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1.
Cell ; 149(3): 525-37, 2012 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-22521361

RESUMO

Balanced chromosomal abnormalities (BCAs) represent a relatively untapped reservoir of single-gene disruptions in neurodevelopmental disorders (NDDs). We sequenced BCAs in patients with autism or related NDDs, revealing disruption of 33 loci in four general categories: (1) genes previously associated with abnormal neurodevelopment (e.g., AUTS2, FOXP1, and CDKL5), (2) single-gene contributors to microdeletion syndromes (MBD5, SATB2, EHMT1, and SNURF-SNRPN), (3) novel risk loci (e.g., CHD8, KIRREL3, and ZNF507), and (4) genes associated with later-onset psychiatric disorders (e.g., TCF4, ZNF804A, PDE10A, GRIN2B, and ANK3). We also discovered among neurodevelopmental cases a profoundly increased burden of copy-number variants from these 33 loci and a significant enrichment of polygenic risk alleles from genome-wide association studies of autism and schizophrenia. Our findings suggest a polygenic risk model of autism and reveal that some neurodevelopmental genes are sensitive to perturbation by multiple mutational mechanisms, leading to variable phenotypic outcomes that manifest at different life stages.


Assuntos
Transtornos Globais do Desenvolvimento Infantil/genética , Aberrações Cromossômicas , Transtorno Autístico/diagnóstico , Transtorno Autístico/genética , Criança , Transtornos Globais do Desenvolvimento Infantil/diagnóstico , Quebra Cromossômica , Deleção Cromossômica , Variações do Número de Cópias de DNA , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Sistema Nervoso/crescimento & desenvolvimento , Esquizofrenia/genética , Análise de Sequência de DNA , Transdução de Sinais
2.
Am J Hum Genet ; 99(5): 1015-1033, 2016 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-27745839

RESUMO

In this exciting era of "next-gen cytogenetics," integrating genomic sequencing into the prenatal diagnostic setting is possible within an actionable time frame and can provide precise delineation of balanced chromosomal rearrangements at the nucleotide level. Given the increased risk of congenital abnormalities in newborns with de novo balanced chromosomal rearrangements, comprehensive interpretation of breakpoints could substantially improve prediction of phenotypic outcomes and support perinatal medical care. Herein, we present and evaluate sequencing results of balanced chromosomal rearrangements in ten prenatal subjects with respect to the location of regulatory chromatin domains (topologically associated domains [TADs]). The genomic material from all subjects was interpreted to be "normal" by microarray analyses, and their rearrangements would not have been detected by cell-free DNA (cfDNA) screening. The findings of our systematic approach correlate with phenotypes of both pregnancies with untoward outcomes (5/10) and with healthy newborns (3/10). Two pregnancies, one with a chromosomal aberration predicted to be of unknown clinical significance and another one predicted to be likely benign, were terminated prior to phenotype-genotype correlation (2/10). We demonstrate that the clinical interpretation of structural rearrangements should not be limited to interruption, deletion, or duplication of specific genes and should also incorporate regulatory domains of the human genome with critical ramifications for the control of gene expression. As detailed in this study, our molecular approach to both detecting and interpreting the breakpoints of structural rearrangements yields unparalleled information in comparison to other commonly used first-tier diagnostic methods, such as non-invasive cfDNA screening and microarray analysis, to provide improved genetic counseling for phenotypic outcome in the prenatal setting.


Assuntos
Aberrações Cromossômicas , Anormalidades Congênitas/genética , Rearranjo Gênico , Nucleotídeos/genética , Diagnóstico Pré-Natal/métodos , Alelos , Mapeamento Cromossômico , Anormalidades Congênitas/diagnóstico , Feminino , Regulação da Expressão Gênica , Testes Genéticos , Genoma Humano , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cariotipagem , Masculino , Gravidez , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Análise de Sequência de DNA , Translocação Genética
3.
Am J Hum Genet ; 96(5): 784-96, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25937446

