RESUMO
Mutations in the gene encoding the CDKL5 kinase are among the most common genetic causes of childhood epilepsy and can also give rise to the severe neurodevelopmental condition CDD (CDKL5 deficiency disorder). Despite its importance for human health, the phosphorylation targets and cellular roles of CDKL5 are poorly understood, especially in the cell nucleus. Here, we report that CDKL5 is recruited to sites of DNA damage in actively transcribed regions of the nucleus. A quantitative phosphoproteomic screen for nuclear CDKL5 substrates reveals a network of transcriptional regulators including Elongin A (ELOA), phosphorylated on a specific CDKL5 consensus motif. Recruitment of CDKL5 and ELOA to damaged DNA, and subsequent phosphorylation of ELOA, requires both active transcription and the synthesis of poly(ADP-ribose) (PAR), to which CDKL5 can bind. Critically, CDKL5 kinase activity is essential for the transcriptional silencing of genes induced by DNA double-strand breaks. Thus, CDKL5 is a DNA damage-sensing, PAR-controlled transcriptional modulator, a finding with implications for understanding the molecular basis of CDKL5-related diseases.
Assuntos
Quebras de DNA de Cadeia Dupla , Dano ao DNA , Elonguina/metabolismo , Neurônios/patologia , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ativação Transcricional , Elonguina/genética , Síndromes Epilépticas/genética , Síndromes Epilépticas/metabolismo , Síndromes Epilépticas/patologia , Humanos , Mutação , Neurônios/metabolismo , Fosfoproteínas/genética , Fosforilação , Poli Adenosina Difosfato Ribose/metabolismo , Proteínas Serina-Treonina Quinases/genética , Espasmos Infantis/genética , Espasmos Infantis/metabolismo , Espasmos Infantis/patologiaRESUMO
CDKL5 Deficiency Disorder (CDD) is a debilitating epileptic encephalopathy disorder affecting young children with no effective treatments. CDD is caused by pathogenic variants in Cyclin-Dependent Kinase-Like 5 (CDKL5), a protein kinase that regulates key phosphorylation events in neurons. For therapeutic intervention, it is essential to understand molecular pathways and phosphorylation targets of CDKL5. Using an unbiased phosphoproteomic approach we identified novel targets of CDKL5, including GTF2I, PPP1R35, GATAD2A and ZNF219 in human iPSC-derived neuronal cells. The phosphoserine residue in the target proteins lies in the CDKL5 consensus motif. We validated direct phosphorylation of GTF2I and PPP1R35 by CDKL5 using complementary approaches. GTF2I controls axon guidance, cell cycle and neurodevelopment by regulating expression of neuronal genes. PPP1R35 is critical for centriole elongation and cilia morphology, processes that are impaired in CDD. PPP1R35 interacts with CEP131, a known CDKL5 phospho-target. GATAD2A and ZNF219 belong to the Nucleosome Remodelling Deacetylase (NuRD) complex, which regulates neuronal activity-dependent genes and synaptic connectivity. In-depth knowledge of molecular pathways regulated by CDKL5 will allow a better understanding of druggable disease pathways to fast-track therapeutic development.
Assuntos
Síndromes Epilépticas , Células-Tronco Pluripotentes Induzidas , Neurônios , Proteínas Serina-Treonina Quinases , Espasmos Infantis , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/metabolismo , Neurônios/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fosforilação , Síndromes Epilépticas/metabolismo , Síndromes Epilépticas/genética , Síndromes Epilépticas/patologia , Espasmos Infantis/metabolismo , Espasmos Infantis/genética , Espasmos Infantis/patologiaRESUMO
WOREE syndrome is an early infantile epileptic encephalopathy characterized by drug-resistant seizures and severe psychomotor developmental delays. We report a case of a WWOX splice-site mutation with uniparental isodisomy. A 1-year and 7-month-old girl presented with nystagmus and epileptic seizures from early infancy, with no fixation or pursuit of vision. Physical examination revealed small deformities, such as swelling of both cheeks, folded fingers, rocking feet, and scoliosis. Brain imaging revealed slight hypoplasia of the cerebrum. Electroencephalogram showed focal paroxysmal discharges during the interictal phase of seizures. Vitamin B6 and zonisamide were administered for early infantile epileptic encephalopathy; however, the seizures were not relieved. Despite altering the type and dosage of antiepileptic drugs and ACTH therapy, the seizures were intractable. Whole-exome analysis revealed the homozygosity of WWOX(NM_016373.4):c.516+1G>A. The WWOX mRNA sequencing using peripheral blood RNA confirmed that exon 5 was homozygously deleted. Based on these results, the patient was diagnosed with WOREE syndrome at 5 months. The WWOX variant found in this study is novel and has never been reported before. WOREE syndrome being extremely rare, further case series and analyses of its pathophysiology are warranted.
