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1.
Proc Natl Acad Sci U S A ; 118(25)2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34161264

RESUMO

Osmotic equilibrium and membrane potential in animal cells depend on concentration gradients of sodium (Na+) and potassium (K+) ions across the plasma membrane, a function catalyzed by the Na+,K+-ATPase α-subunit. Here, we describe ATP1A3 variants encoding dysfunctional α3-subunits in children affected by polymicrogyria, a developmental malformation of the cerebral cortex characterized by abnormal folding and laminar organization. To gain cell-biological insights into the spatiotemporal dynamics of prenatal ATP1A3 expression, we built an ATP1A3 transcriptional atlas of fetal cortical development using mRNA in situ hybridization and transcriptomic profiling of ∼125,000 individual cells with single-cell RNA sequencing (Drop-seq) from 11 areas of the midgestational human neocortex. We found that fetal expression of ATP1A3 is most abundant to a subset of excitatory neurons carrying transcriptional signatures of the developing subplate, yet also maintains expression in nonneuronal cell populations. Moving forward a year in human development, we profiled ∼52,000 nuclei from four areas of an infant neocortex and show that ATP1A3 expression persists throughout early postnatal development, most predominantly in inhibitory neurons, including parvalbumin interneurons in the frontal cortex. Finally, we discovered the heteromeric Na+,K+-ATPase pump complex may form nonredundant cell-type-specific α-ß isoform combinations, including α3-ß1 in excitatory neurons and α3-ß2 in inhibitory neurons. Together, the developmental malformation phenotype of affected individuals and single-cell ATP1A3 expression patterns point to a key role for α3 in human cortex development, as well as a cell-type basis for pre- and postnatal ATP1A3-associated diseases.


Assuntos
Encéfalo/embriologia , Encéfalo/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Adulto , Encéfalo/anormalidades , Encéfalo/diagnóstico por imagem , Criança , Feminino , Feto/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Lactente , Recém-Nascido , Interneurônios/metabolismo , Imageamento por Ressonância Magnética , Masculino , Mutação/genética , Neocórtex/embriologia , Neocórtex/enzimologia , Neurônios/metabolismo , Parvalbuminas/metabolismo , Fenótipo , Polimicrogiria/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Célula Única , ATPase Trocadora de Sódio-Potássio/genética
2.
Genet Med ; 23(6): 1158-1162, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33531666

RESUMO

PURPOSE: The endoplasmic reticulum membrane complex (EMC) is a highly conserved, multifunctional 10-protein complex related to membrane protein biology. In seven families, we identified 13 individuals with highly overlapping phenotypes who harbor a single identical homozygous frameshift variant in EMC10. METHODS: Using exome, genome, and Sanger sequencing, a recurrent frameshift EMC10 variant was identified in affected individuals in an international cohort of consanguineous families. Multiple families were independently identified and connected via Matchmaker Exchange and internal databases. We assessed the effect of the frameshift variant on EMC10 RNA and protein expression and evaluated EMC10 expression in normal human brain tissue using immunohistochemistry. RESULTS: A homozygous variant EMC10 c.287delG (Refseq NM_206538.3, p.Gly96Alafs*9) segregated with affected individuals in each family, who exhibited a phenotypic spectrum of intellectual disability (ID) and global developmental delay (GDD), variable seizures and variable dysmorphic features (elongated face, curly hair, cubitus valgus, and arachnodactyly). The variant arose on two founder haplotypes and results in significantly reduced EMC10 RNA expression and an unstable truncated EMC10 protein. CONCLUSION: We propose that a homozygous loss-of-function variant in EMC10 causes a novel syndromic neurodevelopmental phenotype. Remarkably, the recurrent variant is likely the result of a hypermutable site and arose on distinct founder haplotypes.


Assuntos
Deficiências do Desenvolvimento , Deficiência Intelectual , Criança , Deficiências do Desenvolvimento/genética , Mutação da Fase de Leitura , Homozigoto , Humanos , Deficiência Intelectual/genética , Proteínas de Membrana/genética , Linhagem , Fenótipo , Convulsões/genética
3.
Am J Hum Genet ; 100(3): 537-545, 2017 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-28190459

