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1.
Hum Mol Genet ; 32(9): 1497-1510, 2023 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-36579832

RESUMEN

TBR1 is a neuron-specific transcription factor involved in brain development and implicated in a neurodevelopmental disorder (NDD) combining features of autism spectrum disorder (ASD), intellectual disability (ID) and speech delay. TBR1 has been previously shown to interact with a small number of transcription factors and co-factors also involved in NDDs (including CASK, FOXP1/2/4 and BCL11A), suggesting that the wider TBR1 interactome may have a significant bearing on normal and abnormal brain development. Here, we have identified approximately 250 putative TBR1-interaction partners by affinity purification coupled to mass spectrometry. As well as known TBR1-interactors such as CASK, the identified partners include transcription factors and chromatin modifiers, along with ASD- and ID-related proteins. Five interaction candidates were independently validated using bioluminescence resonance energy transfer assays. We went on to test the interaction of these candidates with TBR1 protein variants implicated in cases of NDD. The assays uncovered disturbed interactions for NDD-associated variants and identified two distinct protein-binding domains of TBR1 that have essential roles in protein-protein interaction.


Asunto(s)
Trastornos del Neurodesarrollo , Proteínas de Dominio T Box , Humanos , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , Unión Proteica/genética , Unión Proteica/fisiología , Proteínas/genética , Proteínas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
2.
Am J Hum Genet ; 105(2): 403-412, 2019 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-31303265

RESUMEN

POU3F3, also referred to as Brain-1, is a well-known transcription factor involved in the development of the central nervous system, but it has not previously been associated with a neurodevelopmental disorder. Here, we report the identification of 19 individuals with heterozygous POU3F3 disruptions, most of which are de novo variants. All individuals had developmental delays and/or intellectual disability and impairments in speech and language skills. Thirteen individuals had characteristic low-set, prominent, and/or cupped ears. Brain abnormalities were observed in seven of eleven MRI reports. POU3F3 is an intronless gene, insensitive to nonsense-mediated decay, and 13 individuals carried protein-truncating variants. All truncating variants that we tested in cellular models led to aberrant subcellular localization of the encoded protein. Luciferase assays demonstrated negative effects of these alleles on transcriptional activation of a reporter with a FOXP2-derived binding motif. In addition to the loss-of-function variants, five individuals had missense variants that clustered at specific positions within the functional domains, and one small in-frame deletion was identified. Two missense variants showed reduced transactivation capacity in our assays, whereas one variant displayed gain-of-function effects, suggesting a distinct pathophysiological mechanism. In bioluminescence resonance energy transfer (BRET) interaction assays, all the truncated POU3F3 versions that we tested had significantly impaired dimerization capacities, whereas all missense variants showed unaffected dimerization with wild-type POU3F3. Taken together, our identification and functional cell-based analyses of pathogenic variants in POU3F3, coupled with a clinical characterization, implicate disruptions of this gene in a characteristic neurodevelopmental disorder.


Asunto(s)
Regulación de la Expresión Génica , Mutación , Trastornos del Neurodesarrollo/etiología , Factores del Dominio POU/genética , Activación Transcripcional , Secuencia de Aminoácidos , Niño , Femenino , Estudios de Asociación Genética , Genotipo , Humanos , Masculino , Trastornos del Neurodesarrollo/patología , Factores del Dominio POU/química , Conformación Proteica , Homología de Secuencia
3.
Hum Mol Genet ; 27(7): 1212-1227, 2018 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-29365100

