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Sequence-based genetic testing identifies causative variants in ~ 50% of individuals with developmental and epileptic encephalopathies (DEEs). Aberrant changes in DNA methylation are implicated in various neurodevelopmental disorders but remain unstudied in DEEs. We interrogate the diagnostic utility of genome-wide DNA methylation array analysis on peripheral blood samples from 582 individuals with genetically unsolved DEEs. We identify rare differentially methylated regions (DMRs) and explanatory episignatures to uncover causative and candidate genetic etiologies in 12 individuals. Using long-read sequencing, we identify DNA variants underlying rare DMRs, including one balanced translocation, three CG-rich repeat expansions, and four copy number variants. We also identify pathogenic variants associated with episignatures. Finally, we refine the CHD2 episignature using an 850 K methylation array and bisulfite sequencing to investigate potential insights into CHD2 pathophysiology. Our study demonstrates the diagnostic yield of genome-wide DNA methylation analysis to identify causal and candidate variants as 2% (12/582) for unsolved DEE cases.
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Variaciones en el Número de Copia de ADN , Metilación de ADN , Epilepsia , Humanos , Metilación de ADN/genética , Femenino , Niño , Masculino , Epilepsia/genética , Epilepsia/diagnóstico , Variaciones en el Número de Copia de ADN/genética , Preescolar , Proteínas de Unión al ADN/genética , Adolescente , Pruebas Genéticas/métodos , LactanteRESUMEN
Efforts to resolve the functional impact of variants of uncertain significance (VUS) have lagged behind the identification of new VUS; as such, there is a critical need for scalable VUS resolution technologies. Computational variant effect predictors (VEPs), once trained, can predict pathogenicity for all missense variants in a gene, set of genes, or the exome. Existing tools have employed information on known pathogenic and benign variants throughout the genome to predict pathogenicity of VUS. We hypothesize that taking a gene-specific approach will improve pathogenicity prediction over globally-trained VEPs. We tested this hypothesis using the gene TSC2, whose loss of function results in tuberous sclerosis, a multisystem mTORopathy affecting about 1 in 6,000 individuals born in the United States. TSC2 has been identified as a high-priority target for VUS resolution, with (1) well-characterized molecular and patient phenotypes associated with loss-of-function variants, and (2) more than 2,700 VUS already documented in ClinVar. We developed Tuberous sclerosis classifier to Resolve variants of Uncertain Significance in T SC2 (TRUST), a machine learning model to predict pathogenicity of TSC2 missense VUS. To test whether these predictions are accurate, we further introduce curated loci prime editing (cliPE) as an accessible strategy for performing scalable multiplexed assays of variant effect (MAVEs). Using cliPE, we tested the effects of more than 200 TSC2 variants, including 106 VUS. It is highly likely this functional data alone would be sufficient to reclassify 92 VUS with most being reclassified as likely benign. We found that TRUST's classifications were correlated with the functional data, providing additional validation for the in silico predictions. We provide our pathogenicity predictions and MAVE data to aid with VUS resolution. In the near future, we plan to host these data on a public website and deposit into relevant databases such as MAVEdb as a community resource. Ultimately, this study provides a framework to complete variant effect maps of TSC1 and TSC2 and adapt this approach to other mTORopathy genes.
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Glutamine synthetase (GS), encoded by GLUL, catalyzes the conversion of glutamate to glutamine. GS is pivotal for the generation of the neurotransmitters glutamate and gamma-aminobutyric acid and is the primary mechanism of ammonia detoxification in the brain. GS levels are regulated post-translationally by an N-terminal degron that enables the ubiquitin-mediated degradation of GS in a glutamine-induced manner. GS deficiency in humans is known to lead to neurological defects and death in infancy, yet how dysregulation of the degron-mediated control of GS levels might affect neurodevelopment is unknown. We ascertained nine individuals with severe developmental delay, seizures, and white matter abnormalities but normal plasma and cerebrospinal fluid biochemistry with de novo variants in GLUL. Seven out of nine were start-loss variants and two out of nine disrupted 5' UTR splicing resulting in splice exclusion of the initiation codon. Using transfection-based expression systems and mass spectrometry, these variants were shown to lead to translation initiation of GS from methionine 18, downstream of the N-terminal degron motif, resulting in a protein that is stable and enzymatically competent but insensitive to negative feedback by glutamine. Analysis of human single-cell transcriptomes demonstrated that GLUL is widely expressed in neuro- and glial-progenitor cells and mature astrocytes but not in post-mitotic neurons. One individual with a start-loss GLUL variant demonstrated periventricular nodular heterotopia, a neuronal migration disorder, yet overexpression of stabilized GS in mice using in utero electroporation demonstrated no migratory deficits. These findings underline the importance of tight regulation of glutamine metabolism during neurodevelopment in humans.