RESUMO

The 16p11.2 600 kb copy-number variants (CNVs) are associated with mirror phenotypes on BMI, head circumference, and brain volume and represent frequent genetic lesions in autism spectrum disorders (ASDs) and schizophrenia. Here we interrogated the transcriptome of individuals carrying reciprocal 16p11.2 CNVs. Transcript perturbations correlated with clinical endophenotypes and were enriched for genes associated with ASDs, abnormalities of head size, and ciliopathies. Ciliary gene expression was also perturbed in orthologous mouse models, raising the possibility that ciliary dysfunction contributes to 16p11.2 pathologies. In support of this hypothesis, we found structural ciliary defects in the CA1 hippocampal region of 16p11.2 duplication mice. Moreover, by using an established zebrafish model, we show genetic interaction between KCTD13, a key driver of the mirrored neuroanatomical phenotypes of the 16p11.2 CNV, and ciliopathy-associated genes. Overexpression of BBS7 rescues head size and neuroanatomical defects of kctd13 morphants, whereas suppression or overexpression of CEP290 rescues phenotypes induced by KCTD13 under- or overexpression, respectively. Our data suggest that dysregulation of ciliopathy genes contributes to the clinical phenotypes of these CNVs.


Assuntos
Transtornos Globais do Desenvolvimento Infantil/genética , Cromossomos Humanos Par 16/genética , Variações do Número de Cópias de DNA/genética , Esquizofrenia/genética , Animais , Encéfalo , Criança , Transtornos Globais do Desenvolvimento Infantil/patologia , Deleção Cromossômica , Corpo Ciliar/metabolismo , Corpo Ciliar/patologia , Regulação da Expressão Gênica , Humanos , Camundongos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Esquizofrenia/patologia , Transcriptoma , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
4.
Genome Res ; 25(2): 155-66, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25561519

RESUMO

RNA polymerase III (Pol III) synthesizes tRNAs and other small noncoding RNAs to regulate protein synthesis. Dysregulation of Pol III transcription has been linked to cancer, and germline mutations in genes encoding Pol III subunits or tRNA processing factors cause neurogenetic disorders in humans, such as hypomyelinating leukodystrophies and pontocerebellar hypoplasia. Here we describe an autosomal recessive disorder characterized by cerebellar hypoplasia and intellectual disability, as well as facial dysmorphic features, short stature, microcephaly, and dental anomalies. Whole-exome sequencing revealed biallelic missense alterations of BRF1 in three families. In support of the pathogenic potential of the discovered alleles, suppression or CRISPR-mediated deletion of brf1 in zebrafish embryos recapitulated key neurodevelopmental phenotypes; in vivo complementation showed all four candidate mutations to be pathogenic in an apparent isoform-specific context. BRF1 associates with BDP1 and TBP to form the transcription factor IIIB (TFIIIB), which recruits Pol III to target genes. We show that disease-causing mutations reduce Brf1 occupancy at tRNA target genes in Saccharomyces cerevisiae and impair cell growth. Moreover, BRF1 mutations reduce Pol III-related transcription activity in vitro. Taken together, our data show that BRF1 mutations that reduce protein activity cause neurodevelopmental anomalies, suggesting that BRF1-mediated Pol III transcription is required for normal cerebellar and cognitive development.


Assuntos
Anormalidades Múltiplas/genética , Deficiência Intelectual/genética , Mutação , RNA Polimerase III/metabolismo , Fatores Associados à Proteína de Ligação a TATA/genética , Transcrição Gênica , Anormalidades Múltiplas/diagnóstico , Adolescente , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Encéfalo/patologia , Proliferação de Células , Criança , Pré-Escolar , Exoma , Fácies , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Deficiência Intelectual/diagnóstico , Imageamento por Ressonância Magnética , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Linhagem , Fenótipo , Conformação Proteica , Isoformas de Proteínas , Irmãos , Síndrome , Fatores Associados à Proteína de Ligação a TATA/química , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Peixe-Zebra
5.
Am J Hum Genet ; 94(6): 870-83, 2014 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-24906019