RESUMO

Congenital muscular dystrophies display a wide phenotypic and genetic heterogeneity. The combination of clinical, biochemical, and molecular genetic findings must be considered to obtain the precise diagnosis and provide appropriate genetic counselling. Here we report five individuals from four families presenting with variable clinical features including muscular dystrophy with a reduction in dystroglycan glycosylation, short stature, intellectual disability, and cataracts, overlapping both the dystroglycanopathies and Marinesco-Sjögren syndrome. Whole-exome sequencing revealed homozygous missense and compound heterozygous mutations in INPP5K in the affected members of each family. INPP5K encodes the inositol polyphosphate-5-phosphatase K, also known as SKIP (skeletal muscle and kidney enriched inositol phosphatase), which is highly expressed in the brain and muscle. INPP5K localizes to both the endoplasmic reticulum and to actin ruffles in the cytoplasm. It has been shown to regulate myoblast differentiation and has also been implicated in protein processing through its interaction with the ER chaperone HSPA5/BiP. We show that morpholino-mediated inpp5k loss of function in the zebrafish results in shortened body axis, microphthalmia with disorganized lens, microcephaly, reduced touch-evoked motility, and highly disorganized myofibers. Altogether these data demonstrate that mutations in INPP5K cause a congenital muscular dystrophy syndrome with short stature, cataracts, and intellectual disability.


Assuntos
Distrofia Muscular do Cíngulo dos Membros/genética , Monoéster Fosfórico Hidrolases/genética , Degenerações Espinocerebelares/genética , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Criança , Modelos Animais de Doenças , Distroglicanas/metabolismo , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Feminino , Estudo de Associação Genômica Ampla , Glicosilação , Transtornos do Crescimento/genética , Humanos , Deficiência Intelectual/genética , Masculino , Microcefalia/genética , Músculo Esquelético/metabolismo , Mutação , Linhagem , Adulto Jovem , Peixe-Zebra/genética
4.
Genome Res ; 27(8): 1323-1335, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28630177

RESUMO

While next-generation sequencing has accelerated the discovery of human disease genes, progress has been largely limited to the "low hanging fruit" of mutations with obvious exonic coding or canonical splice site impact. In contrast, the lack of high-throughput, unbiased approaches for functional assessment of most noncoding variants has bottlenecked gene discovery. We report the integration of transcriptome sequencing (RNA-seq), which surveys all mRNAs to reveal functional impacts of variants at the transcription level, into the gene discovery framework for a unique human disease, microcephaly-micromelia syndrome (MMS). MMS is an autosomal recessive condition described thus far in only a single First Nations population and causes intrauterine growth restriction, severe microcephaly, craniofacial anomalies, skeletal dysplasia, and neonatal lethality. Linkage analysis of affected families, including a very large pedigree, identified a single locus on Chromosome 21 linked to the disease (LOD > 9). Comprehensive genome sequencing did not reveal any pathogenic coding or canonical splicing mutations within the linkage region but identified several nonconserved noncoding variants. RNA-seq analysis detected aberrant splicing in DONSON due to one of these noncoding variants, showing a causative role for DONSON disruption in MMS. We show that DONSON is expressed in progenitor cells of embryonic human brain and other proliferating tissues, is co-expressed with components of the DNA replication machinery, and that Donson is essential for early embryonic development in mice as well, suggesting an essential conserved role for DONSON in the cell cycle. Our results demonstrate the utility of integrating transcriptomics into the study of human genetic disease when DNA sequencing alone is not sufficient to reveal the underlying pathogenic mutation.


Assuntos
Proteínas de Ciclo Celular/genética , Replicação do DNA , Microcefalia/genética , Microcefalia/patologia , Mutação , Proteínas Nucleares/genética , Osteocondrodisplasias/genética , Osteocondrodisplasias/patologia , Transcriptoma , Animais , Mapeamento Cromossômico , Feminino , Ligação Genética , Instabilidade Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Camundongos , Camundongos Knockout , Microcefalia/etiologia , Osteocondrodisplasias/etiologia , Linhagem , Gravidez , Splicing de RNA , Análise de Sequência de RNA , Sequenciamento Completo do Genoma
5.
Genet Med ; 22(6): 1040-1050, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32103185