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Epilepsia Generalizada , Glutamato-Amoníaco Ligasa , Glutamina , Animales , Humanos , Ratones , Encéfalo/metabolismo , Epilepsia Generalizada/genética , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Glutamatos/metabolismo , Glutamina/genética , Glutamina/metabolismoRESUMEN
Genes encoding long non-coding RNAs (lncRNAs) comprise a large fraction of the human genome, yet haploinsufficiency of a lncRNA has not been shown to cause a Mendelian disease. CHASERR is a highly conserved human lncRNA adjacent to CHD2-a coding gene in which de novo loss-of-function variants cause developmental and epileptic encephalopathy. Here we report three unrelated individuals each harboring an ultra-rare heterozygous de novo deletion in the CHASERR locus. We report similarities in severe developmental delay, facial dysmorphisms, and cerebral dysmyelination in these individuals, distinguishing them from the phenotypic spectrum of CHD2 haploinsufficiency. We demonstrate reduced CHASERR mRNA expression and corresponding increased CHD2 mRNA and protein in whole blood and patient-derived cell lines-specifically increased expression of the CHD2 allele in cis with the CHASERR deletion, as predicted from a prior mouse model of Chaserr haploinsufficiency. We show for the first time that de novo structural variants facilitated by Alu-mediated non-allelic homologous recombination led to deletion of a non-coding element (the lncRNA CHASERR) to cause a rare syndromic neurodevelopmental disorder. We also demonstrate that CHD2 has bidirectional dosage sensitivity in human disease. This work highlights the need to carefully evaluate other lncRNAs, particularly those upstream of genes associated with Mendelian disorders.
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Sequence-based genetic testing currently identifies causative genetic variants in â¼50% of individuals with developmental and epileptic encephalopathies (DEEs). Aberrant changes in DNA methylation are implicated in various neurodevelopmental disorders but remain unstudied in DEEs. Rare epigenetic variations ("epivariants") can drive disease by modulating gene expression at single loci, whereas genome-wide DNA methylation changes can result in distinct "episignature" biomarkers for monogenic disorders in a growing number of rare diseases. Here, we interrogate the diagnostic utility of genome-wide DNA methylation array analysis on peripheral blood samples from 516 individuals with genetically unsolved DEEs who had previously undergone extensive genetic testing. We identified rare differentially methylated regions (DMRs) and explanatory episignatures to discover causative and candidate genetic etiologies in 10 individuals. We then used long-read sequencing to identify DNA variants underlying rare DMRs, including one balanced translocation, three CG-rich repeat expansions, and two copy number variants. We also identify pathogenic sequence variants associated with episignatures; some had been missed by previous exome sequencing. Although most DEE genes lack known episignatures, the increase in diagnostic yield for DNA methylation analysis in DEEs is comparable to the added yield of genome sequencing. Finally, we refine an episignature for CHD2 using an 850K methylation array which was further refined at higher CpG resolution using bisulfite sequencing to investigate potential insights into CHD2 pathophysiology. Our study demonstrates the diagnostic yield of genome-wide DNA methylation analysis to identify causal and candidate genetic causes as â¼2% (10/516) for unsolved DEE cases.
