RESUMO
The objective of the study is to evaluate the evolving phenotype and genetic spectrum of patients with succinic semialdehyde dehydrogenase deficiency (SSADHD) in long-term follow-up. Longitudinal clinical and biochemical data of 22 pediatric and 9 adult individuals with SSADHD from the patient registry of the International Working Group on Neurotransmitter related Disorders (iNTD) were studied with in silico analyses, pathogenicity scores and molecular modeling of ALDH5A1 variants. Leading initial symptoms, with onset in infancy, were developmental delay and hypotonia. Year of birth and specific initial symptoms influenced the diagnostic delay. Clinical phenotype of 26 individuals (median 12 years, range 1.8-33.4 years) showed a diversifying course in follow-up: 77% behavioral problems, 76% coordination problems, 73% speech disorders, 58% epileptic seizures and 40% movement disorders. After ataxia, dystonia (19%), chorea (11%) and hypokinesia (15%) were the most frequent movement disorders. Involvement of the dentate nucleus in brain imaging was observed together with movement disorders or coordination problems. Short attention span (78.6%) and distractibility (71.4%) were the most frequently behavior traits mentioned by parents while impulsiveness, problems communicating wishes or needs and compulsive behavior were addressed as strongly interfering with family life. Treatment was mainly aimed to control epileptic seizures and psychiatric symptoms. Four new pathogenic variants were identified. In silico scoring system, protein activity and pathogenicity score revealed a high correlation. A genotype/phenotype correlation was not observed, even in siblings. This study presents the diversifying characteristics of disease phenotype during the disease course, highlighting movement disorders, widens the knowledge on the genotypic spectrum of SSADHD and emphasizes a reliable application of in silico approaches.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Fenótipo , Succinato-Semialdeído Desidrogenase , Humanos , Succinato-Semialdeído Desidrogenase/deficiência , Succinato-Semialdeído Desidrogenase/genética , Criança , Masculino , Feminino , Pré-Escolar , Adulto , Erros Inatos do Metabolismo dos Aminoácidos/genética , Lactente , Adolescente , Adulto Jovem , Deficiências do Desenvolvimento/genética , Transtornos dos Movimentos/genética , Mutação , Hipotonia Muscular/genéticaRESUMO
Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine biosynthesis catalyzing the tetrahydrobiopterin (BH4 )-dependent hydroxylation of tyrosine to L-DOPA. Here, we analyzed 25 TH variants associated with various degrees of dopa-responsive dystonia and evaluate the effect of each variant on protein stability, activity and cellular localization. Furthermore, we investigated the physical interaction between TH and human wildtype (wt) GTP cyclohydrolase 1 (GTPCH) and the effect of variants on this interaction. Our in vitro results classify variants according to their resistance to proteinase K digestion into three groups (stable, intermediate, unstable). Based on their cellular localization, two groups of variants can be identified, variant group one with cytoplasmic distribution and variant group two forming aggregates. These aggregates do not correlate with loss of enzymatic activity but nevertheless might be a good target for molecular chaperones. Unfortunately, no obvious correlation between the half-life of a variant and its enzymatic activity or between solubility, stability and enzymatic activity of a given variant could be found. Excitingly, some variants disrupt the physical interaction between TH and human wildtype GTPCH, thereby interfering with enzymatic activity and offering new druggable targets for therapy. Taken together, our results highlight the importance of an in-depth molecular analysis of each variant in order to be able to classify groups of disease variants and to find specific therapies for each subgroup. Stand-alone in silico analyses predict less precise the effect of specific variants and should be combined with other in vitro analyses in cellular model systems.
RESUMO
PURPOSE: TNR, encoding Tenascin-R, is an extracellular matrix glycoprotein involved in neurite outgrowth and neural cell adhesion, proliferation and migration, axonal guidance, myelination, and synaptic plasticity. Tenascin-R is exclusively expressed in the central nervous system with highest expression after birth. The protein is crucial in the formation of perineuronal nets that ensheath interneurons. However, the role of Tenascin-R in human pathology is largely unknown. We aimed to establish TNR as a human disease gene and unravel the associated clinical spectrum. METHODS: Exome sequencing and an online matchmaking tool were used to identify patients with biallelic variants in TNR. RESULTS: We identified 13 individuals from 8 unrelated families with biallelic variants in TNR sharing a phenotype consisting of spastic para- or tetraparesis, axial muscular hypotonia, developmental delay, and transient opisthotonus. Four homozygous loss-of-function and four different missense variants were identified. CONCLUSION: We establish TNR as a disease gene for an autosomal recessive nonprogressive neurodevelopmental disorder with spasticity and transient opisthotonus and highlight the role of central nervous system extracellular matrix proteins in the pathogenicity of spastic disorders.