RESUMO

PURPOSE: The exocyst complex is a conserved protein complex that mediates fusion of intracellular vesicles to the plasma membrane and is implicated in processes including cell polarity, cell migration, ciliogenesis, cytokinesis, autophagy, and fusion of secretory vesicles. The essential role of these genes in human genetic disorders, however, is unknown. METHODS: We performed homozygosity mapping and exome sequencing of consanguineous families with recessively inherited brain development disorders. We modeled an EXOC7 splice variant in vitro and examined EXOC7 messenger RNA (mRNA) expression in developing mouse and human cortex. We modeled exoc7 loss-of-function in a zebrafish knockout. RESULTS: We report variants in exocyst complex members, EXOC7 and EXOC8, in a novel disorder of cerebral cortex development. In EXOC7, we identified four independent partial loss-of-function (LOF) variants in a recessively inherited disorder characterized by brain atrophy, seizures, and developmental delay, and in severe cases, microcephaly and infantile death. In EXOC8, we found a homozygous truncating variant in a family with a similar clinical disorder. We modeled exoc7 deficiency in zebrafish and found the absence of exoc7 causes microcephaly. CONCLUSION: Our results highlight the essential role of the exocyst pathway in normal cortical development and how its perturbation causes complex brain disorders.


Assuntos
Encefalopatias , Microcefalia , Animais , Proliferação de Células/genética , Homozigoto , Humanos , Camundongos , Microcefalia/genética , Peixe-Zebra/genética
6.
Nature ; 515(7526): 209-15, 2014 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-25363760

RESUMO

The genetic architecture of autism spectrum disorder involves the interplay of common and rare variants and their impact on hundreds of genes. Using exome sequencing, here we show that analysis of rare coding variation in 3,871 autism cases and 9,937 ancestry-matched or parental controls implicates 22 autosomal genes at a false discovery rate (FDR) < 0.05, plus a set of 107 autosomal genes strongly enriched for those likely to affect risk (FDR < 0.30). These 107 genes, which show unusual evolutionary constraint against mutations, incur de novo loss-of-function mutations in over 5% of autistic subjects. Many of the genes implicated encode proteins for synaptic formation, transcriptional regulation and chromatin-remodelling pathways. These include voltage-gated ion channels regulating the propagation of action potentials, pacemaking and excitability-transcription coupling, as well as histone-modifying enzymes and chromatin remodellers-most prominently those that mediate post-translational lysine methylation/demethylation modifications of histones.


Assuntos
Transtornos Globais do Desenvolvimento Infantil/genética , Cromatina/genética , Predisposição Genética para Doença/genética , Mutação/genética , Sinapses/metabolismo , Transcrição Gênica/genética , Sequência de Aminoácidos , Transtornos Globais do Desenvolvimento Infantil/patologia , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Exoma/genética , Feminino , Mutação em Linhagem Germinativa/genética , Humanos , Masculino , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Rede Nervosa/metabolismo , Razão de Chances
7.
Proc Natl Acad Sci U S A ; 113(38): E5598-607, 2016 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-27601654

RESUMO

Mutations that cause neurological phenotypes are highly informative with regard to mechanisms governing human brain function and disease. We report autosomal recessive mutations in the enzyme glutamate pyruvate transaminase 2 (GPT2) in large kindreds initially ascertained for intellectual and developmental disability (IDD). GPT2 [also known as alanine transaminase 2 (ALT2)] is one of two related transaminases that catalyze the reversible addition of an amino group from glutamate to pyruvate, yielding alanine and α-ketoglutarate. In addition to IDD, all affected individuals show postnatal microcephaly and ∼80% of those followed over time show progressive motor symptoms, a spastic paraplegia. Homozygous nonsense p.Arg404* and missense p.Pro272Leu mutations are shown biochemically to be loss of function. The GPT2 gene demonstrates increasing expression in brain in the early postnatal period, and GPT2 protein localizes to mitochondria. Akin to the human phenotype, Gpt2-null mice exhibit reduced brain growth. Through metabolomics and direct isotope tracing experiments, we find a number of metabolic abnormalities associated with loss of Gpt2. These include defects in amino acid metabolism such as low alanine levels and elevated essential amino acids. Also, we find defects in anaplerosis, the metabolic process involved in replenishing TCA cycle intermediates. Finally, mutant brains demonstrate misregulated metabolites in pathways implicated in neuroprotective mechanisms previously associated with neurodegenerative disorders. Overall, our data reveal an important role for the GPT2 enzyme in mitochondrial metabolism with relevance to developmental as well as potentially to neurodegenerative mechanisms.


