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DEP domain-containing 5 protein (DEPDC5) is a repressor of the recently recognized amino acid-sensing branch of the mTORC1 pathway. So far, its function in the brain remains largely unknown. Germline loss-of-function mutations in DEPDC5 have emerged as a major cause of familial refractory focal epilepsies, with case reports of sudden unexpected death in epilepsy (SUDEP). Remarkably, a fraction of patients also develop focal cortical dysplasia (FCD), a neurodevelopmental cortical malformation. We therefore hypothesized that a somatic second-hit mutation arising during brain development may support the focal nature of the dysplasia. Here, using postoperative human tissue, we provide the proof of concept that a biallelic 2-hit - brain somatic and germline - mutational mechanism in DEPDC5 causes focal epilepsy with FCD. We discovered a mutation gradient with a higher rate of mosaicism in the seizure-onset zone than in the surrounding epileptogenic zone. Furthermore, we demonstrate the causality of a Depdc5 brain mosaic inactivation using CRISPR-Cas9 editing and in utero electroporation in a mouse model recapitulating focal epilepsy with FCD and SUDEP-like events. We further unveil a key role of Depdc5 in shaping dendrite and spine morphology of excitatory neurons. This study reveals promising therapeutic avenues for treating drug-resistant focal epilepsies with mTORC1-targeting molecules.

Stéphanie Baulac and colleagues report the identification of mutations in the DEPDC5 gene that cause focal epilepsies. The main familial focal epilepsies are autosomal dominant nocturnal frontal lobe epilepsy, familial temporal lobe epilepsy and familial focal epilepsy with variable foci. A frameshift mutation in the DEPDC5 gene (encoding DEP domain–containing protein 5) was identified in a family with focal epilepsy with variable foci by linkage analysis and exome sequencing. Subsequent pyrosequencing of DEPDC5 in a cohort of 15 additional families with focal epilepsies identified 4 nonsense mutations and 1 missense mutation. Our findings provided evidence of frequent (37%) loss-of-function mutations in DEPDC5 associated with a broad spectrum of focal epilepsies. The implication of a DEP (Dishevelled, Egl-10 and Pleckstrin) domain–containing protein that may be involved in membrane trafficking and/or G protein signaling opens new avenues for research.

Familial Adult Myoclonic Epilepsy (FAME) is a genetically heterogeneous disorder characterized by cortical tremor and seizures. Intronic TTTTA/TTTCA repeat expansions in SAMD12 (FAME1) are the main cause of FAME in Asia. Using genome sequencing and repeat-primed PCR, we identify another site of this repeat expansion, in MARCH6 (FAME3) in four European families. Analysis of single DNA molecules with nanopore sequencing and molecular combing show that expansions range from 3.3 to 14 kb on average. However, we observe considerable variability in expansion length and structure, supporting the existence of multiple expansion configurations in blood cells and fibroblasts of the same individual. Moreover, the largest expansions are associated with micro-rearrangements occurring near the expansion in 20% of cells. This study provides further evidence that FAME is caused by intronic TTTTA/TTTCA expansions in distinct genes and reveals that expansions exhibit an unexpectedly high somatic instability that can ultimately result in genomic rearrangements. Familial cortical myoclonic tremor with epilepsy (FAME) is a slowly progressing cortical tremor mapping to various genomic loci, including intronic expansions in SAMD12 for FAME1. Here, Florian et al. describe mixed intronic TTTTA/TTTCA expansions of various lengths in the first intron of MARCH6 as a cause of FAME3.