Assuntos
Espasticidade Muscular , Transtornos do Neurodesenvolvimento , Sistema Nervoso Central , Matriz Extracelular , Homozigoto , Humanos , Espasticidade Muscular/genética , Transtornos do Neurodesenvolvimento/genéticaRESUMO
Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare, monogenic disorder affecting the degradation of the main inhibitory neurotransmitter γ-amino butyric acid (GABA). Pathogenic variants in the ALDH5A1 gene that cause an enzymatic dysfunction of succinic semialdehyde dehydrogenase (SSADH) lead to an accumulation of potentially toxic metabolites, including γ-hydroxybutyrate (GHB). Here, we present a patient with a severe phenotype of SSADHD caused by a novel genetic variant c.728T > C that leads to an exchange of leucine to proline at residue 243, located within the highly conserved nicotinamide adenine dinucleotide (NAD)+ binding domain of SSADH. Proline harbors a pyrrolidine within its side chain known for its conformational rigidity and disruption of protein secondary structures. We investigate the effect of this novel variant in vivo, in vitro, and in silico. We furthermore examine the mutational spectrum of all previously described disease-causing variants and computationally assess all biologically possible missense variants of ALDH5A1 to identify mutational hotspots.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Simulação por Computador , Deficiências do Desenvolvimento , Mutação de Sentido Incorreto , Succinato-Semialdeído Desidrogenase/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Erros Inatos do Metabolismo dos Aminoácidos/genética , Substituição de Aminoácidos , Deficiências do Desenvolvimento/enzimologia , Deficiências do Desenvolvimento/genética , Células HEK293 , Humanos , Domínios Proteicos , Succinato-Semialdeído Desidrogenase/genética , Succinato-Semialdeído Desidrogenase/metabolismoRESUMO
Neurotransmitter deficiencies are rare neurological disorders with clinical onset during childhood. The disorders are caused by genetic defects in the enzymes involved in synthesis, degradation, or transport of neurotransmitters or by defects in the cofactor biosynthesis such as tetrahydrobiopterin (BH4). With the newly described DNAJC12 deficiency, a chaperon-associated neurotransmitter disorder, the pathophysiological spectrum has been broadened. All deficiencies result in a lack of monoamine neurotransmitters, especially dopamine and its products, with a subset leading to decreased levels of serotonin. Symptoms can occur already in the neonatal period. Classical signs are hypotonia, movement disorders, autonomous dysregulations, and impaired development. Diagnosis depends on quantitative detection of neurotransmitters in cerebrospinal fluid, since peripheral markers in blood or urine are less reliable. Treatment is based on supplementation of the missing neurotransmitter precursors or restoring deficient cofactors for endogenous enzymatic synthesis. In recent years, knowledge about this orphan group of diseases increased substantially among clinicians. However, the difficult task of integrating clinical symptoms and laboratory values still leads to a critical delay in diagnosis and therapy for patients. This review aims at enhancing the understanding of neurotransmitter disorders and should help practicing clinicians to choose useful diagnostic steps on the way to a valid diagnosis.
