Sixty cases of febrile seizures from a Chinese cohort had previously been reported with a strong association between variants in the seizure-related (SEZ) 6 gene and febrile seizures. They found a striking lack of genetic variation in their controls. We found genetic variation in SEZ6 at similar levels at the same DNA sequence positions in our 94 febrile seizure cases as in our 96 unaffected controls. Two of our febrile seizure cases carried rare variants predicted to have damaging consequences. Combined with some of the variants from the Chinese cohort, these data are compatible with a role for SEZ6 as a susceptibility gene for febrile seizures. However, the polygenic determinants underlying most cases of febrile seizures with complex inheritance remain to be determined.

To determine clinical phenotypes, evolution and genetic background of a large family with a combination of two unusual forms of reflex epilepsies.

We apply a novel gene expression network analysis to a cohort of 182 recently reported candidate Epileptic Encephalopathy genes to identify those most likely to be true Epileptic Encephalopathy genes. These candidate genes were identified as having single variants of likely pathogenic significance discovered in a large-scale massively parallel sequencing study. Candidate Epileptic Encephalopathy genes were prioritized according to their co-expression with 29 known Epileptic Encephalopathy genes. We utilized developing brain and adult brain gene expression data from the Allen Human Brain Atlas (AHBA) and compared this to data from Celsius: a large, heterogeneous gene expression data warehouse. We show replicable prioritization results using these three independent gene expression resources, two of which are brain-specific, with small sample size, and the third derived from a heterogeneous collection of tissues with large sample size. Of the nineteen genes that we predicted with the highest likelihood to be true Epileptic Encephalopathy genes, two (GNAO1 and GRIN2B) have recently been independently reported and confirmed. We compare our results to those produced by an established in silico prioritization approach called Endeavour, and finally present gene expression networks for the known and candidate Epileptic Encephalopathy genes. This highlights sub-networks of gene expression, particularly in the network derived from the adult AHBA gene expression dataset. These networks give clues to the likely biological interactions between Epileptic Encephalopathy genes, potentially highlighting underlying mechanisms and avenues for therapeutic targets.

Genetically tractable fruit flies have been used for decades to study seizure disorders. However, there is a paucity of data specifically correlating fly and human seizure phenotypes. We have previously shown that mutation of orthologous PRICKLE genes from flies to humans produce seizures. This study aimed to determine whether the prickle-mediated seizure phenotypes in flies closely parallel the epilepsy syndrome found in PRICKLE patients.

Receptor occupancy (RO) assays provide a means to measure the direct interaction of therapeutics with their cell surface targets. Free receptor assays quantify cell-surface receptors not bound by a therapeutic while total receptor assays quantify the amount of target on the cell surface.

Action myoclonus-renal failure syndrome (AMRF) is an autosomal-recessive disorder with the remarkable combination of focal glomerulosclerosis, frequently with glomerular collapse, and progressive myoclonus epilepsy associated with storage material in the brain. Here, we employed a novel combination of molecular strategies to find the responsible gene and show its effects in an animal model. Utilizing only three unrelated affected individuals and their relatives, we used homozygosity mapping with single-nucleotide polymorphism chips to localize AMRF. We then used microarray-expression analysis to prioritize candidates prior to sequencing. The disorder was mapped to 4q13-21, and microarray-expression analysis identified SCARB2/Limp2, which encodes a lysosomal-membrane protein, as the likely candidate. Mutations in SCARB2/Limp2 were found in all three families used for mapping and subsequently confirmed in two other unrelated AMRF families. The mutations were associated with lack of SCARB2 protein. Reanalysis of an existing Limp2 knockout mouse showed intracellular inclusions in cerebral and cerebellar cortex, and the kidneys showed subtle glomerular changes. This study highlights that recessive genes can be identified with a very small number of subjects. The ancestral lysosomal-membrane protein SCARB2/LIMP-2 is responsible for AMRF. The heterogeneous pathology in the kidney and brain suggests that SCARB2/Limp2 has pleiotropic effects that may be relevant to understanding the pathogenesis of other forms of glomerulosclerosis or collapse and myoclonic epilepsies.

