Epileptic encephalopathy (EE) refers to a clinically and genetically heterogeneous group of severe disorders characterized by seizures, abnormal interictal electro-encephalogram, psychomotor delay, and/or cognitive deterioration. We ascertained two multiplex families (including one consanguineous family) consistent with an autosomal-recessive inheritance pattern of EE. All seven affected individuals developed subclinical seizures as early as the first day of life, severe epileptic disease, and profound developmental delay with no facial dysmorphism. Given the similarity in clinical presentation in the two families, we hypothesized that the observed phenotype was due to mutations in the same gene, and we performed exome sequencing in three affected individuals. Analysis of rare variants in genes consistent with an autosomal-recessive mode of inheritance led to identification of mutations in SLC13A5, which encodes the cytoplasmic sodium-dependent citrate carrier, notably expressed in neurons. Disease association was confirmed by cosegregation analysis in additional family members. Screening of 68 additional unrelated individuals with early-onset epileptic encephalopathy for SLC13A5 mutations led to identification of one additional subject with compound heterozygous mutations of SLC13A5 and a similar clinical presentation as the index subjects. Mutations affected key residues for sodium binding, which is critical for citrate transport. These findings underline the value of careful clinical characterization for genetic investigations in highly heterogeneous conditions such as EE and further highlight the role of citrate metabolism in epilepsy.

Cationic amino acid transporters (CATs) mediate the entry of L-type cationic amino acids (arginine, ornithine and lysine) into the cells including neurons. CAT-3, encoded by the SLC7A3 gene on chromosome X, is one of the three CATs present in the human genome, with selective expression in brain. SLC7A3 is highly intolerant to variation in humans, as attested by the low frequency of deleterious variants in available databases, but the impact on variants in this gene in humans remains undefined. In this study, we identified a missense variant in SLC7A3, encoding the CAT-3 cationic amino acid transporter, on chromosome X by exome sequencing in two brothers with autism spectrum disorder (ASD). We then sequenced the SLC7A3 coding sequence in 148 male patients with ASD and identified three additional rare missense variants in unrelated patients. Functional analyses of the mutant transporters showed that two of the four identified variants cause severe or moderate loss of CAT-3 function due to altered protein stability or abnormal trafficking to the plasma membrane. The patient with the most deleterious SLC7A3 variant had high-functioning autism and epilepsy, and also carries a de novo 16p11.2 duplication possibly contributing to his phenotype. This study shows that rare hypomorphic variants of SLC7A3 exist in male individuals and suggest that SLC7A3 variants possibly contribute to the etiology of ASD in male subjects in association with other genetic factors.

Neurodegeneration with brain iron accumulation (NBIA) is a group of genetic disorders characterized by abnormal iron deposition in the basal ganglia. We report that de novo mutations in WDR45, a gene located at Xp11.23 and encoding a beta-propeller scaffold protein with a putative role in autophagy, cause a distinctive NBIA phenotype. The clinical features include early-onset global developmental delay and further neurological deterioration (parkinsonism, dystonia, and dementia developing by early adulthood). Brain MRI revealed evidence of iron deposition in the substantia nigra and globus pallidus. Males and females are phenotypically similar, an observation that might be explained by somatic mosaicism in surviving males and germline or somatic mutations in females, as well as skewing of X chromosome inactivation. This clinically recognizable disorder is among the more common forms of NBIA, and we suggest that it be named accordingly as beta-propeller protein-associated neurodegeneration.

Developmental and epileptic encephalopathies (DEEs) are severe neurodevelopmental disorders often beginning in infancy or early childhood that are characterized by intractable seizures, abundant epileptiform activity on EEG, and developmental impairment or regression. CACNA1E is highly expressed in the central nervous system and encodes the α1-subunit of the voltage-gated CaV2.3 channel, which conducts high voltage-activated R-type calcium currents that initiate synaptic transmission. Using next-generation sequencing techniques, we identified de novo CACNA1E variants in 30 individuals with DEE, characterized by refractory infantile-onset seizures, severe hypotonia, and profound developmental impairment, often with congenital contractures, macrocephaly, hyperkinetic movement disorders, and early death. Most of the 14, partially recurring, variants cluster within the cytoplasmic ends of all four S6 segments, which form the presumed CaV2.3 channel activation gate. Functional analysis of several S6 variants revealed consistent gain-of-function effects comprising facilitated voltage-dependent activation and slowed inactivation. Another variant located in the domain II S4-S5 linker results in facilitated activation and increased current density. Five participants achieved seizure freedom on the anti-epileptic drug topiramate, which blocks R-type calcium channels. We establish pathogenic variants in CACNA1E as a cause of DEEs and suggest facilitated R-type calcium currents as a disease mechanism for human epilepsy and developmental disorders.