Assuntos
Mutação , Sítios de Splice de RNA , Espasmos Infantis , Dissomia Uniparental , Oxidorredutase com Domínios WW , Humanos , Feminino , Lactente , Oxidorredutase com Domínios WW/genética , Espasmos Infantis/genética , Espasmos Infantis/tratamento farmacológico , Espasmos Infantis/patologia , Dissomia Uniparental/genética , Dissomia Uniparental/patologia , Sítios de Splice de RNA/genética , Mutação/genética , Fenótipo , Sequenciamento do Exoma , Eletroencefalografia , Proteínas Supressoras de TumorRESUMO
The X-linked GRIA3 gene encodes the GLUA3 subunit of AMPA-type glutamate receptors. Pathogenic variants in this gene were previously reported in neurodevelopmental diseases, mostly in male patients but rarely in females. Here we report a de novo pathogenic missense variant in GRIA3 (c.1979G>C; p. R660T) identified in a 1-year-old female patient with severe epilepsy and global developmental delay. When exogenously expressed in human embryonic kidney (HEK) cells, GLUA3_R660T showed slower desensitization and deactivation kinetics compared to wildtype (wt) GLUA3 receptors. Substantial non-desensitized currents were observed with the mutant but not for wt GLUA3 with prolonged exposure to glutamate. When co-expressed with GLUA2, the decay kinetics were similarly slowed in GLUA2/A3_R660T with non-desensitized steady state currents. In cultured cerebellar granule neurons, miniature excitatory postsynaptic currents (mEPSCs) were significantly slower in R660T transfected cells than those expressing wt GLUA3. When overexpressed in hippocampal CA1 neurons by in utero electroporation, the evoked EPSCs and mEPSCs were slower in neurons expressing R660T mutant compared to those expressing wt GLUA3. Therefore our study provides functional evidence that a gain of function (GoF) variant in GRIA3 may cause epileptic encephalopathy and global developmental delay in a female subject by enhancing synaptic transmission.
Assuntos
Proteínas do Ovo/genética , Mutação com Ganho de Função , Proteínas de Membrana/genética , Neurônios/metabolismo , Receptores de AMPA/genética , Espasmos Infantis/genética , Sequência de Aminoácidos , Animais , Cerebelo/metabolismo , Cerebelo/patologia , Pré-Escolar , Proteínas do Ovo/metabolismo , Feminino , Expressão Gênica , Células HEK293 , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Modelos Moleculares , Neurônios/patologia , Cultura Primária de Células , Conformação Proteica , Receptores de AMPA/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espasmos Infantis/metabolismo , Espasmos Infantis/patologiaRESUMO
Ral (Ras-like) GTPases play an important role in the control of cell migration and have been implicated in Ras-mediated tumorigenicity. Recently, variants in RALA were also described as a cause of intellectual disability and developmental delay, indicating the relevance of this pathway to neuropediatric diseases. Here, we report the identification of bi-allelic variants in RALGAPA1 (encoding Ral GTPase activating protein catalytic alpha subunit 1) in four unrelated individuals with profound neurodevelopmental disability, muscular hypotonia, feeding abnormalities, recurrent fever episodes, and infantile spasms . Dysplasia of corpus callosum with focal thinning of the posterior part and characteristic facial features appeared to be unifying findings. RalGAPA1 was absent in the fibroblasts derived from two affected individuals suggesting a loss-of-function effect of the RALGAPA1 variants. Consequently, RalA activity was increased in these cell lines, which is in keeping with the idea that RalGAPA1 deficiency causes a constitutive activation of RalA. Additionally, levels of RalGAPB, a scaffolding subunit of the RalGAP complex, were dramatically reduced, indicating a dysfunctional RalGAP complex. Moreover, RalGAPA1 deficiency clearly increased cell-surface levels of lipid raft components in detached fibroblasts, which might indicate that anchorage-dependence of cell growth signaling is disturbed. Our findings indicate that the dysregulation of the RalA pathway has an important impact on neuronal function and brain development. In light of the partially overlapping phenotype between RALA- and RALGAPA1-associated diseases, it appears likely that dysregulation of the RalA signaling pathway leads to a distinct group of genetic syndromes that we suggest could be named RALopathies.
