RESUMO
Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An example of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy)1. Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons2. Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss3, interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1-deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance.
Assuntos
Autofagia , Estresse do Retículo Endoplasmático , Retículo Endoplasmático , Proteínas Ubiquitinadas , Ubiquitinação , Animais , Humanos , Camundongos , Autofagia/genética , Retículo Endoplasmático/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Ubiquitinadas/metabolismo , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologia , Membranas Intracelulares/metabolismoRESUMO
Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions1-3. Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus4,5. This suggests that mutations in disordered proteins may alter condensate properties and function6-8. Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans.
Assuntos
Nucléolo Celular , Proteína HMGB1 , Humanos , Arginina/genética , Arginina/metabolismo , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Nucléolo Celular/patologia , Proteína HMGB1/química , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Síndrome , Mutação da Fase de Leitura , Transição de FaseRESUMO
Mouse models are a critical tool for studying human diseases, particularly developmental disorders1. However, conventional approaches for phenotyping may fail to detect subtle defects throughout the developing mouse2. Here we set out to establish single-cell RNA sequencing of the whole embryo as a scalable platform for the systematic phenotyping of mouse genetic models. We applied combinatorial indexing-based single-cell RNA sequencing3 to profile 101 embryos of 22 mutant and 4 wild-type genotypes at embryonic day 13.5, altogether profiling more than 1.6 million nuclei. The 22 mutants represent a range of anticipated phenotypic severities, from established multisystem disorders to deletions of individual regulatory regions4,5. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibit changes in dozens of trajectories whereas others exhibit changes in only a few cell types. We also identify differences between widely used wild-type strains, compare phenotyping of gain- versus loss-of-function mutants and characterize deletions of topological associating domain boundaries. Notably, some changes are shared among mutants, suggesting that developmental pleiotropy might be 'decomposable' through further scaling of this approach. Overall, our findings show how single-cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.
Assuntos
Deficiências do Desenvolvimento , Embrião de Mamíferos , Mutação , Fenótipo , Análise da Expressão Gênica de Célula Única , Animais , Camundongos , Núcleo Celular/genética , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/patologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Mutação com Ganho de Função , Genótipo , Mutação com Perda de Função , Modelos Genéticos , Modelos Animais de DoençasRESUMO
Fast and reliable detection of patients with severe and heterogeneous illnesses is a major goal of precision medicine1,2. Patients with leukaemia can be identified using machine learning on the basis of their blood transcriptomes3. However, there is an increasing divide between what is technically possible and what is allowed, because of privacy legislation4,5. Here, to facilitate the integration of any medical data from any data owner worldwide without violating privacy laws, we introduce Swarm Learning-a decentralized machine-learning approach that unites edge computing, blockchain-based peer-to-peer networking and coordination while maintaining confidentiality without the need for a central coordinator, thereby going beyond federated learning. To illustrate the feasibility of using Swarm Learning to develop disease classifiers using distributed data, we chose four use cases of heterogeneous diseases (COVID-19, tuberculosis, leukaemia and lung pathologies). With more than 16,400 blood transcriptomes derived from 127 clinical studies with non-uniform distributions of cases and controls and substantial study biases, as well as more than 95,000 chest X-ray images, we show that Swarm Learning classifiers outperform those developed at individual sites. In addition, Swarm Learning completely fulfils local confidentiality regulations by design. We believe that this approach will notably accelerate the introduction of precision medicine.