RESUMEN

FOXP transcription factors play important roles in neurodevelopment, but little is known about how their transcriptional activity is regulated. FOXP proteins cooperatively regulate gene expression by forming homo- and hetero-dimers with each other. Physical associations with other transcription factors might also modulate the functions of FOXP proteins. However, few FOXP-interacting transcription factors have been identified so far. Therefore, we sought to discover additional transcription factors that interact with the brain-expressed FOXP proteins, FOXP1, FOXP2 and FOXP4, through affinity-purifications of protein complexes followed by mass spectrometry. We identified seven novel FOXP-interacting transcription factors (NR2F1, NR2F2, SATB1, SATB2, SOX5, YY1 and ZMYM2), five of which have well-estabslished roles in cortical development. Accordingly, we found that these transcription factors are co-expressed with FoxP2 in the deep layers of the cerebral cortex and also in the Purkinje cells of the cerebellum, suggesting that they may cooperate with the FoxPs to regulate neural gene expression in vivo. Moreover, we demonstrated that etiological mutations of FOXP1 and FOXP2, known to cause neurodevelopmental disorders, severely disrupted the interactions with FOXP-interacting transcription factors. Additionally, we pinpointed specific regions within FOXP2 sequence involved in mediating these interactions. Thus, by expanding the FOXP interactome we have uncovered part of a broader neural transcription factor network involved in cortical development, providing novel molecular insights into the transcriptional architecture underlying brain development and neurodevelopmental disorders.


Asunto(s)
Factores de Transcripción Forkhead , Regulación de la Expresión Génica , Trastornos del Neurodesarrollo , Células de Purkinje/metabolismo , Proteínas Represoras , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Células HEK293 , Humanos , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , Trastornos del Neurodesarrollo/patología , Células de Purkinje/patología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
4.
Trends Genet ; 33(9): 642-656, 2017 09.
Artículo en Inglés | MEDLINE | ID: mdl-28781152

RESUMEN

Investigation of the biological basis of human speech and language is being transformed by developments in molecular technologies, including high-throughput genotyping and next-generation sequencing of whole genomes. These advances are shedding new light on the genetic architecture underlying language-related disorders (speech apraxia, specific language impairment, developmental dyslexia) as well as that contributing to variation in relevant skills in the general population. We discuss how state-of-the-art methods are uncovering a range of genetic mechanisms, from rare mutations of large effect to common polymorphisms that increase risk in a subtle way, while converging on neurogenetic pathways that are shared between distinct disorders. We consider the future of the field, highlighting the unusual challenges and opportunities associated with studying genomics of language-related traits.


Asunto(s)
Genoma , Lenguaje , Habla , Humanos
5.
PLoS Genet ; 13(3): e1006683, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-28346496

RESUMEN

Schinzel-Giedion syndrome (SGS) is a rare developmental disorder characterized by multiple malformations, severe neurological alterations and increased risk of malignancy. SGS is caused by de novo germline mutations clustering to a 12bp hotspot in exon 4 of SETBP1. Mutations in this hotspot disrupt a degron, a signal for the regulation of protein degradation, and lead to the accumulation of SETBP1 protein. Overlapping SETBP1 hotspot mutations have been observed recurrently as somatic events in leukemia. We collected clinical information of 47 SGS patients (including 26 novel cases) with germline SETBP1 mutations and of four individuals with a milder phenotype caused by de novo germline mutations adjacent to the SETBP1 hotspot. Different mutations within and around the SETBP1 hotspot have varying effects on SETBP1 stability and protein levels in vitro and in in silico modeling. Substitutions in SETBP1 residue I871 result in a weak increase in protein levels and mutations affecting this residue are significantly more frequent in SGS than in leukemia. On the other hand, substitutions in residue D868 lead to the largest increase in protein levels. Individuals with germline mutations affecting D868 have enhanced cell proliferation in vitro and higher incidence of cancer compared to patients with other germline SETBP1 mutations. Our findings substantiate that, despite their overlap, somatic SETBP1 mutations driving malignancy are more disruptive to the degron than germline SETBP1 mutations causing SGS. Additionally, this suggests that the functional threshold for the development of cancer driven by the disruption of the SETBP1 degron is higher than for the alteration in prenatal development in SGS. Drawing on previous studies of somatic SETBP1 mutations in leukemia, our results reveal a genotype-phenotype correlation in germline SETBP1 mutations spanning a molecular, cellular and clinical phenotype.