RESUMO

Reciprocal copy-number variation (CNV) of a 593 kb region of 16p11.2 is a common genetic cause of autism spectrum disorder (ASD), yet it is not completely penetrant and can manifest in a wide array of phenotypes. To explore its molecular consequences, we performed RNA sequencing of cerebral cortex from mouse models with CNV of the syntenic 7qF3 region and lymphoblast lines from 34 members of 7 multiplex ASD-affected families harboring the 16p11.2 CNV. Expression of all genes in the CNV region correlated well with their DNA copy number, with no evidence of dosage compensation. We observed effects on gene expression outside the CNV region, including apparent positional effects in cis and in trans at genomic segments with evidence of physical interaction in Hi-C chromosome conformation data. One of the most significant positional effects was telomeric to the 16p11.2 CNV and includes the previously described "distal" 16p11.2 microdeletion. Overall, 16p11.2 CNV was associated with altered expression of genes and networks that converge on multiple hypotheses of ASD pathogenesis, including synaptic function (e.g., NRXN1, NRXN3), chromatin modification (e.g., CHD8, EHMT1, MECP2), transcriptional regulation (e.g., TCF4, SATB2), and intellectual disability (e.g., FMR1, CEP290). However, there were differences between tissues and species, with the strongest effects being consistently within the CNV region itself. Our analyses suggest that through a combination of indirect regulatory effects and direct effects on nuclear architecture, alteration of 16p11.2 genes disrupts expression networks that involve other genes and pathways known to contribute to ASD, suggesting an overlap in mechanisms of pathogenesis.


Assuntos
Transtorno Autístico/genética , Deleção Cromossômica , Duplicação Cromossômica , Cromossomos Humanos Par 16/genética , Animais , Córtex Cerebral/patologia , Criança , Variações do Número de Cópias de DNA , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Deficiência Intelectual/genética , Masculino , Camundongos , Linhagem , Fenótipo , Análise de Sequência de RNA , Transcrição Gênica
6.
Am J Med Genet A ; 173(2): 395-406, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27759917

RESUMO

We performed whole-genome sequencing on an individual from a family with variable psychiatric phenotypes that had a sensory processing disorder, apraxia, and autism. The proband harbored a maternally inherited balanced translocation (46,XY,t(11;14)(p12;p12)mat) that disrupted LRRC4C, a member of the highly specialized netrin G family of axon guidance molecules. The proband also inherited a paternally derived chromosomal inversion that disrupted DPP6, a potassium channel interacting protein. Copy Number (CN) analysis in 14,077 cases with neurodevelopmental disorders and 8,960 control subjects revealed that 60% of cases with exonic deletions in LRRC4C had a second clinically recognizable syndrome associated with variable clinical phenotypes, including 16p11.2, 1q44, and 2q33.1 CN syndromes, suggesting LRRC4C deletion variants may be modifiers of neurodevelopmental disorders. In vitro, functional assessments modeling patient deletions in LRRC4C suggest a negative regulatory role of these exons found in the untranslated region of LRRC4C, which has a single, terminal coding exon. These data suggest that the proband's autism may be due to the inheritance of disruptions in both DPP6 and LRRC4C, and may highlight the importance of the netrin G family and potassium channel interacting molecules in neurodevelopmental disorders. © 2016 Wiley Periodicals, Inc.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Estudos de Associação Genética , Proteínas do Tecido Nervoso/genética , Transtornos do Neurodesenvolvimento/diagnóstico , Transtornos do Neurodesenvolvimento/genética , Fenótipo , Canais de Potássio/genética , Receptores de Superfície Celular/genética , Regiões 5' não Traduzidas , Adolescente , Adulto , Apraxias/diagnóstico , Apraxias/genética , Transtorno Autístico/diagnóstico , Transtorno Autístico/genética , Criança , Pré-Escolar , Pontos de Quebra do Cromossomo , Inversão Cromossômica , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cariótipo , Masculino , Pessoa de Meia-Idade , Família Multigênica , Linhagem , Translocação Genética , Adulto Jovem
7.
Proc Natl Acad Sci U S A ; 111(42): E4468-77, 2014 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-25294932

RESUMO

Truncating mutations of chromodomain helicase DNA-binding protein 8 (CHD8), and of many other genes with diverse functions, are strong-effect risk factors for autism spectrum disorder (ASD), suggesting multiple mechanisms of pathogenesis. We explored the transcriptional networks that CHD8 regulates in neural progenitor cells (NPCs) by reducing its expression and then integrating transcriptome sequencing (RNA sequencing) with genome-wide CHD8 binding (ChIP sequencing). Suppressing CHD8 to levels comparable with the loss of a single allele caused altered expression of 1,756 genes, 64.9% of which were up-regulated. CHD8 showed widespread binding to chromatin, with 7,324 replicated sites that marked 5,658 genes. Integration of these data suggests that a limited array of direct regulatory effects of CHD8 produced a much larger network of secondary expression changes. Genes indirectly down-regulated (i.e., without CHD8-binding sites) reflect pathways involved in brain development, including synapse formation, neuron differentiation, cell adhesion, and axon guidance, whereas CHD8-bound genes are strongly associated with chromatin modification and transcriptional regulation. Genes associated with ASD were strongly enriched among indirectly down-regulated loci (P < 10(-8)) and CHD8-bound genes (P = 0.0043), which align with previously identified coexpression modules during fetal development. We also find an intriguing enrichment of cancer-related gene sets among CHD8-bound genes (P < 10(-10)). In vivo suppression of chd8 in zebrafish produced macrocephaly comparable to that of humans with inactivating mutations. These data indicate that heterozygous disruption of CHD8 precipitates a network of gene-expression changes involved in neurodevelopmental pathways in which many ASD-associated genes may converge on shared mechanisms of pathogenesis.


