KCNT1- related epilepsy: An international multicenter cohort of 27 pediatric cases

ObjectiveThrough international collaboration, we evaluated the phenotypic aspects of a multiethnic cohort of KCNT1- related epilepsy and explored genotype- phenotype correlations associated with frequently encountered variants.MethodsA cross- sectional analysis of children harboring pathogenic or likely pathogenic KCNT1 variants was completed. Children with one of the two more common recurrent KCNT1 variants were compared with the rest of the cohort for the presence of particular characteristics.ResultsTwenty- seven children (15 males, mean age = 40.8 months) were included. Seizure onset ranged from 1 day to 6 months, and half (48.1%) exhibited developmental plateauing upon onset. Two- thirds had epilepsy of infancy with migrating focal seizures (EIMFS), and focal tonic seizures were common (48.1%). The most frequent recurrent KCNT1 variants were c.2800G>A; p.Ala934Thr (n = 5) and c.862G>A; p.Gly288Ser (n = 4). De novo variants were found in 96% of tested parents (23/24). Sixty percent had abnormal magnetic resonance imaging (MRI) findings. Delayed myelination, thin corpus callosum, and brain atrophy were the most common. One child had gray- white matter interface indistinctness, suggesting a malformation of cortical development. Several antiepileptic drugs (mean = 7.4/patient) were tried, with no consistent response to any one agent. Eleven tried quinidine; 45% had marked (>50% seizure reduction) or some improvement (25%- 50% seizure reduction). Seven used cannabidiol; 71% experienced marked or some improvement. Fourteen tried diet therapies; 57% had marked or some improvement. When comparing the recurrent variants to the rest of the cohort with respect to developmental trajectory, presence of EIMFS, >500 seizures/mo, abnormal MRI, and treatment response, there were no statistically significant differences. Four patients died (15%), none of sudden unexpected death in epilepsy.SignificanceOur cohort reinforces common aspects of this highly pleiotropic entity. EIMFS manifesting with refractory tonic seizures was the most common. Cannabidiol, diet therapy, and quinidine seem to offer the best chances of seizure reduction, although evidence- based practice is still unavailable.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154940/1/epi16480_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154940/2/epi16480.pd

Novel homozygous mutation in the WWOX gene causes seizures and global developmental delay: Report and review

The WWOX gene has a WW domain containing oxidoreductase, which is located at the common fragile site FRA16D at chromosome 16q23. WWOX is a tumor suppressor gene that has been associated with several types of cancer such as hepatic, breast, lung, prostate, gastric, and ovarian. Recently WWOX has been implicated in epilepsy, where studies show homozygous loss-of-function mutation lead to early-infantile epileptic encephalopathy, spinocerebellar ataxia, intractable seizures and developmental delay, and early lethal microcephaly syndrome with epilepsy. Here we investigate two consanguineous Saudi families and we identified three probands with epileptic encephalopathy. Whole exome sequencing revealed a novel homozygous mutation in the WWOX gene in one proband. In addition, we identified a previously reported WWOX mutation in two probands. Later on these findings were confirmed with Sanger sequencing. The underlying mechanism on how WWOX mutations lead to seizure remains elusive. To date very few WWOX mutations have been associated with neurological disorder and our newly identified mutations support the notion that WWOX play an important role in neurons and will aid in better diagnosis and genetic counseling

Seizures Are Regulated by Ubiquitin-specific Peptidase 9 X-linked (USP9X), a De-Ubiquitinase