Focal cortical dysplasia (FCD) causes drug-resistant epilepsy and is associated with pathogenic variants in mTOR pathway genes. How germline variants cause these focal lesions is unclear, however a germline + somatic "2-hit" model is hypothesized. In a boy with drug-resistant epilepsy, FCD, and a germline DEPDC5 pathogenic variant, we show that a second-hit DEPDC5 variant is limited to dysmorphic neurons, and the somatic mutation load correlates with both dysmorphic neuron density and the epileptogenic zone. These findings provide new insights into the molecular and cellular correlates of FCD determining drug-resistant epilepsy and refine conceptualization of the epileptogenic zone.

The CACNA1G gene encodes the low-voltage-activated Cav3.1 channel, which is expressed in various areas of the CNS, including the cerebellum. We studied two missense CACNA1G variants, p.L208P and p.L909F, and evaluated the relationships between the severity of Cav3.1 dysfunction and the clinical phenotype. The presentation was of a developmental and epileptic encephalopathy without evident cerebellar atrophy. Both patients exhibited axial hypotonia, developmental delay, and severe to profound cognitive impairment. The patient with the L909F mutation had initially refractory seizures and cerebellar ataxia, whereas the L208P patient had seizures only transiently but was overall more severely affected. In transfected mammalian cells, we determined the biophysical characteristics of L208P and L909F variants, relative to the wild-type channel and a previously reported gain-of-function Cav3.1 variant. The L208P mutation shifted the activation and inactivation curves to the hyperpolarized direction, slowed the kinetics of inactivation and deactivation, and reduced the availability of Ca2+ current during repetitive stimuli. The L909F mutation impacted channel function less severely, resulting in a hyperpolarizing shift of the activation curve and slower deactivation. These data suggest that L909F results in gain-of-function, whereas L208P exhibits mixed gain-of-function and loss-of-function effects due to opposing changes in the biophysical properties. Our study expands the clinical spectrum associated with CACNA1G mutations, corroborating further the causal association with distinct complex phenotypes.

Trio exome sequencing has been successful in identifying genes with de novo mutations (DNMs) causing epileptic encephalopathy (EE) and other neurodevelopmental disorders. Here, we evaluate how well a case-control collapsing analysis recovers genes causing dominant forms of EE originally implicated by DNM analysis. We performed a genome-wide search for an enrichment of "qualifying variants" in protein-coding genes in 488 unrelated cases compared to 12,151 unrelated controls. These "qualifying variants" were selected to be extremely rare variants predicted to functionally impact the protein to enrich for likely pathogenic variants. Despite modest sample size, three known EE genes (KCNT1, SCN2A, and STXBP1) achieved genome-wide significance (p<2.68×10-6). In addition, six of the 10 most significantly associated genes are known EE genes, and the majority of the known EE genes (17 out of 25) originally implicated in trio sequencing are nominally significant (p<0.05), a proportion significantly higher than the expected (Fisher's exact p = 2.33×10-17). Our results indicate that a case-control collapsing analysis can identify several of the EE genes originally implicated in trio sequencing studies, and clearly show that additional genes would be implicated with larger sample sizes. The case-control analysis not only makes discovery easier and more economical in early onset disorders, particularly when large cohorts are available, but also supports the use of this approach to identify genes in diseases that present later in life when parents are not readily available.

