RESUMO
PURPOSE: Epigenetic dysregulation has been associated with many inherited disorders. RBBP5 (HGNC:9888) encodes a core member of the protein complex that methylates histone 3 lysine-4 (H3K4) and has not been implicated in human disease. METHODS: We identify five unrelated individuals with de novo heterozygous variants in RBBP5. Three nonsense/frameshift and two missense variants were identified in probands with neurodevelopmental symptoms including global developmental delay, intellectual disability, microcephaly, and short stature. Here, we investigate the pathogenicity of the variants through protein structural analysis and transgenic Drosophila models. RESULTS: Both missense p.(T232I) and p.(E296D) variants affect evolutionarily conserved amino acids located at the interface between RBBP5 and the nucleosome. In Drosophila, overexpression analysis identifies partial loss-of-function mechanisms when the variants are expressed using the fly Rbbp5 or human RBBP5 cDNA. Loss of Rbbp5 leads to a reduction in brain size. The human reference or variant transgenes fail to rescue this loss and expression of either missense variant in an Rbbp5 null background results in a less severe microcephaly phenotype than the human reference, indicating both missense variants are partial loss-of-function alleles. CONCLUSION: Haploinsufficiency of RBBP5 observed through de novo null and hypomorphic loss-of-function variants is associated with a syndromic neurodevelopmental disorder.
RESUMO
PURPOSE: We investigated the value of transcriptome sequencing (RNAseq) in ascertaining the consequence of DNA variants on RNA transcripts to improve the diagnostic rate from exome or genome sequencing for undiagnosed Mendelian diseases spanning a wide spectrum of clinical indications. METHODS: From 234 subjects referred to the Undiagnosed Diseases Network, University of California-Los Angeles clinical site between July 2014 and August 2018, 113 were enrolled for high likelihood of having rare undiagnosed, suspected genetic conditions despite thorough prior clinical evaluation. Exome or genome sequencing and RNAseq were performed, and RNAseq data was integrated with genome sequencing data for DNA variant interpretation genome-wide. RESULTS: The molecular diagnostic rate by exome or genome sequencing was 31%. Integration of RNAseq with genome sequencing resulted in an additional seven cases with clear diagnosis of a known genetic disease. Thus, the overall molecular diagnostic rate was 38%, and 18% of all genetic diagnoses returned required RNAseq to determine variant causality. CONCLUSION: In this rare disease cohort with a wide spectrum of undiagnosed, suspected genetic conditions, RNAseq analysis increased the molecular diagnostic rate above that possible with genome sequencing analysis alone even without availability of the most appropriate tissue type to assess.
Assuntos
Doenças Genéticas Inatas/diagnóstico , Patologia Molecular , Doenças Raras/diagnóstico , Transcriptoma/genética , Exoma/genética , Doenças Genéticas Inatas/genética , Testes Genéticos/normas , Humanos , Mutação/genética , RNA-Seq/normas , Doenças Raras/genética , Análise de Sequência de DNA/normas , Sequenciamento do Exoma/normas , Sequenciamento Completo do Genoma/normasRESUMO
In recent years, volatile fatty acid (VFA) production through anaerobic fermentation of sewage sludge, instead of methane production, has been regarded as a high-value and promising roadmap for sludge stabilization and resource recovery. This review first presents the effects of some essential factors that influence VFA production and composition. In the second part, we present an extensive analysis of conventional pretreatment and co-fermentation strategies ultimately addressed to improving VFA production and composition. Also, the effectiveness of these approaches is summarized in terms of sludge degradation, hydrolysis rate, and VFA production and composition. According to published studies, it is concluded that some pretreatments such as alkaline and thermal pretreatment are the most effective ways to enhance VFA production from sewage sludge. The possible reasons for the improvement of VFA production by different methods are also discussed. Finally, this review also highlights several current technical challenges and opportunities in VFA production with spectrum control, and further related research is proposed.