<div><p>Epilepsy is a common disabling disease with complex, multifactorial genetic and environmental etiology. The small fraction of epilepsies subject to Mendelian inheritance offers key insight into epilepsy disease mechanisms; and pathologies brought on by mutations in a single gene can point the way to generalizable therapeutic strategies. Mutations in the PRICKLE genes can cause seizures in humans, zebrafish, mice, and flies, suggesting the seizure-suppression pathway is evolutionarily conserved. This pathway has never been targeted for novel anti-seizure treatments. Here, the mammalian PRICKLE-interactome was defined, identifying prickle-interacting proteins that localize to synapses and a novel interacting partner, USP9X, a substrate-specific de-ubiquitinase. PRICKLE and USP9X interact through their carboxy-termini; and USP9X de-ubiquitinates PRICKLE, protecting it from proteasomal degradation. In forebrain neurons of mice, USP9X deficiency reduced levels of Prickle2 protein. Genetic analysis suggests the same pathway regulates Prickle-mediated seizures. The seizure phenotype was suppressed in <i>prickle</i> mutant flies by the small-molecule USP9X inhibitor, Degrasyn/WP1130, or by reducing the dose of <i>fat facets</i> a <i>USP9X</i> orthologue. <i>USP9X</i> mutations were identified by resequencing a cohort of patients with epileptic encephalopathy, one patient harbored a <i>de novo</i> missense mutation and another a novel coding mutation. Both <i>USP9X</i> variants were outside the PRICKLE-interacting domain. These findings demonstrate that USP9X inhibition can suppress <i>prickle</i>-mediated seizure activity, and that <i>USP9X</i> variants may predispose to seizures. These studies point to a new target for anti-seizure therapy and illustrate the translational power of studying diseases in species across the evolutionary spectrum.</p></div

USP9X stabilizes PRICKLE in HEK293T cells and the mouse brain.

<p> <b>A</b>. Flag-immunoprecipitates from HEK293T cells overexpressing the indicated constructs show PRICKLE and USP9X interact via their carboxyl termini. <b>B, C.</b> USP9X deubiquitinates PRICKLE. Immunoprecipitates from HEK293T cells overexpressing the indicated constructs show PRICKLE ubiquitination in the presence of HA-Ubiquitin and deubiquitination/stabilization by Usp9x. IP (immunoprecipitates), IN (input). <b>D.</b> Loss of Usp9X affects Prickle2 (green) expression. Deletion of Usp9x results in decreased Prickle2 expression in the cortex (I), CA1 (II) and dentate gyrus (III) of 4-week old mice when compared to controls (IV, V, VI). n = 2. Scale bar:20μM. Nuclear stain = DAPI.</p

Genetic and pharmacological suppression of the seizure phenotype in prickle mutant flies.

<p>(<b>A–C</b>) Reducing faf dosage with three separate loss-of-function alleles ((faf^B3, faf^BX3, faf^BX4; graphs A, B and C, respectively) suppresses the pk^sple-mediated seizure phenotype detected by the modified bang sensitivity assay. Six vials per genotype were assayed. <b>D</b>. Inhibition of faf activity with Degrasyn suppresses pk^sple-mediated seizure activity detected using the “fly flip” assay. Three vials per genotype were assayed. Note: +/+ = Oregon-R control flies; sple = pk^sple; sple/faf = pk^sple/+; +/faf. * = p < 0.05. Error bars = standard error. Each vial contains 5 males and 5 females.</p

KCNT1‐related epilepsy: An international multicenter cohort of 27 pediatric cases

ObjectiveThrough international collaboration, we evaluated the phenotypic aspects of a multiethnic cohort of KCNT1- related epilepsy and explored genotype- phenotype correlations associated with frequently encountered variants.MethodsA cross- sectional analysis of children harboring pathogenic or likely pathogenic KCNT1 variants was completed. Children with one of the two more common recurrent KCNT1 variants were compared with the rest of the cohort for the presence of particular characteristics.ResultsTwenty- seven children (15 males, mean age = 40.8 months) were included. Seizure onset ranged from 1 day to 6 months, and half (48.1%) exhibited developmental plateauing upon onset. Two- thirds had epilepsy of infancy with migrating focal seizures (EIMFS), and focal tonic seizures were common (48.1%). The most frequent recurrent KCNT1 variants were c.2800G>A; p.Ala934Thr (n = 5) and c.862G>A; p.Gly288Ser (n = 4). De novo variants were found in 96% of tested parents (23/24). Sixty percent had abnormal magnetic resonance imaging (MRI) findings. Delayed myelination, thin corpus callosum, and brain atrophy were the most common. One child had gray- white matter interface indistinctness, suggesting a malformation of cortical development. Several antiepileptic drugs (mean = 7.4/patient) were tried, with no consistent response to any one agent. Eleven tried quinidine; 45% had marked (>50% seizure reduction) or some improvement (25%- 50% seizure reduction). Seven used cannabidiol; 71% experienced marked or some improvement. Fourteen tried diet therapies; 57% had marked or some improvement. When comparing the recurrent variants to the rest of the cohort with respect to developmental trajectory, presence of EIMFS, >500 seizures/mo, abnormal MRI, and treatment response, there were no statistically significant differences. Four patients died (15%), none of sudden unexpected death in epilepsy.SignificanceOur cohort reinforces common aspects of this highly pleiotropic entity. EIMFS manifesting with refractory tonic seizures was the most common. Cannabidiol, diet therapy, and quinidine seem to offer the best chances of seizure reduction, although evidence- based practice is still unavailable.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154940/1/epi16480_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154940/2/epi16480.pd