Asunto(s)
Anomalías Múltiples/genética , Proteínas Portadoras/genética , Anomalías Craneofaciales/genética , Predisposición Genética a la Enfermedad/genética , Deformidades Congénitas de la Mano/genética , Neoplasias Hematológicas/genética , Discapacidad Intelectual/genética , Mutación , Uñas Malformadas/genética , Proteínas Nucleares/genética , Anomalías Múltiples/metabolismo , Anomalías Múltiples/patología , Western Blotting , Proteínas Portadoras/metabolismo , Línea Celular , Proliferación Celular/genética , Transformación Celular Neoplásica/genética , Niño , Preescolar , Anomalías Craneofaciales/metabolismo , Anomalías Craneofaciales/patología , Femenino , Perfilación de la Expresión Génica , Estudios de Asociación Genética , Mutación de Línea Germinal , Células HEK293 , Deformidades Congénitas de la Mano/metabolismo , Deformidades Congénitas de la Mano/patología , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patología , Humanos , Lactante , Recién Nacido , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Masculino , Uñas Malformadas/metabolismo , Uñas Malformadas/patología , Proteínas Nucleares/metabolismo , Fenotipo
6.
Am J Hum Genet ; 99(2): 253-74, 2016 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-27453576

RESUMEN

Intellectual disability (ID) is a common condition with considerable genetic heterogeneity. Next-generation sequencing of large cohorts has identified an increasing number of genes implicated in ID, but their roles in neurodevelopment remain largely unexplored. Here we report an ID syndrome caused by de novo heterozygous missense, nonsense, and frameshift mutations in BCL11A, encoding a transcription factor that is a putative member of the BAF swi/snf chromatin-remodeling complex. Using a comprehensive integrated approach to ID disease modeling, involving human cellular analyses coupled to mouse behavioral, neuroanatomical, and molecular phenotyping, we provide multiple lines of functional evidence for phenotypic effects. The etiological missense variants cluster in the amino-terminal region of human BCL11A, and we demonstrate that they all disrupt its localization, dimerization, and transcriptional regulatory activity, consistent with a loss of function. We show that Bcl11a haploinsufficiency in mice causes impaired cognition, abnormal social behavior, and microcephaly in accordance with the human phenotype. Furthermore, we identify shared aberrant transcriptional profiles in the cortex and hippocampus of these mouse models. Thus, our work implicates BCL11A haploinsufficiency in neurodevelopmental disorders and defines additional targets regulated by this gene, with broad relevance for our understanding of ID and related syndromes.


Asunto(s)
Proteínas Portadoras/genética , Haploinsuficiencia/genética , Discapacidad Intelectual/genética , Trastornos del Neurodesarrollo/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Transcripción Genética , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Corteza Cerebral/metabolismo , Ensamble y Desensamble de Cromatina/genética , Codón sin Sentido/genética , Trastornos del Conocimiento/genética , Mutación del Sistema de Lectura/genética , Hipocampo/metabolismo , Humanos , Discapacidad Intelectual/patología , Discapacidad Intelectual/psicología , Masculino , Ratones , Microcefalia/genética , Mutación Missense/genética , Trastornos del Neurodesarrollo/patología , Trastornos del Neurodesarrollo/fisiopatología , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fenotipo , Proteínas Represoras , Conducta Social , Síndrome , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transcriptoma
7.
Hum Mol Genet ; 25(3): 546-57, 2016 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-26647308