Assuntos
Encéfalo/crescimento & desenvolvimento , Mitocôndrias/enzimologia , Doenças do Sistema Nervoso/genética , Transaminases/genética , Sequência de Aminoácidos/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Ciclo do Ácido Cítrico/genética , Homozigoto , Humanos , Ácidos Cetoglutáricos/metabolismo , Camundongos , Mitocôndrias/patologia , Mutação de Sentido Incorreto , Doenças do Sistema Nervoso/patologia , Fenótipo , Ácido Pirúvico/metabolismo , Transaminases/metabolismo
8.
Am J Hum Genet ; 96(5): 709-19, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25865492

RESUMO

Despite recent advances in understanding the genetic bases of microcephaly, a large number of cases of microcephaly remain unexplained, suggesting that many microcephaly syndromes and associated genes have yet to be identified. Here, we report mutations in PYCR2, which encodes an enzyme in the proline biosynthesis pathway, as the cause of a unique syndrome characterized by postnatal microcephaly, hypomyelination, and reduced cerebral white-matter volume. Linkage mapping and whole-exome sequencing identified homozygous mutations (c.355C>T [p.Arg119Cys] and c.751C>T [p.Arg251Cys]) in PYCR2 in the affected individuals of two consanguineous families. A lymphoblastoid cell line from one affected individual showed a strong reduction in the amount of PYCR2. When mutant cDNAs were transfected into HEK293FT cells, both variant proteins retained normal mitochondrial localization but had lower amounts than the wild-type protein, suggesting that the variant proteins were less stable. A PYCR2-deficient HEK293FT cell line generated by genome editing with the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that PYCR2 loss of function led to decreased mitochondrial membrane potential and increased susceptibility to apoptosis under oxidative stress. Morpholino-based knockdown of a zebrafish PYCR2 ortholog, pycr1b, recapitulated the human microcephaly phenotype, which was rescued by wild-type human PYCR2 mRNA, but not by mutant mRNAs, further supporting the pathogenicity of the identified variants. Hypomyelination and the absence of lax, wrinkly skin distinguishes this condition from that caused by previously reported mutations in the gene encoding PYCR2's isozyme, PYCR1, suggesting a unique and indispensable role for PYCR2 in the human CNS during development.


Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/deficiência , Antiporters/deficiência , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Microcefalia/genética , Doenças Mitocondriais/genética , Transtornos Psicomotores/genética , Pirrolina Carboxilato Redutases/genética , Sistemas de Transporte de Aminoácidos Acídicos/genética , Antiporters/genética , Feminino , Genótipo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Homozigoto , Humanos , Masculino , Microcefalia/patologia , Doenças Mitocondriais/patologia , Mutação , Fenótipo , Transtornos Psicomotores/patologia , delta-1-Pirrolina-5-Carboxilato Redutase
9.
Am J Med Genet B Neuropsychiatr Genet ; 177(8): 736-745, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30421579

RESUMO

Protein homeostasis is tightly regulated by the ubiquitin proteasome pathway. Disruption of this pathway gives rise to a host of neurological disorders. Through whole exome sequencing (WES) in families with neurodevelopmental disorders, we identified mutations in PSMD12, a core component of the proteasome, underlying a neurodevelopmental disorder with intellectual disability (ID) and features of autism spectrum disorder (ASD). We performed WES on six affected siblings from a multiplex family with ID and autistic features, the affected father, and two unaffected mothers, and a trio from a simplex family with one affected child with ID and periventricular nodular heterotopia. We identified an inherited heterozygous nonsense mutation in PSMD12 (NM_002816: c.367C>T: p.R123X) in the multiplex family and a de novo nonsense mutation in the same gene (NM_002816: c.601C>T: p.R201X) in the simplex family. PSMD12 encodes a non-ATPase regulatory subunit of the 26S proteasome. We confirm the association of PSMD12 with ID, present the first cases of inherited PSMD12 mutation, and demonstrate the heterogeneity of phenotypes associated with PSMD12 mutations.


Assuntos
Deficiência Intelectual/genética , Complexo de Endopeptidases do Proteassoma/genética , Adolescente , Adulto , Transtorno do Espectro Autista/genética , Transtorno Autístico/genética , Criança , Pré-Escolar , Família , Feminino , Predisposição Genética para Doença , Haploinsuficiência/genética , Humanos , Masculino , Mutação , Transtornos do Neurodesenvolvimento/genética , Linhagem , Complexo de Endopeptidases do Proteassoma/metabolismo , Irmãos , Sequenciamento do Exoma
10.
Hum Mutat ; 38(10): 1348-1354, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28493438