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Pathogenic loss-of-function SCN1A variants cause a spectrum of seizure disorders. We previously identified variants in individuals with SCN1A -related epilepsy that fall in or near a poison exon (PE) in SCN1A intron 20 (20N). We hypothesized these variants lead to increased PE inclusion, which introduces a premature stop codon, and, therefore, reduced abundance of the full-length SCN1A transcript and Na v 1.1 protein. We used a splicing reporter assay to interrogate PE inclusion in HEK293T cells. In addition, we used patient-specific induced pluripotent stem cells (iPSCs) differentiated into neurons to quantify 20N inclusion by long and short-read sequencing and Na v 1.1 abundance by western blot. We performed RNA-antisense purification with mass spectrometry to identify RNA-binding proteins (RBPs) that could account for the aberrant PE splicing. We demonstrate that variants in/near 20N lead to increased 20N inclusion by long-read sequencing or splicing reporter assay and decreased Na v 1.1 abundance. We also identified 28 RBPs that differentially interact with variant constructs compared to wild-type, including SRSF1 and HNRNPL. We propose a model whereby 20N variants disrupt RBP binding to splicing enhancers (SRSF1) and suppressors (HNRNPL), to favor PE inclusion. Overall, we demonstrate that SCN1A 20N variants cause haploinsufficiency and SCN1A -related epilepsies. This work provides insights into the complex control of RBP-mediated PE alternative splicing, with broader implications for PE discovery and identification of pathogenic PE variants in other genetic conditions.
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PPFIA3 encodes the Protein-Tyrosine Phosphatase, Receptor-Type, F Polypeptide-Interacting Protein Alpha-3 (PPFIA3), which is a member of the LAR protein-tyrosine phosphatase-interacting protein (liprin) family involved in synaptic vesicle transport and presynaptic active zone assembly. The protein structure and function are well conserved in both invertebrates and vertebrates, but human diseases related to PPFIA3 dysfunction are not yet known. Here, we report 14 individuals with rare mono-allelic PPFIA3 variants presenting with features including developmental delay, intellectual disability, hypotonia, autism, and epilepsy. To determine the pathogenicity of PPFIA3 variants in vivo , we generated transgenic fruit flies expressing either human PPFIA3 wildtype (WT) or variant protein using GAL4-UAS targeted gene expression systems. Ubiquitous expression with Actin-GAL4 showed that the PPFIA3 variants had variable penetrance of pupal lethality, eclosion defects, and anatomical leg defects. Neuronal expression with elav-GAL4 showed that the PPFIA3 variants had seizure-like behaviors, motor defects, and bouton loss at the 3 rd instar larval neuromuscular junction (NMJ). Altogether, in the fly overexpression assays, we found that the PPFIA3 variants in the N-terminal coiled coil domain exhibited stronger phenotypes compared to those in the C-terminal region. In the loss-of-function fly assay, we show that the homozygous loss of fly Liprin- α leads to embryonic lethality. This lethality is partially rescued by the expression of human PPFIA3 WT, suggesting human PPFIA3 protein function is partially conserved in the fly. However, the PPFIA3 variants failed to rescue lethality. Altogether, the human and fruit fly data reveal that the rare PPFIA3 variants are dominant negative loss-of-function alleles that perturb multiple developmental processes and synapse formation.
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The anticoagulant warfarin is commonly used to control and prevent thrombotic disorders, such as venous thromboembolism (VTE), which disproportionately afflicts African Americans. Despite the importance of copy number variants (CNVs), few studies have focused on characterizing and understanding their role in drug response and disease risk among African Americans. In this study, we conduct the first genome-wide analysis of CNVs to more comprehensively account for the contribution of genetic variation in warfarin dose requirement and VTE risk among African Americans. We used hidden Markov models to detect CNVs from high-throughput single-nucleotide polymorphism arrays for 340 African American participants in the International Warfarin Pharmacogenetics Consortium. We identified 11,570 CNVs resulting in 2,038 copy number variable regions (CNVRs) and found 3 CNVRs associated with warfarin dose requirement and 3 CNVRs associated with VTE risk in African Americans. CNVRs 1q31.2del and 6q14.1del were associated with increased warfarin dose requirement (ß = 11.18 and 4.94, respectively; Pemp = < 0.002); CNVR 19p13.31del was associated with decreased warfarin dose requirement (ß = -1.41, Pemp = 0.0004); CNVRs (2p22.1del and 5q35.1-q35.2del) were found to be associated with increased risk of VTE (odds ratios (ORs) = 1.88 and 14.9, respectively; Pemp ≤0.02); and CNVR 10q26.12del was associated with a decreased risk of VTE (OR = 0.6; Pemp = 0.05). Modeling of the 10q26.12del in HepG2 cells revealed that this deletion results in decreased fibrinogen gene expression, decreased fibrinogen and WDR11 protein levels, and decreased secretion of fibrinogen into the extracellular matrix. We found robust evidence that CNVRs could contribute to warfarin dose requirement and risk of VTE in African Americans and for 10q26.3del describe a possible pathogenic mechanism.