Assuntos
Transtornos Globais do Desenvolvimento Infantil/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Neurais/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Axônios/metabolismo , Sítios de Ligação , Transtornos Globais do Desenvolvimento Infantil/metabolismo , Cromatina/metabolismo , DNA Helicases/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Genoma , Heterozigoto , Humanos , Megalencefalia/metabolismo , Mutação , Neoplasias/metabolismo , Neurônios/metabolismo , Ligação Proteica , Fatores de Risco , Análise de Sequência de RNA , Software , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
8.
Am J Hum Genet ; 92(3): 375-86, 2013 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-23472757

RESUMO

NRXN1 microdeletions occur at a relatively high frequency and confer increased risk for neurodevelopmental and neurobehavioral abnormalities. The mechanism that makes NRXN1 a deletion hotspot is unknown. Here, we identified deletions of the NRXN1 region in affected cohorts, confirming a strong association with the autism spectrum and other neurodevelopmental disorders. Interestingly, deletions in both affected and control individuals were clustered in the 5' portion of NRXN1 and its immediate upstream region. To explore the mechanism of deletion, we mapped and analyzed the breakpoints of 32 deletions. At the deletion breakpoints, frequent microhomology (68.8%, 2-19 bp) suggested predominant mechanisms of DNA replication error and/or microhomology-mediated end-joining. Long terminal repeat (LTR) elements, unique non-B-DNA structures, and MEME-defined sequence motifs were significantly enriched, but Alu and LINE sequences were not. Importantly, small-size inverted repeats (minus self chains, minus sequence motifs, and partial complementary sequences) were significantly overrepresented in the vicinity of NRXN1 region deletion breakpoints, suggesting that, although they are not interrupted by the deletion process, such inverted repeats can predispose a region to genomic instability by mediating single-strand DNA looping via the annealing of partially reverse complementary strands and the promoting of DNA replication fork stalling and DNA replication error. Our observations highlight the potential importance of inverted repeats of variable sizes in generating a rearrangement hotspot in which individual breakpoints are not recurrent. Mechanisms that involve short inverted repeats in initiating deletion may also apply to other deletion hotspots in the human genome.


Assuntos
Moléculas de Adesão Celular Neuronais/genética , Variações do Número de Cópias de DNA , Sequências Repetidas Invertidas , Transtornos Mentais/genética , Proteínas do Tecido Nervoso/genética , Deleção de Sequência , Proteínas de Ligação ao Cálcio , Estudos de Coortes , Replicação do DNA/genética , DNA de Forma B/genética , DNA de Cadeia Simples/genética , Éxons , Predisposição Genética para Doença , Instabilidade Genômica , Humanos , Moléculas de Adesão de Célula Nervosa , Sequências Repetidas Terminais
9.
Neurodegener Dis ; 16(3-4): 245-59, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26959244

RESUMO

BACKGROUND: Huntington's disease is induced by CAG expansion in a single gene coding the huntingtin protein. The mutated huntingtin (mtHtt) primarily causes degeneration of neurons in the brain, but it also affects peripheral tissues, including testes. OBJECTIVE: We studied sperm and testes of transgenic boars expressing the N-terminal region of human mtHtt. METHODS: In this study, measures of reproductive parameters and electron microscopy (EM) images of spermatozoa and testes of transgenic (TgHD) and wild-type (WT) boars of F1 (24-48 months old) and F2 (12-36 months old) generations were compared. In addition, immunofluorescence, immunohistochemistry, Western blot, hormonal analysis and whole-genome sequencing were done in order to elucidate the effects of mtHtt. RESULTS: Evidence for fertility failure of both TgHD generations was observed at the age of 13 months. Reproductive parameters declined and progressively worsened with age. EM revealed numerous pathological features in sperm tails and in testicular epithelium from 24- and 36-month-old TgHD boars. Moreover, immunohistochemistry confirmed significantly lower proliferation activity of spermatogonia in transgenic testes. mtHtt was highly expressed in spermatozoa and testes of TgHD boars and localized in all cells of seminiferous tubules. Levels of fertility-related hormones did not differ in TgHD and WT siblings. Genome analysis confirmed that insertion of the lentiviral construct did not interrupt any coding sequence in the pig genome. CONCLUSIONS: The sperm and testicular degeneration of TgHD boars is caused by gain-of-function of the highly expressed mtHtt.