Assuntos
Blockchain , Tomada de Decisão Clínica/métodos , Confidencialidade , Conjuntos de Dados como Assunto , Aprendizado de Máquina , Medicina de Precisão/métodos , COVID-19/diagnóstico , COVID-19/epidemiologia , Surtos de Doenças , Feminino , Humanos , Leucemia/diagnóstico , Leucemia/patologia , Leucócitos/patologia , Pneumopatias/diagnóstico , Aprendizado de Máquina/tendências , Masculino , Software , Tuberculose/diagnósticoRESUMO
ADGRL1 (latrophilin 1), a well-characterized adhesion G protein-coupled receptor, has been implicated in synaptic development, maturation, and activity. However, the role of ADGRL1 in human disease has been elusive. Here, we describe ten individuals with variable neurodevelopmental features including developmental delay, intellectual disability, attention deficit hyperactivity and autism spectrum disorders, and epilepsy, all heterozygous for variants in ADGRL1. In vitro, human ADGRL1 variants expressed in neuroblastoma cells showed faulty ligand-induced regulation of intracellular Ca2+ influx, consistent with haploinsufficiency. In vivo, Adgrl1 was knocked out in mice and studied on two genetic backgrounds. On a non-permissive background, mice carrying a heterozygous Adgrl1 null allele exhibited neurological and developmental abnormalities, while homozygous mice were non-viable. On a permissive background, knockout animals were also born at sub-Mendelian ratios, but many Adgrl1 null mice survived gestation and reached adulthood. Adgrl1-/- mice demonstrated stereotypic behaviors, sexual dysfunction, bimodal extremes of locomotion, augmented startle reflex, and attenuated pre-pulse inhibition, which responded to risperidone. Ex vivo synaptic preparations displayed increased spontaneous exocytosis of dopamine, acetylcholine, and glutamate, but Adgrl1-/- neurons formed synapses in vitro poorly. Overall, our findings demonstrate that ADGRL1 haploinsufficiency leads to consistent developmental, neurological, and behavioral abnormalities in mice and humans.
Assuntos
Transtorno do Espectro Autista , Deficiência Intelectual , Transtornos do Neurodesenvolvimento , Receptores Acoplados a Proteínas G , Receptores de Peptídeos , Adulto , Animais , Transtorno do Espectro Autista/genética , Modelos Animais de Doenças , Haploinsuficiência/genética , Humanos , Deficiência Intelectual/genética , Camundongos , Camundongos Knockout , Transtornos do Neurodesenvolvimento/genéticaRESUMO
Homozygous VPS50 variants have been previously described in two unrelated patients with a neurodevelopmental disorder with microcephaly, seizures and neonatal cholestasis. VPS50 encodes a subunit that is unique to the heterotetrameric endosome-associated recycling protein (EARP) complex. The other subunits of the EARP complex, such as VPS51, VPS52 and VPS53, are also shared by the Golgi-associated retrograde protein complex. We report on an 18-month-old female patient with biallelic VPS50 variants. She carried a paternally inherited heterozygous nonsense c.13A>T; p.(Lys5*) variant. By long-read genome sequencing, we characterised a structural variant with a 4.3 Mb inversion flanked by deletions at both breakpoints on the maternal allele. The ~428 kb deletion at the telomeric inversion breakpoint encompasses the entire VPS50 gene. We demonstrated a deficiency of VPS50 in patient-derived fibroblasts, confirming the loss-of-function nature of both VPS50 variants. VPS53 and VPS52 protein levels were significantly reduced and absent, respectively, in fibroblasts of the patient. These data show that VPS50 and/or EARP deficiency and the associated functional defects underlie the phenotype in patients with VPS50 pathogenic variants. The VPS50-related core phenotype comprises severe developmental delay, postnatal microcephaly, hypoplastic corpus callosum, neonatal low gamma-glutamyl transpeptidase cholestasis and failure to thrive. The disease is potentially fatal in early childhood.
RESUMO
PURPOSE: Neurodevelopmental disorders exhibit clinical and genetic heterogeneity, ergo manifest dysfunction in components of diverse cellular pathways; the precise pathomechanism for the majority remains elusive. METHODS: We studied 5 affected individuals from 3 unrelated families manifesting global developmental delay, postnatal microcephaly, and hypotonia. We used exome sequencing and prioritized variants that were subsequently characterized using immunofluorescence, immunoblotting, pulldown assays, and RNA sequencing. RESULTS: We identified biallelic variants in ZFTRAF1, encoding a protein of yet unknown function. Four affected individuals from 2 unrelated families segregated 2 homozygous frameshift variants in ZFTRAF1, whereas, in the third family, an intronic splice site variant was detected. We investigated ZFTRAF1 at the cellular level and signified it as a nucleocytoplasmic protein in different human cell lines. ZFTRAF1 was completely absent in the fibroblasts of 2 affected individuals. We also identified 110 interacting proteins enriched in mRNA processing and autophagy-related pathways. Based on profiling of autophagy markers, patient-derived fibroblasts show irregularities in the protein degradation process. CONCLUSION: Thus, our findings suggest that biallelic variants of ZFTRAF1 cause a severe neurodevelopmental disorder.
