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1.
J Med Genet ; 51(11): 724-36, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25167861

RESUMO

BACKGROUND: Intellectual disability (ID) is characterised by an extreme genetic heterogeneity. Several hundred genes have been associated to monogenic forms of ID, considerably complicating molecular diagnostics. Trio-exome sequencing was recently proposed as a diagnostic approach, yet remains costly for a general implementation. METHODS: We report the alternative strategy of targeted high-throughput sequencing of 217 genes in which mutations had been reported in patients with ID or autism as the major clinical concern. We analysed 106 patients with ID of unknown aetiology following array-CGH analysis and other genetic investigations. Ninety per cent of these patients were males, and 75% sporadic cases. RESULTS: We identified 26 causative mutations: 16 in X-linked genes (ATRX, CUL4B, DMD, FMR1, HCFC1, IL1RAPL1, IQSEC2, KDM5C, MAOA, MECP2, SLC9A6, SLC16A2, PHF8) and 10 de novo in autosomal-dominant genes (DYRK1A, GRIN1, MED13L, TCF4, RAI1, SHANK3, SLC2A1, SYNGAP1). We also detected four possibly causative mutations (eg, in NLGN3) requiring further investigations. We present detailed reasoning for assigning causality for each mutation, and associated patients' clinical information. Some genes were hit more than once in our cohort, suggesting they correspond to more frequent ID-associated conditions (KDM5C, MECP2, DYRK1A, TCF4). We highlight some unexpected genotype to phenotype correlations, with causative mutations being identified in genes associated to defined syndromes in patients deviating from the classic phenotype (DMD, TCF4, MECP2). We also bring additional supportive (HCFC1, MED13L) or unsupportive (SHROOM4, SRPX2) evidences for the implication of previous candidate genes or mutations in cognitive disorders. CONCLUSIONS: With a diagnostic yield of 25% targeted sequencing appears relevant as a first intention test for the diagnosis of ID, but importantly will also contribute to a better understanding regarding the specific contribution of the many genes implicated in ID and autism.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Técnicas de Diagnóstico Molecular/métodos , Adolescente , Adulto , Criança , Pré-Escolar , Análise Mutacional de DNA/métodos , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Análise de Sequência de DNA/métodos , Adulto Jovem
2.
J Vis Exp ; (197)2023 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-37486110

RESUMO

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of satellite cells from the limb muscle, a tissue with a high content of structural proteins. Dissected limb muscles from adult mice were mechanically disrupted by mincing in medium supplemented with dispase and type I collagenase. Upon digestion, the homogenate was filtered through cell strainers, and cells were suspended in FACS buffer. Viability was determined with fixable viability stain, and immunostained satellite cells were isolated by FACS. Cells were lysed with Triton X-100 and released nuclei were bound to concanavalin A magnetic beads. Nucleus/bead complexes were incubated with antibodies against the transcription factor or histone modifications of interest. After washes, nucleus/bead complexes were incubated with protein A-micrococcal nuclease, and chromatin cleavage was initiated with CaCl2. After DNA extraction, libraries were generated and sequenced, and the profiles for genome-wide transcription factor binding and covalent histone modifications were obtained by bioinformatic analysis. The peaks obtained for the various histone marks showed that the binding events were specific for satellite cells. Moreover, known motif analysis unveiled that the transcription factor was bound to chromatin via its cognate response element. This protocol is therefore adapted to study gene regulation in adult mice limb muscle satellite cells.


Assuntos
Células Satélites de Músculo Esquelético , Camundongos , Animais , Citometria de Fluxo , Estudo de Associação Genômica Ampla , Cromatina , Fatores de Transcrição
3.
J Exp Med ; 218(10)2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34459852

RESUMO

Our understanding of cell fate decisions in hematopoietic stem cells is incomplete. Here, we show that the transcription factor Helios is highly expressed in murine hematopoietic stem and progenitor cells (HSPCs), where it is required to suppress the separation of the platelet/megakaryocyte lineage from the HSPC pool. Helios acts mainly in quiescent cells, where it directly represses the megakaryocyte gene expression program in cells as early as the stem cell stage. Helios binding promotes chromatin compaction, notably at the regulatory regions of platelet-specific genes recognized by the Gata2 and Runx1 transcriptional activators, implicated in megakaryocyte priming. Helios null HSPCs are biased toward the megakaryocyte lineage at the expense of the lymphoid and partially resemble cells of aging animals. We propose that Helios acts as a guardian of HSPC pluripotency by continuously repressing the megakaryocyte fate, which in turn allows downstream lymphoid priming to take place. These results highlight the importance of negative and positive priming events in lineage commitment.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Megacariócitos/fisiologia , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Linfócitos/citologia , Linfócitos/fisiologia , Masculino , Megacariócitos/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Linfócitos T/citologia , Linfócitos T/fisiologia , Fatores de Transcrição/genética
4.
Nat Commun ; 7: 11067, 2016 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-27063795

RESUMO

Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing changes. Cardiac alterations, characterized by conduction delays and arrhythmia, are the second most common cause of death in DM. Using RNA sequencing, here we identify novel splicing alterations in DM heart samples, including a switch from adult exon 6B towards fetal exon 6A in the cardiac sodium channel, SCN5A. We find that MBNL1 regulates alternative splicing of SCN5A mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability compared with the control adult isoform. Importantly, reproducing splicing alteration of Scn5a in mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant features of myotonic dystrophy. In conclusion, misregulation of the alternative splicing of SCN5A may contribute to a subset of the cardiac dysfunctions observed in myotonic dystrophy.