Assuntos
Proteína Huntingtina/metabolismo , Mutação , Espermatozoides/metabolismo , Espermatozoides/patologia , Testículo/metabolismo , Testículo/patologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Animais Geneticamente Modificados , Proliferação de Células/fisiologia , Modelos Animais de Doenças , Vetores Genéticos , Humanos , Proteína Huntingtina/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Lentivirus/genética , Masculino , Contagem de Espermatozoides , Suínos , Porco Miniatura
10.
Am J Hum Genet ; 91(6): 1128-34, 2012 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-23217328

RESUMO

Large intergenic noncoding (linc) RNAs represent a newly described class of ribonucleic acid whose importance in human disease remains undefined. We identified a severely developmentally delayed 16-year-old female with karyotype 46,XX,t(2;11)(p25.1;p15.1)dn in the absence of clinically significant copy number variants (CNVs). DNA capture followed by next-generation sequencing of the translocation breakpoints revealed disruption of a single noncoding gene on chromosome 2, LINC00299, whose RNA product is expressed in all tissues measured, but most abundantly in brain. Among a series of additional, unrelated subjects referred for clinical diagnostic testing who showed CNV affecting this locus, we identified four with exon-crossing deletions in association with neurodevelopmental abnormalities. No disruption of the LINC00299 coding sequence was seen in almost 14,000 control subjects. Together, these subjects with disruption of LINC00299 implicate this particular noncoding RNA in brain development and raise the possibility that, as a class, abnormalities of lincRNAs may play a significant role in human developmental disorders.


Assuntos
Deficiências do Desenvolvimento/genética , Mutação , RNA Longo não Codificante/genética , Adolescente , Processamento Alternativo , Sequência de Bases , Pontos de Quebra do Cromossomo , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 2 , Feminino , Ordem dos Genes , Humanos , Linfócitos/metabolismo , Dados de Sequência Molecular , Células-Tronco Neurais/metabolismo , Translocação Genética
11.
N Engl J Med ; 367(23): 2226-32, 2012 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-23215558

RESUMO

Conventional cytogenetic testing offers low-resolution detection of balanced karyotypic abnormalities but cannot provide the precise, gene-level knowledge required to predict outcomes. The use of high-resolution whole-genome deep sequencing is currently impractical for the purpose of routine clinical care. We show here that whole-genome "jumping libraries" can offer an immediately applicable, nucleotide-level complement to conventional genetic diagnostics within a time frame that allows for clinical action. We performed large-insert sequencing of DNA extracted from amniotic-fluid cells with a balanced de novo translocation. The amniotic-fluid sample was from a patient in the third trimester of pregnancy who underwent amniocentesis because of severe polyhydramnios after multiple fetal anomalies had been detected on ultrasonography. Using a 13-day sequence and analysis pipeline, we discovered direct disruption of CHD7, a causal locus in the CHARGE syndrome (coloboma of the eye, heart anomaly, atresia of the choanae, retardation, and genital and ear anomalies). Clinical findings at birth were consistent with the CHARGE syndrome, a diagnosis that could not have been reliably inferred from the cytogenetic breakpoint. This case study illustrates the potential power of customized whole-genome jumping libraries when used to augment prenatal karyotyping.


