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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.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Células Madre Pluripotentes Inducidas , Humanos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Colforsina/farmacología , Pulmón , Células Epiteliales , OrganoidesRESUMEN
Genetic up-regulation of mitochondrial 2-oxoglutarate dehydrogenase is known to increase reactive oxygen species, being detrimental for cancer cells. Thiamine diphosphate (ThDP, cocarboxylase) is an essential activator of the enzyme and inhibits p53-DNA binding in cancer cells. We hypothesize that the pleiotropic regulator ThDP may be of importance for anticancer therapies. The hypothesis is tested in the present work on lung adenocarcinoma cells A549 possessing the p53-p21 pathway as fully functional or perturbed by p21 knockdown. Molecular mechanisms of ThDP action on cellular viability and their interplay with the cisplatin and p53-p21 pathways are characterized. Despite the well-known antioxidant properties of thiamine, A549 cells exhibit decreases in their reducing power and glutathione level after incubation with 5 mM ThDP, not observed in non-cancer epithelial cells Vero. Moreover, thiamine deficiency elevates glutathione in A549 cells. Viability of the thiamine deficient A549 cells is increased at a low (0.05 mM) ThDP. However, the increase is attenuated by 5 mM ThDP, p21 knockdown, specific inhibitor of the 2-oxoglutarate dehydrogenase complex (OGDHC), or cisplatin. Cellular levels of the catalytically competent ThDP·OGDHC holoenzyme are dysregulated by p21 knockdown and correlate negatively with the A549 viability. The inverse relationship between cellular glutathione and holo-OGDHC is corroborated by their comparison in the A549 and Vero cells. The similarity, non-additivity, and p21 dependence of the dual actions of ThDP and cisplatin on A549 cells manifest a common OGDHC-mediated mechanism of the viability decrease. High ThDP saturation of OGDHC compromises the redox state of A549 cells under the control of p53-p21 axes.
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Adenocarcinoma/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Neoplasias Pulmonares/metabolismo , Tiamina Pirofosfato/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Células A549 , Animales , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Cisplatino/farmacología , Glutatión/metabolismo , Humanos , Oxidación-Reducción , Tiamina/metabolismo , Células VeroRESUMEN
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.
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Sistemas de Transporte de Aminoácidos Neutros/genética , Cistinosis/genética , Mutación , Preescolar , Consanguinidad , Cisteamina/uso terapéutico , Depletores de Cistina/uso terapéutico , Cistinosis/tratamiento farmacológico , Cistinosis/metabolismo , Análisis Mutacional de ADN , Exones/genética , Fibroblastos/química , Humanos , Lactante , Intrones/genética , Masculino , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
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.
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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.
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Progeria , Recién Nacido , Femenino , Humanos , Niño , Progeria/genética , Progeria/diagnóstico , Progeria/patología , Retardo del Crecimiento Fetal/patología , Mutación , Federación de Rusia , ARN Polimerasa III/genéticaRESUMEN
Skin fibroblasts obtained from a 5-year-old girl with genetically proven (two heterozygous mutations in ARSB gene) and clinically manifested mucopolysaccharidosis type VI were successfully transformed into induced pluripotent stem cells by using Sendai virus-based reprogramming vectors including the four Yamanaka factors namely SOX2, OCT3/4, KLF4, and c-MYC. These iPSCs expressed pluripotency markers, had a normal karyotype and the potential to differentiate into three germ layers in spontaneous differentiation assay. The line may be used for cell differentiation and pharmacological investigations, and also may provide a model for development of a personalized treatment including drug screening and genome editing.
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Células Madre Pluripotentes Inducidas , Mucopolisacaridosis VI , Femenino , Humanos , Preescolar , Células Madre Pluripotentes Inducidas/metabolismo , Mucopolisacaridosis VI/metabolismo , Factor 4 Similar a Kruppel , Diferenciación Celular/genética , Fibroblastos/metabolismo , Reprogramación CelularRESUMEN
Urine cells obtained from a 14-year-old man with genetically proven (ACVR1: c.6176G > A) and clinically manifested fibrodysplasia ossificans progressiva were successfully transformed into induced pluripotent stem cells by using Sendai virus-based reprogramming vectors including the four Yamanaka factors such as OCT3/4, SOX2, KLF4, and c-MYC. These iPSCs expressed pluripotency markers, exhibited the potential to differentiate into three germ layers in spontaneous differentiation assay and had a normal karyotype. The iPSC line may provide a model for development of a personalized treatment including genome editing and drug screening, may be used for disease modelling, cell differentiation and pharmacological investigations. .
