The Molecular Genetic Investigation of Epilepsy of Infancy with Migrating Focal Seizures

Epilepsy of infancy with migrating focal seizures (EIMFS) is characterised by the onset of frequent focal seizures in the first 6 months of life, a typical migratory EEG pattern and severe developmental delay. In this thesis, I report a cohort of patients with EIMFS, delineate clinical features and investigate the molecular genetic basis of this syndrome. In 2012, heterozygous mutations in the sodium-gated potassium channel KCNT1, were described in patients with EIMFS. Using a variety of genetic techniques, I have identified 12 patients with mutations in this gene. Four are novel, previously unreported mutations. Functional investigations, including protein homology modelling and electrophysiology in a xenopus oocyte model showed that all novel KCNT1 variants were gain-of-function mutations. In addition, I describe a new genetic cause of EIMFS. Within my cohort, I identified a consanguineous family with two affected children. Autozygosity mapping and whole exome sequencing revealed a novel, homozygous mutation in SLC12A5. SLC12A5 encodes KCC2, the neuronal potassium chloride co-transporter that determines the direction and polarity of GABA-mediated signalling. Through international collaboration, I found a second family with two affected children harbouring compound heterozygous SLC12A5 mutations. All three SLC12A5 variants were investigated using an overexpression HEK293 cell model. Immunoblotting and immunohistochemistry revealed decreased cell surface expression of mutant KCC2. Electrophysiology experiments showed a depolarization of the chloride reversal potential and a delayed response to chloride loading. Taken together, these results indicate that loss of KCC2 function is likely to result in abnormal neuronal inhibition in this form of EIMFS. The genetic heterogeneity in EIMFS is strong evidence that a wide variety of different pathogenic mechanisms can result in the severe epilepsy and abnormal neurodevelopment observed in this condition. Further elucidation of causative genes in both animal and cell models is needed to identify novel therapeutic targets for this devastating disorder

Cerebral Organoids and Antisense Oligonucleotide Therapeutics: Challenges and Opportunities

The advent of stem cell-derived cerebral organoids has already advanced our understanding of disease mechanisms in neurological diseases. Despite this, many remain without effective treatments, resulting in significant personal and societal health burden. Antisense oligonucleotides (ASOs) are one of the most widely used approaches for targeting RNA and modifying gene expression, with significant advancements in clinical trials for epilepsy, neuromuscular disorders and other neurological conditions. ASOs have further potential to address the unmet need in other neurological diseases for novel therapies which directly target the causative genes, allowing precision treatment. Induced pluripotent stem cell (iPSC) derived cerebral organoids represent an ideal platform in which to evaluate novel ASO therapies. In patient-derived organoids, disease-causing mutations can be studied in the native genetic milieu, opening the door to test personalized ASO therapies and n-of-1 approaches. In addition, CRISPR-Cas9 can be used to generate isogenic iPSCs to assess the effects of ASOs, by either creating disease-specific mutations or correcting available disease iPSC lines. Currently, ASO therapies face a number of challenges to wider translation, including insufficient uptake by distinct and preferential cell types in central nervous system and inability to cross the blood brain barrier necessitating intrathecal administration. Cerebral organoids provide a practical model to address and improve these limitations. In this review we will address the current use of organoids to test ASO therapies, opportunities for future applications and challenges including those inherent to cerebral organoids, issues with organoid transfection and choice of appropriate read-outs

Factor associated with the occurrence of epilepsy in autism: A systematic review

This systematic review aimed to identify factors significantly associated with the occurrence of epilepsy in autistic individuals and to consider the impact of study quality on findings. Electronic databases were systematically searched on October 2nd, 2020 and records retrieved were limited to those published from 2000 onwards. Study quality was categorised as 'good', 'moderate' or 'weak'. Fifty-three studies were included and in studies where the prevalence of epilepsy was reported (n = 257,892), 18,254 (7%) had co-occurring epilepsy. Intellectual disability/cognitive impairment was the most commonly reported risk factor associated with occurrence of epilepsy in autistic individuals. The evidence supporting other, potentially relevant factors was weak and inconsistent and requires further evaluation. Only 9/53 studies were considered 'good' quality