Brain glucose transport is dependent on glucose transporter 1 (GLUT1), encoded by the solute carrier family 2 member 1 (SLC2A1) gene. Mutations in SLC2A1 cause GLUT1 deficiency which is characterized by a broad spectrum of neurological phenotypes including generalized epilepsy, motor disorders, developmental delay and microcephaly. Recent case reports suggest SLC2A1 mutations can contribute to non-acquired focal epilepsy (NAFE) but interrogation of a large patient cohort has not been reported. We studied 200 patients with NAFE (126 with temporal lobe epilepsy) comprising 104 females and 96 males with a mean age of onset of 18 years. Polymerase chain reaction (PCR) and Sanger sequencing was performed to detect variants in all 10 coding exons and splice site regions of the SLC2A1 gene. We did not detect any pathogenic mutations in SLC2A1 in this cohort. Our data suggests that the frequency of GLUT1 mutations in NAFE is low. Limitations of this study include the mean age of onset and cohort size. Future research should focus on subpopulations of focal epilepsy with lower age of seizure onset particularly with co-existent movement disorders in which GLUT1 mutations may play a more important role.

Animal models of COVID-19 have been rapidly reported after the start of the pandemic. We aimed to assess whether the newly created models reproduce the full spectrum of human COVID-19.

To compare the frequency and impact on the channel function of KCNH2 variants in SUDEP patients with epilepsy controls comprising patients older than 50 years, a group with low SUDEP risk, and establish loss-of-function KCNH2 variants as predictive biomarkers of SUDEP risk.

Maffucci syndrome is a rare, highly variable somatic mosaic condition, and well-known cancer-related gain-of-function variants in either the IDH1 or IDH2 genes have been found in the affected tissues of most reported individuals. Features include benign enchondroma and spindle-cell hemangioma, with a recognized increased risk of various malignancies. Fewer than 200 affected individuals have been reported; therefore, accurate estimates of malignancy risk are difficult to quantify and recommended surveillance guidelines are not available. The same gain-of-function IDH1 and IDH2 variants are also implicated in a variety of other benign and malignant tumors. An adult male presented with several soft palpable lesions on the right upper limb. Imaging and histopathology raised the possibility of Maffucci syndrome. DNA was extracted from peripheral blood lymphocytes and tissue surgically resected from a spindle-cell hemangioma. Sanger sequencing and droplet digital polymerase chain reaction (PCR) analysis of the IDH1 gene were performed. We identified a somatic mosaic c.394C > T (p.R132C) variant in exon 5 of IDH1, in DNA derived from hemangioma tissue at ∼17% variant allele fraction. This variant was absent in DNA derived from blood. This variant has been identified in the affected tissue of most reported individuals with Maffucci syndrome. Although this individual has a potentially targetable variant, and there is a recognized risk of malignant transformation in this condition, a decision was made not to intervene with an IDH1 inhibitor. The reasons and prospects for therapy in this condition are discussed.

The vacuolar H+-ATPase is a large multi-subunit proton pump, composed of an integral membrane V0 domain, involved in proton translocation, and a peripheral V1 domain, catalysing ATP hydrolysis. This complex is widely distributed on the membrane of various subcellular organelles, such as endosomes and lysosomes, and plays a critical role in cellular processes ranging from autophagy to protein trafficking and endocytosis. Variants in ATP6V0A1, the brain-enriched isoform in the V0 domain, have been recently associated with developmental delay and epilepsy in four individuals. Here, we identified 17 individuals from 14 unrelated families with both with new and previously characterized variants in this gene, representing the largest cohort to date. Five affected subjects with biallelic variants in this gene presented with a phenotype of early-onset progressive myoclonus epilepsy with ataxia, while 12 individuals carried de novo missense variants and showed severe developmental and epileptic encephalopathy. The R740Q mutation, which alone accounts for almost 50% of the mutations identified among our cases, leads to failure of lysosomal hydrolysis by directly impairing acidification of the endolysosomal compartment, causing autophagic dysfunction and severe developmental defect in Caenorhabditis elegans. Altogether, our findings further expand the neurological phenotype associated with variants in this gene and provide a direct link with endolysosomal acidification in the pathophysiology of ATP6V0A1-related conditions.

Levetiracetam (LEV) is an effective antiseizure medicine, but 10%-20% of people treated with LEV report psychiatric side-effects, and up to 1% may have psychotic episodes. Pharmacogenomic predictors of these adverse drug reactions (ADRs) have yet to be identified. We sought to determine the contribution of both common and rare genetic variation to psychiatric and behavioral ADRs associated with LEV.