Assuntos
Mutação com Perda de Função , Microcefalia , Hipotonia Muscular , Transtornos do Neurodesenvolvimento , Linhagem , Humanos , Microcefalia/genética , Microcefalia/patologia , Hipotonia Muscular/genética , Hipotonia Muscular/patologia , Masculino , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Feminino , Pré-Escolar , Mutação com Perda de Função/genética , Alelos , Criança , Lactente , Sequenciamento do Exoma , Fibroblastos/metabolismo , Fibroblastos/patologia , Autofagia/genéticaRESUMO
The voltage-gated sodium channel NaV1.8 is prominently expressed in the soma and axons of small-caliber sensory neurons, and pathogenic variants of the corresponding gene SCN10A are associated with peripheral pain and autonomic dysfunction. While most disease-associated SCN10A variants confer gain-of-function properties to NaV1.8, resulting in hyperexcitability of sensory neurons, a few affect afferent excitability through a loss-of-function mechanism. Using whole-exome sequencing, we here identify a rare heterozygous SCN10A missense variant resulting in alteration p.V1287I in NaV1.8 in a patient with a 15-year history of progressively worsening temperature dysregulation in the distal extremities, particularly in the feet. Further symptoms include increasingly intensifying tingling and numbness in the fingers and increased sweating. To assess the impact of p.V1287I on channel function, we performed voltage-clamp recordings demonstrating that the alteration confers loss- and gain-of-function characteristics to NaV1.8 characterized by a right-shifted voltage dependence of channel activation and inactivation. Current-clamp recordings from transfected mouse dorsal root ganglion neurons further revealed that NaV1.8-V1287I channels broaden the action potentials of sensory neurons and increase their firing rates in response to depolarizing current stimulations, indicating a gain-of-function mechanism of the variant at the cellular level in a heterozygous setting. The data support the hypothesis that the properties of NaV1.8 p.V1287I are causative for the patient's symptoms and that nonpainful peripheral paresthesias should be considered part of the clinical spectrum of NaV1.8-associated disorders.
RESUMO
Chromosome conformation capture methods have identified subchromosomal structures of higher-order chromatin interactions called topologically associated domains (TADs) that are separated from each other by boundary regions. By subdividing the genome into discrete regulatory units, TADs restrict the contacts that enhancers establish with their target genes. However, the mechanisms that underlie partitioning of the genome into TADs remain poorly understood. Here we show by chromosome conformation capture (capture Hi-C and 4C-seq methods) that genomic duplications in patient cells and genetically modified mice can result in the formation of new chromatin domains (neo-TADs) and that this process determines their molecular pathology. Duplications of non-coding DNA within the mouse Sox9 TAD (intra-TAD) that cause female to male sex reversal in humans, showed increased contact of the duplicated regions within the TAD, but no change in the overall TAD structure. In contrast, overlapping duplications that extended over the next boundary into the neighbouring TAD (inter-TAD), resulted in the formation of a new chromatin domain (neo-TAD) that was isolated from the rest of the genome. As a consequence of this insulation, inter-TAD duplications had no phenotypic effect. However, incorporation of the next flanking gene, Kcnj2, in the neo-TAD resulted in ectopic contacts of Kcnj2 with the duplicated part of the Sox9 regulatory region, consecutive misexpression of Kcnj2, and a limb malformation phenotype. Our findings provide evidence that TADs are genomic regulatory units with a high degree of internal stability that can be sculptured by structural genomic variations. This process is important for the interpretation of copy number variations, as these variations are routinely detected in diagnostic tests for genetic disease and cancer. This finding also has relevance in an evolutionary setting because copy-number differences are thought to have a crucial role in the evolution of genome complexity.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Variações do Número de Cópias de DNA/genética , Doença/genética , Duplicação Gênica/genética , Animais , DNA/genética , Fácies , Feminino , Fibroblastos , Dedos/anormalidades , Deformidades Congênitas do Pé/genética , Expressão Gênica , Genômica , Deformidades Congênitas da Mão/genética , Masculino , Camundongos , Fenótipo , Fatores de Transcrição SOX9/genéticaRESUMO
Axonopathies are neurodegenerative disorders caused by axonal degeneration, affecting predominantly the longest neurons. Several of these axonopathies are caused by genetic defects in proteins involved in the shaping and dynamics of the endoplasmic reticulum (ER); however, it is unclear how these defects impinge on neuronal survival. Given its central and widespread position within a cell, the ER is a pivotal player in inter-organelle communication. Here, we demonstrate that defects in the ER fusion protein ATL3, which were identified in patients suffering from hereditary sensory and autonomic neuropathy, result in an increased number of ER-mitochondria contact sites both in HeLa cells and in patient-derived fibroblasts. This increased contact is reflected in higher phospholipid metabolism, upregulated autophagy and augmented Ca2+ crosstalk between both organelles. Moreover, the mitochondria in these cells display lowered motility, and the number of axonal mitochondria in neurons expressing disease-causing mutations in ATL3 is strongly decreased. These results underscore the functional interdependence of subcellular organelles in health and disease and show that disorders caused by ER-shaping defects are more complex than previously assumed.