Assuntos
Ácidos Graxos Voláteis , Fermentação , Eliminação de Resíduos Líquidos/métodos , Anaerobiose , Hidrólise , Metano , EsgotosRESUMO
Disturbed mitochondrial fusion and fission have been linked to various neurodegenerative disorders. In siblings from two unrelated families who died soon after birth with a profound neurodevelopmental disorder characterized by pontocerebellar hypoplasia and apnoea, we discovered a missense mutation and an exonic deletion in the SLC25A46 gene encoding a mitochondrial protein recently implicated in optic atrophy spectrum disorder. We performed functional studies that confirmed the mitochondrial localization and pro-fission properties of SLC25A46. Knockdown of slc24a46 expression in zebrafish embryos caused brain malformation, spinal motor neuron loss, and poor motility. At the cellular level, we observed abnormally elongated mitochondria, which was rescued by co-injection of the wild-type but not the mutant slc25a46 mRNA. Conversely, overexpression of the wild-type protein led to mitochondrial fragmentation and disruption of the mitochondrial network. In contrast to mutations causing non-lethal optic atrophy, missense mutations causing lethal congenital pontocerebellar hypoplasia markedly destabilize the protein. Indeed, the clinical severity appears inversely correlated with the relative stability of the mutant protein. This genotype-phenotype correlation underscores the importance of SLC25A46 and fine tuning of mitochondrial fission and fusion in pontocerebellar hypoplasia and central neurodevelopment in addition to optic and peripheral neuropathy across the life span.
Assuntos
Doenças Cerebelares/genética , Predisposição Genética para Doença/genética , Proteínas Mitocondriais/genética , Mutação/genética , Proteínas de Transporte de Fosfato/genética , Polimorfismo de Nucleotídeo Único/genética , Aminoácidos/genética , Animais , Animais Geneticamente Modificados , Encéfalo/anormalidades , Linhagem Celular Transformada , Células Cultivadas , Doenças Cerebelares/diagnóstico por imagem , Estudos de Coortes , Embrião não Mamífero , Feminino , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Dinâmica Mitocondrial/genética , Modelos Moleculares , Peixe-ZebraRESUMO
Autosomal dominant retinal vasculopathy with cerebral leukodystrophy is a microvascular endotheliopathy with middle-age onset. In nine families, we identified heterozygous C-terminal frameshift mutations in TREX1, which encodes a 3'-5' exonuclease. These truncated proteins retain exonuclease activity but lose normal perinuclear localization. These data have implications for the maintenance of vascular integrity in the degenerative cerebral microangiopathies leading to stroke and dementias.
Assuntos
Encefalopatias/genética , Exodesoxirribonucleases/genética , Mutação , Fosfoproteínas/genética , Doenças Retinianas/genética , Sequência de Aminoácidos , Encefalopatias/enzimologia , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Genes Dominantes , Predisposição Genética para Doença , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Doenças Retinianas/enzimologia , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
In Duchenne muscular dystrophy, dystrophin loss leads to chronic muscle damage, dysregulation of repair, fibro-fatty replacement, and weakness. We develop methodology to efficiently isolate individual nuclei from minute quantities of frozen skeletal muscle, allowing single nuclei sequencing of irreplaceable archival samples and from very small samples. We apply this method to identify cell and gene expression dynamics within human DMD and mdx mouse muscle, characterizing effects of dystrophin rescue by exon skipping therapy at single nuclei resolution. DMD exon 23 skipping events are directly observed and increased in myonuclei from treated mice. We describe partial rescue of type IIa and IIx myofibers, expansion of an MDSC-like myeloid population, recovery of repair/remodeling M2-macrophage, and repression of inflammatory POSTN1 + fibroblasts in response to exon skipping and partial dystrophin restoration. Use of this method enables exploration of cellular and transcriptomic mechanisms of dystrophin loss and repair within an intact muscle environment. Our initial findings will scaffold our future work to more directly examine muscular dystrophies and putative recovery pathways.
Assuntos
Distrofina , Distrofia Muscular de Duchenne , Animais , Distrofina/genética , Humanos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , TranscriptomaRESUMO
De novo truncations in Interferon Regulatory Factor 2 Binding Protein Like (IRF2BPL) lead to severe childhood-onset neurodegenerative disorders. To determine how loss of IRF2BPL causes neural dysfunction, we examined its function in Drosophila and zebrafish. Overexpression of either IRF2BPL or Pits, the Drosophila ortholog, represses Wnt transcription in flies. In contrast, neuronal depletion of Pits leads to increased wingless (wg) levels in the brain and is associated with axonal loss, whereas inhibition of Wg signaling is neuroprotective. Moreover, increased neuronal expression of wg in flies is sufficient to cause age-dependent axonal loss, similar to reduction of Pits. Loss of irf2bpl in zebrafish also causes neurological defects with an associated increase in wnt1 transcription and downstream signaling. WNT1 is also increased in patient-derived astrocytes, and pharmacological inhibition of Wnt suppresses the neurological phenotypes. Last, IRF2BPL and the Wnt antagonist, CKIα, physically and genetically interact, showing that IRF2BPL and CkIα antagonize Wnt transcription and signaling.