RESUMO

Aminoacyl-transfer RNA (tRNA) synthetases ligate amino acids to specific tRNAs and are essential for protein synthesis. Although alanyl-tRNA synthetase (AARS) is a synthetase implicated in a wide range of neurological disorders from Charcot-Marie-Tooth disease to infantile epileptic encephalopathy, there have been limited data on their pathogenesis. Here, we report loss-of-function mutations in AARS in two siblings with progressive microcephaly with hypomyelination, intractable epilepsy, and spasticity. Whole-exome sequencing identified that the affected individuals were compound heterozygous for mutations in AARS gene, c.2067dupC (p.Tyr690Leufs*3) and c.2738G>A (p.Gly913Asp). A lymphoblastoid cell line developed from one of the affected individuals showed a strong reduction in AARS abundance. The mutations decrease aminoacylation efficiency by 70%-90%. The p.Tyr690Leufs*3 mutation also abolished editing activity required for hydrolyzing misacylated tRNAs, thereby increasing errors during aminoacylation. Our study has extended potential mechanisms underlying AARS-related disorders to include destabilization of the protein, aminoacylation dysfunction, and defective editing activity.


Assuntos
Alanina-tRNA Ligase/genética , Síndrome de Lennox-Gastaut/genética , Microcefalia/genética , Espasmos Infantis/genética , Paraplegia Espástica Hereditária/genética , Sequência de Aminoácidos/genética , Aminoacilação/genética , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Pré-Escolar , Eletroencefalografia , Feminino , Humanos , Lactente , Síndrome de Lennox-Gastaut/complicações , Síndrome de Lennox-Gastaut/diagnóstico , Síndrome de Lennox-Gastaut/patologia , Microcefalia/diagnóstico por imagem , Microcefalia/patologia , Mutação/genética , Biossíntese de Proteínas/genética , Irmãos , Espasmos Infantis/complicações , Espasmos Infantis/diagnóstico por imagem , Espasmos Infantis/patologia , Paraplegia Espástica Hereditária/complicações , Paraplegia Espástica Hereditária/patologia , Sequenciamento do Exoma
11.
Am J Hum Genet ; 94(4): 547-58, 2014 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-24656866

RESUMO

Progressive microcephaly is a heterogeneous condition with causes including mutations in genes encoding regulators of neuronal survival. Here, we report the identification of mutations in QARS (encoding glutaminyl-tRNA synthetase [QARS]) as the causative variants in two unrelated families affected by progressive microcephaly, severe seizures in infancy, atrophy of the cerebral cortex and cerebellar vermis, and mild atrophy of the cerebellar hemispheres. Whole-exome sequencing of individuals from each family independently identified compound-heterozygous mutations in QARS as the only candidate causative variants. QARS was highly expressed in the developing fetal human cerebral cortex in many cell types. The four QARS mutations altered highly conserved amino acids, and the aminoacylation activity of QARS was significantly impaired in mutant cell lines. Variants p.Gly45Val and p.Tyr57His were located in the N-terminal domain required for QARS interaction with proteins in the multisynthetase complex and potentially with glutamine tRNA, and recombinant QARS proteins bearing either substitution showed an over 10-fold reduction in aminoacylation activity. Conversely, variants p.Arg403Trp and p.Arg515Trp, each occurring in a different family, were located in the catalytic core and completely disrupted QARS aminoacylation activity in vitro. Furthermore, p.Arg403Trp and p.Arg515Trp rendered QARS less soluble, and p.Arg403Trp disrupted QARS-RARS (arginyl-tRNA synthetase 1) interaction. In zebrafish, homozygous qars loss of function caused decreased brain and eye size and extensive cell death in the brain. Our results highlight the importance of QARS during brain development and that epilepsy due to impairment of QARS activity is unusually severe in comparison to other aminoacyl-tRNA synthetase disorders.


Assuntos
Aminoacil-tRNA Sintetases/genética , Encefalopatias/genética , Predisposição Genética para Doença , Microcefalia/genética , Mutação , Convulsões/genética , Aminoacilação , Animais , Pré-Escolar , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Microcefalia/patologia , Linhagem , Peixe-Zebra
12.
Hum Mol Genet ; 23(13): 3456-66, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24501276