To delineate the phenotype associated with biallelic ATAD1 variants.

Variants in IQSEC2, escaping X inactivation, cause X-linked intellectual disability with frequent epilepsy in males and females. We aimed to investigate sex-specific differences.

We delineate a KMT2E-related neurodevelopmental disorder on the basis of 38 individuals in 36 families. This study includes 31 distinct heterozygous variants in KMT2E (28 ascertained from Matchmaker Exchange and three previously reported), and four individuals with chromosome 7q22.2-22.23 microdeletions encompassing KMT2E (one previously reported). Almost all variants occurred de novo, and most were truncating. Most affected individuals with protein-truncating variants presented with mild intellectual disability. One-quarter of individuals met criteria for autism. Additional common features include macrocephaly, hypotonia, functional gastrointestinal abnormalities, and a subtle facial gestalt. Epilepsy was present in about one-fifth of individuals with truncating variants and was responsive to treatment with anti-epileptic medications in almost all. More than 70% of the individuals were male, and expressivity was variable by sex; epilepsy was more common in females and autism more common in males. The four individuals with microdeletions encompassing KMT2E generally presented similarly to those with truncating variants, but the degree of developmental delay was greater. The group of four individuals with missense variants in KMT2E presented with the most severe developmental delays. Epilepsy was present in all individuals with missense variants, often manifesting as treatment-resistant infantile epileptic encephalopathy. Microcephaly was also common in this group. Haploinsufficiency versus gain-of-function or dominant-negative effects specific to these missense variants in KMT2E might explain this divergence in phenotype, but requires independent validation. Disruptive variants in KMT2E are an under-recognized cause of neurodevelopmental abnormalities.

To evaluate the role that chromosomal micro-rearrangements play in patients with both corpus callosum abnormality and intellectual disability, we analyzed copy number variations (CNVs) in patients with corpus callosum abnormality/intellectual disability STUDY DESIGN: We screened 149 patients with corpus callosum abnormality/intellectual disability using Illumina SNP arrays.

No abstract available

Subtelomeric 1q43q44 microdeletions cause a syndrome associating intellectual disability, microcephaly, seizures and anomalies of the corpus callosum. Despite several previous studies assessing genotype-phenotype correlations, the contribution of genes located in this region to the specific features of this syndrome remains uncertain. Among those, three genes, AKT3, HNRNPU and ZBTB18 are highly expressed in the brain and point mutations in these genes have been recently identified in children with neurodevelopmental phenotypes. In this study, we report the clinical and molecular data from 17 patients with 1q43q44 microdeletions, four with ZBTB18 mutations and seven with HNRNPU mutations, and review additional data from 37 previously published patients with 1q43q44 microdeletions. We compare clinical data of patients with 1q43q44 microdeletions with those of patients with point mutations in HNRNPU and ZBTB18 to assess the contribution of each gene as well as the possibility of epistasis between genes. Our study demonstrates that AKT3 haploinsufficiency is the main driver for microcephaly, whereas HNRNPU alteration mostly drives epilepsy and determines the degree of intellectual disability. ZBTB18 deletions or mutations are associated with variable corpus callosum anomalies with an incomplete penetrance. ZBTB18 may also contribute to microcephaly and HNRNPU to thin corpus callosum, but with a lower penetrance. Co-deletion of contiguous genes has additive effects. Our results confirm and refine the complex genotype-phenotype correlations existing in the 1qter microdeletion syndrome and define more precisely the neurodevelopmental phenotypes associated with genetic alterations of AKT3, ZBTB18 and HNRNPU in humans.

A-type lamins, the intermediate filament proteins participating in nuclear structure and function, are encoded by LMNA. LMNA mutations can lead to laminopathies such as lipodystrophies, premature aging syndromes (progeria) and muscular dystrophies. Here, we identified a novel heterozygous LMNA p.R388P de novo mutation in a patient with a non-previously described severe phenotype comprising congenital muscular dystrophy (L-CMD) and lipodystrophy. In culture, the patient's skin fibroblasts entered prematurely into senescence, and some nuclei showed a lamina honeycomb pattern. C2C12 myoblasts were transfected with a construct carrying the patient's mutation; R388P-lamin A (LA) predominantly accumulated within the nucleoplasm and was depleted at the nuclear periphery, altering the anchorage of the inner nuclear membrane protein emerin and the nucleoplasmic protein LAP2-alpha. The mutant LA triggered a frequent and severe nuclear dysmorphy that occurred independently of prelamin A processing, as well as increased histone H3K9 acetylation. Nuclear dysmorphy was not significantly improved when transfected cells were treated with drugs disrupting microtubules or actin filaments or modifying the global histone acetylation pattern. Therefore, releasing any force exerted at the nuclear envelope by the cytoskeleton or chromatin did not rescue nuclear shape, in contrast to what was previously shown in Hutchinson-Gilford progeria due to other LMNA mutations. Our results point to the specific cytotoxic effect of the R388P-lamin A mutant, which is clinically related to a rare and severe multisystemic laminopathy phenotype.