Assuntos
Síndrome CHARGE/genética , Transtornos Cromossômicos/diagnóstico , Testes Genéticos/métodos , Biblioteca Genômica , Cardiopatias Congênitas/genética , Análise de Sequência com Séries de Oligonucleotídeos , Diagnóstico Pré-Natal/métodos , Adulto , Síndrome CHARGE/diagnóstico , Aberrações Cromossômicas , Feminino , Doenças Fetais/diagnóstico , Genoma Humano , Cardiopatias Congênitas/diagnóstico por imagem , Humanos , Cariótipo , Mutação , Gravidez , Translocação Genética , Ultrassonografia Pré-Natal
12.
Hum Genet ; 132(5): 537-52, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23354975

RESUMO

We describe a female subject (DGAP100) with a 46,X,t(X;5)(p11.3;q35.3)inv(5)(q35.3q35.1)dn, severe psychomotor retardation with hypotonia, global postnatal growth restriction, microcephaly, globally reduced cerebral volume, seizures, facial dysmorphia and cleft palate. Fluorescence in situ hybridization and whole-genome sequencing demonstrated that the X chromosome breakpoint disrupts KDM6A in the second intron. No genes were directly disrupted on chromosome 5. KDM6A is a histone 3 lysine 27 demethylase and a histone 3 lysine 4 methyltransferase. Expression of KDM6A is significantly reduced in DGAP100 lymphoblastoid cells compared to control samples. We identified nine additional cases with neurodevelopmental delay and various other features consistent with the DGAP100 phenotype with copy number variation encompassing KDM6A from microarray databases. We evaluated haploinsufficiency of kdm6a in a zebrafish model. kdm6a is expressed in the pharyngeal arches and ethmoid plate of the developing zebrafish, while a kdm6a morpholino knockdown exhibited craniofacial defects. We conclude KDM6A dosage regulation is associated with severe and diverse structural defects and developmental abnormalities.


Assuntos
Anormalidades Múltiplas/genética , Cromossomos Humanos Par 5 , Haploinsuficiência/genética , Histona Desmetilases/genética , Proteínas Nucleares/genética , Cromossomo X , Animais , Região Branquial/enzimologia , Linhagem Celular , Cromossomos Humanos Par 5/genética , Fissura Palatina/genética , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Feminino , Técnicas de Silenciamento de Genes , Histona Desmetilases/metabolismo , Humanos , Deficiência Intelectual/genética , Cariotipagem , Microcefalia/genética , Hipotonia Muscular/genética , Proteínas Nucleares/metabolismo , Fenótipo , Transtornos Psicomotores/genética , Convulsões/genética , Translocação Genética , Cromossomo X/genética , Adulto Jovem , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
14.
Hum Mutat ; 33(4): 728-40, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22290657

RESUMO

SOX5 encodes a transcription factor involved in the regulation of chondrogenesis and the development of the nervous system. Despite its important developmental roles, SOX5 disruption has yet to be associated with human disease. We report one individual with a reciprocal translocation breakpoint within SOX5, eight individuals with intragenic SOX5 deletions (four are apparently de novo and one inherited from an affected parent), and seven individuals with larger 12p12 deletions encompassing SOX5. Common features in these subjects include prominent speech delay, intellectual disability, behavior abnormalities, and dysmorphic features. The phenotypic impact of the deletions may depend on the location of the deletion and, consequently, which of the three major SOX5 protein isoforms are affected. One intragenic deletion, involving only untranslated exons, was present in a more mildly affected subject, was inherited from a healthy parent and grandparent, and is similar to a deletion found in a control cohort. Therefore, some intragenic SOX5 deletions may have minimal phenotypic effect. Based on the location of the deletions in the subjects compared to the controls, the de novo nature of most of these deletions, and the phenotypic similarities among cases, SOX5 appears to be a dosage-sensitive, developmentally important gene.


Assuntos
Transtornos Dismórficos Corporais/genética , Deficiências do Desenvolvimento/genética , Haploinsuficiência , Transtornos do Desenvolvimento da Linguagem/genética , Transtornos Mentais/genética , Fatores de Transcrição SOXD/genética , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Cromossomos Humanos Par 12 , Feminino , Humanos , Masculino
15.
Adv Healthc Mater ; 11(9): e2101944, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34889072

RESUMO

Engineered immune cells are an exciting therapeutic modality, which survey and attack tumors. Backpacking strategies exploit cell targeting capabilities for delivery of drugs to combat tumors and their immune-suppressive environments. Here, a new platform for arming cell therapeutics through dual receptor and polymeric prodrug engineering is developed. Macrophage and T cell therapeutics are engineered to express a bioorthogonal single chain variable fragment receptor. The receptor binds a fluorescein ligand that directs cell loading with ligand-tagged polymeric prodrugs, termed "drugamers." The fluorescein ligand facilitates stable binding of drugamer to engineered macrophages over 10 days with 80% surface retention. Drugamers also incorporate prodrug monomers of the phosphoinositide-3-kinase inhibitor, PI-103. The extended release of PI-103 from the drugamer sustains antiproliferative activity against a glioblastoma cell line compared to the parent drug. The versatility and modularity of this cell arming system is demonstrated by loading T cells with a second fluorescein-drugamer. This drugamer incorporates a small molecule estrogen analog, CMP8, which stabilizes a degron-tagged transgene to provide temporal regulation of protein activity in engineered T cells. These results demonstrate that this bioorthogonal receptor and drugamer system can be used to arm multiple immune cell classes with both antitumor and transgene-activating small molecule prodrugs.