RESUMEN

De novo disruptions of the neural transcription factor FOXP1 are a recently discovered, rare cause of sporadic intellectual disability (ID). We report three new cases of FOXP1-related disorder identified through clinical whole-exome sequencing. Detailed phenotypic assessment confirmed that global developmental delay, autistic features, speech/language deficits, hypotonia and mild dysmorphic features are core features of the disorder. We expand the phenotypic spectrum to include sensory integration disorder and hypertelorism. Notably, the etiological variants in these cases include two missense variants within the DNA-binding domain of FOXP1. Only one such variant has been reported previously. The third patient carries a stop-gain variant. We performed functional characterization of the three missense variants alongside our stop-gain and two previously described truncating/frameshift variants. All variants severely disrupted multiple aspects of protein function. Strikingly, the missense variants had similarly severe effects on protein function as the truncating/frameshift variants. Our findings indicate that a loss of transcriptional repression activity of FOXP1 underlies the neurodevelopmental phenotype in FOXP1-related disorder. Interestingly, the three novel variants retained the ability to interact with wild-type FOXP1, suggesting these variants could exert a dominant-negative effect by interfering with the normal FOXP1 protein. These variants also retained the ability to interact with FOXP2, a paralogous transcription factor disrupted in rare cases of speech and language disorder. Thus, speech/language deficits in these individuals might be worsened through deleterious effects on FOXP2 function. Our findings highlight that de novo FOXP1 variants are a cause of sporadic ID and emphasize the importance of this transcription factor in neurodevelopment.


Asunto(s)
Discapacidades del Desarrollo/genética , Factores de Transcripción Forkhead/genética , Hipertelorismo/genética , Discapacidad Intelectual/genética , Trastornos del Desarrollo del Lenguaje/genética , Mutación Missense , Proteínas Represoras/genética , Adolescente , Secuencia de Bases , Niño , ADN/genética , ADN/metabolismo , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Exoma , Femenino , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hipertelorismo/metabolismo , Hipertelorismo/patología , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Trastornos del Desarrollo del Lenguaje/metabolismo , Trastornos del Desarrollo del Lenguaje/patología , Masculino , Datos de Secuencia Molecular , Linaje , Unión Proteica , Proteínas Represoras/metabolismo , Transducción de Señal , Transcripción Genética
8.
Hum Mutat ; 38(11): 1542-1554, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28741757

RESUMEN

The closely related paralogues FOXP2 and FOXP1 encode transcription factors with shared functions in the development of many tissues, including the brain. However, while mutations in FOXP2 lead to a speech/language disorder characterized by childhood apraxia of speech (CAS), the clinical profile of FOXP1 variants includes a broader neurodevelopmental phenotype with global developmental delay, intellectual disability, and speech/language impairment. Using clinical whole-exome sequencing, we report an identical de novo missense FOXP1 variant identified in three unrelated patients. The variant, p.R514H, is located in the forkhead-box DNA-binding domain and is equivalent to the well-studied p.R553H FOXP2 variant that cosegregates with CAS in a large UK family. We present here for the first time a direct comparison of the molecular and clinical consequences of the same mutation affecting the equivalent residue in FOXP1 and FOXP2. Detailed functional characterization of the two variants in cell model systems revealed very similar molecular consequences, including aberrant subcellular localization, disruption of transcription factor activity, and deleterious effects on protein interactions. Nonetheless, clinical manifestations were broader and more severe in the three cases carrying the p.R514H FOXP1 variant than in individuals with the p.R553H variant related to CAS, highlighting divergent roles of FOXP2 and FOXP1 in neurodevelopment.


Asunto(s)
Discapacidades del Desarrollo/diagnóstico , Discapacidades del Desarrollo/genética , Factores de Transcripción Forkhead/genética , Mutación Missense , Fenotipo , Proteínas Represoras/genética , Sustitución de Aminoácidos , Línea Celular , Niño , Preescolar , Femenino , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/metabolismo , Expresión Génica , Genes Reporteros , Estudios de Asociación Genética , Genotipo , Humanos , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Mutación con Pérdida de Función , Imagen por Resonancia Magnética , Masculino , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Secuenciación del Exoma
10.
EMBO J ; 30(15): 3065-77, 2011 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-21743439