Pediatric epilepsy surgery from 2000 to 2018: Changes in referral and surgical volumes, patient characteristics, genetic testing, and postsurgical outcomes

OBJECTIVE: Neurosurgery is a safe and effective form of treatment for select children with drug-resistant epilepsy. Still, there is concern that it remains underutilized, and that seizure freedom rates have not improved over time. We investigated referral and surgical practices, patient characteristics, and postoperative outcomes over the past two decades. METHODS: We performed a retrospective cohort study of children referred for epilepsy surgery at a tertiary center between 2000 and 2018. We extracted information from medical records and analyzed temporal trends using regression analyses. RESULTS: A total of 1443 children were evaluated for surgery. Of these, 859 (402 females) underwent surgical resection or disconnection at a median age of 8.5 years (interquartile range [IQR] = 4.6-13.4). Excluding palliative procedures, 67% of patients were seizure-free and 15% were on no antiseizure medication (ASM) at 1-year follow-up. There was an annual increase in the number of referrals (7%, 95% confidence interval [CI] = 5.3-8.6; p < .001) and surgeries (4% [95% CI = 2.9-5.6], p < .001) over time. Duration of epilepsy and total number of different ASMs trialed from epilepsy onset to surgery were, however, unchanged, and continued to exceed guidelines. Seizure freedom rates were also unchanged overall but showed improvement (odds ratio [OR] 1.09, 95% CI = 1.01-1.18; p = .027) after adjustment for an observed increase in complex cases. Children who underwent surgery more recently were more likely to be off ASMs postoperatively (OR 1.04, 95% CI = 1.01-1.08; p = .013). There was a 17% annual increase (95% CI = 8.4-28.4, p < .001) in children identified to have a genetic cause of epilepsy, which was associated with poor outcome. SIGNIFICANCE: Children with drug-resistant epilepsy continue to be put forward for surgery late, despite national and international guidelines urging prompt referral. Seizure freedom rates have improved over the past decades, but only after adjustment for a concurrent increase in complex cases. Finally, genetic testing in epilepsy surgery patients has expanded considerably over time and shows promise in identifying patients in whom surgery is less likely to be successful

TBC1D24 Mutations in a Sibship with Multifocal Polymyoclonus

Background: Advances in molecular genetic technologies have improved our understanding of genetic causes of rare neurological disorders with features of myoclonus. Case Report: A family with two affected siblings, presenting with multifocal polymyoclonus and neurodevelopmental delay, was recruited for whole-exome sequencing following unyielding diagnostic neurometabolic investigations. Compound heterozygous mutations in TBC1D24, a gene previously associated with various epilepsy phenotypes and hearing loss, were identified in both siblings. The mutations included a missense change c.457G>A (p.Glu157Lys), and a novel frameshift mutation c.545del (p.Thr182Serfs*6). Discussion: We propose that TBC1D24-related diseases should be in the differential diagnosis for children with polymyoclonus

<i>TBC1D24</i> Mutations in a Sibship with Multifocal Polymyoclonus

<p><strong>Background:</strong>&nbsp;Advances in molecular genetic technologies have improved our understanding of genetic causes of rare neurological disorders with features of myoclonus.</p><p><strong>Case Report:</strong>&nbsp;A family with two affected siblings, presenting with multifocal polymyoclonus and neurodevelopmental delay, was recruited for whole-exome sequencing following unyielding diagnostic neurometabolic investigations. Compound heterozygous mutations in&nbsp;<em>TBC1D24</em>, a gene previously associated with various epilepsy phenotypes and hearing loss, were identified in both siblings. The mutations included a missense change c.457G&gt;A (p.Glu157Lys), and a novel frameshift mutation c.545del (p.Thr182Serfs*6).</p><p><strong>Discussion:</strong>&nbsp;We propose that&nbsp;<em>TBC1D24-</em>related diseases should be in the differential diagnosis for children with polymyoclonus.</p><p>&nbsp;</p