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Células Madre Pluripotentes Inducidas , Miositis Osificante , Masculino , Humanos , Adolescente , Células Madre Pluripotentes Inducidas/metabolismo , Miositis Osificante/metabolismo , Factor 4 Similar a Kruppel , Diferenciación Celular/genética , Virus Sendai/genética , Reprogramación CelularRESUMEN
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.
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Paraplejía Espástica Hereditaria , Masculino , Humanos , Preescolar , Paraplejía Espástica Hereditaria/genética , Paraplejía Espástica Hereditaria/diagnóstico , Mutación , Paraplejía/genética , Exones , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Morquio B disease (MBD) is an ultra-rare lysosomal storage disease, which represents the relatively mild form of GLB1-associated disorders. In this article, we present the unique case of "pure" MBD associated with an insertion of the mobile genetic element from the class of retrotransposons. Using whole-genome sequencing (WGS), we identified an integration of the processed pseudogene NPM1 deep in the intron 5 of GLB1. The patient's mRNA analysis and the detailed functional analysis revealed the underlying molecular genetic mechanism of pathogenesis, which is an alteration of the GLB1 normal splicing. By co-expression of minigenes and antisense splice-modulating oligonucleotides (ASMOs), we demonstrated that pseudogene-derived splicing regulatory motifs contributed to an activation of the cryptic exon located 36 bp upstream of the integration site. Blocking the cryptic exon with ASMOs incorporated in the modified U7 small nuclear RNA (modU7snRNA) almost completely restored the wild-type splicing in the model cell line, that could be further extended toward the personalized genetic therapy. To our knowledge, this is the second reported case of the processed pseudogene insertion for monogenic disorders. Our data emphasizes the unique role of WGS in identification of such rare and probably underrepresented in literature types of disease-associated genetic variants.
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Skin fibroblasts obtained from a 20-year-old woman with clinically manifested and genetically proven (F508del/CFTRdele2.3) cystic fibrosis were successfully transformed into induced pluripotent stem cells (iPSCs) by using Sendai virus-based reprogramming vectors including the four Yamanaka factors, OCT3/4, SOX2, KLF4, and c-MYC. The iPSCs showed a normal karyotype, expressed pluripotency markers and exhibited the potential to differentiate into three germ layers in spontaneous differentiation assay. This iPSC line may be used for development of a personalized treatment including genome editing, disease modelling, cell differentiation and organoid formation, pharmacological investigations and drug screening.
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Fibrosis Quística , Células Madre Pluripotentes Inducidas , Adulto , Diferenciación Celular/genética , Reprogramación Celular , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Femenino , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación/genética , Adulto JovenRESUMEN
Induced pluripotent stem cells (iPSCs) was successfully generated from skin fibroblast obtained from patient with cystic fibrosis by using non-integrating, viral CytoTune™-iPS 2.0 Sendai Reprogramming Kit, which contain three vectors preparation: polycistronic Klf4-Oct3/4-Sox2, cMyc, and Klf4. Created iPSC lines showed a normal karyotype, expressed pluripotency markers and demonstrated the potential to differentiate into three germ layers in spontaneous differentiation assay.
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Fibrosis Quística , Células Madre Pluripotentes Inducidas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Mutación , Diferenciación Celular , Fibroblastos/metabolismoRESUMEN
Niemann-Pick disease type C (NP-C) (OMIM#257220) is a rare lysosomal storage disorder caused by pathogenic variants in either the NPC1 or NPC2 genes. It manifests with a wide spectrum of clinical symptoms and variable age of onset. We studied the impact of the frequent polymorphic variant c.2793 C > T (p.Asn931 = ), located in the donor splice site (SS) of NPC1 exon 18 on the penetrance of the rare synonymous variant c.2727 C > T (p.Cys909 = ), identified in two 55 y.o. twins with an adult onset form of NP-C. The patients' diagnosis was supported by biochemical analysis and positive filipin test. Analysis of the patients' cDNA showed that the c.2727 C > T variant leads to cryptic donor SS activation and frameshift deletion in the NPC1 exon 18. However, the minigene assay demonstrated that this exon shortening takes place only in the presence of the frequent polymorphic variant c.2793 C > T. Results of the transcript specific qPCR showed that only the presence in the NPC1 exon 18 of both variants leads to significant decrease of wild type (WT) transcript isoform.