RESUMO

Whereas many genes associated with intellectual disability (ID) encode synaptic proteins, transcriptional defects leading to ID are less well understood. We studied a large, consanguineous pedigree of Arab origin with seven members affected with ID and mild dysmorphic features. Homozygosity mapping and linkage analysis identified a candidate region on chromosome 17 with a maximum multipoint logarithm of odds score of 6.01. Targeted high-throughput sequencing of the exons in the candidate region identified a homozygous 4-bp deletion (c.169_172delCACT) in the METTL23 (methyltransferase like 23) gene, which is predicted to result in a frameshift and premature truncation (p.His57Valfs*11). Overexpressed METTL23 protein localized to both nucleus and cytoplasm, and physically interacted with GABPA (GA-binding protein transcription factor, alpha subunit). GABP, of which GABPA is a component, is known to regulate the expression of genes such as THPO (thrombopoietin) and ATP5B (ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide) and is implicated in a wide variety of important cellular functions. Overexpression of METTL23 resulted in increased transcriptional activity at the THPO promoter, whereas knockdown of METTL23 with siRNA resulted in decreased expression of ATP5B, thus revealing the importance of METTL23 as a regulator of GABPA function. The METTL23 mutation highlights a new transcriptional pathway underlying human intellectual function.


Assuntos
Metilases de Modificação do DNA/metabolismo , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Metilases de Modificação do DNA/genética , Feminino , Fator de Transcrição de Proteínas de Ligação GA/genética , Genótipo , Humanos , Imunoprecipitação , Masculino , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Ligação Proteica , RNA Interferente Pequeno , Trombopoetina/genética , Trombopoetina/metabolismo , Técnicas do Sistema de Duplo-Híbrido
13.
Nat Genet ; 39(8): 957-9, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17632512

RESUMO

Donnai-Barrow syndrome is associated with agenesis of the corpus callosum, congenital diaphragmatic hernia, facial dysmorphology, ocular anomalies, sensorineural hearing loss and developmental delay. By studying multiplex families, we mapped this disorder to chromosome 2q23.3-31.1 and identified LRP2 mutations in six families with Donnai-Barrow syndrome and one family with facio-oculo-acoustico-renal syndrome. LRP2 encodes megalin, a multiligand uptake receptor that regulates levels of diverse circulating compounds. This work implicates a pathway with potential pharmacological therapeutic targets.


Assuntos
Anormalidades Múltiplas/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Agenesia do Corpo Caloso , Cromossomos Humanos Par 2 , Anormalidades Craniofaciais/genética , Oftalmopatias Hereditárias/genética , Família , Perda Auditiva Neurossensorial/genética , Hérnia Diafragmática/genética , Humanos , Rim/anormalidades , Mutação , Síndrome
14.
Am J Hum Genet ; 91(3): 541-7, 2012 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-22958903

RESUMO

Whole-exome sequencing (WES), which analyzes the coding sequence of most annotated genes in the human genome, is an ideal approach to studying fully penetrant autosomal-recessive diseases, and it has been very powerful in identifying disease-causing mutations even when enrollment of affected individuals is limited by reduced survival. In this study, we combined WES with homozygosity analysis of consanguineous pedigrees, which are informative even when a single affected individual is available, to identify genetic mutations responsible for Walker-Warburg syndrome (WWS), a genetically heterogeneous autosomal-recessive disorder that severely affects the development of the brain, eyes, and muscle. Mutations in seven genes are known to cause WWS and explain 50%-60% of cases, but multiple additional genes are expected to be mutated because unexplained cases show suggestive linkage to diverse loci. Using WES in consanguineous WWS-affected families, we found multiple deleterious mutations in GTDC2 (also known as AGO61). GTDC2's predicted role as an uncharacterized glycosyltransferase is consistent with the function of other genes that are known to be mutated in WWS and that are involved in the glycosylation of the transmembrane receptor dystroglycan. Therefore, to explore the role of GTDC2 loss of function during development, we used morpholino-mediated knockdown of its zebrafish ortholog, gtdc2. We found that gtdc2 knockdown in zebrafish replicates all WWS features (hydrocephalus, ocular defects, and muscular dystrophy), strongly suggesting that GTDC2 mutations cause WWS.


Assuntos
Glicosiltransferases/genética , Síndrome de Walker-Warburg/genética , Exoma , Humanos , Mutação
15.
PLoS Genet ; 8(4): e1002635, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22511880

RESUMO

Although autism has a clear genetic component, the high genetic heterogeneity of the disorder has been a challenge for the identification of causative genes. We used homozygosity analysis to identify probands from nonconsanguineous families that showed evidence of distant shared ancestry, suggesting potentially recessive mutations. Whole-exome sequencing of 16 probands revealed validated homozygous, potentially pathogenic recessive mutations that segregated perfectly with disease in 4/16 families. The candidate genes (UBE3B, CLTCL1, NCKAP5L, ZNF18) encode proteins involved in proteolysis, GTPase-mediated signaling, cytoskeletal organization, and other pathways. Furthermore, neuronal depolarization regulated the transcription of these genes, suggesting potential activity-dependent roles in neurons. We present a multidimensional strategy for filtering whole-exome sequence data to find candidate recessive mutations in autism, which may have broader applicability to other complex, heterogeneous disorders.