Assuntos
Neoplasias , Pró-Fármacos , Fluoresceínas , Humanos , Ligantes , Polímeros/química , Pró-Fármacos/química , Pró-Fármacos/farmacologia
16.
Hepatology ; 52(4): 1350-9, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20803559

RESUMO

UNLABELLED: Immune-mediated liver injury in hepatitis is due to activated T cells producing interferon-γ (IFN-γ). It is important to identify negative feedback immune mechanisms that can regulate T cell activity. In this study, we demonstrate that liver inflammation mediated by type 1 T helper (Th1) cells can induce the accumulation of myeloid-derived suppressor cells (MDSCs), pleiomorphic cells capable of modulating T cell-mediated immunity, that heretofore have been studied almost exclusively in the context of tumor-associated inflammation. Mice deficient in the gene encoding transforming growth factor-ß1 (Tgfb1(-/-) mice) acutely develop liver necroinflammation caused by IFN-γ-producing clusters of differentiation 4-positive (CD4(+)) T cells. Liver Th1 cell accumulation was accompanied by myeloid cells expressing CD11b and Gr1, phenotypic hallmarks of MDSCs. Isolated Tgfb1(-/-) liver CD11b(+)Gr1(+) cells were functional MDSCs, readily suppressing T cell proliferation in vitro. Pharmacologic inhibitors of inducible nitric oxide (NO) synthase completely eliminated suppressor function. Suppressor function and the production of NO were dependent on cell-cell contact between MDSCs and T cells, and upon IFN-γ, and were specifically associated with the "monocytic" CD11b(+)Ly6G(-) Ly6C(hi) subset of liver Tgfb1(-/-) CD11b(+) cells. The rapid accumulation of CD11b(+)Gr1(+) cells in Tgfb1(-/-) liver was abrogated when mice were either depleted of CD4(+) T cells or rendered unable to produce IFN-γ, showing that Th1 activity induces MDSC accumulation. CONCLUSION: Th1 liver inflammation mobilizes an MDSC response that, through the production of NO, can inhibit T cell proliferation. We propose that MDSCs serve an important negative feedback function in liver immune homeostasis, and that insufficient or inappropriate activity of this cell population may contribute to inflammatory liver pathology.


Assuntos
Antígeno CD11b/fisiologia , Hepatite/imunologia , Interferon gama/fisiologia , Fator de Crescimento Transformador beta1/fisiologia , Animais , Linfócitos T CD4-Positivos/imunologia , Comunicação Celular , Proliferação de Células/efeitos dos fármacos , Fígado/imunologia , Camundongos , Camundongos Knockout , Células Mieloides/imunologia , Células Progenitoras Mieloides/fisiologia , Óxido Nítrico/fisiologia , Receptores de Quimiocinas/biossíntese , Células Th1/imunologia
17.
FEBS Open Bio ; 11(3): 705-713, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33480189

RESUMO

Combinations of human lysozyme (hLYS) and antimicrobial peptides (AMPs) are known to exhibit either additive or synergistic activity, and as a result, they have therapeutic potential for persistent and antibiotic-resistant infections. We examined hLYS activity against Pseudomonas aeruginosa when combined with six different AMPs. In contrast to prior reports, we discovered that some therapeutically relevant AMPs manifest striking antagonistic interactions with hLYS across particular concentration ranges. We further found that the synthetic AMP Tet009 can inhibit hLYS-mediated bacterial lysis. To the best of our knowledge, these results represent the first observations of antagonism between hLYS and AMPs, and they advise that future development of lytic enzyme and AMP combination therapies considers the potential for antagonistic interactions.