Assuntos
Transtornos dos Movimentos/diagnóstico , Transtornos dos Movimentos/metabolismo , Doenças do Sistema Nervoso/diagnóstico , Doenças do Sistema Nervoso/metabolismo , Neurotransmissores/deficiência , Animais , Dopamina/deficiência , Dopamina/uso terapêutico , Humanos , Transtornos dos Movimentos/tratamento farmacológico , Doenças do Sistema Nervoso/tratamento farmacológico , Neurotransmissores/uso terapêutico , Serotonina/deficiência , Serotonina/uso terapêuticoRESUMO
The ability to reprogram somatic cells to induced pluripotent stem cells (iPSCs) has revolutionized the way of modeling human disease. Especially for the modeling of rare human monogenetic diseases with limited numbers of patients available worldwide and limited access to the mostly affected tissues, iPSCs have become an invaluable tool. To study rare diseases affecting neurotransmitter biosynthesis and neurotransmission, stem cell models carrying patient-specific mutations have become highly important as most of the cell types present in the human brain and the central nervous system (CNS), including motoneurons, neurons, oligodendrocytes, astrocytes, and microglia, can be differentiated from iPSCs following distinct developmental programs. Differentiation can be performed using classical 2D differentiation protocols, thereby generating specific subtypes of neurons or glial cells in a dish. On the other side, 3D differentiation into "organoids" opened new ways to study misregulated developmental processes associated with rare neurological and neurometabolic diseases. For the analysis of defects in neurotransmission associated with rare neurometabolic diseases, different types of brain organoids have been made available during the last years including forebrain, midbrain and cerebral organoids. In this review, we illustrate reprogramming of somatic cells to iPSCs, differentiation in 2D and 3D, as well as already available disease-specific iPSC models, and discuss current and future applications of these techniques.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Erros Inatos do Metabolismo , Modelos Neurológicos , Animais , Diferenciação Celular , Técnicas de Cultura , Humanos , Microglia/citologia , Neurônios/citologia , Neurotransmissores/metabolismo , Oligodendroglia/citologiaRESUMO
LINE-1 (L1) retrotransposons are mobile genetic elements whose extensive proliferation resulted in the generation of ≈ 34% of the human genome. They have been shown to be a cause of single-gene diseases. Moreover, L1-encoded endonuclease can elicit double-strand breaks that may lead to genomic instability. Mammalian cells adopted strategies restricting mobility and deleterious consequences of uncontrolled retrotransposition. The human APOBEC3 protein family of polynucleotide cytidine deaminases contributes to intracellular defense against retroelements. APOBEC3 members inhibit L1 retrotransposition by 35-99%. However, genomic L1 retrotransposition events that occurred in the presence of L1-restricting APOBEC3 proteins are devoid of detectable G-to-A hypermutations, suggesting one or multiple deaminase-independent L1 restricting mechanisms. We set out to uncover the mechanism of APOBEC3C (A3C)-mediated L1 inhibition and found that it is deaminase independent, requires an intact dimerization site and the RNA-binding pocket mutation R122A abolishes L1 restriction by A3C. Density gradient centrifugation of L1 ribonucleoprotein particles, subcellular co-localization of L1-ORF1p and A3C and co-immunoprecipitation experiments indicate that an RNA-dependent physical interaction between L1 ORF1p and A3C dimers is essential for L1 restriction. Furthermore, we demonstrate that the amount of L1 complementary DNA synthesized by L1 reverse transcriptase is reduced by ≈ 50% if overexpressed A3C is present.
Assuntos
Citidina Desaminase/metabolismo , Elementos Nucleotídeos Longos e Dispersos , Proteínas/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas de Transporte/análise , Citidina Desaminase/química , Citidina Desaminase/genética , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/enzimologia , DNA Helicases , Células HeLa , Humanos , Mutação , Proteínas de Ligação a Poli-ADP-Ribose , Multimerização Proteica , Proteínas/análise , Proteínas/química , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNARESUMO
SINE-VNTR-Alu (SVA) elements are non-autonomous, hominid-specific non-LTR retrotransposons and distinguished by their organization as composite mobile elements. They represent the evolutionarily youngest, currently active family of human non-LTR retrotransposons, and sporadically generate disease-causing insertions. Since preexisting, genomic SVA sequences are characterized by structural hallmarks of Long Interspersed Elements 1 (LINE-1, L1)-mediated retrotransposition, it has been hypothesized for several years that SVA elements are mobilized by the L1 protein machinery in trans. To test this hypothesis, we developed an SVA retrotransposition reporter assay in cell culture using three different human-specific SVA reporter elements. We demonstrate that SVA elements are mobilized in HeLa cells only in the presence of both L1-encoded proteins, ORF1p and ORF2p. SVA trans-mobilization rates exceeded pseudogene formation frequencies by 12- to 300-fold in HeLa-HA cells, indicating that SVA elements represent a preferred substrate for L1 proteins. Acquisition of an AluSp element increased the trans-mobilization frequency of the SVA reporter element by ~25-fold. Deletion of (CCCTCT)(n) repeats and Alu-like region of a canonical SVA reporter element caused significant attenuation of the SVA trans-mobilization rate. SVA de novo insertions were predominantly full-length, occurred preferentially in G+C-rich regions, and displayed all features of L1-mediated retrotransposition which are also observed in preexisting genomic SVA insertions.