Assuntos
Axônios/metabolismo , Retículo Endoplasmático/genética , GTP Fosfo-Hidrolases/genética , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Autofagia/genética , Axônios/patologia , Cálcio/metabolismo , Sinalização do Cálcio/genética , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Células HeLa , Neuropatias Hereditárias Sensoriais e Autônomas/metabolismo , Neuropatias Hereditárias Sensoriais e Autônomas/patologia , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Neurônios/metabolismo , Neurônios/patologiaRESUMO
The extensive clinical and genetic heterogeneity of congenital limb malformation calls for comprehensive genome-wide analysis of genetic variation. Genome sequencing (GS) has the potential to identify all genetic variants. Here we aim to determine the diagnostic potential of GS as a comprehensive one-test-for-all strategy in a cohort of undiagnosed patients with congenital limb malformations. We collected 69 cases (64 trios, 1 duo, 5 singletons) with congenital limb malformations with no molecular diagnosis after standard clinical genetic testing and performed genome sequencing. We also developed a framework to identify potential noncoding pathogenic variants. We identified likely pathogenic/disease-associated variants in 12 cases (17.4%) including four in known disease genes, and one repeat expansion in HOXD13. In three unrelated cases with ectrodactyly, we identified likely pathogenic variants in UBA2, establishing it as a novel disease gene. In addition, we found two complex structural variants (3%). We also identified likely causative variants in three novel high confidence candidate genes. We were not able to identify any noncoding variants. GS is a powerful strategy to identify all types of genomic variants associated with congenital limb malformation, including repeat expansions and complex structural variants missed by standard diagnostic approaches. In this cohort, no causative noncoding SNVs could be identified.
Assuntos
Heterogeneidade Genética , Proteínas de Homeodomínio/genética , Deformidades Congênitas dos Membros/genética , Mutação , Fatores de Transcrição/genética , Enzimas Ativadoras de Ubiquitina/genética , Sequência de Bases , Estudos de Coortes , Variações do Número de Cópias de DNA , Expressão Gênica , Testes Genéticos , Humanos , Lactente , Deformidades Congênitas dos Membros/metabolismo , Deformidades Congênitas dos Membros/patologia , Masculino , Linhagem , Fatores de Transcrição/deficiência , Enzimas Ativadoras de Ubiquitina/deficiência , Sequenciamento Completo do GenomaRESUMO
The muscle specific isoform of the supervillin protein (SV2), encoded by the SVIL gene, is a large sarcolemmal myosin II- and F-actin-binding protein. Supervillin (SV2) binds and co-localizes with costameric dystrophin and binds nebulin, potentially attaching the sarcolemma to myofibrillar Z-lines. Despite its important role in muscle cell physiology suggested by various in vitro studies, there are so far no reports of any human disease caused by SVIL mutations. We here report four patients from two unrelated, consanguineous families with a childhood/adolescence onset of a myopathy associated with homozygous loss-of-function mutations in SVIL. Wide neck, anteverted shoulders and prominent trapezius muscles together with variable contractures were characteristic features. All patients showed increased levels of serum creatine kinase but no or minor muscle weakness. Mild cardiac manifestations were observed. Muscle biopsies showed complete loss of large supervillin isoforms in muscle fibres by western blot and immunohistochemical analyses. Light and electron microscopic investigations revealed a structural myopathy with numerous lobulated muscle fibres and considerable myofibrillar alterations with a coarse and irregular intermyofibrillar network. Autophagic vacuoles, as well as frequent and extensive deposits of lipoproteins, including immature lipofuscin, were observed. Several sarcolemma-associated proteins, including dystrophin and sarcoglycans, were partially mis-localized. The results demonstrate the importance of the supervillin (SV2) protein for the structural integrity of muscle fibres in humans and show that recessive loss-of-function mutations in SVIL cause a distinctive and novel myopathy.