RESUMEN

Prion diseases are associated with the conversion of cellular prion protein (PrP(C)) to toxic ß-sheet isoforms (PrP(Sc)), which are reported to inhibit the ubiquitin-proteasome system (UPS). Accordingly, UPS substrates accumulate in prion-infected mouse brains, suggesting impairment of the 26S proteasome. A direct interaction between its 20S core particle and PrP isoforms was demonstrated by immunoprecipitation. ß-PrP aggregates associated with the 20S particle, but did not impede binding of the PA26 complex, suggesting that the aggregates do not bind to its ends. Aggregated ß-PrP reduced the 20S proteasome's basal peptidase activity, and the enhanced activity induced by C-terminal peptides from the 19S ATPases or by the 19S regulator itself, including when stimulated by polyubiquitin conjugates. However, the 20S proteasome was not inhibited when the gate in the α-ring was open due to a truncation mutation or by association with PA26/PA28. These PrP aggregates inhibit by stabilising the closed conformation of the substrate entry channel. A similar inhibition of substrate entry into the proteasome may occur in other neurodegenerative diseases where misfolded ß-sheet-rich proteins accumulate.


Asunto(s)
Proteínas PrPSc/metabolismo , Inhibidores de Proteasoma , Mapeo de Interacción de Proteínas , Animales , Humanos , Inmunoprecipitación , Ratones , Ratones Transgénicos , Modelos Moleculares , Unión Proteica
11.
Neuropsychol Rev ; 25(1): 3-26, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25597031

RESUMEN

The human capacity to acquire sophisticated language is unmatched in the animal kingdom. Despite the discontinuity in communicative abilities between humans and other primates, language is built on ancient genetic foundations, which are being illuminated by comparative genomics. The genetic architecture of the language faculty is also being uncovered by research into neurodevelopmental disorders that disrupt the normally effortless process of language acquisition. In this article, we discuss the strategies that researchers are using to reveal genetic factors contributing to communicative abilities, and review progress in identifying the relevant genes and genetic variants. The first gene directly implicated in a speech and language disorder was FOXP2. Using this gene as a case study, we illustrate how evidence from genetics, molecular cell biology, animal models and human neuroimaging has converged to build a picture of the role of FOXP2 in neurodevelopment, providing a framework for future endeavors to bridge the gaps between genes, brains and behavior.


Asunto(s)
Encéfalo/fisiología , Comunicación , Variación Genética , Desarrollo del Lenguaje , Animales , Humanos , Trastornos del Lenguaje/genética
12.
Biochim Biophys Acta ; 1782(12): 713-22, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18644436

RESUMEN

Prion diseases are fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in animals. They are unique in terms of their biology because they are caused by the conformational re-arrangement of a normal host-encoded prion protein, PrPC, to an abnormal infectious isoform, PrPSc. Currently the precise mechanism behind prion-mediated neurodegeneration remains unclear. It is hypothesised than an unknown toxic gain of function of PrPSc, or an intermediate oligomeric form, underlies neuronal death. Increasing evidence suggests a role for the ubiquitin proteasome system (UPS) in prion disease. Both wild-type PrPC and disease-associated PrP isoforms accumulate in cells after proteasome inhibition leading to increased cell death, and abnormal beta-sheet-rich PrP isoforms have been shown to inhibit the catalytic activity of the proteasome. Here we review potential interactions between prions and the proteasome outlining how the UPS may be implicated in prion-mediated neurodegeneration.