Assuntos
Endonucleases/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Retroelementos , Ribonucleoproteínas/metabolismo , Elementos Alu , Genes Reporter , Engenharia Genética , Células HeLa , Humanos , Transdução GenéticaRESUMO
Brain organoids represent a useful tool for modeling of neurodevelopmental disorders and can recapitulate brain volume alterations such as microcephaly. To monitor organoid growth, brightfield microscopy images are frequently used and evaluated manually which is time-consuming and prone to observer-bias. Recent software applications for organoid evaluation address this issue using classical or AI-based methods. These pipelines have distinct strengths and weaknesses that are not evident to external observers. We provide a dataset of more than 1,400 images of 64 trackable brain organoids from four clones differentiated from healthy and diseased patients. This dataset is especially powerful to test and compare organoid analysis pipelines because of (1) trackable organoids (2) frequent imaging during development (3) clone diversity (4) distinct clone development (5) cross sample imaging by two different labs (6) common imaging distractors, and (6) pixel-level ground truth organoid annotations. Therefore, this dataset allows to perform differentiated analyses to delineate strengths, weaknesses, and generalizability of automated organoid analysis pipelines as well as analysis of clone diversity and similarity.
Assuntos
Encéfalo , Organoides , Organoides/citologia , Encéfalo/diagnóstico por imagem , Encéfalo/citologia , HumanosRESUMO
Cerebral organoids recapitulate the structure and function of the developing human brain in vitro, offering a large potential for personalized therapeutic strategies. The enormous growth of this research area over the past decade with its capability for clinical translation makes a non-invasive, automated analysis pipeline of organoids highly desirable. This work presents a novel non-invasive approach to monitor and analyze cerebral organoids over time using high-field magnetic resonance imaging and state-of-the-art tools for automated image analysis. Three specific objectives are addressed, (I) organoid segmentation to investigate organoid development over time, (II) global cysticity classification and (III) local cyst segmentation for organoid quality assessment. We show that organoid growth can be monitored reliably over time and cystic and non-cystic organoids can be separated with high accuracy, with on par or better performance compared to state-of-the-art tools applied to brightfield imaging. Local cyst segmentation is feasible but could be further improved in the future. Overall, these results highlight the potential of the pipeline for clinical application to larger-scale comparative organoid analysis.
Assuntos
Cistos , Organoides , Humanos , Organoides/patologia , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Cistos/patologia , Inteligência ArtificialRESUMO
TUBB2A tubulinopathy is a rare neurodevelopmental disorder with developmental delay, epilepsy, and less frequent malformations of cortical development compared to other tubulinopathies. Peripheral blood mononuclear cells (PBMCs) from a male subject harboring the heterozygous de novo TUBB2A variant c.[743C>T] (p.[Ala248Val]) were reprogrammed to induced pluripotent stem cells (iPSCs) using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Invitrogen). Generated iPSCs showed a normal karyotype, expression of pluripotency markers, spontaneous in vitro differentiation in all three germ layers, and are a suitable human disease model to analyze pathomechanisms underlying TUBB2A tubulinopathy and potential therapeutic targets.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Masculino , Diferenciação Celular , Reprogramação Celular , Heterozigoto , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Leucócitos Mononucleares/metabolismo , Tubulina (Proteína)RESUMO
Variants in different neuronal tubulin isotypes cause severe neurodevelopmental disorders with cerebral malformations accompanied by developmental delay, motor impairment, and epilepsy, known as tubulinopathies. Induced pluripotent stem cells were generated from peripheral blood mononuclear cells from a female subject carrying the heterozygous de novo variant c.[521C > T] (p.[Ala174Val]) in the TUBA1A gene. PBMCs were reprogrammed using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Invitrogen) and showed a normal karyotype, expression of pluripotency markers, and spontaneous in vitro differentiation into all three germ layers. The generated iPSCs represent a useful tool to study the pathophysiology of TUBA1A tubulinopathy.
