Aneuploidy is Linked to Neurological Phenotypes Through Oxidative Stress.

Aneuploidy, having an aberrant genome, is gaining increasing attention in neurodegenerative diseases. It gives rise to proteotoxic stress as well as a stereotypical oxidative shift which makes these cells sensitive to internal and environmental stresses. A growing body of research from numerous laboratories suggests that many neurodegenerative disorders, especially Alzheimer's disease and frontotemporal dementia, are characterised by neuronal aneuploidy and the ensuing apoptosis, which may contribute to neuronal loss. Using Drosophila as a model, we investigated the effect of induced aneuploidy in GABAergic neurons. We found an increased proportion of aneuploidy due to Mad2 depletion in the third-instar larval brain and increased cell death. Depletion of Mad2 in GABAergic neurons also gave a defective climbing and seizure phenotype. Feeding animals an antioxidant rescued the climbing and seizure phenotype. These findings suggest that increased aneuploidy leads to higher oxidative stress in GABAergic neurons which causes cell death, climbing defects, and seizure phenotype. Antioxidant feeding represents a potential therapy to reduce the aneuploidy-driven neurological phenotype.

Drosophila expressing mutant human KCNT1 transgenes make an effective tool for targeted drug screening in a whole animal model of KCNT1-epilepsy.

Mutations in the KCNT1 potassium channel cause severe forms of epilepsy which are poorly controlled with current treatments. In vitro studies have shown that KCNT1-epilepsy mutations are gain of function, significantly increasing K+ current amplitudes. To investigate if Drosophila can be used to model human KCNT1 epilepsy, we generated Drosophila melanogaster lines carrying human KCNT1 with the patient mutation G288S, R398Q or R928C. Expression of each mutant channel in GABAergic neurons gave a seizure phenotype which responded either positively or negatively to 5 frontline epilepsy drugs most commonly administered to patients with KCNT1-epilepsy, often with little or no improvement of seizures. Cannabidiol showed the greatest reduction of the seizure phenotype while some drugs increased the seizure phenotype. Our study shows that Drosophila has the potential to model human KCNT1- epilepsy and can be used as a tool to assess new treatments for KCNT1- epilepsy.

Chromosomal Instability Causes Sensitivity to Polyamines and One-Carbon Metabolism.

Aneuploidy, or having a disrupted genome, is an aberration commonly found in tumours but rare in normal tissues. It gives rise to proteotoxic stress as well as a stereotypical oxidative shift, which makes these cells sensitive to internal and environmental stresses. Using Drosophila as a model, we investigated the changes in transcription in response to ongoing changes to ploidy (chromosomal instability, CIN). We noticed changes in genes affecting one-carbon metabolism, specifically those affecting the production and use of s-adenosyl methionine (SAM). The depletion of several of these genes has led to cell death by apoptosis in CIN cells but not in normal proliferating cells. We found that CIN cells are particularly sensitive to SAM metabolism at least partly because of its role in generating polyamines. Feeding animals spermine was seen to rescue the cell death caused by the loss of SAM synthase in CIN tissues. The loss of polyamines led to decreased rates of autophagy and sensitivity to reactive oxygen species (ROS), which we have shown to contribute significantly to cell death in CIN cells. These findings suggest that a well-tolerated metabolic intervention such as polyamine inhibition has the potential to target CIN tumours via a relatively well-characterised mechanism.

Investigating genetic variants in microRNA regulators of Neurokinin-1 receptor in sudden infant death syndrome.

Sudden infant death syndrome (SIDS) occurs more often in male than in female infants, suggesting involvement of the X-chromosome. Histopathological studies have suggested that altered expression of the Neurokinin-1 receptor may also play a role in the pathogenesis of SIDS. It was hypothesised that genetic variants in three X-chromosome-encoded microRNA (miRNA/miR), known to down-regulate expression of the Neurokinin-1 receptor, may contribute to SIDS. AIM: To identify sequence variants in the miRNAs within a study cohort (27 cases of SIDS and 28 controls) and determine if there was a difference in the frequencies in male and female SIDS infants. METHODS: Genomic DNA prepared from stored blood spots was amplified and sequenced to identify genetic variants in miR500A, miR500B and miR320D2. RESULTS: No novel variants in the miRNAs were identified in our study cohort. We identified one known single-nucleotide polymorphism (SNP) in miR320D2: rs5907732 G/T, in both cases and controls. No significant difference in the SNP frequency was observed between male and female SIDS cases. CONCLUSION: This pilot study suggests that sequence variants in three miRNAs do not contribute to the reported higher prevalence of SIDS in male infants and do not contribute to the pathogenesis of SIDS in our cohort.