Asunto(s)
Enfermedades por Prión/metabolismo , Priones/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Humanos
13.
Lancet Neurol ; 8(1): 57-66, 2009 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19081515

RESUMEN

BACKGROUND: Human and animal prion diseases are under genetic control, but apart from PRNP (the gene that encodes the prion protein), we understand little about human susceptibility to bovine spongiform encephalopathy (BSE) prions, the causal agent of variant Creutzfeldt-Jakob disease (vCJD). METHODS: We did a genome-wide association study of the risk of vCJD and tested for replication of our findings in samples from many categories of human prion disease (929 samples) and control samples from the UK and Papua New Guinea (4254 samples), including controls in the UK who were genotyped by the Wellcome Trust Case Control Consortium. We also did follow-up analyses of the genetic control of the clinical phenotype of prion disease and analysed candidate gene expression in a mouse cellular model of prion infection. FINDINGS: The PRNP locus was strongly associated with risk across several markers and all categories of prion disease (best single SNP [single nucleotide polymorphism] association in vCJD p=2.5 x 10(-17); best haplotypic association in vCJD p=1 x 10(-24)). Although the main contribution to disease risk was conferred by PRNP polymorphic codon 129, another nearby SNP conferred increased risk of vCJD. In addition to PRNP, one technically validated SNP association upstream of RARB (the gene that encodes retinoic acid receptor beta) had nominal genome-wide significance (p=1.9 x 10(-7)). A similar association was found in a small sample of patients with iatrogenic CJD (p=0.030) but not in patients with sporadic CJD (sCJD) or kuru. In cultured cells, retinoic acid regulates the expression of the prion protein. We found an association with acquired prion disease, including vCJD (p=5.6 x 10(-5)), kuru incubation time (p=0.017), and resistance to kuru (p=2.5 x 10(-4)), in a region upstream of STMN2 (the gene that encodes SCG10). The risk genotype was not associated with sCJD but conferred an earlier age of onset. Furthermore, expression of Stmn2 was reduced 30-fold post-infection in a mouse cellular model of prion disease. INTERPRETATION: The polymorphic codon 129 of PRNP was the main genetic risk factor for vCJD; however, additional candidate loci have been identified, which justifies functional analyses of these biological pathways in prion disease.


Asunto(s)
Síndrome de Creutzfeldt-Jakob/epidemiología , Síndrome de Creutzfeldt-Jakob/genética , Adulto , Edad de Inicio , Anciano , Alelos , Cromosomas Humanos/genética , ADN/genética , Interpretación Estadística de Datos , Femenino , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Kuru/epidemiología , Desequilibrio de Ligamiento/genética , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Papúa Nueva Guinea/epidemiología , Polimorfismo de Nucleótido Simple , Vigilancia de la Población , Proteínas Priónicas , Priones/genética , Control de Calidad , Factores de Riesgo , Estatmina , Reino Unido/epidemiología
14.
Sci Rep ; 8(1): 14279, 2018 09 24.
Artículo en Inglés | MEDLINE | ID: mdl-30250039

RESUMEN

Recurrent de novo variants in the TBR1 transcription factor are implicated in the etiology of sporadic autism spectrum disorders (ASD). Disruptions include missense variants located in the T-box DNA-binding domain and previous work has demonstrated that they disrupt TBR1 protein function. Recent screens of thousands of simplex families with sporadic ASD cases uncovered additional T-box variants in TBR1 but their etiological relevance is unclear. We performed detailed functional analyses of de novo missense TBR1 variants found in the T-box of ASD cases, assessing many aspects of protein function, including subcellular localization, transcriptional activity and protein-interactions. Only two of the three tested variants severely disrupted TBR1 protein function, despite in silico predictions that all would be deleterious. Furthermore, we characterized a putative interaction with BCL11A, a transcription factor that was recently implicated in a neurodevelopmental syndrome involving developmental delay and language deficits. Our findings enhance understanding of molecular functions of TBR1, as well as highlighting the importance of functional testing of variants that emerge from next-generation sequencing, to decipher their contributions to neurodevelopmental disorders like ASD.