Assuntos
Proteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Doenças Musculares/genética , Doenças Musculares/patologia , Adolescente , Idade de Início , Autofagia , Criança , Feminino , Humanos , Mutação com Perda de Função , Masculino , Músculo Esquelético/patologia , Linhagem , Vacúolos/patologiaRESUMO
In pleiotropic diseases, multiple organ systems are affected causing a variety of clinical manifestations. Here, we report a pleiotropic disorder with a unique constellation of neurological, endocrine, exocrine, and haematological findings that is caused by biallelic MADD variants. MADD, the mitogen-activated protein kinase (MAPK) activating death domain protein, regulates various cellular functions, such as vesicle trafficking, activity of the Rab3 and Rab27 small GTPases, tumour necrosis factor-α (TNF-α)-induced signalling and prevention of cell death. Through national collaboration and GeneMatcher, we collected 23 patients with 21 different pathogenic MADD variants identified by next-generation sequencing. We clinically evaluated the series of patients and categorized the phenotypes in two groups. Group 1 consists of 14 patients with severe developmental delay, endo- and exocrine dysfunction, impairment of the sensory and autonomic nervous system, and haematological anomalies. The clinical course during the first years of life can be potentially fatal. The nine patients in Group 2 have a predominant neurological phenotype comprising mild-to-severe developmental delay, hypotonia, speech impairment, and seizures. Analysis of mRNA revealed multiple aberrant MADD transcripts in two patient-derived fibroblast cell lines. Relative quantification of MADD mRNA and protein in fibroblasts of five affected individuals showed a drastic reduction or loss of MADD. We conducted functional tests to determine the impact of the variants on different pathways. Treatment of patient-derived fibroblasts with TNF-α resulted in reduced phosphorylation of the extracellular signal-regulated kinases 1 and 2, enhanced activation of the pro-apoptotic enzymes caspase-3 and -7 and increased apoptosis compared to control cells. We analysed internalization of epidermal growth factor in patient cells and identified a defect in endocytosis of epidermal growth factor. We conclude that MADD deficiency underlies multiple cellular defects that can be attributed to alterations of TNF-α-dependent signalling pathways and defects in vesicular trafficking. Our data highlight the multifaceted role of MADD as a signalling molecule in different organs and reveal its physiological role in regulating the function of the sensory and autonomic nervous system and endo- and exocrine glands.
Assuntos
Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Deficiências do Desenvolvimento/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Doenças do Sistema Nervoso/genética , Humanos , Mutação , Fenótipo , Transporte Proteico/genética , Transdução de Sinais/genéticaRESUMO
The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and autophagy has an important role in this process. However, its underlying regulatory mechanisms remain unexplained. Here we show that members of the FAM134 reticulon protein family are ER-resident receptors that bind to autophagy modifiers LC3 and GABARAP, and facilitate ER degradation by autophagy ('ER-phagy'). Downregulation of FAM134B protein in human cells causes an expansion of the ER, while FAM134B overexpression results in ER fragmentation and lysosomal degradation. Mutant FAM134B proteins that cause sensory neuropathy in humans are unable to act as ER-phagy receptors. Consistently, disruption of Fam134b in mice causes expansion of the ER, inhibits ER turnover, sensitizes cells to stress-induced apoptotic cell death and leads to degeneration of sensory neurons. Therefore, selective ER-phagy via FAM134 proteins is indispensable for mammalian cell homeostasis and controls ER morphology and turnover in mice and humans.
Assuntos
Autofagia/fisiologia , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose , Biomarcadores/metabolismo , Linhagem Celular , Retículo Endoplasmático/química , Feminino , Deleção de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisossomos/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Fagossomos/metabolismo , Ligação Proteica , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologiaRESUMO
De novo pathogenic variants in CNOT3 have recently been reported in a developmental delay disorder (intellectual developmental disorder with speech delay, autism, and dysmorphic facies [IDDSADF, OMIM: #618672]). The patients present with a variable degree of developmental delay and behavioral problems. To date, all reported disease-causing variants occurred de novo and no parent-child transmission was observed. We report for the first time autosomal dominant transmissions of the CNOT3-associated developmental disorder in two unrelated families. The clinical characteristics in our patients match the IDDSADF features reported so far and suggest substantial variability of the phenotype within the same family.