Assuntos
Transtornos da Alimentação e da Ingestão de Alimentos/etiologia , Proteínas Ativadoras de GTPase/genética , Hipotonia Muscular/etiologia , Mutação , Proteínas do Tecido Nervoso/genética , Transtornos do Neurodesenvolvimento/etiologia , Espasmos Infantis/etiologia , Alelos , Movimento Celular , Proliferação de Células , Pré-Escolar , Família , Transtornos da Alimentação e da Ingestão de Alimentos/patologia , Feminino , Humanos , Lactente , Masculino , Hipotonia Muscular/patologia , Transtornos do Neurodesenvolvimento/patologia , Fenótipo , Espasmos Infantis/patologiaRESUMO
Cyclin-dependent kinase-like 5 (CDKL5), a serine-threonine kinase encoded by an X-linked gene, is highly expressed in the mammalian forebrain. Mutations in this gene cause CDKL5 deficiency disorder, a neurodevelopmental encephalopathy characterized by early-onset seizures, motor dysfunction, and intellectual disability. We previously found that mice lacking CDKL5 exhibit hyperlocomotion and increased impulsivity, resembling the core symptoms in attention-deficit hyperactivity disorder (ADHD). Here, we report the potential neural mechanisms and treatment for hyperlocomotion induced by CDKL5 deficiency. Our results showed that loss of CDKL5 decreases the proportion of phosphorylated dopamine transporter (DAT) in the rostral striatum, leading to increased levels of extracellular dopamine and hyperlocomotion. Administration of methylphenidate (MPH), a DAT inhibitor clinically effective to improve symptoms in ADHD, significantly alleviated the hyperlocomotion phenotype in Cdkl5 null mice. In addition, the improved behavioral effects of MPH were accompanied by a region-specific restoration of phosphorylated dopamine- and cAMP-regulated phosphoprotein Mr 32 kDa, a key signaling protein for striatal motor output. Finally, mice carrying a Cdkl5 deletion selectively in DAT-expressing dopaminergic neurons, but not dopamine receptive neurons, recapitulated the hyperlocomotion phenotype found in Cdkl5 null mice. Our findings suggest that CDKL5 is essential to control locomotor behavior by regulating region-specific dopamine content and phosphorylation of dopamine signaling proteins in the striatum. The direct, as well as indirect, target proteins regulated by CDKL5 may play a key role in movement control and the therapeutic development for hyperactivity disorders.
Assuntos
Síndromes Epilépticas/genética , Hipercinese/genética , Proteínas Serina-Treonina Quinases/genética , Espasmos Infantis/genética , Animais , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Síndromes Epilépticas/patologia , Transtornos Neurológicos da Marcha/genética , Transtornos Neurológicos da Marcha/metabolismo , Transtornos Neurológicos da Marcha/patologia , Humanos , Hipercinese/metabolismo , Hipercinese/patologia , Metilfenidato/metabolismo , Camundongos , Camundongos Knockout , Espasmos Infantis/patologiaRESUMO
Mutations in the gene encoding the protein kinase CDKL5 cause a debilitating neurodevelopmental disease termed CDKL5 disorder. The impact of these mutations on CDKL5 function is poorly understood because the substrates and cellular processes controlled by CDKL5 are unclear. Here, we describe a quantitative phosphoproteomic screening which identified MAP1S, CEP131 and DLG5-regulators of microtubule and centrosome function-as cellular substrates of CDKL5. Antibodies against MAP1S phospho-Ser900 and CEP131 phospho-Ser35 confirmed CDKL5-dependent phosphorylation of these targets in human cells. The phospho-acceptor serine residues in MAP1S, CEP131 and DLG5 lie in the motif RPXSA, although CDKL5 can tolerate residues other than Ala immediately C-terminal to the phospho-acceptor serine. We provide insight into the control of CDKL5 activity and show that pathogenic mutations in CDKL5 cause a major reduction in CDKL5 activity in vitro and in cells. These data reveal the first cellular substrates of CDKL5, which may represent important biomarkers in the diagnosis and treatment of CDKL5 disorder, and illuminate the functions of this poorly characterized kinase.
Assuntos
Síndromes Epilépticas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espasmos Infantis/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Motivos de Aminoácidos , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Centrossomo/metabolismo , Proteínas do Citoesqueleto , Síndromes Epilépticas/genética , Síndromes Epilépticas/patologia , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas dos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteômica , Espasmos Infantis/genética , Espasmos Infantis/patologia , Proteínas Supressoras de Tumor/genéticaRESUMO
Different parts of a gene can be of differential importance to development and health. This regional heterogeneity is also apparent in the distribution of disease-associated mutations, which often cluster in particular regions of disease-associated genes. The ability to precisely estimate functionally important sub-regions of genes will be key in correctly deciphering relationships between genetic variation and disease. Previous methods have had some success using standing human variation to characterize this variability in importance by measuring sub-regional intolerance, i.e., the depletion in functional variation from expectation within a given region of a gene. However, the ability to precisely estimate local intolerance was restricted by the fact that only information within a given sub-region is used, leading to instability in local estimates, especially for small regions. We show that borrowing information across regions using a Bayesian hierarchical model stabilizes estimates, leading to lower variability and improved predictive utility. Specifically, our approach more effectively identifies regions enriched for ClinVar pathogenic variants. We also identify significant correlations between sub-region intolerance and the distribution of pathogenic variation in disease-associated genes, with AUCs for classifying de novo missense variants in Online Mendelian Inheritance in Man (OMIM) genes of up to 0.86 using exonic sub-regions and 0.91 using sub-regions defined by protein domains. This result immediately suggests that considering the intolerance of regions in which variants are found may improve diagnostic interpretation. We also illustrate the utility of integrating regional intolerance into gene-level disease association tests with a study of known disease-associated genes for epileptic encephalopathy.