Asunto(s)
Trastorno del Espectro Autista/genética , Discapacidades del Desarrollo/genética , Trastornos del Neurodesarrollo/genética , Proteínas de Dominio T Box/genética , Trastorno del Espectro Autista/fisiopatología , Discapacidades del Desarrollo/fisiopatología , Exoma/genética , Regulación de la Expresión Génica/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación Missense/genética , Trastornos del Neurodesarrollo/fisiopatología , Conformación Proteica , Proteínas de Dominio T Box/química , Secuenciación del Exoma
15.
Sci Rep ; 6: 20911, 2016 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-26867680

RESUMEN

Mutations affecting the transcription factor FOXP2 cause a rare form of severe speech and language disorder. Although it is clear that sufficient FOXP2 expression is crucial for normal brain development, little is known about how this transcription factor is regulated. To investigate post-translational mechanisms for FOXP2 regulation, we searched for protein interaction partners of FOXP2, and identified members of the PIAS family as novel FOXP2 interactors. PIAS proteins mediate post-translational modification of a range of target proteins with small ubiquitin-like modifiers (SUMOs). We found that FOXP2 can be modified with all three human SUMO proteins and that PIAS1 promotes this process. An aetiological FOXP2 mutation found in a family with speech and language disorder markedly reduced FOXP2 SUMOylation. We demonstrate that FOXP2 is SUMOylated at a single major site, which is conserved in all FOXP2 vertebrate orthologues and in the paralogues FOXP1 and FOXP4. Abolishing this site did not lead to detectable changes in FOXP2 subcellular localization, stability, dimerization or transcriptional repression in cellular assays, but the conservation of this site suggests a potential role for SUMOylation in regulating FOXP2 activity in vivo.


Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Lenguaje , Procesamiento Proteico-Postraduccional , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Secuencia de Aminoácidos , Factores de Transcripción Forkhead/química , Células HEK293 , Humanos , Lisina/metabolismo , Proteínas Mutantes/metabolismo , Unión Proteica , Proteínas Inhibidoras de STAT Activados/metabolismo , Sumoilación , Ubiquitina-Proteína Ligasas/metabolismo
16.
J Neurodev Disord ; 8: 44, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27933109

RESUMEN

BACKGROUND: Heterozygous disruption of FOXP2 causes a rare form of speech and language impairment. Screens of the FOXP2 sequence in individuals with speech/language-related disorders have identified several rare protein-altering variants, but their phenotypic relevance is often unclear. FOXP2 encodes a transcription factor with a forkhead box DNA-binding domain, but little is known about the functions of protein regions outside this domain. METHODS: We performed detailed functional analyses of seven rare FOXP2 variants found in affected cases, including three which have not been previously characterized, testing intracellular localization, transcriptional regulation, dimerization, and interaction with other proteins. To shed further light on molecular functions of FOXP2, we characterized the interaction between this transcription factor and co-repressor proteins of the C-terminal binding protein (CTBP) family. Finally, we analysed the functional significance of the polyglutamine tracts in FOXP2, since tract length variations have been reported in cases of neurodevelopmental disorder. RESULTS: We confirmed etiological roles of multiple FOXP2 variants. Of three variants that have been suggested to cause speech/language disorder, but never before been characterized, only one showed functional effects. For the other two, we found no effects on protein function in any assays, suggesting that they are incidental to the phenotype. We identified a CTBP-binding region within the N-terminal portion of FOXP2. This region includes two amino acid substitutions that occurred on the human lineage following the split from chimpanzees. However, we did not observe any effects of these amino acid changes on CTBP binding or other core aspects of FOXP2 function. Finally, we found that FOXP2 variants with reduced polyglutamine tracts did not exhibit altered behaviour in cellular assays, indicating that such tracts are non-essential for core aspects of FOXP2 function, and that tract variation is unlikely to be a highly penetrant cause of speech/language disorder. CONCLUSIONS: Our findings highlight the importance of functional characterization of novel rare variants in FOXP2 in assessing the contribution of such variants to speech/language disorder and provide further insights into the molecular function of the FOXP2 protein.