Assuntos
Transtorno Autístico/genética , Éxons , Genes Recessivos , Mutação , Neurônios , Proteínas Adaptadoras de Transdução de Sinal/genética , Cadeias Pesadas de Clatrina/genética , Éxons/genética , Genoma Humano , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Fatores de Transcrição Kruppel-Like/genética , Neurônios/metabolismo , Neurônios/fisiologia , Proteínas Oncogênicas/genética , Transcrição Gênica , Ubiquitina-Proteína Ligases/genética
16.
Am J Hum Genet ; 88(5): 536-47, 2011 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-21529751

RESUMO

Genes disrupted in human microcephaly (meaning "small brain") define key regulators of neural progenitor proliferation and cell-fate specification. In comparison, genes mutated in human lissencephaly (lissos means smooth and cephalos means brain) highlight critical regulators of neuronal migration. Here, we report two families with extreme microcephaly and grossly simplified cortical gyral structure, a condition referred to as microlissencephaly, and show that they carry homozygous frameshift mutations in NDE1, which encodes a multidomain protein that localizes to the centrosome and mitotic spindle poles. Both human mutations in NDE1 truncate the C-terminal NDE1domains, which are essential for interactions with cytoplasmic dynein and thus for regulation of cytoskeletal dynamics in mitosis and for cell-cycle-dependent phosphorylation of NDE1 by Cdk1. We show that the patient NDE1 proteins are unstable, cannot bind cytoplasmic dynein, and do not localize properly to the centrosome. Additionally, we show that CDK1 phosphorylation at T246, which is within the C-terminal region disrupted by the mutations, is required for cell-cycle progression from the G2 to the M phase. The role of NDE1 in cell-cycle progression probably contributes to the profound neuronal proliferation defects evident in Nde1-null mice and patients with NDE1 mutations, demonstrating the essential role of NDE1 in human cerebral cortical neurogenesis.


Assuntos
Mutação da Fase de Leitura , Lisencefalia/genética , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/genética , Animais , Proteína Quinase CDC2/metabolismo , Diferenciação Celular , Linhagem Celular , Movimento Celular , Centrossomo/metabolismo , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Criança , Pré-Escolar , Feminino , Ligação Genética , Homozigoto , Humanos , Lactente , Masculino , Camundongos , Camundongos Knockout , Neurônios/citologia , Fosforilação , Estabilidade Proteica , Fuso Acromático/metabolismo , Transfecção
17.
Am J Hum Genet ; 87(6): 882-9, 2010 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-21109224

RESUMO

The tight junction, or zonula occludens, is a specialized cell-cell junction that regulates epithelial and endothelial permeability, and it is an essential component of the blood-brain barrier in the cerebrovascular endothelium. In addition to functioning as a diffusion barrier, tight junctions are also involved in signal transduction. In this study, we identified a homozygous mutation in the tight-junction protein gene JAM3 in a large consanguineous family from the United Arab Emirates. Some members of this family had a rare autosomal-recessive syndrome characterized by severe hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts. Their clinical presentation overlaps with some reported cases of pseudo-TORCH syndrome as well as with cases involving mutations in occludin, another component of the tight-junction complex. However, massive intracranial hemorrhage distinguishes these patients from others. Homozygosity mapping identified the disease locus in this family on chromosome 11q25 with a maximum multipoint LOD score of 6.15. Sequence analysis of genes in the candidate interval uncovered a mutation in the canonical splice-donor site of intron 5 of JAM3. RT-PCR analysis of a patient lymphoblast cell line confirmed abnormal splicing, leading to a frameshift mutation with early termination. JAM3 is known to be present in vascular endothelium, although its roles in cerebral vasculature have not been implicated. Our results suggest that JAM3 is essential for maintaining the integrity of the cerebrovascular endothelium as well as for normal lens development in humans.