<i>TBC1D24</i> Mutations in a Sibship with Multifocal Polymyoclonus

<p><strong>Background:</strong>&nbsp;Advances in molecular genetic technologies have improved our understanding of genetic causes of rare neurological disorders with features of myoclonus.</p><p><strong>Case Report:</strong>&nbsp;A family with two affected siblings, presenting with multifocal polymyoclonus and neurodevelopmental delay, was recruited for whole-exome sequencing following unyielding diagnostic neurometabolic investigations. Compound heterozygous mutations in&nbsp;<em>TBC1D24</em>, a gene previously associated with various epilepsy phenotypes and hearing loss, were identified in both siblings. The mutations included a missense change c.457G&gt;A (p.Glu157Lys), and a novel frameshift mutation c.545del (p.Thr182Serfs*6).</p><p><strong>Discussion:</strong>&nbsp;We propose that&nbsp;<em>TBC1D24-</em>related diseases should be in the differential diagnosis for children with polymyoclonus.</p><p>&nbsp;</p

Emergency treatment with levetiracetam or phenytoin in status epilepticus in children-the EcLiPSE study: Study protocol for a randomised controlled trial

© The Author(s). 2017. Background: Convulsive status epilepticus (CSE) is the most common life-threatening neurological emergency in childhood. These children are also at risk of significant morbidity, with acute and chronic impact on the family and the health and social care systems. The current recommended first-choice, second-line treatment in children aged 6 months and above is intravenous phenytoin (fosphenytoin in the USA), although there is a lack of evidence for its use and it is associated with significant side effects. Emerging evidence suggests that intravenous levetiracetam may be effective as a second-line agent for CSE, and fewer adverse effects have been described. This trial therefore aims to determine whether intravenous phenytoin or levetiracetam is more effective, and safer, in treating childhood CSE. Methods/design: This is a phase IV, multi-centre, parallel group, randomised controlled, open-label trial. Following treatment for CSE with first-line treatment, children with ongoing seizures are randomised to receive either phenytoin (20 mg/kg, maximum 2 g) or levetiracetam (40 mg/kg, maximum 2.5 g) intravenously. The primary outcome measure is the cessation of all visible signs of CSE as determined by the treating clinician. Secondary outcome measures include the need for further anti-seizure medications or rapid sequence induction for ongoing CSE, admission to critical care areas, and serious adverse reactions. Patients are recruited without prior consent, with deferred consent sought at an appropriate time for the family. The primary analysis will be by intention-to-treat. The primary outcome is a time to event outcome and a sample size of 140 participants in each group will have 80% power to detect an increase in CSE cessation rates from 60% to 75%. Our total sample size of 308 randomised and treated participants will allow for 10% loss to follow-up. Discussion: This clinical trial will determine whether phenytoin or levetiracetam is more effective as an intravenous second-line agent for CSE, and provide evidence for management recommendations. In addition, this trial will also provide data on which of these therapies is safer in this setting

Evaluation of the feasibility, diagnostic yield, and clinical utility of rapid genome sequencing in infantile epilepsy (Gene-STEPS): an international, multicentre, pilot cohort study