Autism spectrum disorders affect millions of individuals worldwide, but their heterogeneity complicates therapeutic intervention that is essentially symptomatic. A versatile yet relevant model to rationally screen among hundreds of therapeutic options would help improving clinical practice. Here we investigated whether neurons differentiated from pluripotent stem cells can provide such a tool using SHANK3 haploinsufficiency as a proof of principle. A library of compounds was screened for potential to increase SHANK3 mRNA content in neurons differentiated from control human embryonic stem cells. Using induced pluripotent stem cell technology, active compounds were then evaluated for efficacy in correcting dysfunctional networks of neurons differentiated from individuals with deleterious point mutations of SHANK3. Among 202 compounds tested, lithium and valproic acid showed the best efficacy at corrected SHANK3 haploinsufficiency associated phenotypes in cellulo. Lithium pharmacotherapy was subsequently provided to one patient and, after one year, an encouraging decrease in autism severity was observed. This demonstrated that pluripotent stem cell-derived neurons provide a novel cellular paradigm exploitable in the search for specific disease-modifying treatments.

Phelan-McDermid syndrome (PMS) is characterized by a variety of clinical symptoms with heterogeneous degrees of severity, including intellectual disability (ID), absent or delayed speech, and autism spectrum disorders (ASD). It results from a deletion of the distal part of chromosome 22q13 that in most cases includes the SHANK3 gene. SHANK3 is considered a major gene for PMS, but the factors that modulate the severity of the syndrome remain largely unknown. In this study, we investigated 85 patients with different 22q13 rearrangements (78 deletions and 7 duplications). We first explored the clinical features associated with PMS, and provide evidence for frequent corpus callosum abnormalities in 28% of 35 patients with brain imaging data. We then mapped several candidate genomic regions at the 22q13 region associated with high risk of clinical features, and suggest a second locus at 22q13 associated with absence of speech. Finally, in some cases, we identified additional clinically relevant copy-number variants (CNVs) at loci associated with ASD, such as 16p11.2 and 15q11q13, which could modulate the severity of the syndrome. We also report an inherited SHANK3 deletion transmitted to five affected daughters by a mother without ID nor ASD, suggesting that some individuals could compensate for such mutations. In summary, we shed light on the genotype-phenotype relationship of patients with PMS, a step towards the identification of compensatory mechanisms for a better prognosis and possibly treatments of patients with neurodevelopmental disorders.

Corpus callosum agenesis (CCA) is a brain malformation associated with a wide clinical spectrum including intellectual disability (ID) and an etiopathological complexity. We identified a novel missense G424R mutation in the X-linked p21-activated kinase 3 (PAK3) gene in a boy presenting with severe ID, microcephaly and CCA and his fetal sibling with CCA and severe hydrocephaly. PAK3 kinase is known to control synaptic plasticity and dendritic spine dynamics but its implication is less characterized in brain ontogenesis. In order to identify developmental functions of PAK3 impacted by mutations responsible for CCA, we compared the biochemical and biological effects of three PAK3 mutations localized in the catalytic domain. These mutations include two "severe" G424R and K389N variants (responsible for severe ID and CCA) and the "mild" A365E variant (responsible for nonsyndromic mild ID). Whereas they suppressed kinase activity, only the two severe variants displayed normal protein stability. Furthermore, they increased interactions between PAK3 and the guanine exchange factor αPIX/ARHGEF6, disturbed adhesion point dynamics and cell spreading, and severely impacted cell migration. Our findings highlight new molecular defects associated with mutations responsible for severe clinical phenotypes with developmental brain defects.