Functional Effects of Epilepsy Associated KCNT1 Mutations Suggest Pathogenesis via Aberrant Inhibitory Neuronal Activity.

KCNT1 (K+ channel subfamily T member 1) is a sodium-activated potassium channel highly expressed in the nervous system which regulates neuronal excitability by contributing to the resting membrane potential and hyperpolarisation following a train of action potentials. Gain of function mutations in the KCNT1 gene are the cause of neurological disorders associated with different forms of epilepsy. To gain insights into the underlying pathobiology we investigated the functional effects of 9 recently published KCNT1 mutations, 4 previously studied KCNT1 mutations, and one previously unpublished KCNT1 variant of unknown significance. We analysed the properties of KCNT1 potassium currents and attempted to find a correlation between the changes in KCNT1 characteristics due to the mutations and severity of the neurological disorder they cause. KCNT1 mutations identified in patients with epilepsy were introduced into the full length human KCNT1 cDNA using quick-change site-directed mutagenesis protocol. Electrophysiological properties of different KCNT1 constructs were investigated using a heterologous expression system (HEK293T cells) and patch clamping. All mutations studied, except T314A, increased the amplitude of KCNT1 currents, and some mutations shifted the voltage dependence of KCNT1 open probability, increasing the proportion of channels open at the resting membrane potential. The T314A mutation did not affect KCNT1 current amplitude but abolished its voltage dependence. We observed a positive correlation between the severity of the neurological disorder and the KCNT1 channel open probability at resting membrane potential. This suggests that gain of function KCNT1 mutations cause epilepsy by increasing resting potassium conductance and suppressing the activity of inhibitory neurons. A reduction in action potential firing in inhibitory neurons due to excessively high resting potassium conductance leads to disinhibition of neural circuits, hyperexcitability and seizures.

KCNT1-related epilepsies and epileptic encephalopathies: phenotypic and mutational spectrum.

Variants in KCNT1, encoding a sodium-gated potassium channel (subfamily T member 1), have been associated with a spectrum of epilepsies and neurodevelopmental disorders. These range from familial autosomal dominant or sporadic sleep-related hypermotor epilepsy to epilepsy of infancy with migrating focal seizures (EIMFS) and include developmental and epileptic encephalopathies. This study aims to provide a comprehensive overview of the phenotypic and genotypic spectrum of KCNT1 mutation-related epileptic disorders in 248 individuals, including 66 previously unpublished and 182 published cases, the largest cohort reported so far. Four phenotypic groups emerged from our analysis: (i) EIMFS (152 individuals, 33 previously unpublished); (ii) developmental and epileptic encephalopathies other than EIMFS (non-EIMFS developmental and epileptic encephalopathies) (37 individuals, 17 unpublished); (iii) autosomal dominant or sporadic sleep-related hypermotor epilepsy (53 patients, 14 unpublished); and (iv) other phenotypes (six individuals, two unpublished). In our cohort of 66 new cases, the most common phenotypic features were: (i) in EIMFS, heterogeneity of seizure types, including epileptic spasms, epilepsy improvement over time, no epilepsy-related deaths; (ii) in non-EIMFS developmental and epileptic encephalopathies, possible onset with West syndrome, occurrence of atypical absences, possible evolution to developmental and epileptic encephalopathies with sleep-related hypermotor epilepsy features; one case of sudden unexplained death in epilepsy; (iii) in autosomal dominant or sporadic sleep-related hypermotor epilepsy, we observed a high prevalence of drug-resistance, although seizure frequency improved with age in some individuals, appearance of cognitive regression after seizure onset in all patients, no reported severe psychiatric disorders, although behavioural/psychiatric comorbidities were reported in ∼50% of the patients, sudden unexplained death in epilepsy in one individual; and (iv) other phenotypes in individuals with mutation of KCNT1 included temporal lobe epilepsy, and epilepsy with tonic-clonic seizures and cognitive regression. Genotypic analysis of the whole cohort of 248 individuals showed only missense mutations and one inframe deletion in KCNT1. Although the KCNT1 mutations in affected individuals were seen to be distributed among the different domains of the KCNT1 protein, genotype-phenotype considerations showed many of the autosomal dominant or sporadic sleep-related hypermotor epilepsy-associated mutations to be clustered around the RCK2 domain in the C terminus, distal to the NADP domain. Mutations associated with EIMFS/non-EIMFS developmental and epileptic encephalopathies did not show a particular pattern of distribution in the KCNT1 protein. Recurrent KCNT1 mutations were seen to be associated with both severe and less severe phenotypes. Our study further defines and broadens the phenotypic and genotypic spectrums of KCNT1-related epileptic conditions and emphasizes the increasingly important role of this gene in the pathogenesis of early onset developmental and epileptic encephalopathies as well as of focal epilepsies, namely autosomal dominant or sporadic sleep-related hypermotor epilepsy.