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Enfermedad de Niemann-Pick Tipo C/genética , Penetrancia , Mutación Puntual , Sitios de Empalme de ARN , Células Cultivadas , Fibroblastos/metabolismo , Mutación del Sistema de Lectura , Humanos , Persona de Mediana Edad , Proteína Niemann-Pick C1/genética , Proteína Niemann-Pick C1/metabolismo , Enfermedad de Niemann-Pick Tipo C/patología , Polimorfismo de Nucleótido Simple , Gemelos DicigóticosRESUMEN
Introduction: Pathogenic variants in the pyruvate carboxylase (PC) gene cause a wide spectrum of recessive phenotypes, ranging from the early-onset fatal encephalopathy to the adult-onset benign form. Results: Patient 1 is a 6 y.o. boy with ataxia, hypoglycemia and episodes of lactic acidosis. WGS revealed the novel heterozygous missense variant c.1372A > G (p.Asn458Asp) in the PC gene. Additional analysis revealed discordant reads mapped to chromosomes 11 and 1, so a reciprocal translocation disrupted the PC gene was suspected. The translocation was validated via FISH-analysis and Sanger sequencing of its boundaries.Patient 2 is a 13 y.o. girl with psychomotor delay, episodes of lactic acidosis and ketonuria. WES revealed the novel homozygous intronic variant c.1983-116C > T. The PC's mRNA analysis demonstrated the exonization of several intron 16 sequences and some residual amount of WT mRNA isoform.Two other patients had more severe course of the disease. Their genotype represents missense variants in compound heterozygous and homozygous state (c.1876C > T (p.Arg626Trp), c.2606G > C (p.Gly869Ala), c.2435C > A (p.Ala812Asp). Conclusion: In patients with metabolic crises, lactic acidosis and hypoglycemia analysis of PC gene is recommended. WGS with deep bioinformatic analysis should be taken into consideration when none or the only one pathogenic variant in the PC gene is found.
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Skin fibroblasts obtained from a 28-year-old man with clinically manifested and genetically proven (F508del/W1282X) cystic fibrosis were successfully transformed into induced pluripotent stem cells (iPSCs) by using non-viral, non-integrating, self-replicating RNA reprogramming vectorthat contains five reprogramming factors: OCT4, KLF4, SOX2, GLIS1, and c-MYC as well as a puromycin-resistance gene. Two iPSC lines showed a normal karyotype, expressed pluripotency markers and exhibited the potential to differentiate into three germ layers in spontaneous differentiation assay. These iPSC lines may be subsequently used for development of a personalized etiotropic treatment,disease modelling, cell differentiation and organoid formation, pharmacological investigations and drug screening.
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Fibrosis Quística , Células Madre Pluripotentes Inducidas , Adulto , Diferenciación Celular , Reprogramación Celular , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibroblastos , Humanos , Factor 4 Similar a Kruppel , Masculino , MutaciónRESUMEN
Pitt-Hopkins syndrome is a rare neurodevelopment disorder caused by haploinsufficiency of the transcription factor 4 (TCF4). The main clinical symptoms of Pitt-Hopkins syndrome are severe development delay, intellectual disability, characteristic facial phenotype, and breathing abnormalities, including episodic hyperventilation. Different pathogenic variants can lead to Pitt-Hopkins syndrome. The most common are large deletions at 18q21 encompassing the TCF4 gene and frameshifting/nonsense single nucleotide variants. However, variants in noncoding regions can also lead to Pitt-Hopkins syndrome by disrupting the normal pre-mRNA splicing machinery. Here we describe three patients with Pitt-Hopkins syndrome caused by a large deletion in chromosome 18, a nonsense variant, and a novel variant located in intron 11 of TCF4 c.922+5G > A. Using RT-PCR analysis and minigene splicing assay we showed that this intronic variant leads to exon 11 skipping resulting in a formation of a premature stop codon. To our knowledge, this is the first functional annotation of a splicing variant in Pitt-Hopkins syndrome.