Assuntos
Proteínas de Drosophila , Animais , Proteínas de Transporte/metabolismo , Criança , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Humanos , Fator Regulador 2 de Interferon/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/genética , Via de Sinalização Wnt , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
This study investigated the combination of low-thermal and sodium dodecylbenzene sulfonate (SDBS) as a pretreatment method to improve the volatile fatty acids (VFA) production from waste activated sludge (WAS). The results showed that the maximum VFA yield of 320 ± 7.7 mg COD/g VS was obtained in the combined pretreatment (0.01 g SDBS/g TS + 70 °C for 60 min), which was 1.8, 1.7 and 4.0 times of that from sole low-thermal, sole SDBS and the control test. The mechanism study revealed the combined pretreatment had synergetic effect on enhancement of disintegration of WAS. Moreover, low-thermal exhibited greater performance on releasing organic matters, and SDBS accelerated hydrolysis and acidogenesis, thus contributing to the enhancement of VFA production in the combined pretreatment. The microbial community analysis demonstrated that the combined pretreatment increased the abundance of phyla Firmicutes which might be responsible for the improvement of VFA production.
Assuntos
Ácidos Graxos Voláteis , Esgotos , Derivados de Benzeno , Fermentação , Hidrólise , SódioRESUMO
Primary episodic ataxias (EAs) are a group of dominantly inherited disorders characterized by transient recurrent incoordination and truncal instability, often triggered by physical exertion and emotional stress, variably associated with progressive baseline ataxia. There are now eight designated subtypes based largely on genetic loci. Mutations have been identified in multiple individuals and families with EA1, EA2, and EA6, mostly with onset before adulthood. EA1 and EA2 are prototypical neurologic channelopathies. EA1 is caused by heterozygous mutations in KCNA1, which encodes the α1 subunit of a neuronal voltage-gated potassium channel, Kv1.1. EA2, the most common and best characterized, is caused by heterozygous mutations in CACNA1A, which encodes the α1A subunit of a neuronal voltage-gated calcium channel, Cav2.1. EA6 is caused by heterozygous mutations in SLC1A3, which encodes a subunit of a glial excitatory amino acid transporter, EAAT1. The other EA subtypes were defined in single families awaiting gene identification and further confirmation. This chapter focuses on the best-characterized EA syndromes, the clinical assessment and genetic diagnosis of EA, and the management of EA, as well as newly recognized allelic disorders that have greatly expanded the clinical spectrum of EA2. Illustrative cases are discussed, with a focus on sporadic patients with congenital features without episodic ataxia who present diagnostic and therapeutic challenges.
Assuntos
Ataxia/genética , Canal de Potássio Kv1.1/genética , Mutação/genética , Ataxia/classificação , Canalopatias/etiologia , Canalopatias/genética , Transportador 1 de Aminoácido Excitatório/genética , HumanosRESUMO
The familial episodic ataxias (EAs) are prototypical channelopathies in the central nervous system clinically characterized by attacks of imbalance and incoordination variably associated with progressive ataxia and variable interictal features. EA1, EA2, and EA6 are caused by mutations in ion channel- and transporter-encoding genes that regulate neuronal excitability and neurotransmission.
Assuntos
Ataxia/genética , Canalopatias/genética , Canais Iônicos/genética , Mutação/genética , Ataxia/fisiopatologia , HumanosRESUMO
SCL25A46 is a mitochondrial carrier protein that surprisingly localizes to the outer membrane and is distantly related to Ugo1. Here we show that a subset of SLC25A46 interacts with mitochondrial dynamics components and the MICOS complex. Decreased expression of SLC25A46 results in increased stability and oligomerization of MFN1 and MFN2 on mitochondria, promoting mitochondrial hyperfusion. A mutation at L341P causes rapid degradation of SLC25A46, which manifests as a rare disease, pontocerebellar hypoplasia. The E3 ubiquitin ligases MULAN and MARCH5 coordinate ubiquitylation of SLC25A46 L341P, leading to degradation by organized activities of P97 and the proteasome. Whereas outer mitochondrial membrane-associated degradation is typically associated with apoptosis or a specialized type of autophagy termed mitophagy, SLC25A46 degradation operates independently of activation of outer membrane stress pathways. Thus SLC25A46 is a new component in mitochondrial dynamics that serves as a regulator for MFN1/2 oligomerization. Moreover, SLC25A46 is selectively degraded from the outer membrane independently of mitophagy and apoptosis, providing a framework for mechanistic studies in the proteolysis of outer membrane proteins.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Dinâmica Mitocondrial/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Apoptose/fisiologia , Autofagia/fisiologia , Células HEK293 , Células HeLa , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