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Tromboembolia Venosa , Warfarina , Humanos , Warfarina/efectos adversos , Negro o Afroamericano/genética , Tromboembolia Venosa/genética , Variaciones en el Número de Copia de ADN , Anticoagulantes/efectos adversos , Estudio de Asociación del Genoma Completo , Polimorfismo de Nucleótido SimpleRESUMEN
PURPOSE: Sub-Saharan Africa bears the highest burden of epilepsy worldwide. A presumed proportion is genetic, but this etiology is buried under the burden of infections and perinatal insults in a setting of limited awareness and few options for testing. Children with developmental and epileptic encephalopathies (DEEs) are most severely affected by this diagnostic gap in Africa, because the rate of actionable findings is highest in DEE-associated genes. METHODS: We tested 234 genetically naive South African children diagnosed with/possible DEE using gene panels, exome sequencing, and chromosomal microarray. Statistical comparison of electroclinical features in children with and children without candidate variants was performed to identify characteristics most likely predictive of a positive genetic finding. RESULTS: Of the 41 (of 234) children with likely/pathogenic variants, 26 had variants supporting precision therapy. Multivariate regression modeling highlighted neonatal or infantile-onset seizures and movement abnormalities as predictive of a positive genetic finding. We used this, coupled with an emphasis on precision medicine outcomes, to propose the pragmatic "Think-Genetics" strategy for early recognition of a possible genetic etiology. CONCLUSION: Our findings emphasize the importance of an early genetic diagnosis in DEE. We designed the Think-Genetics strategy for early recognition, appropriate interim management, and genetic testing for DEE in resource-constrained settings.
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Epilepsia , Medicina de Precisión , Niño , Recién Nacido , Humanos , Configuración de Recursos Limitados , Epilepsia/diagnóstico , Epilepsia/epidemiología , Epilepsia/genética , Pruebas Genéticas , ÁfricaRESUMEN
Focal cortical dysplasia (FCD) is a common cause of focal epilepsy that typically results from brain mosaic mutations in the mTOR cell signaling pathway. To identify new FCD genes, we developed an in vitro CRISPRi screen in human neurons and used FACS enrichment based on the FCD biomarker, phosphorylated S6 ribosomal protein (pS6). Using whole-genome (110,000 gRNAs) and candidate (129 gRNAs) libraries, we discovered 12 new genes that significantly increase pS6 levels. Interestingly, positive hits were enriched for brain-specific genes, highlighting the effectiveness of using human iPSC-derived induced neurons (iNeurons) in our screen. We investigated the signaling pathways of six candidate genes: LRRC4, EIF3A, TSN, HIP1, PIK3R3, and URI1. All six genes increased phosphorylation of S6. However, only two genes, PIK3R3 and HIP1, caused hyperphosphorylation more proximally in the AKT/mTOR/S6 signaling pathway. Importantly, these two genes have recently been found independently to be mutated in resected brain tissue from FCD patients, supporting the predictive validity of our screen. Knocking down each of the other four genes (LRRC4, EIF3A, TSN, and URI1) in iNeurons caused them to become resistant to the loss of growth factor signaling; without growth factor stimulation, pS6 levels were comparable to growth factor stimulated controls. Our data markedly expand the set of genes that are likely to regulate mTOR pathway signaling in neurons and provide additional targets for identifying somatic gene variants that cause FCD.
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AIM: To identify additional genes associated with infantile spasms using a cohort with defined infantile spasms. METHOD: Whole-exome sequencing (WES) was performed on 21 consented individuals with infantile spasms and their unaffected parents (a trio-based study). Clinical history and imaging were reviewed. Potentially deleterious exonic variants were identified and segregated. To refine potential candidates, variants were further prioritized on the basis of evidence for relevance to disease phenotype or known associations with infantile spasms, epilepsy, or neurological disease. RESULTS: Likely pathogenic de novo variants were identified in NR2F1, GNB1, NEUROD2, GABRA2, and NDUFAF5. Suggestive dominant and recessive candidate variants were identified in PEMT, DYNC1I1, ASXL1, RALGAPB, and STRADA; further confirmation is required to support their relevance to disease etiology. INTERPRETATION: This study supports the utility of WES in uncovering the genetic etiology in undiagnosed individuals with infantile spasms with an overall yield of five out of 21. High-priority candidates were identified in an additional five individuals. WES provides additional support for previously described disease-associated genes and expands their already broad mutational and phenotypic spectrum.