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Haploinsuficiencia , Hiperventilación/genética , Discapacidad Intelectual/genética , Factor de Transcripción 4/genética , Preescolar , Deleción Cromosómica , Cromosomas Humanos Par 18/genética , Codón sin Sentido , Facies , Femenino , Células HEK293 , Humanos , Hiperventilación/patología , Lactante , Discapacidad Intelectual/patología , Factor de Transcripción 4/metabolismoRESUMEN
Skin fibroblasts obtained from a 27-year-old man with clinically manifested and genetically proven (F508del/F508del) cystic fibrosis were successfully transformed into induced pluripotent stem cells (iPSCs) by using Sendai virus-based reprogramming vectors including the four Yamanaka factors, OCT3/4, SOX2, KLF4, and c-MYC. The iPSCs showed a normal karyotype, expressed pluripotency markers and exhibited the potential to differentiate into three germ layers in spontaneous differentiation assay. This iPSC line may be subsequently used for development of a personalized etiotropic treatment including genome editing, and for disease modelling and drug screening.
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Fibrosis Quística , Células Madre Pluripotentes Inducidas , Adulto , Diferenciación Celular , Reprogramación Celular/genética , Fibrosis Quística/genética , Fibroblastos , Humanos , Factor 4 Similar a Kruppel , Masculino , Mutación , Virus SendaiRESUMEN
In this article we present clinical, molecular and biochemical investigations of three patients with LHON caused by rare point substitutions in mtDNA. One patient harbours the known mtDNA mutation (m.13513 G>A), the others have new variants (m.13379 A>G in MT-ND5 gene and m.14597 A>G in MT-ND6 gene, which has never been previously associated with LHON). NGS analysis of a whole mtDNA derived from patient's blood revealed a low mutation load (24%, 47%, 23% respectively). Our data, including family segregation analysis, measurement of reactive oxygen species (ROS) production and cytotoxic effect of paraquat and high-resolution respirometry, showed that nucleotide variant m.14597 A>G can be classified as pathogenic mutation.
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ADN Mitocondrial/genética , Heteroplasmia , Atrofia Óptica Hereditaria de Leber/genética , Mutación Puntual , Adulto , Células Cultivadas , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Herbicidas/farmacología , Humanos , Potencial de la Membrana Mitocondrial/fisiología , Paraquat/farmacología , Adulto JovenRESUMEN
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.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Edición Génica/métodos , Sistemas CRISPR-Cas , Células Cultivadas , Reparación del ADN , Humanos , Células Madre Pluripotentes Inducidas/metabolismoRESUMEN
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.
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Encefalopatías/genética , Epilepsia/genética , Genes Mitocondriales , Enfermedades Genéticas Congénitas/genética , Mutación Puntual , ARN Mitocondrial/genética , ARN de Transferencia de Fenilalanina/genética , Adolescente , Femenino , Humanos , Mitocondrias/genéticaRESUMEN
Here we present a case report of collagen VI related myopathy in a patient, 8â¯y.o. boy, with intermediate phenotype between severe Ullrich congenital muscular dystrophy and milder Bethlem myopathy. Whole exome sequencing revealed two novel single nucleotide variants in COL6A3 gene: paternal p.Glu2402Ter, resulting in premature translation termination codon and degradation of mRNA from this allele probably due to nonsense-mediated decay, and maternal p.Arg1660Cys leading to amino-acid substitution in N2-terminal domain. COL6A3 expression analysis of proband's fibroblasts reveals functional homozygosity of the latter variant. Paternal fibroblasts showed only WT allele expression, which could lead to a reduction in mature transcript level, while maternal fibroblasts expressed both alleles. Functional assay of immunofluorescent staining of COL6A3 protein in fibroblasts culture reveals profound changes in COL6A3 localization and reduction of protein level in studied cultures when comparing with the controls. This study not only broadens the allelic spectrum of pathogenic COL6A3 variants in myopathy but also gives an additional support to Ullrich congenital muscular dystrophy and Bethlem myopathy clinical continuum.