Assuntos
Componentes do Gene/genética , Modelos Genéticos , Mutação/genética , Espasmos Infantis/genética , Espasmos Infantis/patologia , Teorema de Bayes , Éxons/genética , HumanosRESUMO
OBJECTIVE: Approximately 50% of patients with tuberous sclerosis complex develop infantile spasms, a sudden onset epilepsy syndrome associated with poor neurological outcomes. An increased burden of tubers confers an elevated risk of infantile spasms, but it remains unknown whether some tuber locations confer higher risk than others. Here, we test whether tuber location and connectivity are associated with infantile spasms. METHODS: We segmented tubers from 123 children with (n = 74) and without (n = 49) infantile spasms from a prospective observational cohort. We used voxelwise lesion symptom mapping to test for an association between spasms and tuber location. We then used lesion network mapping to test for an association between spasms and connectivity with tuber locations. Finally, we tested the discriminability of identified associations with logistic regression and cross-validation as well as statistical mediation. RESULTS: Tuber locations associated with infantile spasms were heterogenous, and no single location was significantly associated with spasms. However, >95% of tuber locations associated with spasms were functionally connected to the globi pallidi and cerebellar vermis. These connections were specific compared to tubers in patients without spasms. Logistic regression found that globus pallidus connectivity was a stronger predictor of spasms (odds ratio [OR] = 1.96, 95% confidence interval [CI] = 1.10-3.50, p = 0.02) than tuber burden (OR = 1.65, 95% CI = 0.90-3.04, p = 0.11), with a mean receiver operating characteristic area under the curve of 0.73 (±0.1) during repeated cross-validation. INTERPRETATION: Connectivity between tuber locations and the bilateral globi pallidi is associated with infantile spasms. Our findings lend insight into spasm pathophysiology and may identify patients at risk. ANN NEUROL 2021;89:726-739.
Assuntos
Hamartoma/diagnóstico por imagem , Rede Nervosa/diagnóstico por imagem , Espasmos Infantis/diagnóstico por imagem , Esclerose Tuberosa/diagnóstico por imagem , Idade de Início , Mapeamento Encefálico , Núcleos Cerebelares/diagnóstico por imagem , Núcleos Cerebelares/patologia , Pré-Escolar , Conectoma , Feminino , Globo Pálido/diagnóstico por imagem , Globo Pálido/patologia , Hamartoma/patologia , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Rede Nervosa/patologia , Estudos Prospectivos , Curva ROC , Espasmos Infantis/patologia , Esclerose Tuberosa/patologiaRESUMO
Mutations in the X-linked CDKL5 gene cause CDKL5 deficiency disorder (CDD), a severe neurodevelopmental condition mainly characterized by infantile epileptic encephalopathy, intellectual disability, and autistic features. The molecular mechanisms underlying the clinical symptoms remain largely unknown and the identification of reliable biomarkers in animal models will certainly contribute to increase our comprehension of CDD as well as to assess the efficacy of therapeutic strategies. Here, we used different Magnetic Resonance (MR) methods to disclose structural, functional, or metabolic signatures of Cdkl5 deficiency in the brain of adult mice. We found that loss of Cdkl5 does not cause cerebral atrophy but affects distinct brain areas, particularly the hippocampus. By in vivo proton-MR spectroscopy (MRS), we revealed in the Cdkl5 null brain a metabolic dysregulation indicative of mitochondrial dysfunctions. Accordingly, we unveiled a significant reduction in ATP levels and a decrease in the expression of complex IV of mitochondrial electron transport chain. Conversely, the number of mitochondria appeared preserved. Importantly, we reported a significant defect in the activation of one of the major regulators of cellular energy balance, the adenosine monophosphate-activated protein kinase (AMPK), that might contribute to the observed metabolic impairment and become an interesting therapeutic target for future preclinical trials. In conclusion, MRS revealed in the Cdkl5 null brain the presence of a metabolic dysregulation suggestive of a mitochondrial dysfunction that permitted to foster our comprehension of Cdkl5 deficiency and brought our interest towards targeting mitochondria as therapeutic strategy for CDD.
