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1.
J Clin Invest ; 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33001864

RESUMO

BACKGROUND: Transcriptome sequencing (RNA-seq) improves diagnostic rates in individuals with suspected Mendelian conditions to varying degrees, primarily by directing the prioritization of candidate DNA variants identified on exome or genome sequencing (ES/GS). Here we implemented an RNA-seq guided method to diagnose individuals across a wide range of ages and clinical phenotypes. METHODS: One hundred fifteen undiagnosed adult and pediatric patients with diverse phenotypes and 67 family members (182 total individuals) underwent RNA-seq from whole blood and fibroblasts at the Baylor College of Medicine (BCM) Undiagnosed Diseases Network (UDN) clinical site from 2014-2020. We implemented a workflow to detect outliers in gene expression and splicing for cases that remained undiagnosed despite standard genomic and transcriptomic analysis. RESULTS: The transcriptome-directed approach resulted in a diagnostic rate of 12% across the entire cohort, or 17% after excluding cases solved on ES/GS alone. Newly diagnosed conditions included Koolen-de Vries syndrome (KANSL1), Renpenning syndrome (PQBP1), TBCK-associated encephalopathy, NSD2- and CLTC-related intellectual disability, and others, all with negative conventional genomic testing, including ES and chromosomal microarray (CMA). Fibroblasts exhibited higher and more consistent expression of clinically relevant genes than whole blood. In solved cases with RNA-seq from both tissues, the causative defect was missed in blood in half the cases but none from fibroblasts. CONCLUSION: For our cohort of undiagnosed individuals with suspected Mendelian conditions, transcriptome-directed genomic analysis facilitated diagnoses, primarily through the identification of variants missed on ES and CMA.

2.
Am J Hum Genet ; 106(5): 717-725, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32330417

RESUMO

We identified three unrelated individuals with de novo missense variants in CDK19, encoding a cyclin-dependent kinase protein family member that predominantly regulates gene transcription. These individuals presented with hypotonia, global developmental delay, epileptic encephalopathy, and dysmorphic features. CDK19 is conserved between vertebrate and invertebrate model organisms, but currently abnormalities in CDK19 are not known to be associated with a human disorder. Loss of Cdk8, the fly homolog of CDK19, causes larval lethality, which is suppressed by expression of human CDK19 reference cDNA. In contrast, the CDK19 p.Tyr32His and p.Thr196Ala variants identified in the affected individuals fail to rescue the loss of Cdk8 and behave as null alleles. Additionally, neuronal RNAi-mediated knockdown of Cdk8 in flies results in semi-lethality. The few eclosing flies exhibit severe seizures and a reduced lifespan. Both phenotypes are fully suppressed by moderate expression of the CDK19 reference cDNA but not by expression of the two variants. Finally, loss of Cdk8 causes an obvious loss of boutons and synapses at larval neuromuscular junctions (NMJs). Together, our findings demonstrate that human CDK19 fully replaces the function of Cdk8 in the fly, the human disease-associated CDK19 variants behave as strong loss-of-function variants, and deleterious CDK19 variants underlie a syndromic neurodevelopmental disorder.


Assuntos
Encefalopatias/genética , Quinases Ciclina-Dependentes/genética , Epilepsia Generalizada/genética , Deficiência Intelectual/genética , Mutação de Sentido Incorreto/genética , Adulto , Sequência de Aminoácidos , Animais , Pré-Escolar , Quinase 8 Dependente de Ciclina/deficiência , Quinase 8 Dependente de Ciclina/genética , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Junção Neuromuscular , Doenças Raras/genética , Convulsões/genética , Síndrome , Adulto Jovem
3.
Am J Hum Genet ; 106(4): 570-583, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32197074