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Espasmos Infantiles , Hormona Adrenocorticotrópica , Humanos , Mutación/genética , Fenotipo , Espasmos Infantiles/tratamiento farmacológico , Espasmos Infantiles/genética , Secuenciación del ExomaRESUMEN
An increasing number of epilepsies are being attributed to variants in genes with epigenetic functions. The products of these genes include factors that regulate the structure and function of chromatin and the placing, reading and removal of epigenetic marks, as well as other epigenetic processes. In this Review, we provide an overview of the various epigenetic processes, structuring our discussion around five function-based categories: DNA methylation, histone modifications, histone-DNA crosstalk, non-coding RNAs and chromatin remodelling. We provide background information on each category, describing the general mechanism by which each process leads to altered gene expression. We also highlight key clinical and mechanistic aspects, providing examples of genes that strongly associate with epilepsy within each class. We consider the practical applications of these findings, including tissue-based and biofluid-based diagnostics and precision medicine-based treatments. We conclude that variants in epigenetic genes are increasingly found to be causally involved in the epilepsies, with implications for disease mechanisms, treatments and diagnostics.
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Epigénesis Genética , Epilepsia , Metilación de ADN/genética , Epigénesis Genética/genética , Epilepsia/genética , Histonas/genética , Histonas/metabolismo , Humanos , Procesamiento Proteico-PostraduccionalRESUMEN
Biallelic pathogenic variants in SZT2 result in a neurodevelopmental disorder with shared features, including early-onset epilepsy, developmental delay, macrocephaly, and corpus callosum abnormalities. SZT2 is as a critical scaffolding protein in the amino acid sensing arm of the mTORC1 signalling pathway. Due to its large size (3432 amino acids), lack of crystal structure, and absence of functional domains, it is difficult to determine the pathogenicity of SZT2 missense and in-frame deletions, but these variants are increasingly detected and reported by clinical genetic testing in individuals with epilepsy. To exemplify this latter point, here we describe a cohort of 12 individuals with biallelic SZT2 variants and phenotypic overlap with SZT2-related neurodevelopmental disorders. However, the majority of individuals carried one or more SZT2 variants of uncertain significance (VUS), highlighting the need for functional characterization to determine, which, if any, of these VUS were pathogenic. Thus, we developed a novel individualized platform to identify SZT2 loss-of-function variants in the context of mTORC1 signalling and reclassify VUS. Using this platform, we identified a recurrent in-frame deletion (SZT2 p.Val1984del) which was determined to be a loss-of-function variant and therefore likely pathogenic. Haplotype analysis revealed that this single in-frame deletion is a founder variant in those of Ashkenazi Jewish ancestry. Moreover, this approach allowed us to tentatively reclassify all of the VUS in our cohort of 12 individuals, identifying five individuals with biallelic pathogenic or likely pathogenic variants. Clinical features of these five individuals consisted of early-onset seizures (median 24 months), focal seizures, developmental delay and macrocephaly similar to previous reports. However, we also show a widening of the phenotypic spectrum, as none of the five individuals had corpus callosum abnormalities, in contrast to previous reports. Overall, we present a rapid assay to resolve VUS in SZT2, identify a founder variant in individuals of Ashkenazi Jewish ancestry, and demonstrate that corpus callosum abnormalities is not a hallmark feature of this condition. Our approach is widely applicable to other mTORopathies including the most common causes of the focal genetic epilepsies, DEPDC5, TSC1/2, MTOR and NPRL2/3.