Assuntos
Peptídeos Antimicrobianos/farmacologia , Muramidase/efeitos adversos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Peptídeos Antimicrobianos/química , Bacteriólise/efeitos dos fármacos , Antagonismo de Drogas , Humanos , Pseudomonas aeruginosa/efeitos dos fármacos
18.
Sci Rep ; 7: 41120, 2017 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-28120936

RESUMO

Integration of exogenous DNA into a host genome represents an important route to generate animal and cellular models for exploration into human disease and therapeutic development. In most models, little is known concerning structural integrity of the transgene, precise site of integration, or its impact on the host genome. We previously used whole-genome and targeted sequencing approaches to reconstruct transgene structure and integration sites in models of Huntington's disease, revealing complex structural rearrangements that can result from transgenesis. Here, we demonstrate in the R6/2 mouse, a widely used Huntington's disease model, that integration of a rearranged transgene with coincident deletion of 5,444 bp of host genome within the gene Gm12695 has striking molecular consequences. Gm12695, the function of which is unknown, is normally expressed at negligible levels in mouse brain, but transgene integration has resulted in cortical expression of a partial fragment (exons 8-11) 3' to the transgene integration site in R6/2. This transcript shows significant expression among the extensive network of differentially expressed genes associated with this model, including synaptic transmission, cell signalling and transcription. These data illustrate the value of sequence-level resolution of transgene insertions and transcription analysis to inform phenotypic characterization of transgenic models utilized in therapeutic research.


Assuntos
Modelos Animais de Doenças , Doença de Huntington/patologia , Camundongos Transgênicos , Animais , Perfilação da Expressão Gênica , Rearranjo Gênico , Humanos , Camundongos , Análise de Sequência de DNA , Deleção de Sequência
19.
Nat Genet ; 49(1): 36-45, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27841880

RESUMO

Despite the clinical significance of balanced chromosomal abnormalities (BCAs), their characterization has largely been restricted to cytogenetic resolution. We explored the landscape of BCAs at nucleotide resolution in 273 subjects with a spectrum of congenital anomalies. Whole-genome sequencing revised 93% of karyotypes and demonstrated complexity that was cryptic to karyotyping in 21% of BCAs, highlighting the limitations of conventional cytogenetic approaches. At least 33.9% of BCAs resulted in gene disruption that likely contributed to the developmental phenotype, 5.2% were associated with pathogenic genomic imbalances, and 7.3% disrupted topologically associated domains (TADs) encompassing known syndromic loci. Remarkably, BCA breakpoints in eight subjects altered a single TAD encompassing MEF2C, a known driver of 5q14.3 microdeletion syndrome, resulting in decreased MEF2C expression. We propose that sequence-level resolution dramatically improves prediction of clinical outcomes for balanced rearrangements and provides insight into new pathogenic mechanisms, such as altered regulation due to changes in chromosome topology.


Assuntos
Aberrações Cromossômicas , Anormalidades Congênitas/genética , Rearranjo Gênico , Marcadores Genéticos/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Feminino , Humanos , Masculino
20.
Nat Neurosci ; 19(3): 517-22, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26829649

RESUMO

Recurrent, reciprocal genomic disorders resulting from non-allelic homologous recombination (NAHR) between near-identical segmental duplications (SDs) are a major cause of human disease, often producing phenotypically distinct syndromes. The genomic architecture of flanking SDs presents a challenge for modeling these syndromes; however, the capability to efficiently generate reciprocal copy number variants (CNVs) that mimic NAHR would represent a valuable modeling tool. We describe here a CRISPR/Cas9 genome engineering method, single-guide CRISPR/Cas targeting of repetitive elements (SCORE), to model reciprocal genomic disorders and demonstrate its capabilities by generating reciprocal CNVs of 16p11.2 and 15q13.3, including alteration of one copy-equivalent of the SDs that mediate NAHR in vivo. The method is reproducible, and RNA sequencing reliably clusters transcriptional signatures from human subjects with in vivo CNVs and their corresponding in vitro models. This new approach will provide broad applicability for the study of genomic disorders and, with further development, may also permit efficient correction of these defects.


Assuntos
Transtorno Autístico/genética , Sistemas CRISPR-Cas/genética , Transtornos Cromossômicos/genética , Variações do Número de Cópias de DNA/genética , Engenharia Genética/métodos , Deficiência Intelectual/genética , Duplicações Segmentares Genômicas/genética , Convulsões/genética , Deleção de Sequência/genética , Deleção Cromossômica , Cromossomos Humanos Par 15/genética , Cromossomos Humanos Par 16/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Humanos
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