RESUMO

EIF2AK1 and EIF2AK2 encode members of the eukaryotic translation initiation factor 2 alpha kinase (EIF2AK) family that inhibits protein synthesis in response to physiologic stress conditions. EIF2AK2 is also involved in innate immune response and the regulation of signal transduction, apoptosis, cell proliferation, and differentiation. Despite these findings, human disorders associated with deleterious variants in EIF2AK1 and EIF2AK2 have not been reported. Here, we describe the identification of nine unrelated individuals with heterozygous de novo missense variants in EIF2AK1 (1/9) or EIF2AK2 (8/9). Features seen in these nine individuals include white matter alterations (9/9), developmental delay (9/9), impaired language (9/9), cognitive impairment (8/9), ataxia (6/9), dysarthria in probands with verbal ability (6/9), hypotonia (7/9), hypertonia (6/9), and involuntary movements (3/9). Individuals with EIF2AK2 variants also exhibit neurological regression in the setting of febrile illness or infection. We use mammalian cell lines and proband-derived fibroblasts to further confirm the pathogenicity of variants in these genes and found reduced kinase activity. EIF2AKs phosphorylate eukaryotic translation initiation factor 2 subunit 1 (EIF2S1, also known as EIF2α), which then inhibits EIF2B activity. Deleterious variants in genes encoding EIF2B proteins cause childhood ataxia with central nervous system hypomyelination/vanishing white matter (CACH/VWM), a leukodystrophy characterized by neurologic regression in the setting of febrile illness and other stressors. Our findings indicate that EIF2AK2 missense variants cause a neurodevelopmental syndrome that may share phenotypic and pathogenic mechanisms with CACH/VWM.


Assuntos
Deficiências do Desenvolvimento/genética , Variação Genética/genética , Leucoencefalopatias/genética , Malformações do Sistema Nervoso/genética , eIF-2 Quinase/genética , Adolescente , Ataxia/genética , Criança , Pré-Escolar , Feminino , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Humanos , Lactente , Masculino , Substância Branca/patologia
4.
Elife ; 92020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-32073399

RESUMO

Mutations in genes encoding synaptic proteins cause many neurodevelopmental disorders, with the majority affecting postsynaptic apparatuses and much fewer in presynaptic proteins. Syntaxin-binding protein 1 (STXBP1, also known as MUNC18-1) is an essential component of the presynaptic neurotransmitter release machinery. De novo heterozygous pathogenic variants in STXBP1 are among the most frequent causes of neurodevelopmental disorders including intellectual disabilities and epilepsies. These disorders, collectively referred to as STXBP1 encephalopathy, encompass a broad spectrum of neurologic and psychiatric features, but the pathogenesis remains elusive. Here we modeled STXBP1 encephalopathy in mice and found that Stxbp1 haploinsufficiency caused cognitive, psychiatric, and motor dysfunctions, as well as cortical hyperexcitability and seizures. Furthermore, Stxbp1 haploinsufficiency reduced cortical inhibitory neurotransmission via distinct mechanisms from parvalbumin-expressing and somatostatin-expressing interneurons. These results demonstrate that Stxbp1 haploinsufficient mice recapitulate cardinal features of STXBP1 encephalopathy and indicate that GABAergic synaptic dysfunction is likely a crucial contributor to disease pathogenesis.

6.
J Vis Exp ; (150)2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31498321

RESUMO

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient's condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are "humanized" by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.


Assuntos
Drosophila melanogaster/fisiologia , Genes/genética , Genômica/métodos , Mutação , Fenótipo , Algoritmos , Alelos , Animais , Drosophila melanogaster/genética , Exoma , Testes Genéticos , Humanos
7.
Genet Med ; 21(12): 2755-2764, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31263215