Assuntos
Encéfalo/metabolismo , Síndromes Epilépticas , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/genética , Espasmos Infantis , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Síndromes Epilépticas/metabolismo , Síndromes Epilépticas/patologia , Espectroscopia de Ressonância Magnética , Metaboloma , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/patologia , Espasmos Infantis/metabolismo , Espasmos Infantis/patologiaRESUMO
Characterized by early-onset seizures, global developmental delay and severe motor deficits, CDKL5 deficiency disorder is caused by pathogenic variants in the cyclin-dependent kinase-like 5 gene. Previous efforts to investigate genotype-phenotype relationships have been limited due to small numbers of recurrent mutations and small cohort sizes. Using data from the International CDKL5 Disorder Database we examined genotype-phenotype relationships for 13 recurrent CDKL5 variants and the previously analyzed historic variant groupings. We have applied the CDKL5 Developmental Score (CDS) and an adapted version of the CDKL5 Clinical Severity Assessment (CCSA), to grade the severity of phenotype and developmental outcomes for 285 individuals with CDKL5 variants. Comparisons of adapted CCSA and CDS between recurrent variants and variant groups were performed using multiple linear regression adjusting for age and sex. Individuals with the missense variant, p.Arg178Trp, had the highest mean adapted CCSA and lowest mean developmental scores. Other variants producing severe phenotypes included p.Arg559* and p.Arg178Gln. Variants producing milder phenotypes included p.Arg134*, p.Arg550*, and p.Glu55Argfs*20. There are observed differences in phenotype severity and developmental outcomes for individuals with different CDKL5 variants. However, the historic variant groupings did not seem to reflect differences in phenotype severity or developmental outcomes as clearly as analyzed by individual variants.
Assuntos
Epilepsia/genética , Síndromes Epilépticas/genética , Estudos de Associação Genética , Proteínas Serina-Treonina Quinases/genética , Espasmos Infantis/genética , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , Epilepsia/patologia , Síndromes Epilépticas/patologia , Feminino , Genótipo , Humanos , Masculino , Mutação de Sentido Incorreto/genética , Transtornos do Neurodesenvolvimento/diagnóstico , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Fenótipo , Síndrome de Rett/genética , Síndrome de Rett/patologia , Convulsões/genética , Convulsões/patologia , Espasmos Infantis/patologiaRESUMO
Adenosine-to-inosine (A-to-I) editing is one of the most prevalent post-transcriptional RNA modifications in metazoan. This reaction is catalysed by enzymes called adenosine deaminases acting on RNA (ADARs). RNA editing is involved in the regulation of protein function and gene expression. The numerous A-to-I editing sites have been identified in both coding and non-coding RNA transcripts. These editing sites are also found in various genes expressed in the central nervous system (CNS) and play an important role in neurological development and brain function. Aberrant regulation of RNA editing has been associated with the pathogenesis of neurological and psychiatric disorders, suggesting the physiological significance of RNA editing in the CNS. In this review, we discuss the current knowledge of editing on neurological disease and development.
Assuntos
Esclerose Lateral Amiotrófica/genética , Astrocitoma/genética , Transtorno Bipolar/genética , Neoplasias do Sistema Nervoso Central/genética , Epilepsia/genética , Edição de RNA , Esquizofrenia/genética , Espasmos Infantis/genética , Adenosina/genética , Adenosina/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Astrocitoma/metabolismo , Astrocitoma/patologia , Transtorno Bipolar/metabolismo , Transtorno Bipolar/patologia , Neoplasias do Sistema Nervoso Central/metabolismo , Neoplasias do Sistema Nervoso Central/patologia , Epilepsia/metabolismo , Epilepsia/patologia , Humanos , Inosina/genética , Inosina/metabolismo , Canal de Potássio Kv1.1/genética , Canal de Potássio Kv1.1/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Receptor 5-HT2C de Serotonina/genética , Receptor 5-HT2C de Serotonina/metabolismo , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Esquizofrenia/metabolismo , Esquizofrenia/patologia , Espasmos Infantis/metabolismo , Espasmos Infantis/patologiaRESUMO
Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene cause a complex neurological disorder, characterized by infantile seizures, impairment of cognitive and motor skills and autistic features. Loss of Cdkl5 in mice affects dendritic spine maturation and dynamics but the underlying molecular mechanisms are still far from fully understood. Here we show that Cdkl5 deficiency in primary hippocampal neurons leads to deranged expression of the alpha-amino-3-hydroxy-5-methyl-4-iso-xazole propionic acid receptors (AMPA-R). In particular, a dramatic reduction of expression of the GluA2 subunit occurs concomitantly with its hyper-phosphorylation on Serine 880 and increased ubiquitination. Consequently, Cdkl5 silencing skews the composition of membrane-inserted AMPA-Rs towards the GluA2-lacking calcium-permeable form. Such derangement is likely to contribute, at least in part, to the altered synaptic functions and cognitive impairment linked to loss of Cdkl5. Importantly, we find that tianeptine, a cognitive enhancer and antidepressant drug, known to recruit and stabilise AMPA-Rs at the synaptic sites, can normalise the expression of membrane inserted AMPA-Rs as well as the number of PSD-95 clusters, suggesting its therapeutic potential for patients with mutations in CDKL5.