BACKGROUND: Most neonatal and infantile-onset epilepsies have presumed genetic aetiologies, and early genetic diagnoses have the potential to inform clinical management and improve outcomes. We therefore aimed to determine the feasibility, diagnostic yield, and clinical utility of rapid genome sequencing in this population. METHODS: We conducted an international, multicentre, cohort study (Gene-STEPS), which is a pilot study of the International Precision Child Health Partnership (IPCHiP). IPCHiP is a consortium of four paediatric centres with tertiary-level subspecialty services in Australia, Canada, the UK, and the USA. We recruited infants with new-onset epilepsy or complex febrile seizures from IPCHiP centres, who were younger than 12 months at seizure onset. We excluded infants with simple febrile seizures, acute provoked seizures, known acquired cause, or known genetic cause. Blood samples were collected from probands and available biological parents. Clinical data were collected from medical records, treating clinicians, and parents. Trio genome sequencing was done when both parents were available, and duo or singleton genome sequencing was done when one or neither parent was available. Site-specific protocols were used for DNA extraction and library preparation. Rapid genome sequencing and analysis was done at clinically accredited laboratories, and results were returned to families. We analysed summary statistics for cohort demographic and clinical characteristics and the timing, diagnostic yield, and clinical impact of rapid genome sequencing. FINDINGS: Between Sept 1, 2021, and Aug 31, 2022, we enrolled 100 infants with new-onset epilepsy, of whom 41 (41%) were girls and 59 (59%) were boys. Median age of seizure onset was 128 days (IQR 46-192). For 43 (43% [binomial distribution 95% CI 33-53]) of 100 infants, we identified genetic diagnoses, with a median time from seizure onset to rapid genome sequencing result of 37 days (IQR 25-59). Genetic diagnosis was associated with neonatal seizure onset versus infantile seizure onset (14 [74%] of 19 vs 29 [36%] of 81; p=0·0027), referral setting (12 [71%] of 17 for intensive care, 19 [44%] of 43 non-intensive care inpatient, and 12 [28%] of 40 outpatient; p=0·0178), and epilepsy syndrome (13 [87%] of 15 for self-limited epilepsies, 18 [35%] of 51 for developmental and epileptic encephalopathies, 12 [35%] of 34 for other syndromes; p=0·001). Rapid genome sequencing revealed genetic heterogeneity, with 34 unique genes or genomic regions implicated. Genetic diagnoses had immediate clinical utility, informing treatment (24 [56%] of 43), additional evaluation (28 [65%]), prognosis (37 [86%]), and recurrence risk counselling (all cases). INTERPRETATION: Our findings support the feasibility of implementation of rapid genome sequencing in the clinical care of infants with new-onset epilepsy. Longitudinal follow-up is needed to further assess the role of rapid genetic diagnosis in improving clinical, quality-of-life, and economic outcomes. FUNDING: American Academy of Pediatrics, Boston Children's Hospital Children's Rare Disease Cohorts Initiative, Canadian Institutes of Health Research, Epilepsy Canada, Feiga Bresver Academic Foundation, Great Ormond Street Hospital Charity, Medical Research Council, Murdoch Children's Research Institute, National Institute of Child Health and Human Development, National Institute for Health and Care Research Great Ormond Street Hospital Biomedical Research Centre, One8 Foundation, Ontario Brain Institute, Robinson Family Initiative for Transformational Research, The Royal Children's Hospital Foundation, University of Toronto McLaughlin Centre

De Novo Mutations in SLC1A2 and CACNA1A Are Important Causes of Epileptic Encephalopathies

Epileptic encephalopathies (EEs) are the most clinically important group of severe early-onset epilepsies. Next-generation sequencing has highlighted the crucial contribution of de novo mutations to the genetic architecture of EEs as well as to their underlying genetic heterogeneity. Our previous whole-exome sequencing study of 264 parent-child trios revealed more than 290 candidate genes in which only a single individual had a de novo variant. We sought to identify additional pathogenic variants in a subset (n = 27) of these genes via targeted sequencing in an unsolved cohort of 531 individuals with a diverse range of EEs. We report 17 individuals with pathogenic variants in seven of the 27 genes, defining a genetic etiology in 3.2% of this unsolved cohort. Our results provide definitive evidence that de novo mutations in SLC1A2 and CACNA1A cause specific EEs and expand the compendium of clinically relevant genotypes for GABRB3. We also identified EEs caused by genetic variants in ALG13, DNM1, and GNAO1 and report a mutation in IQSEC2. Notably, recurrent mutations accounted for 7/17 of the pathogenic variants identified. As a result of high-depth coverage, parental mosaicism was identified in two out of 14 cases tested with mutant allelic fractions of 5%–6% in the unaffected parents, carrying significant reproductive counseling implications. These results confirm that dysregulation in diverse cellular neuronal pathways causes EEs, and they will inform the diagnosis and management of individuals with these devastating disorders