The zebrafish has significant advantages for studying the morphological development of the brain. However, little is known about the functional development of the zebrafish brain. We used patch clamp electrophysiology in live animals to investigate the emergence of excitability in cerebellar Purkinje cells, functional maturation of the cerebellar circuit, and establishment of sensory input to the cerebellum. Purkinje cells are born at 3 days post-fertilization (dpf). By 4 dpf, Purkinje cells spontaneously fired action potentials in an irregular pattern. By 5 dpf, the frequency and regularity of tonic firing had increased significantly and most cells fired complex spikes in response to climbing fiber activation. Our data suggest that, as in mammals, Purkinje cells are initially innervated by multiple climbing fibers that are winnowed to a single input. To probe the development of functional sensory input to the cerebellum, we investigated the response of Purkinje cells to a visual stimulus consisting of a rapid change in light intensity. At 4 dpf, sudden darkness increased the rate of tonic firing, suggesting that afferent pathways carrying visual information are already active by this stage. By 5 dpf, visual stimuli also activated climbing fibers, increasing the frequency of complex spiking. Our results indicate that the electrical properties of zebrafish and mammalian Purkinje cells are highly conserved and suggest that the same ion channels, Nav1.6 and Kv3.3, underlie spontaneous pacemaking activity. Interestingly, functional development of the cerebellum is temporally correlated with the emergence of complex, visually-guided behaviors such as prey capture. Because of the rapid formation of an electrically-active cerebellum, optical transparency, and ease of genetic manipulation, the zebrafish has great potential for functionally mapping cerebellar afferent and efferent pathways and for investigating cerebellar control of motor behavior.
Assuntos
Cerebelo/crescimento & desenvolvimento , Cerebelo/fisiologia , Células de Purkinje/fisiologia , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/fisiologia , Potenciais de Ação/fisiologia , Vias Aferentes/crescimento & desenvolvimento , Vias Aferentes/fisiologia , Animais , Animais Geneticamente Modificados , Canal de Sódio Disparado por Voltagem NAV1.6/metabolismo , Núcleo Olivar/crescimento & desenvolvimento , Núcleo Olivar/fisiologia , Técnicas de Patch-Clamp , Estimulação Luminosa , Canais de Potássio Shaw/metabolismo , Percepção Visual/fisiologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
RNA exosomes are multi-subunit complexes conserved throughout evolution and are emerging as the major cellular machinery for processing, surveillance and turnover of a diverse spectrum of coding and noncoding RNA substrates essential for viability. By exome sequencing, we discovered recessive mutations in EXOSC3 (encoding exosome component 3) in four siblings with infantile spinal motor neuron disease, cerebellar atrophy, progressive microcephaly and profound global developmental delay, consistent with pontocerebellar hypoplasia type 1 (PCH1; MIM 607596). We identified mutations in EXOSC3 in an additional 8 of 12 families with PCH1. Morpholino knockdown of exosc3 in zebrafish embryos caused embryonic maldevelopment, resulting in small brain size and poor motility, reminiscent of human clinical features, and these defects were largely rescued by co-injection with wild-type but not mutant exosc3 mRNA. These findings represent the first example of an RNA exosome core component gene that is responsible for a human disease and further implicate dysregulation of RNA processing in cerebellar and spinal motor neuron maldevelopment and degeneration.
Assuntos
Exossomos , Neurônios Motores , Degeneração Neural/genética , Atrofias Olivopontocerebelares/genética , Ponte/patologia , Proteínas de Ligação a RNA/genética , Nervos Espinhais , Animais , Cerebelo/patologia , Complexo Multienzimático de Ribonucleases do Exossomo , Técnicas de Silenciamento de Genes , Humanos , Degeneração Neural/patologia , Atrofias Olivopontocerebelares/patologia , RNA/análise , Nervos Espinhais/patologia , Peixe-Zebra/embriologiaRESUMO
Episodic ataxia (EA) syndromes are heritable diseases characterized by dramatic episodes of imbalance and incoordination. EA type 2 (EA2), the most common and the best characterized subtype, is caused by mostly nonsense, splice site, small indel, and sometimes missense mutations in CACNA1A. Direct sequencing of CACNA1A fails to identify mutations in some patients with EA2-like features, possibly due to incomplete interrogation of CACNA1A or defects in other EA genes not yet defined. Previous reports described genomic deletions between 4 and 40 kb in EA2. In 47 subjects with EA (26 with EA2-like features) who tested negative for mutations in the known EA genes, we used multiplex ligation-dependent probe amplification to analyze CACNA1A for exonic copy number variations. Breakpoints were further defined by long-range PCR. We identified distinct multi-exonic deletions in three probands with classic EA2-like features: episodes of prolonged vertigo and ataxia triggered by stress and fatigue, interictal nystagmus, with onset during infancy or early childhood. The breakpoints in all three probands are located in Alu sequences, indicating errors in homologous recombination of Alu sequences as the underlying mechanism. The smallest deletion spanned exons 39 and 40, while the largest deletion spanned 200 kb, missing all but the first three exons. One deletion involving exons 39 through 47 arose spontaneously. The search for mutations in CACNA1A appears most fruitful in EA patients with interictal nystagmus and onset early in life. The finding of large heterozygous deletions suggests haploinsufficiency as a possible pathomechanism of EA2.