Prickle interactome.

<p>We combined findings from our proteomics interaction experiment and public databases to generate a prickle interactome. We used the MetaCore (MetaCore, GeneGO Inc., St. Joseph, MI, USA) networking function and String database 9.1 to curate interaction maps of the proteins identified. Information for identified interactions is obtained from several sources including but not limited to genomic context, database imports (PPI and pathway databases), high-throughput experiments, co-expression, and text mining. We uploaded our lists of proteins from LC-MS/MS into the software programs and exported the networks into Cytoscape 2.7.0 for manipulation of the network appearance. (Nodes, circles; Edges, lines). Red lines correspond to interactions observed by our labs using yeast-2-hybrid and IP-MS approaches. The extended interactome was generated as we have previously described. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005022#pgen.1005022.ref001" target="_blank">1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005022#pgen.1005022.ref006" target="_blank">6</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005022#pgen.1005022.ref009" target="_blank">9</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005022#pgen.1005022.ref018" target="_blank">18</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005022#pgen.1005022.ref019" target="_blank">19</a>] Prickle1 and Prickle2 interact with known synaptic proteins. The interaction with USP9X is novel.</p

PRICKLE interacts with USP9X via its carboxyl terminal.

<p> <b>A.</b> PRICKLE1 and PRICKLE2 interact with endogenous Bcr, Tanc2, and Usp9x in NGF-differentiated PC12 cells. GFP immunoprecipitates from stable lines expressing GFP, GFP-PRICKLE1 or GFP-PRICKLE2 confirm that PRICKLE interacts with Bcr, Usp9x and Tanc2. <b>B</b>. Flag immunoprecipitates, from HEK293T cells overexpressing flag-tagged PRICKLE1 or PRICKLE2, show endogenous USP9X physically interacts with PRICKLE. <b>C, D.</b> Schematic of PRICKLE1(C) and PRICKLE2(D) constructs. Flag-immunoprecipitates from HEK293T cells overexpressing the indicated constructs were analyzed by anti-USP9X Western blotting. Both PRICKLE 1 and 2 interact with USP9X via their C-termini while BCR binding mapped to their N-termini.</p

PRICKLE1 Interaction with SYNAPSIN I Reveals a Role in Autism Spectrum Disorders

<div><p>The frequent comorbidity of Autism Spectrum Disorders (ASDs) with epilepsy suggests a shared underlying genetic susceptibility; several genes, when mutated, can contribute to both disorders. Recently, <i>PRICKLE1</i> missense mutations were found to segregate with ASD. However, the mechanism by which mutations in this gene might contribute to ASD is unknown. To elucidate the role of PRICKLE1 in ASDs, we carried out studies in <i>Prickle1^+/−</i> mice and <i>Drosophila</i>, yeast, and neuronal cell lines. We show that mice with <i>Prickle1</i> mutations exhibit ASD-like behaviors. To find proteins that interact with PRICKLE1 in the central nervous system, we performed a yeast two-hybrid screen with a human brain cDNA library and isolated a peptide with homology to SYNAPSIN I (SYN1), a protein involved in synaptogenesis, synaptic vesicle formation, and regulation of neurotransmitter release. Endogenous Prickle1 and Syn1 co-localize in neurons and physically interact via the <i>SYN1</i> region mutated in ASD and epilepsy. Finally, a mutation in <i>PRICKLE1</i> disrupts its ability to increase the size of dense-core vesicles in PC12 cells. Taken together, these findings suggest <i>PRICKLE1</i> mutations contribute to ASD by disrupting the interaction with SYN1 and regulation of synaptic vesicles.</p></div