Assuntos
Processamento Alternativo/genética , Arritmias Cardíacas/complicações , Arritmias Cardíacas/genética , Sistema de Condução Cardíaco/fisiopatologia , Distrofia Miotônica/complicações , Distrofia Miotônica/genética , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Adulto , Idoso , Animais , Sequência de Bases , Sítios de Ligação , Simulação por Computador , Fenômenos Eletrofisiológicos , Éxons/genética , Feminino , Células HEK293 , Sistema de Condução Cardíaco/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Motivos de Nucleotídeos/genética , Proteínas de Ligação a RNA/metabolismo , Canais de Sódio/metabolismo , Xenopus
5.
Res Microbiol ; 166(3): 205-14, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25753102

RESUMO

Pseudomonas xanthomarina S11 is an arsenite-oxidizing bacterium isolated from an arsenic-contaminated former gold mine in Salsigne, France. This bacterium showed high resistance to arsenite and was able to oxidize arsenite to arsenate at concentrations up to 42.72 mM As[III]. The genome of this strain was sequenced and revealed the presence of three ars clusters. One of them is located on a plasmid and is organized as an "arsenic island" harbouring an aio operon and genes involved in phosphorous metabolism, in addition to the ars genes. Neither the aioXRS genes nor a specific sigma-54-dependent promoter located upstream of aioBA genes, both involved in regulation of arsenite oxidase expression in other arsenite-oxidizing bacteria, could be identified in the genome. This observation is in accordance with the fact that no difference was observed in expression of arsenite oxidase in P. xanthomarina S11, whether or not the strain was grown in the presence of As[III].


Assuntos
Arsênio/metabolismo , Proteínas de Bactérias/genética , Oxirredutases/genética , Oxirredutases/metabolismo , Pseudomonas/genética , Arseniatos/metabolismo , Arsênio/farmacologia , ATPases Transportadoras de Arsenito/genética , ATPases Transportadoras de Arsenito/metabolismo , Arsenitos/metabolismo , Arsenitos/farmacologia , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Bacteriano , Farmacorresistência Bacteriana , França , Regulação Bacteriana da Expressão Gênica , Mineração , Óperon , Oxirredução , Filogenia , Plasmídeos , Pseudomonas/enzimologia , Pseudomonas/crescimento & desenvolvimento , Pseudomonas/isolamento & purificação
6.
PLoS One ; 9(2): e87365, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24498316

RESUMO

Collagen 6A3 (Col6a3), a component of extracellular matrix, is often up-regulated in tumours and is believed to play a pro-oncogenic role. However the mechanisms of its tumorigenic activity are poorly understood. We show here that Col6a3 is highly expressed in densely growing mouse embryonic fibroblasts (MEFs). In MEFs where the TAF4 subunit of general transcription factor IID (TFIID) has been inactivated, elevated Col6a3 expression prevents contact inhibition promoting their 3 dimensional growth as foci and fibrospheres. Analyses of gene expression in densely growing Taf4(-/-) MEFs revealed repression of the Hippo pathway and activation of Wnt signalling. The Hippo activator Kibra/Wwc1 is repressed under dense conditions in Taf4(-/-) MEFs, leading to nuclear accumulation of the proliferation factor YAP1 in the cells forming 3D foci. At the same time, Wnt9a is activated and the Sfrp2 antagonist of Wnt signalling is repressed. Surprisingly, treatment of Taf4(-/-) MEFs with all-trans retinoic acid (ATRA) restores contact inhibition suppressing 3D growth. ATRA represses Col6a3 expression independently of TAF4 expression and Col6a3 silencing is sufficient to restore contact inhibition in Taf4(-/-) MEFs and to suppress 3D growth by reactivating Kibra expression to induce Hippo signalling and by inducing Sfrp2 expression to antagonize Wnt signalling. All together, these results reveal a critical role for Col6a3 in regulating both Hippo and Wnt signalling to promote 3D growth, and show that the TFIID subunit TAF4 is essential to restrain the growth promoting properties of Col6a3. Our data provide new insight into the role of extra cellular matrix components in regulating cell growth.


Assuntos
Proliferação de Células , Colágeno Tipo VI/metabolismo , Fibroblastos/metabolismo , Fator de Transcrição TFIID/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Proteínas de Ciclo Celular , Células Cultivadas , Colágeno Tipo VI/genética , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Expressão Gênica , Via de Sinalização Hippo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Microscopia Confocal , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Fator de Transcrição TFIID/genética , Tretinoína/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética , Proteínas de Sinalização YAP
7.
Genome Announc ; 1(5)2013 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-24115546

RESUMO

We report the genome sequence of Halomonas sp. strain A3H3, a bacterium with a high tolerance to arsenite, isolated from multicontaminated sediments of the l'Estaque harbor in Marseille, France. The genome is composed of a 5,489,893-bp chromosome and a 157,085-bp plasmid.

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