RESUMO

PURPOSE: Haploinsufficiency of DYRK1A causes a recognizable clinical syndrome. The goal of this paper is to investigate congenital anomalies of the kidney and urinary tract (CAKUT) and genital defects (GD) in patients with DYRK1A variants. METHODS: A large database of clinical exome sequencing (ES) was queried for de novo DYRK1A variants and CAKUT/GD phenotypes were characterized. Xenopus laevis (frog) was chosen as a model organism to assess Dyrk1a's role in renal development. RESULTS: Phenotypic details and variants of 19 patients were compiled after an initial observation that one patient with a de novo pathogenic variant in DYRK1A had GD. CAKUT/GD data were available from 15 patients, 11 of whom presented with CAKUT/GD. Studies in Xenopus embryos demonstrated that knockdown of Dyrk1a, which is expressed in forming nephrons, disrupts the development of segments of embryonic nephrons, which ultimately give rise to the entire genitourinary (GU) tract. These defects could be rescued by coinjecting wild-type human DYRK1A RNA, but not with DYRK1AR205* or DYRK1AL245R RNA. CONCLUSION: Evidence supports routine GU screening of all individuals with de novo DYRK1A pathogenic variants to ensure optimized clinical management. Collectively, the reported clinical data and loss-of-function studies in Xenopus substantiate a novel role for DYRK1A in GU development.


Assuntos
Deficiência Intelectual/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Anormalidades Urogenitais/genética , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Bases de Dados Genéticas , Modelos Animais de Doenças , Exoma/genética , Feminino , Haploinsuficiência/genética , Humanos , Deficiência Intelectual/complicações , Rim/anormalidades , Rim/embriologia , Masculino , Néfrons/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sistema Urinário/embriologia , Sistema Urinário/metabolismo , Sequenciamento Completo do Exoma/métodos , Xenopus laevis/genética , Xenopus laevis/metabolismo , Adulto Jovem
8.
Am J Med Genet A ; 179(3): 475-479, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30569621

RESUMO

Genetic alterations leading to overactivation of mammalian target of rapamycin (mTOR) signaling result in brain overgrowth syndromes such as focal cortical dysplasia (FCD) and megalencephaly. Megalencephaly with cutis tri-color of the Blaschko-linear type pigmentary mosaicism and intellectual disability is a rare neurodevelopmental disorder attributed to the recurrent mosaic c.5930C > T (p.Thr1977Ile) MTOR variant. This variant was previously reported at low to intermediate levels of mosaicism in the peripheral blood of three unrelated individuals with consistent clinical findings. We report a fourth case of a 3-year-old female presenting with megalencephaly, obstructive hydrocephalus due to cerebral aqueductal stenosis, asymmetric polymicrogyria, dysgenesis of the corpus callosum, hypotonia, developmental delay, and cutaneous pigmentary mosaicism. Oligonucleotide and SNP chromosomal microarray (CMA), karyotype, and trio whole exome sequencing (WES) in the peripheral blood, as well as a targeted gene variant panel from fibroblasts derived from hyperpigmented and non-hyperpigmented skin did not detect any abnormalities in MTOR or other genes associated with brain overgrowth syndromes. Unlike the previously reported cases, the de novo c.5930C > T (p.Thr1977Ile) MTOR variant was detected at 32% mosaicism in our patient only after WES was performed on fibroblast-derived DNA from the hyperpigmented skin. This case demonstrates the tissue variability in mosaic expression of the recurrent p.Thr1977Ile MTOR variant, emphasizes the need for skin biopsies in the genetic evaluation of patients with skin pigmentary mosaicism, and expands the clinical phenotype associated with this pathogenic MTOR variant.


Assuntos
Malformações do Desenvolvimento Cortical/diagnóstico , Malformações do Desenvolvimento Cortical/genética , Megalencefalia/diagnóstico , Megalencefalia/genética , Mutação , Transtornos da Pigmentação/diagnóstico , Transtornos da Pigmentação/genética , Serina-Treonina Quinases TOR/genética , Alelos , Substituição de Aminoácidos , Pré-Escolar , Feminino , Genótipo , Humanos , Imagem por Ressonância Magnética , Mosaicismo , Fenótipo , Polimorfismo de Nucleotídeo Único
9.
Genetics ; 207(1): 9-27, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28874452