RESUMO
BACKGROUND: Episodic ataxia (EA) is variably associated with additional neurologic symptoms. At least 4 genes have been implicated. Recently, a mutation in the SLC1A3 gene encoding the glutamate transporter EAAT1 was identified in a patient with severe episodic and progressive ataxia, seizures, alternating hemiplegia, and migraine headache. The mutant EAAT1 showed severely reduced uptake of glutamate. The syndrome was designated EA6 and shares overlapping clinical features with EA2, which is caused by mutations in CACNA1A. OBJECTIVE: To test the role of the SLC1A3 gene in EA. DESIGN: Genetic and functional studies. We analyzed the coding region of the SLC1A3 gene by direct sequencing. SETTING: Academic research. PATIENTS: DNA samples from 20 patients with EA (with or without interictal nystagmus) negative for CACNA1A mutations were analyzed. MAIN OUTCOME MEASURES: We identified 1 novel EAAT1 mutation in a family with EA and studied the functional consequences of this mutation using glutamate uptake assay. RESULTS: We identified a missense C186S mutation that segregated with EA in 3 family members. The mutant EAAT1 showed a modest but significant reduction of glutamate uptake. CONCLUSIONS: We broadened the clinical spectrum associated with SLC1A3 mutations to include milder manifestations of EA without seizures or alternating hemiplegia. The severity of EA6 symptoms appears to be correlated with the extent of glutamate transporter dysfunction.
Assuntos
Transportador 1 de Aminoácido Excitatório/genética , Predisposição Genética para Doença/genética , Ácido Glutâmico/metabolismo , Mutação/genética , Degenerações Espinocerebelares/genética , Degenerações Espinocerebelares/metabolismo , Adulto , Química Encefálica/genética , Cerebelo/metabolismo , Cerebelo/fisiopatologia , Análise Mutacional de DNA , Feminino , Marcadores Genéticos/genética , Testes Genéticos , Genótipo , Hemiplegia/genética , Hemiplegia/fisiopatologia , Humanos , Padrões de Herança/genética , Masculino , Pessoa de Meia-Idade , Transtornos de Enxaqueca/genética , Transtornos de Enxaqueca/fisiopatologia , Linhagem , Fenótipo , Convulsões/genética , Convulsões/fisiopatologia , Degenerações Espinocerebelares/fisiopatologiaRESUMO
We discovered intronic mutations in two episodic ataxia type 2 (EA2) families: a four-nucleotide GAGT deletion at IVS41+(3-6) and a single nucleotide insertion (insT) at IVS24+3. We expressed minigenes harboring the mutations in cell lines to demonstrate exon skipping from the deletion mutation and the activation of a cryptic splice donor site from the insertion mutation. The identification of these disease-causing mutations expands the spectrum of EA2 mutations and emphasizes the importance of intronic sequences in regulating gene expression.
Assuntos
Ataxia/genética , Íntrons , Mutação , Proteínas do Tecido Nervoso/genética , Adolescente , Adulto , Animais , Células COS , Canais de Cálcio Tipo L , Criança , Chlorocebus aethiops , Análise Mutacional de DNA/métodos , Éxons , Saúde da Família , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Splicing de RNA/fisiologia , RNA Nuclear Pequeno/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transfecção/métodosRESUMO
The mechanisms controlling axon guidance are of fundamental importance in understanding brain development. Growing corticospinal and somatosensory axons cross the midline in the medulla to reach their targets and thus form the basis of contralateral motor control and sensory input. The motor and sensory projections appeared uncrossed in patients with horizontal gaze palsy with progressive scoliosis (HGPPS). In patients affected with HGPPS, we identified mutations in the ROBO3 gene, which shares homology with roundabout genes important in axon guidance in developing Drosophila, zebrafish, and mouse. Like its murine homolog Rig1/Robo3, but unlike other Robo proteins, ROBO3 is required for hindbrain axon midline crossing.