Assuntos
Células-Tronco Pluripotentes Induzidas , Diferenciação Celular/fisiologia , Reprogramação Celular , Diástase Muscular , Feminino , Heterozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Mutations in the PKHD1 gene, encoding for the ciliary protein fibrocystin, play a major role in the cystogenesis in autosomal recessive polycystic kidney disease (ARPKD), a severe pediatric kidney disorder. Peripheral blood mononuclear cells (PBMCs) from a female patient carrying a compound heterozygous PKHD1 mutation (c.6331A>G(;)7717C>T) were obtained and reprogrammed by viral transduction using the Cytotune®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen). The resulting iPSCs display a normal karyotype, express pluripotency markers, and show the potential for spontaneous differentiation in vitro, offering a useful tool for studying ARPKD pathomechanisms and drug screening.
RESUMO
Autosomal recessive polycystic kidney disease (ARPKD) is a severe pediatric kidney disorder primarily caused by mutations in the fibrocystin-encoding PKHD1 gene. It is characterized by the progressive development of cysts, eventually leading to renal failure. In order to create patient specific iPSCs, peripheral blood mononuclear cells (PBMCs) from a female patient carrying a homozygous PKHD1 mutation (c.8285A>T(;)(8285A>T)) were reprogrammed using the non-integral Cytotune®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen). Morphology and karyotype of the cells are normal. Pluripotency hallmarks as well as the potential to spontaneously differentiate into all three germ layers were shown by immunofluorescence staining and RT-PCR.
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Succinic semialdehyde dehydrogenase deficiency (SSADH-D) is a genetic disorder that results from the aberrant metabolism of the neurotransmitter γ-amino butyric acid (GABA). The disease is caused by impaired activity of the mitochondrial enzyme succinic semialdehyde dehydrogenase. SSADH-D manifests as varying degrees of mental retardation, autism, ataxia, and epileptic seizures, but the clinical picture is highly heterogeneous. So far, there is no approved curative therapy for this disease. In this review, we briefly summarize the molecular genetics of SSADH-D, the past and ongoing clinical trials, and the emerging features of the molecular pathogenesis, including redox imbalance and mitochondrial dysfunction. The main aim of this review is to discuss the potential of further therapy approaches that have so far not been tested in SSADH-D, such as pharmacological chaperones, read-through drugs, and gene therapy. Special attention will also be paid to elucidating the role of patient advocacy organizations in facilitating research and in the communication between researchers and patients.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/tratamento farmacológico , Erros Inatos do Metabolismo dos Aminoácidos/genética , Deficiências do Desenvolvimento/tratamento farmacológico , Deficiências do Desenvolvimento/genética , Terapia de Reposição de Enzimas/métodos , Terapia Genética/métodos , Terapia de Alvo Molecular/métodos , Succinato-Semialdeído Desidrogenase/deficiência , Adulto , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Erros Inatos do Metabolismo dos Aminoácidos/fisiopatologia , Animais , Criança , Ensaios Clínicos como Assunto , Deficiências do Desenvolvimento/enzimologia , Deficiências do Desenvolvimento/fisiopatologia , Modelos Animais de Doenças , Humanos , Camundongos , Mutação , Succinato-Semialdeído Desidrogenase/química , Succinato-Semialdeído Desidrogenase/genética , Ácido gama-Aminobutírico/metabolismoRESUMO
Inborn errors of monoamine neurotransmitter biosynthesis and degradation belong to the rare inborn errors of metabolism. They are caused by monogenic variants in the genes encoding the proteins involved in (1) neurotransmitter biosynthesis (like tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC)), (2) in tetrahydrobiopterin (BH4) cofactor biosynthesis (GTP cyclohydrolase 1 (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), sepiapterin reductase (SPR)) and recycling (pterin-4a-carbinolamine dehydratase (PCD), dihydropteridine reductase (DHPR)), or (3) in co-chaperones (DNAJC12). Clinically, they present early during childhood with a lack of monoamine neurotransmitters, especially dopamine and its products norepinephrine and epinephrine. Classical symptoms include autonomous dysregulations, hypotonia, movement disorders, and developmental delay. Therapy is predominantly based on supplementation of missing cofactors or neurotransmitter precursors. However, diagnosis is difficult and is predominantly based on quantitative detection of neurotransmitters, cofactors, and precursors in cerebrospinal fluid (CSF), urine, and blood. This review aims at summarizing the diverse analytical tools routinely used for diagnosis to determine quantitatively the amounts of neurotransmitters and cofactors in the different types of samples used to identify patients suffering from these rare diseases.