RESUMO

Efforts to identify the genetic underpinnings of rare undiagnosed diseases increasingly involve the use of next-generation sequencing and comparative genomic hybridization methods. These efforts are limited by a lack of knowledge regarding gene function, and an inability to predict the impact of genetic variation on the encoded protein function. Diagnostic challenges posed by undiagnosed diseases have solutions in model organism research, which provides a wealth of detailed biological information. Model organism geneticists are by necessity experts in particular genes, gene families, specific organs, and biological functions. Here, we review the current state of research into undiagnosed diseases, highlighting large efforts in North America and internationally, including the Undiagnosed Diseases Network (UDN) (Supplemental Material, File S1) and UDN International (UDNI), the Centers for Mendelian Genomics (CMG), and the Canadian Rare Diseases Models and Mechanisms Network (RDMM). We discuss how merging human genetics with model organism research guides experimental studies to solve these medical mysteries, gain new insights into disease pathogenesis, and uncover new therapeutic strategies.


Assuntos
Modelos Animais de Doenças , Doenças Genéticas Inatas/genética , Doenças Raras/genética , Animais , Drosophila/genética , Exoma , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/terapia , Testes Genéticos/métodos , Humanos , Doenças Raras/diagnóstico , Doenças Raras/terapia , Peixe-Zebra/genética
10.
Genome Med ; 9(1): 83, 2017 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-28934986

RESUMO

BACKGROUND: Exon-targeted microarrays can detect small (<1000 bp) intragenic copy number variants (CNVs), including those that affect only a single exon. This genome-wide high-sensitivity approach increases the molecular diagnosis for conditions with known disease-associated genes, enables better genotype-phenotype correlations, and facilitates variant allele detection allowing novel disease gene discovery. METHODS: We retrospectively analyzed data from 63,127 patients referred for clinical chromosomal microarray analysis (CMA) at Baylor Genetics laboratories, including 46,755 individuals tested using exon-targeted arrays, from 2007 to 2017. Small CNVs harboring a single gene or two to five non-disease-associated genes were identified; the genes involved were evaluated for a potential disease association. RESULTS: In this clinical population, among rare CNVs involving any single gene reported in 7200 patients (11%), we identified 145 de novo autosomal CNVs (117 losses and 28 intragenic gains), 257 X-linked deletion CNVs in males, and 1049 inherited autosomal CNVs (878 losses and 171 intragenic gains); 111 known disease genes were potentially disrupted by de novo autosomal or X-linked (in males) single-gene CNVs. Ninety-one genes, either recently proposed as candidate disease genes or not yet associated with diseases, were disrupted by 147 single-gene CNVs, including 37 de novo deletions and ten de novo intragenic duplications on autosomes and 100 X-linked CNVs in males. Clinical features in individuals with de novo or X-linked CNVs encompassing at most five genes (224 bp to 1.6 Mb in size) were compared to those in individuals with larger-sized deletions (up to 5 Mb in size) in the internal CMA database or loss-of-function single nucleotide variants (SNVs) detected by clinical or research whole-exome sequencing (WES). This enabled the identification of recently published genes (BPTF, NONO, PSMD12, TANGO2, and TRIP12), novel candidate disease genes (ARGLU1 and STK3), and further confirmation of disease association for two recently proposed disease genes (MEIS2 and PTCHD1). Notably, exon-targeted CMA detected several pathogenic single-exon CNVs missed by clinical WES analyses. CONCLUSIONS: Together, these data document the efficacy of exon-targeted CMA for detection of genic and exonic CNVs, complementing and extending WES in clinical diagnostics, and the potential for discovery of novel disease genes by genome-wide assay.