Assuntos
Transtorno Autístico/genética , Deficiência Intelectual/genética , Transtornos do Desenvolvimento da Linguagem/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Transtorno Autístico/complicações , Transtorno Autístico/diagnóstico , Transtorno Autístico/diagnóstico por imagem , Criança , Pré-Escolar , Fácies , Feminino , Predisposição Genética para Doença , Humanos , Deficiência Intelectual/complicações , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/diagnóstico por imagem , Transtornos do Desenvolvimento da Linguagem/complicações , Transtornos do Desenvolvimento da Linguagem/diagnóstico , Transtornos do Desenvolvimento da Linguagem/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Fenótipo , Sequenciamento do Exoma , Adulto JovemRESUMO
Atlastins (ATLs) are membrane-bound GTPases involved in shaping of the endoplasmic reticulum (ER). Mutations in ATL1 and ATL3 cause spastic paraplegia and hereditary sensory neuropathy. We here show that the sensory neuropathy causing ATL3 Y192C mutation reduces the complexity of the tubular ER-network. ATL3 Y192C delays ER-export by reducing the number of ER exit sites, reduces autophagy, fragments the Golgi and causes malformation of the nucleus. In cultured primary neurons, ATL3 Y192C does not localize to the growing axon, resulting in axon growth deficits. Patient-derived fibroblasts possess a tubular ER with reduced complexity and have a reduced number of autophagosomes. The data suggest that the disease-causing ATL3 Y192C mutation affects multiple ER-related pathways, possibly as a consequence of the distorted ER morphology.
Assuntos
Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Animais , Autofagossomos , Autofagia , Axônios/metabolismo , Células Cultivadas , Retículo Endoplasmático/patologia , GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Neurônios/citologia , Neurônios/metabolismoRESUMO
Hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders, characterized by a progressive sensory neuropathy often complicated by ulcers and amputations, with variable motor and autonomic involvement. Several pathways have been implicated in the pathogenesis of neuronal degeneration in HSAN, while recent observations point to an emerging role of cytoskeleton organization and function. Here, we report novel biallelic mutations in the DST gene encoding dystonin, a large cytolinker protein of the plakin family, in an adult form of HSAN type VI. Affected individuals harbored the premature termination codon variant p.(Lys4330*) in trans with the p.(Ala203Glu) change affecting a highly conserved residue in an isoform-specific N-terminal region of dystonin. Functional studies showed defects in actin cytoskeleton organization and consequent delayed cell adhesion, spreading and migration, while recombinant p.Ala203Glu dystonin loses the ability to bind actin. Our data aid in the clinical and molecular delineation of HSAN-VI and suggest a central role for cell-motility and cytoskeletal defects in its pathogenesis possibly interfering with the neuronal outgrowth and guidance processes.
Assuntos
Citoesqueleto de Actina/patologia , Distonina/genética , Genes Recessivos , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Mutação/genética , Neurônios/metabolismo , Actinas/metabolismo , Adulto , Idoso , Sequência de Aminoácidos , Animais , Células COS , Adesão Celular , Movimento Celular , Chlorocebus aethiops , Derme/patologia , Distonina/química , Família , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Isoformas de Proteínas/genéticaRESUMO
Pain is necessary to alert us to actual or potential tissue damage. Specialized nerve cells in the body periphery, so called nociceptors, are fundamental to mediate pain perception and humans without pain perception are at permanent risk for injuries, burns and mutilations. Pain insensitivity can be caused by sensory neurodegeneration which is a hallmark of hereditary sensory and autonomic neuropathies (HSANs). Although mutations in several genes were previously associated with sensory neurodegeneration, the etiology of many cases remains unknown. Using next generation sequencing in patients with congenital loss of pain perception, we here identify bi-allelic mutations in the FLVCR1 (Feline Leukemia Virus subgroup C Receptor 1) gene, which encodes a broadly expressed heme exporter. Different FLVCR1 isoforms control the size of the cytosolic heme pool required to sustain metabolic activity of different cell types. Mutations in FLVCR1 have previously been linked to vision impairment and posterior column ataxia in humans, but not to HSAN. Using fibroblasts and lymphoblastoid cell lines from patients with sensory neurodegeneration, we here show that the FLVCR1-mutations reduce heme export activity, enhance oxidative stress and increase sensitivity to programmed cell death. Our data link heme metabolism to sensory neuron maintenance and suggest that intracellular heme overload causes early-onset degeneration of pain-sensing neurons in humans.