Mutations in SQSTM1 encoding the sequestosome 1/p62 protein have recently been identified in familial and sporadic cases of amyotrophic lateral sclerosis (ALS). p62 is a component of the ubiquitin inclusions detected in degenerating neurons in ALS patients. We sequenced SQSTM1 in 90 French patients with familial ALS (FALS) and 74 autopsied ALS cases with sporadic ALS (SALS). We identified, at the heterozygote state, one missense c.1175C>T, p.Pro392Leu (exon 8) in one of our FALS and one substitution in intron 7 (the c.1165+1G>A, previously called IVS7+1 G-A, A390X) affecting the exon 7 splicing site in one SALS. These mutations that are located in the ubiquitin-associated domain (UBA domain) of the p62 protein have already been described in Paget’s disease and ALS patients carrying these mutations had both concomitant Paget’s disease. However, we also identified two novel missense mutations in two SALS: the c.259A>G, p.Met87Val in exon 2 and the c.304A>G, p.Lys102Glu in exon 3. These mutations that were not detected in 360 control subjects are possibly pathogenic. Neuropathology analysis of three patients carrying SQSTM1 variants revealed the presence of large round p62 inclusions in motor neurons, and immunoblot analysis showed an increased p62 and TDP-43 protein levels in the spinal cord. Our results confirm that SQSTM1 gene mutations could be the cause or genetic susceptibility factor of ALS in some patients.

PurposeARF1 was previously implicated in periventricular nodular heterotopia (PVNH) in only five individuals and systematic clinical characterisation was not available. The aim of this study is to provide a comprehensive description of the phenotypic and genotypic spectrum of ARF1-related neurodevelopmental disorder.MethodsWe collected detailed phenotypes of an international cohort of individuals (n=17) with ARF1 variants assembled through the GeneMatcher platform. Missense variants were structurally modelled, and the impact of several were functionally validated.ResultsDe novo variants (10 missense, 1 frameshift, 1 splice altering resulting in 9 residues insertion) in ARF1 were identified among 17 unrelated individuals. Detailed phenotypes included intellectual disability (ID), microcephaly, seizures and PVNH. No specific facial characteristics were consistent across all cases, however microretrognathia was common. Various hearing and visual defects were recurrent, and interestingly, some inflammatory features were reported. MRI of the brain frequently showed abnormalities consistent with a neuronal migration disorder.ConclusionWe confirm the role of ARF1 in an autosomal dominant syndrome with a phenotypic spectrum including severe ID, microcephaly, seizures and PVNH due to impaired neuronal migration.

Dravet syndrome (DS) is a genetically determined epileptic encephalopathy mainly caused by de novo mutations in the SCN1A gene. Since 2003, we have performed molecular analyses in a large series of patients with DS, 27% of whom were negative for mutations or rearrangements in SCN1A. In order to identify new genes responsible for the disorder in the SCN1A-negative patients, 41 probands were screened for micro-rearrangements with Illumina high-density SNP microarrays. A hemizygous deletion on chromosome Xq22.1, encompassing the PCDH19 gene, was found in one male patient. To confirm that PCDH19 is responsible for a Dravet-like syndrome, we sequenced its coding region in 73 additional SCN1A-negative patients. Nine different point mutations (four missense and five truncating mutations) were identified in 11 unrelated female patients. In addition, we demonstrated that the fibroblasts of our male patient were mosaic for the PCDH19 deletion. Patients with PCDH19 and SCN1A mutations had very similar clinical features including the association of early febrile and afebrile seizures, seizures occurring in clusters, developmental and language delays, behavioural disturbances, and cognitive regression. There were, however, slight but constant differences in the evolution of the patients, including fewer polymorphic seizures (in particular rare myoclonic jerks and atypical absences) in those with PCDH19 mutations. These results suggest that PCDH19 plays a major role in epileptic encephalopathies, with a clinical spectrum overlapping that of DS. This disorder mainly affects females. The identification of an affected mosaic male strongly supports the hypothesis that cellular interference is the pathogenic mechanism.