Progressive myoclonus epilepsies-Residual unsolved cases have marked genetic heterogeneity including dolichol-dependent protein glycosylation pathway genes.

Progressive myoclonus epilepsies (PMEs) comprise a group of clinically and genetically heterogeneous rare diseases. Over 70% of PME cases can now be molecularly solved. Known PME genes encode a variety of proteins, many involved in lysosomal and endosomal function. We performed whole-exome sequencing (WES) in 84 (78 unrelated) unsolved PME-affected individuals, with or without additional family members, to discover novel causes. We identified likely disease-causing variants in 24 out of 78 (31%) unrelated individuals, despite previous genetic analyses. The diagnostic yield was significantly higher for individuals studied as trios or families (14/28) versus singletons (10/50) (OR = 3.9, p value = 0.01, Fisher's exact test). The 24 likely solved cases of PME involved 18 genes. First, we found and functionally validated five heterozygous variants in NUS1 and DHDDS and a homozygous variant in ALG10, with no previous disease associations. All three genes are involved in dolichol-dependent protein glycosylation, a pathway not previously implicated in PME. Second, we independently validate SEMA6B as a dominant PME gene in two unrelated individuals. Third, in five families, we identified variants in established PME genes; three with intronic or copy-number changes (CLN6, GBA, NEU1) and two very rare causes (ASAH1, CERS1). Fourth, we found a group of genes usually associated with developmental and epileptic encephalopathies, but here, remarkably, presenting as PME, with or without prior developmental delay. Our systematic analysis of these cases suggests that the small residuum of unsolved cases will most likely be a collection of very rare, genetically heterogeneous etiologies.

Mild malformations of cortical development in sleep-related hypermotor epilepsy due to KCNT1 mutations.

Mutations in the sodium-activated potassium channel gene KCNT1 have been associated with nonlesional sleep-related hypermotor epilepsy (SHE). We report the co-occurrence of mild malformation of cortical development (mMCD) and KCNT1 mutations in four patients with SHE. Focal cortical dysplasia type I was neuropathologically diagnosed after epilepsy surgery in three unrelated MRI-negative patients, periventricular nodular heterotopia was detected in one patient by MRI. Our findings suggest that KCNT1 epileptogenicity may result not only from dysregulated excitability by controlling Na+K+ transport, but also from mMCD. Therefore, pathogenic variants in KCNT1 may encompass both lesional and nonlesional epilepsies.

Knockout of the epilepsy gene Depdc5 in mice causes severe embryonic dysmorphology with hyperactivity of mTORC1 signalling.

DEPDC5 mutations have recently been shown to cause epilepsy in humans. Evidence from in vitro studies has implicated DEPDC5 as a negative regulator of mTORC1 during amino acid insufficiency as part of the GATOR1 complex. To investigate the role of DEPDC5 in vivo we generated a null mouse model using targeted CRISPR mutagenesis. Depdc5 homozygotes display severe phenotypic defects between 12.5-15.5 dpc, including hypotrophy, anaemia, oedema, and cranial dysmorphology as well as blood and lymphatic vascular defects. mTORC1 hyperactivity was observed in the brain of knockout embryos and in fibroblasts and neurospheres isolated from knockout embryos and cultured in nutrient deprived conditions. Heterozygous mice appeared to be normal and we found no evidence of increased susceptibility to seizures or tumorigenesis. Together, these data support mTORC1 hyperactivation as the likely pathogenic mechanism that underpins DEPDC5 loss of function in humans and highlights the potential utility of mTORC1 inhibitors in the treatment of DEPDC5-associated epilepsy.