Assuntos
Síndromes Epilépticas/tratamento farmacológico , Proteínas Serina-Treonina Quinases/genética , Receptores de AMPA/genética , Espasmos Infantis/tratamento farmacológico , Tiazepinas/administração & dosagem , Animais , Antidepressivos/administração & dosagem , Proteína 4 Homóloga a Disks-Large/genética , Síndromes Epilépticas/genética , Síndromes Epilépticas/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/fisiopatologia , Humanos , Camundongos , Mutação , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fosforilação , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/deficiência , Espasmos Infantis/genética , Espasmos Infantis/patologia , Sinapses/efeitos dos fármacos , Sinapses/genéticaRESUMO
Calcineurin is a calcium (Ca2+)/calmodulin-regulated protein phosphatase that mediates Ca2+-dependent signal transduction. Here, we report six heterozygous mutations in a gene encoding the alpha isoform of the calcineurin catalytic subunit (PPP3CA). Notably, mutations were observed in different functional domains: in addition to three catalytic domain mutations, two missense mutations were found in the auto-inhibitory (AI) domain. One additional frameshift insertion that caused premature termination was also identified. Detailed clinical evaluation of the six individuals revealed clinically unexpected consequences of the PPP3CA mutations. First, the catalytic domain mutations and frameshift mutation were consistently found in patients with nonsyndromic early onset epileptic encephalopathy. In contrast, the AI domain mutations were associated with multiple congenital abnormalities including craniofacial dysmorphism, arthrogryposis and short stature. In addition, one individual showed severe skeletal developmental defects, namely, severe craniosynostosis and gracile bones (severe bone slenderness and perinatal fractures). Using a yeast model system, we showed that the catalytic and AI domain mutations visibly result in decreased and increased calcineurin signaling, respectively. These findings indicate that different functional effects of PPP3CA mutations are associated with two distinct disorders and suggest that functional approaches using a simple cellular system provide a tool for resolving complex genotype-phenotype correlations.
Assuntos
Artrogripose/genética , Calcineurina/genética , Anormalidades Craniofaciais/genética , Nanismo/genética , Mutação com Ganho de Função , Mutação com Perda de Função , Espasmos Infantis/genética , Sequência de Aminoácidos , Artrogripose/metabolismo , Artrogripose/patologia , Sequência de Bases , Calcineurina/química , Calcineurina/metabolismo , Criança , Pré-Escolar , Anormalidades Craniofaciais/metabolismo , Anormalidades Craniofaciais/patologia , Nanismo/metabolismo , Nanismo/patologia , Feminino , Regulação da Expressão Gênica , Estudos de Associação Genética , Humanos , Masculino , Modelos Moleculares , Linhagem , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espasmos Infantis/metabolismo , Espasmos Infantis/patologia , Adulto JovemRESUMO
Exome sequencing has readily enabled the discovery of the genetic mutations responsible for a wide range of diseases. This success has been particularly remarkable in the severe epilepsies and other neurodevelopmental diseases for which rare, often de novo, mutations play a significant role in disease risk. Despite significant progress, the high genetic heterogeneity of these disorders often requires large sample sizes to identify a critical mass of individuals with disease-causing mutations in a single gene. By pooling genetic findings across multiple studies, we have identified six individuals with severe developmental delay (6/6), refractory seizures (5/6), and similar dysmorphic features (3/6), each harboring a de novo mutation in PPP3CA. PPP3CA encodes the alpha isoform of a subunit of calcineurin. Calcineurin encodes a calcium- and calmodulin-dependent serine/threonine protein phosphatase that plays a role in a wide range of biological processes, including being a key regulator of synaptic vesicle recycling at nerve terminals. Five individuals with de novo PPP3CA mutations were identified among 4,760 trio probands with neurodevelopmental diseases; this is highly unlikely to occur by chance (p = 1.2 × 10-8) given the size and mutability of the gene. Additionally, a sixth individual with a de novo mutation in PPP3CA was connected to this study through GeneMatcher. Based on these findings, we securely implicate PPP3CA in early-onset refractory epilepsy and further support the emerging role for synaptic dysregulation in epilepsy.