Assuntos
Calcinose/genética , Catarata/congênito , Moléculas de Adesão Celular/genética , Hemorragia Cerebral/genética , Epêndima/patologia , Homozigoto , Mutação , Junções Íntimas/metabolismo , Catarata/genética , Criança , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Linhagem
18.
Nat Genet ; 36(1): 69-76, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14647276

RESUMO

Disruption of human neural precursor proliferation can give rise to a small brain (microcephaly), and failure of neurons to migrate properly can lead to an abnormal arrest of cerebral cortical neurons in proliferative zones near the lateral ventricles (periventricular heterotopia). Here we show that an autosomal recessive condition characterized by microcephaly and periventricular heterotopia maps to chromosome 20 and is caused by mutations in the gene ADP-ribosylation factor guanine nucleotide-exchange factor-2 (ARFGEF2). By northern-blot analysis, we found that mouse Arfgef2 mRNA levels are highest during embryonic periods of ongoing neuronal proliferation and migration, and by in situ hybridization, we found that the mRNA is widely distributed throughout the embryonic central nervous system (CNS). ARFGEF2 encodes the large (>200 kDa) brefeldin A (BFA)-inhibited GEF2 protein (BIG2), which is required for vesicle and membrane trafficking from the trans-Golgi network (TGN). Inhibition of BIG2 by BFA, or by a dominant negative ARFGEF2 cDNA, decreases cell proliferation in vitro, suggesting a cell-autonomous regulation of neural expansion. Inhibition of BIG2 also disturbed the intracellular localization of such molecules as E-cadherin and beta-catenin by preventing their transport from the Golgi apparatus to the cell surface. Our findings show that vesicle trafficking is an important regulator of proliferation and migration during human cerebral cortical development.


Assuntos
Fatores de Ribosilação do ADP/genética , Córtex Cerebral/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas de Saccharomyces cerevisiae , Adolescente , Sequência de Aminoácidos , Animais , Divisão Celular , Movimento Celular , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Imageamento por Ressonância Magnética , Camundongos , Dados de Sequência Molecular , Mutação , Neurônios/fisiologia
19.
Nat Genet ; 36(9): 1008-13, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15322546

RESUMO

Joubert syndrome is a congenital brain malformation of the cerebellar vermis and brainstem with abnormalities of axonal decussation (crossing in the brain) affecting the corticospinal tract and superior cerebellar peduncles. Individuals with Joubert syndrome have motor and behavioral abnormalities, including an inability to walk due to severe clumsiness and 'mirror' movements, and cognitive and behavioral disturbances. Here we identified a locus associated with Joubert syndrome, JBTS3, on chromosome 6q23.2-q23.3 and found three deleterious mutations in AHI1, the first gene to be associated with Joubert syndrome. AHI1 is most highly expressed in brain, particularly in neurons that give rise to the crossing axons of the corticospinal tract and superior cerebellar peduncles. Comparative genetic analysis of AHI1 indicates that it has undergone positive evolutionary selection along the human lineage. Therefore, changes in AHI1 may have been important in the evolution of human-specific motor behaviors.


Assuntos
Anormalidades Múltiplas/genética , Cerebelo/anormalidades , Deficiências do Desenvolvimento/genética , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Animais , Encéfalo/anormalidades , Encéfalo/embriologia , Encéfalo/metabolismo , Tronco Encefálico/anormalidades , Feminino , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Camundongos , Dados de Sequência Molecular , Linhagem , Filogenia , Síndrome
20.
Am J Hum Genet ; 85(6): 897-902, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20004763

RESUMO

Although autosomal genes are increasingly recognized as important causes of intellectual disability, very few of them are known. We identified a genetic locus for autosomal-recessive nonsyndromic intellectual disability associated with variable postnatal microcephaly through homozygosity mapping of a consanguineous Israeli Arab family. Sequence analysis of genes in the candidate interval identified a nonsense nucleotide change in the gene that encodes TRAPPC9 (trafficking protein particle complex 9, also known as NIBP), which has been implicated in NF-kappaB activation and possibly in intracellular protein trafficking. TRAPPC9 is highly expressed in the postmitotic neurons of the cerebral cortex, and MRI analysis of affected patients shows defects in axonal connectivity. This suggests essential roles of TRAPPC9 in human brain development, possibly through its effect on NF-kappaB activation and protein trafficking in the postmitotic neurons of the cerebral cortex.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Deficiência Intelectual/genética , Microcefalia/genética , Mutação , Animais , Encéfalo/metabolismo , Mapeamento Cromossômico , Consanguinidade , Regulação da Expressão Gênica no Desenvolvimento , Genes Recessivos , Homozigoto , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Imageamento por Ressonância Magnética/métodos , Camundongos , Mitose , NF-kappa B/genética , Neurônios/metabolismo
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