Assuntos
Catecolaminas/análise , Erros Inatos do Metabolismo/metabolismo , Proteínas de Transporte de Neurotransmissores/metabolismo , Pterinas/análise , Animais , Humanos , Erros Inatos do Metabolismo/diagnósticoRESUMO
Skin fibroblasts were isolated from a male patient with DNAJC12 deficiency and reprogrammed to iPSCs using the Cytotune®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen). Two clones, DHMCi003-A and DHMCi003-B, were characterized for expression of pluripotency marker genes (Oct4, Nanog, Lin28, SSEA-4, TRA-1-60) and differentiated into all three germ layers using embryoid body (EB) formation. Karyotype of both clones was normal and presence of the homozygous mutation in the DNAJC12 gene was verified by PCR and Sanger sequencing. Both clones represent a useful tool to study the pathomechanisms underlying the deficiency.
Assuntos
Células Clonais , Células-Tronco Pluripotentes Induzidas , Proteínas Repressoras/genética , Diferenciação Celular , Técnicas de Reprogramação Celular , Fibroblastos , Genótipo , Homozigoto , Humanos , Cariótipo , Masculino , PeleRESUMO
Variants in SCYL1 can cause a syndrome with low γ-glutamyl-transferase cholestasis, acute liver failure, and neurodegeneration (CALFAN). The encoded protein is involved in intracellular trafficking between Golgi and ER, specific mechanisms are still to be elucidated. We reprogrammed fibroblasts of a 2â¯years old male patient with CALFAN Syndrome due to a homozygous nonsense variant in SCYL1 (c.[1882Câ¯>â¯T]; c.[1882Câ¯>â¯T]/p.[Gln628*]; p.[Gln628*]) and generated DHMCi005-A using the Cytotune®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen). Cells showed a normal karyotype. Pluripotency was proven using immunohistochemistry, RT-PCR, and flow cytometry. Differentiation into all germ layers was shown using the STEMdiff™ Trilineage Differentiation Kit (Stemcell Technologies).
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Diferenciação Celular , Reprogramação Celular , Proteínas de Ligação a DNA/genética , Fibroblastos/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Falência Hepática Aguda/genética , Mutação , Células Cultivadas , Pré-Escolar , Colestase/genética , Colestase/patologia , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Falência Hepática Aguda/patologia , Masculino , Degeneração Neural/genética , Degeneração Neural/patologia , gama-Glutamiltransferase/deficiênciaRESUMO
Fibroblasts of a patient with Infantile Liver Failure Syndrome 2 (OMIM #616483) due to a homozygous missense variant in the neuroblastoma amplified sequence gene (NBAS; c.[2708T>G]; c.[2708T>G]/p.[Leu903Arg]; p.[Leu903Arg]) were reprogrammed to iPSCs using the Cytotune®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen) delivering the reprogramming factors Oct3/4, Sox2, c-Myc and Klf4. Cells showed a normal karyotype. Pluripotency of DHMCi004-A was proven using immunohistochemistry, RT-PCR analysis, flow cytometry and differentiation into all three germ layers using the STEMdiff™ Trilineage Differentiation Kit (Stemcell Technologies). DHMCi004-A represents the first iPS-based cell model system to elucidate the pathomechanism underlying this disease.
Assuntos
Linhagem Celular , Doenças Genéticas Inatas , Células-Tronco Pluripotentes Induzidas , Falência Hepática , Mutação de Sentido Incorreto , Proteínas de Neoplasias , Pré-Escolar , Feminino , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Doenças Genéticas Inatas/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Fator 4 Semelhante a Kruppel , Falência Hepática/genética , Falência Hepática/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , SíndromeRESUMO
This Article contains an error in the author affiliations. The correct affiliation for author Ruchi Shukla is 'MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK', and is not 'Mater Research Institute - University of Queensland, TRI Building, Woolloongabba QLD 4102, Australia'.