Assuntos
Variações do Número de Cópias de DNA , Éxons , Doenças Genéticas Inatas , Estudos de Coortes , Genoma Humano , Proteínas de Homeodomínio/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Transtornos do Neurodesenvolvimento/genética , Proteínas Serina-Treonina Quinases/genética , Estudos Retrospectivos , Fatores de Transcrição/genética , Sequenciamento Completo do Genoma
11.
Am J Med Genet A ; 173(10): 2680-2689, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28815871

RESUMO

DNA alterations in the 1q43-q44 region are associated with syndromic neurodevelopmental disorders characterized by global developmental delay, intellectual disability, dysmorphic features, microcephaly, seizures, and agenesis of the corpus callosum. HNRNPU is located within the 1q43-q44 region and mutations in the gene have been reported in patients with early infantile epileptic encephalopathy. Here, we report on the clinical presentation of four patients with de novo heterozygous HNRNPU loss-of-function mutations detected by clinical whole exome sequencing: c.651_660del (p.Gly218Alafs*118), c.1089G>A (p.Trp363*), c.1714C>T (p.Arg572*), and c.2270_2271del (p.Pro757Argfs*7). All patients shared similar clinical features as previously reported including seizures, global developmental delay, intellectual disability, variable neurologic regression, behavior issues, and dysmorphic facial features. Features including heart defects and kidney abnormalities were not reported in our patients. These findings expands the clinical spectrum of HNRNPU-related disorder and shows that HNRNPU contributes to a subset of the clinical phenotypes associated with the contiguous 1q43-q44 deletion syndrome.


Assuntos
Deleção Cromossômica , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/genética , Transtornos do Neurodesenvolvimento/genética , Criança , Feminino , Haploinsuficiência , Humanos , Lactente , Masculino , Transtornos do Neurodesenvolvimento/patologia , Linhagem , Fenótipo
12.
Semin Cell Dev Biol ; 70: 49-57, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28579453

RESUMO

Today, biomedical science is equipped with an impressive array of technologies and genetic resources that bolster our basic understanding of fundamental biology and enhance the practice of modern medicine by providing clinicians with a diverse toolkit to diagnose, prognosticate, and treat a plethora of conditions. Many significant advances in our understanding of disease mechanisms and therapeutic interventions have arisen from fruitful dialogues between clinicians and biomedical research scientists. However, the increasingly specialized scientific and medical disciplines, globalization of science and technology, and complex datasets often hinder the development of effective interdisciplinary collaborations between clinical medicine and biomedical research. The goal of this review is to provide examples of diverse strategies to enhance communication and collaboration across diverse disciplines. First, we discuss examples of efforts to foster interdisciplinary collaborations at institutional and multi-institutional levels. Second, we explore resources and tools for clinicians and research scientists to facilitate effective bi-directional dialogues. Third, we use our experiences in neurobiology and human genetics to highlight how communication between clinical medicine and biomedical research lead to effective implementation of cross-species model organism approaches to uncover the biological underpinnings of health and disease.


Assuntos
Pesquisa Biomédica/métodos , Drosophila/genética , Comunicação Interdisciplinar , Doenças Neurodegenerativas/terapia , Pesquisa Médica Translacional/métodos , Peixe-Zebra/genética , Animais , Pesquisa Biomédica/economia , Relações Comunidade-Instituição , Modelos Animais de Doenças , Drosophila/metabolismo , Humanos , Camundongos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Participação do Paciente/estatística & dados numéricos , Rede Social , Pesquisa Médica Translacional/economia , Peixe-Zebra/metabolismo
13.
Am J Hum Genet ; 100(6): 843-853, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28502612

RESUMO

One major challenge encountered with interpreting human genetic variants is the limited understanding of the functional impact of genetic alterations on biological processes. Furthermore, there remains an unmet demand for an efficient survey of the wealth of information on human homologs in model organisms across numerous databases. To efficiently assess the large volume of publically available information, it is important to provide a concise summary of the most relevant information in a rapid user-friendly format. To this end, we created MARRVEL (model organism aggregated resources for rare variant exploration). MARRVEL is a publicly available website that integrates information from six human genetic databases and seven model organism databases. For any given variant or gene, MARRVEL displays information from OMIM, ExAC, ClinVar, Geno2MP, DGV, and DECIPHER. Importantly, it curates model organism-specific databases to concurrently display a concise summary regarding the human gene homologs in budding and fission yeast, worm, fly, fish, mouse, and rat on a single webpage. Experiment-based information on tissue expression, protein subcellular localization, biological process, and molecular function for the human gene and homologs in the seven model organisms are arranged into a concise output. Hence, rather than visiting multiple separate databases for variant and gene analysis, users can obtain important information by searching once through MARRVEL. Altogether, MARRVEL dramatically improves efficiency and accessibility to data collection and facilitates analysis of human genes and variants by cross-disciplinary integration of 18 million records available in public databases to facilitate clinical diagnosis and basic research.