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Epilepsias Parciales , Epilepsia , Megalencefalia , Epilepsia/genética , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Megalencefalia/genética , Proteínas del Tejido Nervioso/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
This review summarizes the pathogenic mechanisms that underpin the monogenic epilepsies and discusses the potential of novel precision therapeutics to treat these disorders. Pathogenic mechanisms of epilepsy include recessive (null alleles), haploinsufficiency, imprinting, gain-of-function, and dominant negative effects. Understanding which pathogenic mechanism(s) that underlie each genetic epilepsy is pivotal to design precision therapies that are most likely to be beneficial for the patient. Novel therapeutics discussed include gene therapy, gene editing, antisense oligonucleotides, and protein replacement. Discussions are illustrated and reinforced with examples from the literature.
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Epilepsia/genética , Mutación con Ganancia de Función/genética , Edición Génica/métodos , Terapia Genética/métodos , Haploinsuficiencia/genética , Medicina de Precisión/métodos , Epilepsia/terapia , Mutación con Ganancia de Función/efectos de los fármacos , Haploinsuficiencia/efectos de los fármacos , Humanos , Oligonucleótidos Antisentido/uso terapéuticoRESUMEN
Kainate receptors (KARs) are glutamate-gated cation channels with diverse roles in the central nervous system. Bi-allelic loss of function of the KAR-encoding gene GRIK2 causes a nonsyndromic neurodevelopmental disorder (NDD) with intellectual disability and developmental delay as core features. The extent to which mono-allelic variants in GRIK2 also underlie NDDs is less understood because only a single individual has been reported previously. Here, we describe an additional eleven individuals with heterozygous de novo variants in GRIK2 causative for neurodevelopmental deficits that include intellectual disability. Five children harbored recurrent de novo variants (three encoding p.Thr660Lys and two p.Thr660Arg), and four children and one adult were homozygous for a previously reported variant (c.1969G>A [p.Ala657Thr]). Individuals with shared variants had some overlapping behavioral and neurological dysfunction, suggesting that the GRIK2 variants are likely pathogenic. Analogous mutations introduced into recombinant GluK2 KAR subunits at sites within the M3 transmembrane domain (encoding p.Ala657Thr, p.Thr660Lys, and p.Thr660Arg) and the M3-S2 linker domain (encoding p.Ile668Thr) had complex effects on functional properties and membrane localization of homomeric and heteromeric KARs. Both p.Thr660Lys and p.Thr660Arg mutant KARs exhibited markedly slowed gating kinetics, similar to p.Ala657Thr-containing receptors. Moreover, we observed emerging genotype-phenotype correlations, including the presence of severe epilepsy in individuals with the p.Thr660Lys variant and hypomyelination in individuals with either the p.Thr660Lys or p.Thr660Arg variant. Collectively, these results demonstrate that human GRIK2 variants predicted to alter channel function are causative for early childhood development disorders and further emphasize the importance of clarifying the role of KARs in early nervous system development.
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Encéfalo/metabolismo , Discapacidades del Desarrollo/genética , Epilepsia/genética , Discapacidad Intelectual/genética , Mutación , Receptores de Ácido Kaínico/genética , Adolescente , Adulto , Alelos , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Niño , Preescolar , Discapacidades del Desarrollo/diagnóstico por imagen , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Epilepsia/diagnóstico por imagen , Epilepsia/metabolismo , Epilepsia/patología , Potenciales Evocados/fisiología , Regulación del Desarrollo de la Expresión Génica , Estudios de Asociación Genética , Heterocigoto , Homocigoto , Humanos , Discapacidad Intelectual/diagnóstico por imagen , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Activación del Canal Iónico , Masculino , Modelos Moleculares , Neuronas/metabolismo , Neuronas/patología , Conformación Proteica , Receptores de Ácido Kaínico/química , Receptores de Ácido Kaínico/metabolismo , Receptor de Ácido Kaínico GluK2RESUMEN
AIM: To determine whether genes that cause developmental and epileptic encephalopathies (DEEs) are more commonly implicated in intellectual disability with epilepsy as a comorbid feature than in intellectual disability only. METHOD: We performed targeted resequencing of 18 genes commonly implicated in DEEs in a cohort of 830 patients with intellectual disability (59% male) and 393 patients with DEEs (52% male). RESULTS: We observed a significant enrichment of pathogenic/likely pathogenic variants in patients with epilepsy and intellectual disability (16 out of 159 in seven genes) compared with intellectual disability only (2 out of 671) (p<1.86×10-10 , odds ratio 37.22, 95% confidence interval 8.60-337.0). INTERPRETATION: We identified seven genes that are more likely to cause epilepsy and intellectual disability than intellectual disability only. Conversely, two genes, GRIN2B and SCN2A, can be implicated in intellectual disability without epilepsy; in these instances intellectual disability is not a secondary consequence of ongoing seizures but rather a primary cause. What this paper adds A subset of genes are more commonly implicated in epilepsy than other neurodevelopmental disorders. GRIN2B and SCN2A are implicated in intellectual disability and epilepsy independently.