Assuntos
Calcineurina/genética , Epilepsia/genética , Mutação , Transtornos do Neurodesenvolvimento/genética , Transmissão Sináptica/fisiologia , Adolescente , Adulto , Calcineurina/metabolismo , Criança , Pré-Escolar , Estudos de Coortes , Epilepsia/patologia , Exoma/genética , Feminino , Humanos , Lactente , Recém-Nascido , Síndrome de Lennox-Gastaut/patologia , Masculino , Transtornos do Neurodesenvolvimento/patologia , Análise de Sequência de DNA , Índice de Gravidade de Doença , Espasmos Infantis/genética , Espasmos Infantis/patologia , Adulto JovemRESUMO
We analyzed our two new cases of infantile-onset epilepsy with developmental delay with de novo variant in TUBB2A and review the related literatures. Our two probands were both girls with infantile-onset epilepsy and global developmental delay. Case 1 had a novel de novo heterozygous missense variant: c.728C>T [p.Pro243Leu] (NM_001069.2). Her brain magnetic resonance imaging (MRI) showed nonspecific white matter myelination delay and slightly enlarged anterior horn of lateral ventricle. Her epilepsy had been controlled by TPM monotherapy. Case 2 had a reported de novo variant c.743C>T [p.Ala248Val] (NM_001069.2). Her brain MRI showed bilateral microgyria and corpus callosum dysplasia. A total of seven TUBB2A mutations cases had been published previously in five papers, therefore, until now, there were nine patients with TUBB2A mutations. All patients had developmental delay, among them seven cases also with infantile-onset epilepsy, one case with abnormal EEG but without clinical seizures. There are six cases that have different degree of cortical dysplasia, one case with cerebellar vermis atrophy and brainstem sacsinopathy, the rest two cases have no obvious brain structural abnormalities. There was one case with variant c.1249G>A (p.D417N) that had atypical clinical presentation, including prominent progressive spastic ataxia, sensory motor axonal neuropathy, and bilateral optic macular dystrophy, but relatively mild intellectual disability, his MRI showed cerebellar atrophy, thinning of the corpus callosum and pons sacsinopathy, but no cortical malformation. The p.A248V mutation was the most common mutation occurred in three patients (3/9). The clinical phenotypes of these three patients were similar, all of them had global developmental delay with no language and corpus callosum dysplasia, two cases with epilepsy and the other one only have EEG epileptic discharges without clinical seizure, two cases with cortical dysplasia and the other one without obvious brain malformation. In brief, global developmental delay was the most common phenotype of TUBB2A mutation-related disease, most cases also had infantile-onset epilepsy and cortical dysplasia and corpus callosum dysplasia. The region between seventh and eighth alpha-helix of TUBB2A may be a "hot spot" mutation domain.
Assuntos
Deficiências do Desenvolvimento/genética , Epilepsia/genética , Espasmos Infantis/genética , Tubulina (Proteína)/genética , Idade de Início , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Deficiências do Desenvolvimento/diagnóstico por imagem , Deficiências do Desenvolvimento/patologia , Eletroencefalografia , Epilepsia/diagnóstico por imagem , Epilepsia/patologia , Feminino , Humanos , Recém-Nascido , Deficiência Intelectual/diagnóstico por imagem , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Imageamento por Ressonância Magnética , Espasticidade Muscular/diagnóstico por imagem , Espasticidade Muscular/genética , Espasticidade Muscular/patologia , Mutação de Sentido Incorreto/genética , Atrofia Óptica/diagnóstico por imagem , Atrofia Óptica/genética , Atrofia Óptica/patologia , Convulsões/diagnóstico por imagem , Convulsões/genética , Convulsões/patologia , Espasmos Infantis/diagnóstico por imagem , Espasmos Infantis/patologia , Ataxias Espinocerebelares/diagnóstico por imagem , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologiaRESUMO
TATA-box binding protein associated factor, RNA polymerase I subunit C (TAF1C) is a component of selectivity factor 1 belonging to RNA polymerase I (Pol I) transcription machinery. We report two unrelated patients with homozygous TAF1C missense variants and an early onset neurological phenotype with severe global developmental delay. Clinical features included lack of speech and ambulation and epilepsy. MRI of the brain demonstrated widespread cerebral atrophy and frontal periventricular white matter hyperintensity. The phenotype resembled that of a previously described variant of UBTF, which encodes another transcription factor of Pol I. TAF1C variants were located in two conserved amino acid positions and were predicted to be deleterious. In patient-derived fibroblasts, TAF1C mRNA and protein expression levels were substantially reduced compared with healthy controls. We propose that the variants impairing TAF1C expression are likely pathogenic and relate to a novel neurological disease. This study expands the disease spectrum related to Pol I transcription machinery, associating the TAF1C missense variants with a severe neurological phenotype for the first time.