Assuntos
Variação Genética , Genoma Humano , Anotação de Sequência Molecular , Software , Bases de Dados Genéticas , Humanos
14.
Am J Hum Genet ; 100(1): 128-137, 2017 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-28017372

RESUMO

Early B cell factor 3 (EBF3) is a member of the highly evolutionarily conserved Collier/Olf/EBF (COE) family of transcription factors. Prior studies on invertebrate and vertebrate animals have shown that EBF3 homologs are essential for survival and that loss-of-function mutations are associated with a range of nervous system developmental defects, including perturbation of neuronal development and migration. Interestingly, aristaless-related homeobox (ARX), a homeobox-containing transcription factor critical for the regulation of nervous system development, transcriptionally represses EBF3 expression. However, human neurodevelopmental disorders related to EBF3 have not been reported. Here, we describe three individuals who are affected by global developmental delay, intellectual disability, and expressive speech disorder and carry de novo variants in EBF3. Associated features seen in these individuals include congenital hypotonia, structural CNS malformations, ataxia, and genitourinary abnormalities. The de novo variants affect a single conserved residue in a zinc finger motif crucial for DNA binding and are deleterious in a fly model. Our findings indicate that mutations in EBF3 cause a genetic neurodevelopmental syndrome and suggest that loss of EBF3 function might mediate a subset of neurologic phenotypes shared by ARX-related disorders, including intellectual disability, abnormal genitalia, and structural CNS malformations.


Assuntos
Mutação , Transtornos do Neurodesenvolvimento/genética , Fatores de Transcrição/genética , Anormalidades Múltiplas/genética , Ataxia/genética , Sistema Nervoso Central/anormalidades , Criança , Pré-Escolar , Deficiências do Desenvolvimento/genética , Feminino , Genitália/anormalidades , Humanos , Lactente , Recém-Nascido , Deficiência Intelectual/genética , Masculino , Hipotonia Muscular/genética , Distúrbios da Fala/genética , Síndrome , Dedos de Zinco/genética
15.
J Neurosci ; 34(3): 855-68, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24431444

RESUMO

Neural circuits are composed of mainly glutamatergic and GABAergic neurons, which communicate through synaptic connections. Many factors instruct the formation and function of these synapses; however, it is difficult to dissect the contribution of intrinsic cell programs from that of extrinsic environmental effects in an intact network. Here, we perform paired recordings from two-neuron microculture preparations of mouse hippocampal glutamatergic and GABAergic neurons to investigate how synaptic input and output of these two principal cells develop. In our reduced preparation, we found that glutamatergic neurons showed no change in synaptic output or input regardless of partner neuron cell type or neuronal activity level. In contrast, we found that glutamatergic input caused the GABAergic neuron to modify its output by way of an increase in synapse formation and a decrease in synaptic release efficiency. These findings are consistent with aspects of GABAergic synapse maturation observed in many brain regions. In addition, changes in GABAergic output are cell wide and not target-cell specific. We also found that glutamatergic neuronal activity determined the AMPA receptor properties of synapses on the partner GABAergic neuron. All modifications of GABAergic input and output required activity of the glutamatergic neuron. Because our system has reduced extrinsic factors, the changes we saw in the GABAergic neuron due to glutamatergic input may reflect initiation of maturation programs that underlie the formation and function of in vivo neural circuits.