Genetic epilepsy with febrile seizures plus: Refining the spectrum.

OBJECTIVE: Following our original description of generalized epilepsy with febrile seizures plus (GEFS+) in 1997, we analyze the phenotypic spectrum in 409 affected individuals in 60 families (31 new families) and expand the GEFS+ spectrum. METHODS: We performed detailed electroclinical phenotyping on all available affected family members. Genetic analysis of known GEFS+ genes was carried out where possible. We compared our phenotypic and genetic data to those published in the literature over the last 19 years. RESULTS: We identified new phenotypes within the GEFS+ spectrum: focal seizures without preceding febrile seizures (16/409 [4%]), classic genetic generalized epilepsies (22/409 [5%]), and afebrile generalized tonic-clonic seizures (9/409 [2%]). Febrile seizures remains the most frequent phenotype in GEFS+ (178/409 [44%]), followed by febrile seizures plus (111/409 [27%]). One third (50/163 [31%]) of GEFS+ families tested have a pathogenic variant in a known GEFS+ gene. CONCLUSION: As 37/409 (9%) affected individuals have focal epilepsies, we suggest that GEFS+ be renamed genetic epilepsy with febrile seizures plus rather than generalized epilepsy with febrile seizures plus. The phenotypic overlap between GEFS+ and the classic generalized epilepsies is considerably greater than first thought. The clinical and molecular data suggest that the 2 major groups of generalized epilepsies share genetic determinants.

GOSR2: a progressive myoclonus epilepsy gene.

GOSR2-associated PME is associated with a homozygous mutation in GOSR2 (c.430G>T, p.Gly144Trp), a Golgi vesicle transport gene. The functional effect of this mutation is a loss of function that results in failure of the GOSR2 protein to localize to the cis-Golgi. The main clinical features of the GOSR2-associated PME are early-onset ataxia, areflexia, action myoclonus and seizures, scoliosis, elevated creatine kinase levels, relative preservation of cognitive function until the late stages of the disease, and relentless disease course. Severe photosensitive myoclonus is a common feature. GOSR2-associated PME is a rare disease with very few cases reported so far and it can be expected that the identification of further patients will contribute to expanding the phenotype and genotype of this condition.

SCARB2/LIMP2 deficiency in action myoclonus-renal failure syndrome.

Action myoclonus-renal failure syndrome (AMRF) is an autosomal recessive progressive myoclonus epilepsy (PME) associated with renal dysfunction that appears in the second or third decade of life and that is caused by loss-of-function mutations in the SCARB2 gene encoding lysosomal integral membrane protein type 2 (LIMP2). Recent reports have documented cases with PME associated with SCARB2 mutations without renal compromise. Additional neurological features can be demyelinating peripheral neuropathy, hearing loss and dementia. The course of the disease in relentlessly progressive. In this paper we provide an updated overview of the clinical and genetic features of SCARB2-related PME and on the functions of the LIMP2 protein.

BRAT1-associated neurodegeneration: Intra-familial phenotypic differences in siblings.

Recessive mutations in BRAT1 cause lethal neonatal rigidity and multifocal seizure syndrome, a phenotype characterized by neonatal microcephaly, hypertonia, and refractory epilepsy with premature death by age 2 years. Recently, attenuated disease variants have been described, suggesting that a wider clinical spectrum of BRAT1-associated neurodegeneration exists than was previously thought. Here, we report two affected siblings with compound heterozygous truncating mutations in BRAT1 and intra-familial phenotypic heterogeneity, with a less severe disease course in the female sibling. This phenotypic variability should be taken into account when treating patients with BRAT1-associated neurodegenerative disease. Mildly affected individuals with BRAT1 mutations show that BRAT1 must be considered as a cause in childhood refractory epilepsy and microcephaly with survival beyond infancy. © 2016 Wiley Periodicals, Inc.