Assuntos
Epilepsia/genética , RNA Polimerase I/genética , Espasmos Infantis/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genética , Pré-Escolar , Epilepsia/diagnóstico por imagem , Epilepsia/patologia , Feminino , Fibroblastos/metabolismo , Homozigoto , Humanos , Lactente , Recém-Nascido , Imageamento por Ressonância Magnética , Masculino , Mutação de Sentido Incorreto/genética , Fenótipo , Espasmos Infantis/diagnóstico por imagem , Espasmos Infantis/patologiaRESUMO
Biallelic mutations in the PLCB1 gene, encoding for a phospholipase C beta isoform strongly expressed in the brain, have been reported to cause infantile epileptic encephalopathy in only four children to date. We report here three additional patients to delineate the phenotypic and genotypic characteristics of the disease. Our three patients were one sporadic case with an intragenic homozygous deletion and two cousins with the homozygous p.(Arg222*) nonsense variant in PLCB1. These patients had severe to profound intellectual disability, epileptic spasms at age 3-5 months concomitant with developmental arrest or regression, other seizure types and drug-resistant epilepsy. With this report, we expand the clinical, radiologic and electroencephalographic knowledge about the extremely rare PLCB1-related encephalopathy. Since the first report in 2010, the overall number of reported patients with our additional patients is currently limited to seven. All seven patients had epileptic encephalopathy, mainly infantile spasms and 6/7 had profound intellectual disability, with one only being able to walk. Truncal hypotonia was the most frequent neurological sign, sometimes associated with pyramidal and/or extrapyramidal hypertonia of limbs. Microcephaly was inconstant. In conclusion, the phenotypical spectrum of PLCB1-related encephalopathy is relatively narrow, comprises infantile spasms and severe to profound intellectual disability, and does not seem to define a recognizable clinical entity.
Assuntos
Fosfolipase C beta/genética , Convulsões/genética , Espasmos Infantis/genética , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Criança , Pré-Escolar , Feminino , Genótipo , Homozigoto , Humanos , Lactente , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Masculino , Fenótipo , Convulsões/patologia , Deleção de Sequência/genética , Espasmos Infantis/diagnóstico por imagem , Espasmos Infantis/patologiaRESUMO
We investigated seven children from six families to expand the phenotypic spectrum associated with an early infantile epileptic encephalopathy caused by biallelic pathogenic variants in the phosphatidylinositol glycan anchor biosynthesis class Q (PIGQ) gene. The affected children were all identified by clinical or research exome sequencing. Clinical data, including EEGs and MRIs, was comprehensively reviewed and flow cytometry and transfection experiments were performed to investigate PIGQ function. Pathogenic biallelic PIGQ variants were associated with increased mortality. Epileptic seizures, axial hypotonia, developmental delay and multiple congenital anomalies were consistently observed. Seizure onset occurred between 2.5 months and 7 months of age and varied from treatable seizures to recurrent episodes of status epilepticus. Gastrointestinal issues were common and severe, two affected individuals had midgut volvulus requiring surgical correction. Cardiac anomalies including arrythmias were observed. Flow cytometry using granulocytes and fibroblasts from affected individuals showed reduced expression of glycosylphosphatidylinositol (GPI)-anchored proteins. Transfection of wildtype PIGQ cDNA into patient fibroblasts rescued this phenotype. We expand the phenotypic spectrum of PIGQ-related disease and provide the first functional evidence in human cells of defective GPI-anchoring due to pathogenic variants in PIGQ.
Assuntos
Anormalidades Múltiplas/genética , Proteínas de Membrana/genética , Hipotonia Muscular/genética , Convulsões/genética , Espasmos Infantis/genética , Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/metabolismo , Criança , Pré-Escolar , Evolução Fatal , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Hipotonia Muscular/patologia , Mutação de Sentido Incorreto , Fenótipo , Convulsões/diagnóstico , Convulsões/metabolismo , Espasmos Infantis/metabolismo , Espasmos Infantis/patologia , Sequenciamento do ExomaRESUMO
AIM: We aimed to examine the occurrence of cerebral palsy (CP) in children with West syndrome (WS), to estimate the possible causative factors by analyzing the neuroimaging examinations of patients, to evaluate their cognitive/motor function and epileptic status and to compare the prognosis of children with double pathology of WS and CP and of those without CP. METHODS: The clinical and magnetic resonance imaging (MRI) data of 62 patients with West syndrome were evaluated. A total of 39 of 62 patients (63%) suffered from CP (CP group). The non-CP group included 23 patients. RESULTS: Abnormal MRI was found in 55/62 (89%) patients. Main anomalies were: brain malformation (21), hypoxic-ischemic encephalopathy (13), cerebrovascular insult (8), infection (7), and other anomalies (6). In the CP group, the most common MRI abnormalities included pre/perinatal hypoxia/ischemia, brain malformation, cerebrovascular insult, and infection. In the non-CP group, brain malformations were the most frequent. Significantly more negative MRIs were found in the non-CP group. More than 60% of the patients were severely cognitively impaired, almost 90% of them had CP. Not only the occurrence of intellectual disability was lower in the non-CP group, but its severity was milder as well. A total of 78% of the children with CP had a very severe motor disability. Fifty-four percent in the CP and 67% in the non-CP group had therapy-resistant epilepsy. CONCLUSION: WS has an especially unfavorable prognosis: cerebral anomaly was confirmed in 89% of our patients. CP was present in almost two-thirds of the children with WS, most of them had severe cognitive and motor deficits.