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Discapacidad Intelectual/genética , Mutación , Canal de Sodio Activado por Voltaje NAV1.2/genética , Fenotipo , Receptores de N-Metil-D-Aspartato/genética , Espasmos Infantiles/genética , Adolescente , Niño , Exoma , Femenino , Humanos , Lactante , MasculinoRESUMEN
OBJECTIVE: The MAST family of microtubule-associated serine-threonine kinases (STKs) have distinct expression patterns in the developing and mature human and mouse brain. To date, only MAST1 has been conclusively associated with neurological disease, with de novo variants in individuals with a neurodevelopmental disorder, including a mega corpus callosum. METHODS: Using exome sequencing, we identify MAST3 missense variants in individuals with epilepsy. We also assess the effect of these variants on the ability of MAST3 to phosphorylate the target gene product ARPP-16 in HEK293T cells. RESULTS: We identify de novo missense variants in the STK domain in 11 individuals, including 2 recurrent variants p.G510S (n = 5) and p.G515S (n = 3). All 11 individuals had developmental and epileptic encephalopathy, with 8 having normal development prior to seizure onset at <2 years of age. All patients developed multiple seizure types, 9 of 11 patients had seizures triggered by fever and 9 of 11 patients had drug-resistant seizures. In vitro analysis of HEK293T cells transfected with MAST3 cDNA carrying a subset of these patient-specific missense variants demonstrated variable but generally lower expression, with concomitant increased phosphorylation of the MAST3 target, ARPP-16, compared to wild-type. These findings suggest the patient-specific variants may confer MAST3 gain-of-function. Moreover, single-nuclei RNA sequencing and immunohistochemistry shows that MAST3 expression is restricted to excitatory neurons in the cortex late in prenatal development and postnatally. INTERPRETATION: In summary, we describe MAST3 as a novel epilepsy-associated gene with a potential gain-of-function pathogenic mechanism that may be primarily restricted to excitatory neurons in the cortex. ANN NEUROL 2021;90:274-284.
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Epilepsia/diagnóstico por imagen , Epilepsia/genética , Variación Genética/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Serina-Treonina Quinasas/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Animales , Niño , Estudios de Cohortes , Epilepsia/metabolismo , Femenino , Estudios de Seguimiento , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas Serina-Treonina Quinasas/biosíntesis , Adulto JovenRESUMEN
Developmental and epileptic encephalopathies (DEEs) describe a subset of neurodevelopmental disorders categorized by refractory epilepsy that is often associated with intellectual disability and autism spectrum disorder. The majority of DEEs are now known to have a genetic basis with de novo coding variants accounting for the majority of cases. More recently, a small number of individuals have been identified with intronic SCN1A variants that result in alternative splicing events that lead to ectopic inclusion of poison exons (PEs). PEs are short highly conserved exons that contain a premature truncation codon, and when spliced into the transcript, lead to premature truncation and subsequent degradation by nonsense-mediated decay. The reason for the inclusion/exclusion of these PEs is not entirely clear, but research suggests an autoregulatory role in gene expression and protein abundance. This is seen in proteins such as RNA-binding proteins and serine/arginine-rich proteins. Recent studies have focused on targeting these PEs as a method for therapeutic intervention. Targeting PEs using antisense oligonucleotides (ASOs) has shown to be effective in modulating alternative splicing events by decreasing the amount of transcripts harboring PEs, thus increasing the abundance of full-length transcripts and thereby the amount of protein in haploinsufficient genes implicated in DEE. In the age of personalized medicine, cellular and animal models of the genetic epilepsies have become essential in developing and testing novel precision therapeutics, including PE-targeting ASOs in a subset of DEEs.