17.
Eur J Hum Genet ; 23(12): 1702-7, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25853299

RESUMEN

FOXP1 (forkhead box protein P1) is a transcription factor involved in the development of several tissues, including the brain. An emerging phenotype of patients with protein-disrupting FOXP1 variants includes global developmental delay, intellectual disability and mild to severe speech/language deficits. We report on a female child with a history of severe hypotonia, autism spectrum disorder and mild intellectual disability with severe speech/language impairment. Clinical exome sequencing identified a heterozygous de novo FOXP1 variant c.1267_1268delGT (p.V423Hfs*37). Functional analyses using cellular models show that the variant disrupts multiple aspects of FOXP1 activity, including subcellular localization and transcriptional repression properties. Our findings highlight the importance of performing functional characterization to help uncover the biological significance of variants identified by genomics approaches, thereby providing insight into pathways underlying complex neurodevelopmental disorders. Moreover, our data support the hypothesis that de novo variants represent significant causal factors in severe sporadic disorders and extend the phenotype seen in individuals with FOXP1 haploinsufficiency.


Asunto(s)
Trastorno Autístico/genética , Factores de Transcripción Forkhead/genética , Discapacidad Intelectual/genética , Trastornos del Lenguaje/genética , Mutación , Proteínas Represoras/genética , Adolescente , Trastorno Autístico/diagnóstico , Femenino , Factores de Transcripción Forkhead/metabolismo , Células HEK293 , Humanos , Discapacidad Intelectual/diagnóstico , Trastornos del Lenguaje/diagnóstico , Transporte de Proteínas , Proteínas Represoras/metabolismo , Síndrome
18.
J Vis Exp ; (87)2014 May 26.
Artículo en Inglés | MEDLINE | ID: mdl-24893771

RESUMEN

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.


Asunto(s)
Proteínas Bacterianas/química , Luciferasas/química , Mediciones Luminiscentes/métodos , Proteínas Luminiscentes/química , Mapas de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Animales , Proteínas Bacterianas/metabolismo , Transferencia de Energía , Transferencia Resonante de Energía de Fluorescencia/métodos , Células HEK293 , Humanos , Luciferasas/metabolismo , Proteínas Luminiscentes/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Renilla/enzimología
19.
Nat Commun ; 5: 4954, 2014 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-25232744

RESUMEN

Next-generation sequencing recently revealed that recurrent disruptive mutations in a few genes may account for 1% of sporadic autism cases. Coupling these novel genetic data to empirical assays of protein function can illuminate crucial molecular networks. Here we demonstrate the power of the approach, performing the first functional analyses of TBR1 variants identified in sporadic autism. De novo truncating and missense mutations disrupt multiple aspects of TBR1 function, including subcellular localization, interactions with co-regulators and transcriptional repression. Missense mutations inherited from unaffected parents did not disturb function in our assays. We show that TBR1 homodimerizes, that it interacts with FOXP2, a transcription factor implicated in speech/language disorders, and that this interaction is disrupted by pathogenic mutations affecting either protein. These findings support the hypothesis that de novo mutations in sporadic autism have severe functional consequences. Moreover, they uncover neurogenetic mechanisms that bridge different neurodevelopmental disorders involving language deficits.


Asunto(s)
Trastorno del Espectro Autista/genética , Mutación , Proteínas de Dominio T Box/genética , Secuencia de Aminoácidos , Línea Celular Tumoral , Niño , Preescolar , Dimerización , Femenino , Factores de Transcripción Forkhead/metabolismo , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Trastornos del Lenguaje/genética , Masculino , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Missense , Mapeo de Interacción de Proteínas , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos Híbridos
20.
Genome Biol ; 14(4): 204, 2013 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-23597266

RESUMEN

Next-generation sequencing is set to transform the discovery of genes underlying neurodevelopmental disorders, and so offer important insights into the biological bases of spoken language. Success will depend on functional assessments in neuronal cell lines, animal models and humans themselves.


Asunto(s)
Apraxias/genética , Genoma Humano , Tartamudeo/genética , Evolución Molecular , Redes Reguladoras de Genes , Humanos , Polimorfismo Genético
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