case demonstrating the molecular genetics of a patient with Wiedemann–Rautenstrauch syndrome from the Russian Federation. A limited number of clinical cases have been documented until this moment; therefore, broadening the linkage between phenotype and molecular changes in the POLR3A gene will significantly contribute to the comprehensive understanding of the molecular basis of POLR3A-related disorders