Assuntos
Neurônios GABAérgicos/fisiologia , Ácido Glutâmico/fisiologia , Rede Nervosa/crescimento & desenvolvimento , Neurogênese/fisiologia , Receptores de AMPA/fisiologia , Sinapses/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Hipocampo/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/fisiologia , Transmissão Sináptica/fisiologia
16.
J Neurosci ; 33(50): 19518-33, 2013 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-24336718

RESUMO

MECP2 duplication syndrome is a childhood neurological disorder characterized by intellectual disability, autism, motor abnormalities, and epilepsy. The disorder is caused by duplications spanning the gene encoding methyl-CpG-binding protein-2 (MeCP2), a protein involved in the modulation of chromatin and gene expression. MeCP2 is thought to play a role in maintaining the structural integrity of neuronal circuits. Loss of MeCP2 function causes Rett syndrome and results in abnormal dendritic spine morphology and decreased pyramidal dendritic arbor complexity and spine density. The consequences of MeCP2 overexpression on dendritic pathophysiology remain unclear. We used in vivo two-photon microscopy to characterize layer 5 pyramidal neuron spine turnover and dendritic arborization as a function of age in transgenic mice expressing the human MECP2 gene at twice the normal levels of MeCP2 (Tg1; Collins et al., 2004). We found that spine density in terminal dendritic branches is initially higher in young Tg1 mice but falls below control levels after postnatal week 12, approximately correlating with the onset of behavioral symptoms. Spontaneous spine turnover rates remain high in older Tg1 animals compared with controls, reflecting the persistence of an immature state. Both spine gain and loss rates are higher, with a net bias in favor of spine elimination. Apical dendritic arbors in both simple- and complex-tufted layer 5 Tg1 pyramidal neurons have more branches of higher order, indicating that MeCP2 overexpression induces dendritic overgrowth. P70S6K was hyperphosphorylated in Tg1 somatosensory cortex, suggesting that elevated mTOR signaling may underlie the observed increase in spine turnover and dendritic growth.


Assuntos
Dendritos/metabolismo , Espinhas Dendríticas/metabolismo , Retardo Mental Ligado ao Cromossomo X/metabolismo , Animais , Modelos Animais de Doenças , Retardo Mental Ligado ao Cromossomo X/genética , Retardo Mental Ligado ao Cromossomo X/fisiopatologia , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Neurônios/metabolismo , Córtex Somatossensorial/metabolismo , Córtex Somatossensorial/fisiopatologia
18.
Nature ; 468(7321): 263-9, 2010 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-21068835

RESUMO

Mutations in the X-linked MECP2 gene, which encodes the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2), cause Rett syndrome and several neurodevelopmental disorders including cognitive disorders, autism, juvenile-onset schizophrenia and encephalopathy with early lethality. Rett syndrome is characterized by apparently normal early development followed by regression, motor abnormalities, seizures and features of autism, especially stereotyped behaviours. The mechanisms mediating these features are poorly understood. Here we show that mice lacking Mecp2 from GABA (γ-aminobutyric acid)-releasing neurons recapitulate numerous Rett syndrome and autistic features, including repetitive behaviours. Loss of MeCP2 from a subset of forebrain GABAergic neurons also recapitulates many features of Rett syndrome. MeCP2-deficient GABAergic neurons show reduced inhibitory quantal size, consistent with a presynaptic reduction in glutamic acid decarboxylase 1 (Gad1) and glutamic acid decarboxylase 2 (Gad2) levels, and GABA immunoreactivity. These data demonstrate that MeCP2 is critical for normal function of GABA-releasing neurons and that subtle dysfunction of GABAergic neurons contributes to numerous neuropsychiatric phenotypes.