BackgroundMutations in SOD1, ANG, VAPB, TARDBP and FUS genes have been identified in amyotrophic lateral sclerosis (ALS).MethodsThe relative contributions of the different mutations to ALS were estimated by systematically screening a cohort of 162 families enrolled in France and 500 controls (1000 chromosomes) using molecular analysis techniques and performing phenotype–genotype correlations.Results31 pathogenic missense mutations were found in 36 patients (20 SOD1, 1 ANG, 1 VAPB, 7 TARDBP and 7 FUS). Surprisingly two FUS mutation carriers also harboured ANG variants. One family of Japanese origin with the P56S VAPB mutation was identified. Seven novel mutations (three in SOD1, two in TARDBP, two in FUS) were found. None of them was detected in controls. Segregation of detected mutations with the disease was confirmed in 11 families including five pedigrees carrying the novel mutations. Clinical comparison of SOD1, TARDBP, FUS and other familial ALS patients (with no mutation in the screened genes) revealed differences in site of onset (predominantly lower limbs for SOD1 and upper limbs for TARDBP mutations), age of onset (younger with FUS mutations), and in lifespan (shorter for FUS carriers). One third of SOD1 patients survived more than 7 years: these patients had earlier disease onset than those presenting with a more typical course. Differences were also observed among FUS mutations, with the R521H FUS mutation being associated with longer disease duration.ConclusionsThis study identifies new genetic associations with ALS and provides phenotype–genotype correlations with both previously reported and novel mutations.

The secreted leucine-rich glioma inactivated 1 (LGI1) protein is an important actor for human seizures of both genetic and autoimmune etiology: mutations in LGI1 cause inherited temporal lobe epilepsy, while LGI1 is involved in antibody-mediated encephalitis. Remarkably, Lgi1- deficient ( Lgi1 −/− ) mice recapitulate the epileptic disorder and display early-onset spontaneous seizures. To understand how Lgi1-deficiency leads to seizures during postnatal development, we here investigated the early functional and structural defects occurring before seizure onset in Lgi1 −/− mice. We found an increased excitatory synaptic transmission in hippocampal slices from Lgi1 −/− mice. No structural alteration in the morphology of pyramidal cell dendrites and synapses was observed at this stage, indicating that Lgi1-deficiency is unlikely to trigger early developmental abnormalities. Consistent with the presynaptic subcellular localization of the protein, Lgi1-deficiency caused presynaptic defects, with no alteration in postsynaptic AMPA receptor activity in Lgi1 −/− pyramidal cells before seizure onset. Presynaptic dysfunction led to increased synaptic glutamate levels, which were associated with hyperexcitable neuronal networks. Altogether, these data show that Lgi1 acts presynaptically as a negative modulator of excitatory synaptic transmission during early postnatal development. We therefore here reveal that increased presynaptic glutamate release is a key early event resulting from Lgi1-deficiency, which likely contributes to epileptogenesis.

Genetic generalized epilepsy (GGE) including childhood absence epilepsy, juvenile absence epilepsy, juvenile myoclonic epilepsy (JME), and GGE with tonic–clonic seizures (TCS) (GGE-TCS), is genetically influenced with a two- to four- fold increased risk in the first-degree relatives of patients. Since large families with GGE are very rare, international studies have focused on sporadic GGE patients using whole exome sequencing, suggesting that GGE are highly genetically heterogeneous and rather involve rare or ultra-rare variants. Moreover, a polygenic mode of inheritance is suspected in most cases. We performed SNP microarrays and whole exome sequencing in 20 families from Sudan, focusing on those with at least four affected members. Standard genetic filters and Endeavour algorithm for functional prioritization of genes selected likely susceptibility variants in FAT1, DCHS1 or ASTN2 genes. FAT1 and DCHS1 are adhesion transmembrane proteins interacting during brain development, while ASTN2 is involved in dendrite development. Our approach on familial forms of GGE is complementary to large-scale collaborative consortia studies of sporadic cases. Our study reinforces the hypothesis that GGE is genetically heterogeneous, even in a relatively limited geographic area, and mainly oligogenic, as supported by the low familial penetrance of GGE and by the Bayesian algorithm that we developed in a large pedigree with JME. Since populations with founder effect and endogamy are appropriate to study autosomal recessive pathologies, they would be also adapted to decipher genetic components of complex diseases, using the reported bayesian model. Graphical Abstract

Rare loss-of-function variants in ITSN1 were recently reported to confer a high risk for Parkinson’s disease (PD). From our local large exome sequencing dataset of PD cases, we identified five carriers from three families. Clinical features of ITSN1 -PD are typical and responsive to standard treatments. Additionally, we discuss whether ITSN1 loss-of-function variants should only be considered as a high-risk factor or a Mendelian PD gene.