KCNT1 mutations in seizure disorders: the phenotypic spectrum and functional effects.

Mutations in the sodium-gated potassium channel subunit gene KCNT1 have recently emerged as a cause of several different epileptic disorders. This review describes the mutational and phenotypic spectrum associated with the gene and discusses the comorbidities found in patients, which include intellectual disability and psychiatric features. The gene may also be linked with cardiac disorders. KCNT1 missense mutations have been found in 39% of patients with the epileptic encephalopathy malignant migrating focal seizures of infancy (MMFSI), making it the most significant MMFSI disease-causing gene identified to date. Mutations in KCNT1 have also been described in eight unrelated cases of sporadic and familial autosomal-dominant nocturnal frontal lobe epilepsy (ADNFLE). These patients have a high frequency of associated intellectual disability and psychiatric features. Two mutations in KCNT1 have been associated with both ADNFLE and MMFSI, suggesting that the genotype-phenotype relationship for KCNT1 mutations is not straightforward. Mutations have also been described in several patients with infantile epileptic encephalopathies other than MMFSI. Notably, all mutations in KCNT1 described to date are missense mutations, and electrophysiological studies have shown that they result in increased potassium current. Together, these genetic and electrophysiological studies raise the possibility of delivering precision medicine by treating patients with KCNT1 mutations using drugs that alter the action of potassium channels to specifically target the biological effects of their disease-causing mutation. Such trials are now in progress. Better understanding of the mechanisms underlying KCNT1-related disease will produce further improvements in treatment of the associated severe seizure disorders.

Multiplex families with epilepsy: Success of clinical and molecular genetic characterization.

OBJECTIVE: To analyze the clinical syndromes and inheritance patterns of multiplex families with epilepsy toward the ultimate aim of uncovering the underlying molecular genetic basis. METHODS: Following the referral of families with 2 or more relatives with epilepsy, individuals were classified into epilepsy syndromes. Families were classified into syndromes where at least 2 family members had a specific diagnosis. Pedigrees were analyzed and molecular genetic studies were performed as appropriate. RESULTS: A total of 211 families were ascertained over an 11-year period in Israel. A total of 169 were classified into broad familial epilepsy syndrome groups: 61 generalized, 22 focal, 24 febrile seizure syndromes, 33 special syndromes, and 29 mixed. A total of 42 families remained unclassified. Pathogenic variants were identified in 49/211 families (23%). The majority were found in established epilepsy genes (e.g., SCN1A, KCNQ2, CSTB), but in 11 families, this cohort contributed to the initial discovery (e.g., KCNT1, PCDH19, TBC1D24). We expand the phenotypic spectrum of established epilepsy genes by reporting a familial LAMC3 homozygous variant, where the predominant phenotype was epilepsy with myoclonic-atonic seizures, and a pathogenic SCN1A variant in a family where in 5 siblings the phenotype was broadly consistent with Dravet syndrome, a disorder that usually occurs sporadically. CONCLUSION: A total of 80% of families were successfully classified, with pathogenic variants identified in 23%. The successful characterization of familial electroclinical and inheritance patterns has highlighted the value of studying multiplex families and their contribution towards uncovering the genetic basis of the epilepsies.

Benign infantile seizures and paroxysmal dyskinesia caused by an SCN8A mutation.