Assuntos
Proteínas de Membrana Transportadoras/genética , Degeneração Neural/genética , Estresse Oxidativo/genética , Dor/genética , Receptores Virais/genética , Apoptose/genética , Linhagem Celular , Exoma/genética , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Mutação da Fase de Leitura/genética , Heme/genética , Humanos , Imunoprecipitação , Masculino , Degeneração Neural/patologia , Nociceptores/metabolismo , Nociceptores/patologia , Dor/patologia , Cultura Primária de Células , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologiaRESUMO
Two polymorphisms in the promoter region of macrophage migration inhibitory factor (MIF) - rs755622 and rs5844572 - exhibit prognostic relevance in inflammatory diseases. The aim of this study was to investigate a correlation between these MIF promoter polymorphisms and the severity of hepatitis C virus (HCV)-induced liver fibrosis. Our analysis included two independent patient cohorts with HCV-induced liver fibrosis (504 and 443 patients, respectively). The genotype of the single nucleotide polymorphism (SNP) -173 G/C and the repeat number of the microsatellite polymorphism -794 CATT5-8 were determined in DNA samples and correlated with fibrosis severity. In the first cohort, homozygous carriers of the C allele in the rs755622 had lower fibrosis stages compared to heterozygous carriers or wild types (1.25 vs. 2.0 vs. 2.0; p = 0.03). Additionally, ≥7 microsatellite repeats were associated with lower fibrosis stages (Assuntos
Carcinoma Hepatocelular/genética
, Predisposição Genética para Doença
, Hepatite C Crônica/genética
, Oxirredutases Intramoleculares/genética
, Cirrose Hepática/genética
, Neoplasias Hepáticas/genética
, Fatores Inibidores da Migração de Macrófagos/genética
, Adulto
, Idoso
, Idoso de 80 Anos ou mais
, Alelos
, Carcinoma Hepatocelular/diagnóstico por imagem
, Carcinoma Hepatocelular/etiologia
, Carcinoma Hepatocelular/virologia
, Estudos de Coortes
, Técnicas de Imagem por Elasticidade
, Feminino
, Expressão Gênica
, Frequência do Gene
, Hepacivirus/patogenicidade
, Hepacivirus/fisiologia
, Hepatite C Crônica/complicações
, Hepatite C Crônica/diagnóstico por imagem
, Hepatite C Crônica/virologia
, Heterozigoto
, Homozigoto
, Humanos
, Fígado/metabolismo
, Fígado/patologia
, Fígado/virologia
, Cirrose Hepática/diagnóstico por imagem
, Cirrose Hepática/etiologia
, Cirrose Hepática/virologia
, Neoplasias Hepáticas/diagnóstico por imagem
, Neoplasias Hepáticas/etiologia
, Neoplasias Hepáticas/virologia
, Masculino
, Repetições de Microssatélites
, Pessoa de Meia-Idade
, Polimorfismo Genético
, Regiões Promotoras Genéticas
, Índice de Gravidade de Doença
RESUMO
BACKGROUND: The causes for neuropathic pain are manifold and remain unexplained in the majority of cases. In recent years a growing number of pain syndromes have been attributed to mutations in genes encoding voltage-gated sodium channels. Hence, this group of rare diseases should be considered in the differential diagnostics of neuropathic pain. MATERIAL AND METHODS: Evaluation of topic-related literature and discussion of own experiences as well as consideration of current guidelines. RESULTS: Alterations in the electrical excitability of nociceptive neurons by pathogenic mutations in sodium channels lead to disease patterns, such as small fiber neuropathy and various pain syndromes. This article summarizes the knowledge on these genetic diseases and discusses the differential diagnosis of neuropathic pain. Current treatment concepts are presented and the predominantly experimental approaches to targeted modulation of sodium channels are discussed. CONCLUSION: The treatment of patients with chronic neuropathic pain requires interdisciplinary cooperation and is often difficult due to an unsatisfactory treatment response. Increasing knowledge on rare genetically determined channelopathies can contribute to the development of novel pharmaceuticals since ion channels are central players in the processing of pain.