De novo germline mutations in the RNA helicase DDX3X account for 1%-3% of unexplained intellectual disability (ID) cases in females and are associated with autism, brain malformations, and epilepsy. Yet, the developmental and molecular mechanisms by which DDX3X mutations impair brain function are unknown. Here, we use human and mouse genetics and cell biological and biochemical approaches to elucidate mechanisms by which pathogenic DDX3X variants disrupt brain development. We report the largest clinical cohort to date with DDX3X mutations (n = 107), demonstrating a striking correlation between recurrent dominant missense mutations, polymicrogyria, and the most severe clinical outcomes. We show that Ddx3x controls cortical development by regulating neuron generation. Severe DDX3X missense mutations profoundly disrupt RNA helicase activity, induce ectopic RNA-protein granules in neural progenitors and neurons, and impair translation. Together, these results uncover key mechanisms underlying DDX3X syndrome and highlight aberrant RNA metabolism in the pathogenesis of neurodevelopmental disease.

Disease gene discovery on chromosome (chr) X is challenging owing to its unique modes of inheritance. We undertook a systematic analysis of human chrX genes. We observe a higher proportion of disorder-associated genes and an enrichment of genes involved in cognition, language, and seizures on chrX compared to autosomes. We analyze gene constraints, exon and promoter conservation, expression, and paralogues, and report 127 genes sharing one or more attributes with known chrX disorder genes. Using machine learning classifiers trained to distinguish disease-associated from dispensable genes, we classify 247 genes, including 115 of the 127, as having high probability of being disease-associated. We provide evidence of an excess of variants in predicted genes in existing databases. Finally, we report damaging variants in CDK16 and TRPC5 in patients with intellectual disability or autism spectrum disorders. This study predicts large-scale gene-disease associations that could be used for prioritization of X-linked pathogenic variants.

To investigate the effect of PLXNA1 variants on the phenotype of patients with autosomal dominant and recessive inheritance patterns and to functionally characterize the zebrafish homologs plxna1a and plxna1b during development.

Massively parallel sequencing has revealed many de novo mutations in the etiology of developmental and epileptic encephalopathies (EEs), highlighting their genetic heterogeneity. Additional candidate genes have been prioritized in silico by their co-expression in the brain. Here, we evaluate rare coding variability in 20 candidates nominated with the use of a reference gene set of 51 established EE-associated genes. Variants within the 20 candidate genes were extracted from exome-sequencing data of 42 subjects with EE and no previous genetic diagnosis. We identified 7 rare non-synonymous variants in 7 of 20 genes and performed Sanger sequence validation in affected probands and parental samples. De novo variants were found only in SLC1A2 (aka EAAT2 or GLT1) (c.244G>A [p.Gly82Arg]) and YWHAG (aka 14-3-3γ) (c.394C>T [p.Arg132Cys]), highlighting the potential cause of EE in 5% (2/42) of subjects. Seven additional subjects with de novo variants in SLC1A2 (n = 1) and YWHAG (n = 6) were subsequently identified through online tools. We identified a highly significant enrichment of de novo variants in YWHAG, establishing their role in early-onset epilepsy, and we provide additional support for the prior assignment of SLC1A2. Hence, in silico modeling of brain co-expression is an efficient method for nominating EE-associated genes to further elucidate the disorder's etiology and genotype-phenotype correlations.

Germline WWOX pathogenic variants have been associated with disorder of sex differentiation (DSD), spinocerebellar ataxia (SCA), and WWOX-related epileptic encephalopathy (WOREE syndrome). We review clinical and molecular data on WWOX-related disorders, further describing WOREE syndrome and phenotype/genotype correlations.

Mutations in several genes encoding components of the SWI/SNF chromatin remodeling complex cause neurodevelopmental disorders (NDDs). Here, we report on five individuals with mutations in SMARCD1; the individuals present with developmental delay, intellectual disability, hypotonia, feeding difficulties, and small hands and feet. Trio exome sequencing proved the mutations to be de novo in four of the five individuals. Mutations in other SWI/SNF components cause Coffin-Siris syndrome, Nicolaides-Baraitser syndrome, or other syndromic and non-syndromic NDDs. Although the individuals presented here have dysmorphisms and some clinical overlap with these syndromes, they lack their typical facial dysmorphisms. To gain insight into the function of SMARCD1 in neurons, we investigated the Drosophila ortholog Bap60 in postmitotic memory-forming neurons of the adult Drosophila mushroom body (MB). Targeted knockdown of Bap60 in the MB of adult flies causes defects in long-term memory. Mushroom-body-specific transcriptome analysis revealed that Bap60 is required for context-dependent expression of genes involved in neuron function and development in juvenile flies when synaptic connections are actively being formed in response to experience. Taken together, we identify an NDD caused by SMARCD1 mutations and establish a role for the SMARCD1 ortholog Bap60 in the regulation of neurodevelopmental genes during a critical time window of juvenile adult brain development when neuronal circuits that are required for learning and memory are formed.