The objectives of this study were to define the clinical and biochemical spectrum of spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME) and to determine if aberrant cellular ceramide accumulation could be normalized by enzyme replacement.

To describe clinical and EEG phenotypes of a family with an unusual familial epilepsy syndrome characterized by myoclonus and dystonia.

Febrile seizures (FS) are the most common seizure syndrome and are potentially a prelude to more severe epilepsy. Although zinc (Zn(2+)) metabolism has previously been implicated in FS, whether or not variation in proteins essential for Zn(2+) homeostasis contributes to susceptibility is unknown. Synaptic Zn(2+) is co-released with glutamate and modulates neuronal excitability. SLC30A3 encodes the zinc transporter 3 (ZNT3), which is primarily responsible for moving Zn(2+) into synaptic vesicles. Here we sequenced SLC30A3 and discovered a rare variant (c.892C > T; p.R298C) enriched in FS populations but absent in population-matched controls. Functional analysis revealed a significant loss-of-function of the mutated protein resulting from a trafficking deficit. Furthermore, mice null for ZnT3 were more sensitive than wild-type to hyperthermia-induced seizures that model FS. Together our data suggest that reduced synaptic Zn(2+) increases the risk of FS and more broadly support the idea that impaired synaptic Zn(2+) homeostasis can contribute to neuronal hyperexcitability.

Mutations of the intrinsic lysosomal membrane protein SCARB2 cause action myoclonus-renal failure syndrome (AMRF syndrome), a rare disease characterized by renal and neurological manifestations. In this study, examination of Cos7 cells transfected with SCARB2 cDNA derived from two patients with AMRF syndrome showed that the resultant protein was truncated and was not incorporated into vesicular structures, as occurred with full-length SCARB2 cDNA. Mutant SCARB2 protein failed to colocalize with lysosomes and was found in the endoplasmic reticulum or the cytosol indicating a loss of function. Cultured skin fibroblast and Epstein-Barr virus-transformed lymphoblastoid B cell lines (LCLs) were created from these two patients. Despite the loss of SCARB2 function, studies with lysosomal-associated membrane protein (LAMP) 1 and LAMP2 demonstrated normal lysosomal numbers in fibroblasts and LCLs. Immunofluorescence microscopy using anti-LAMP1 and anti-LAMP2 antibodies also showed normal lysosomal structures in fibroblasts. There was no change in the morphology of fibroblasts examined by electron microscopy compared with cells from unaffected individuals. By contrast, LCLs from individuals bearing SCARB2 mutations had large intracellular vesicles that resembled autophagosomes and contained heterogeneous cellular debris. Some of the autophagosomes were seen to be extruding cellular contents into the media. Furthermore, LCLs had elevated levels of microtubule-associated protein light chain 3-II, consistent with increased autophagy. These data demonstrate that SCARB2 mutations are associated with an inability to process autophagosomes in B lymphocytes, suggesting a novel function for SCARB2 in immune function.

Epileptic encephalopathies are a devastating group of epilepsies with poor prognosis, of which the majority are of unknown etiology. We perform targeted massively parallel resequencing of 19 known and 46 candidate genes for epileptic encephalopathy in 500 affected individuals (cases) to identify new genes involved and to investigate the phenotypic spectrum associated with mutations in known genes. Overall, we identified pathogenic mutations in 10% of our cohort. Six of the 46 candidate genes had 1 or more pathogenic variants, collectively accounting for 3% of our cohort. We show that de novo CHD2 and SYNGAP1 mutations are new causes of epileptic encephalopathies, accounting for 1.2% and 1% of cases, respectively. We also expand the phenotypic spectra explained by SCN1A, SCN2A and SCN8A mutations. To our knowledge, this is the largest cohort of cases with epileptic encephalopathies to undergo targeted resequencing. Implementation of this rapid and efficient method will change diagnosis and understanding of the molecular etiologies of these disorders.