OBJECTIVE: Benign familial infantile seizures (BFIS), paroxysmal kinesigenic dyskinesia (PKD), and their combination-known as infantile convulsions and paroxysmal choreoathetosis (ICCA)-are related autosomal dominant diseases. PRRT2 (proline-rich transmembrane protein 2 gene) has been identified as the major gene in all 3 conditions, found to be mutated in 80 to 90% of familial and 30 to 35% of sporadic cases. METHODS: We searched for the genetic defect in PRRT2-negative, unrelated families with BFIS or ICCA using whole exome or targeted gene panel sequencing, and performed a detailed cliniconeurophysiological workup. RESULTS: In 3 families with a total of 16 affected members, we identified the same, cosegregating heterozygous missense mutation (c.4447G>A; p.E1483K) in SCN8A, encoding a voltage-gated sodium channel. A founder effect was excluded by linkage analysis. All individuals except 1 had normal cognitive and motor milestones, neuroimaging, and interictal neurological status. Fifteen affected members presented with afebrile focal or generalized tonic-clonic seizures during the first to second year of life; 5 of them experienced single unprovoked seizures later on. One patient had seizures only at school age. All patients stayed otherwise seizure-free, most without medication. Interictal electroencephalogram (EEG) was normal in all cases but 2. Five of 16 patients developed additional brief paroxysmal episodes in puberty, either dystonic/dyskinetic or "shivering" attacks, triggered by stretching, motor initiation, or emotional stimuli. In 1 case, we recorded typical PKD spells by video-EEG-polygraphy, documenting a cortical involvement. INTERPRETATION: Our study establishes SCN8A as a novel gene in which a recurrent mutation causes BFIS/ICCA, expanding the clinical-genetic spectrum of combined epileptic and dyskinetic syndromes.

Exome-based analysis of cardiac arrhythmia, respiratory control, and epilepsy genes in sudden unexpected death in epilepsy.

OBJECTIVE: The leading cause of epilepsy-related premature mortality is sudden unexpected death in epilepsy (SUDEP). The cause of SUDEP remains unknown. To search for genetic risk factors in SUDEP cases, we performed an exome-based analysis of rare variants. METHODS: Demographic and clinical information of 61 SUDEP cases were collected. Exome sequencing and rare variant collapsing analysis with 2,936 control exomes were performed to test for genes enriched with damaging variants. Additionally, cardiac arrhythmia, respiratory control, and epilepsy genes were screened for variants with frequency of <0.1% and predicted to be pathogenic with multiple in silico tools. RESULTS: The 61 SUDEP cases were categorized as definite SUDEP (n = 54), probable SUDEP (n = 5), and definite SUDEP plus (n = 2). We identified de novo mutations, previously reported pathogenic mutations, or candidate pathogenic variants in 28 of 61 (46%) cases. Four SUDEP cases (7%) had mutations in common genes responsible for the cardiac arrhythmia disease, long QT syndrome (LQTS). Nine cases (15%) had candidate pathogenic variants in dominant cardiac arrhythmia genes. Fifteen cases (25%) had mutations or candidate pathogenic variants in dominant epilepsy genes. No gene reached genome-wide significance with rare variant collapsing analysis; however, DEPDC5 (p = 0.00015) and KCNH2 (p = 0.0037) were among the top 30 genes, genome-wide. INTERPRETATION: A sizeable proportion of SUDEP cases have clinically relevant mutations in cardiac arrhythmia and epilepsy genes. In cases with an LQTS gene mutation, SUDEP may occur as a result of a predictable and preventable cause. Understanding the genetic basis of SUDEP may inform cascade testing of at-risk family members.

Mutations in the mammalian target of rapamycin pathway regulators NPRL2 and NPRL3 cause focal epilepsy.

OBJECTIVE: Focal epilepsies are the most common form observed and have not generally been considered to be genetic in origin. Recently, we identified mutations in DEPDC5 as a cause of familial focal epilepsy. In this study, we investigated whether mutations in the mammalian target of rapamycin (mTOR) regulators, NPRL2 and NPRL3, also contribute to cases of focal epilepsy. METHODS: We used targeted capture and next-generation sequencing to analyze 404 unrelated probands with focal epilepsy. We performed exome sequencing on two families with multiple members affected with focal epilepsy and linkage analysis on one of these. RESULTS: In our cohort of 404 unrelated focal epilepsy patients, we identified five mutations in NPRL2 and five in NPRL3. Exome sequencing analysis of two families with focal epilepsy identified NPRL2 and NPRL3 as the top candidate-causative genes. Some patients had focal epilepsy associated with brain malformations. We also identified 18 new mutations in DEPDC5. INTERPRETATION: We have identified NPRL2 and NPRL3 as two new focal epilepsy genes that also play a role in the mTOR-signaling pathway. Our findings show that mutations in GATOR1 complex genes are the most significant cause of familial focal epilepsy identified to date, including cases with brain malformations. It is possible that deregulation of cellular growth control plays a more important role in epilepsy than is currently recognized.