Assuntos
Transtorno Autístico/fisiopatologia , Proteína 2 de Ligação a Metil-CpG/deficiência , Proteína 2 de Ligação a Metil-CpG/metabolismo , Síndrome de Rett/fisiopatologia , Transdução de Sinais , Transtorno de Movimento Estereotipado/fisiopatologia , Ácido gama-Aminobutírico/metabolismo , Animais , Transtorno Autístico/complicações , Transtorno Autístico/genética , Transtorno Autístico/patologia , Encéfalo/citologia , Comportamento Compulsivo/complicações , Comportamento Compulsivo/genética , Comportamento Compulsivo/fisiopatologia , Modelos Animais de Doenças , Eletroencefalografia , Genótipo , Glutamato Descarboxilase/metabolismo , Hipocampo/patologia , Hipocampo/fisiopatologia , Proteínas de Homeodomínio/genética , Potenciais Pós-Sinápticos Inibidores , Potenciação de Longa Duração , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Transgênicos , Inibição Neural , Plasticidade Neuronal , Neurônios/metabolismo , Fenótipo , Terminações Pré-Sinápticas/metabolismo , Transtornos Psicomotores/complicações , Transtornos Psicomotores/genética , Transtornos Psicomotores/fisiopatologia , Reflexo de Sobressalto/genética , Respiração , Síndrome de Rett/complicações , Síndrome de Rett/genética , Síndrome de Rett/patologia , Comportamento Autodestrutivo/complicações , Comportamento Autodestrutivo/genética , Comportamento Autodestrutivo/fisiopatologia , Transtorno de Movimento Estereotipado/complicações , Transtorno de Movimento Estereotipado/genética , Transtorno de Movimento Estereotipado/patologia , Taxa de Sobrevida , Transmissão Sináptica , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética
19.
Nat Struct Mol Biol ; 17(5): 568-75, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20400951

RESUMO

Complexins facilitate and inhibit neurotransmitter release through distinct domains, and their function was proposed to be coupled to the Ca(2+) sensor synaptotagmin-1 (Syt1). However, the mechanisms underlying complexin function remain unclear. We now uncover an interaction between the complexin N terminus and the SNARE complex C terminus, and we show that disrupting this interaction abolishes the facilitatory function of complexins in mouse neurons. Analyses of hypertonically induced exocytosis show that complexins enhance synaptic-vesicle fusogenicity. Genetic experiments crossing complexin- and Syt1-null mice indicate a functional interaction between these proteins but also show that complexins can promote Ca(2+)-triggered release in the absence of Syt1. We propose that the complexin N terminus stabilizes the SNARE complex C terminus and/or helps release the inhibitory function of complexins, thereby activating the fusion machinery in a manner that may cooperate with Syt1 but does not require Syt1.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas SNARE/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Células Cultivadas , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas do Tecido Nervoso/genética , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Ratos , Proteínas SNARE/química , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismo
20.
Proc Natl Acad Sci U S A ; 106(51): 21966-71, 2009 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-20007372

RESUMO

Rett syndrome (RTT) is characterized by specific motor, cognitive, and behavioral deficits. Because several of these abnormalities occur in other disease states associated with alterations in aminergic neurotransmitters, we investigated the contribution of such alterations to RTT pathogenesis. We found that both individuals with RTT and Mecp2-null mice have lower-than-normal levels of aminergic metabolites and content. Deleting Mecp2 from either TH-positive dopaminergic and noradrenergic neurons or PET1-positive serotonergic neurons in mice decreased corresponding neurotransmitter concentration and specific phenotypes, likely through MeCP2 regulation of rate-limiting enzymes involved in aminergic neurotransmitter production. These data support a cell-autonomous, MeCP2-dependent mechanism for the regulation of aminergic neurotransmitter synthesis contributing to unique behavioral phenotypes.


Assuntos
Aminas/metabolismo , Ácido Homovanílico/metabolismo , Ácido Hidroxi-Indolacético/metabolismo , Transtornos Mentais/metabolismo , Proteína 2 de Ligação a Metil-CpG/metabolismo , Neurônios/metabolismo , Animais , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Knockout , Neurônios/enzimologia , Fenótipo , Triptofano Hidroxilase/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo
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