1 Seizure freedom after surgical resection of diffusion-weighted MRI abnormalities
3 Gene editing for Dravet syndrome
4 Modelling PRRT2 loss-of-function in human brain slice cultures
5 Astrocyte dysfunction in CDKL5 deficiency disorder
2 Mapping epileptic activity and eloquent cortex in children with epilepsy using OPM-MEG
3 Physiological drivers of circadian variability in interictal epileptiform discharges
1 Gene-STEPS: shifting the paradigm of genetic testing in early onset epilepsies
2 Design of a non-invasive breath test for epileptic seizures: the VIBES study
3 Multimodal investigation of the thalamus in children with temporal lobe epilepsy
Grant F. Marshall, The University of Edinburgh
Andrés Correa-Sánchez, Nucleic Acid Therapy Accelerator; Peter Oliver, Nucleic Acid Therapy Accelerator; Stuart Cobb, The University of Edinburgh; Catherine M. Abbott, The University of Edinburgh
Background: Heterozygous missense mutations in gene encoding neuromuscular translation elongation factor eEF1A2 cause early onset epilepsy and neurodevelopmental disorders (NDDs) with variable presentation and no specific treatments. Over 60 distinct missense mutations have been found with the most common being E122K, which is associated with a consistently severe phenotype. We modelled this mutation in mice and found that heterozygous (E122K/+) mice exhibited several relevant phenotypes, including electrographic abnormalities and early motor delays. Importantly, comparative phenotyping of mice carrying E122K with mice carrying null mutations on the same genetic background revealed that E122K confers a toxic GOF/ dominant negative effect. We are therefore working with the Nucleic Acid Therapy Accelerator to develop allele-specific antisense oligonucleotides (ASOs) to specifically knock down the E122K transcript, with the aim of alleviating this toxicity.
Methods: As an initial screen, we performed a dose-response study for a series of ASOs in HEK293 cells transiently transfected with wildtype and mutant eEF1A2, each with different epitope tags. This allowed us to independently assay changes in wildtype and mutant proteins at different ASO concentrations. Lead molecules were then taken forward for testing in primary neuronal cultures isolated from mice in our E122K line (+/+, E122K/+ and E122K/E122K).
Results: Western blot data from HEK293 cells showed that a number of ASOs lead to significant knockdown of mutant eEF1A2 (to almost undetectable levels) with minimal change in wildtype eEF1A2 levels. These specific ASOs also show efficacy and allele-specificity against endogenous Eef1a2 in primary neurons, with almost no impact on cell viability.
Conclusions: These data show that ASOs can efficiently and specifically target mutant Eef1a2 transcripts on the basis of single nucleotide differences, making them a potentially viable strategy for treating EEF1A2-related NDD. We are now testing lead molecules in vivo in our mouse line and assessing phenotypic correction.
Jonathan D. Lippiat, University of Leeds
Emily A. Caseley, University of Leeds; Bethan A. Cole, University of Leeds; Stephen P. Muench, University of Leeds; Katie J. Simmons, University of Leeds
Background: Heterozygous dominant gain-of-function KCNT1 variants are associated with drug-resistant paediatric developmental and epileptic encephalopathies. Proposed stratified therapeutic approaches involve suppressing overactive KNa1.1 sodium-activated potassium channels encoded by KCNT1 through pharmacological inhibition. Until 2020, the only known KNa1.1 inhibitors were cardiac-acting, non-selective ion channel inhibitors, such as quinidine.
Methods: Using published KNa1.1 protein structures and high-performance computing, we have conducted docking-based virtual high-throughput screening with various libraries containing up to millions of commercially available compounds to identify novel inhibitors. Top-ranking compounds were selected for purchase (approx. 100 overall), which were assessed at 10 μM in thallium-flux fluorescence assays and a cell line expressing both wild-type and Y796H KNa1.1 subunits. Compounds that exhibited KNa1.1 inhibition were followed up with concentration-inhibition analysis and patch clamp electrophysiology.
Results: Using this workflow, approx. 20% of the compounds tested inhibited KNa1.1 channels with potencies (IC50) ranging between 1 and 20 μM. The active compounds are structurally diverse, and each are a potential starting point for developing a series to optimise potency, selectivity, and pharmacokinetic properties.
Conclusions: Structure-based virtual screening, assisted by high-performance computing to increase capacity, is a viable and economic approach to explore chemical space in the process of identifying potential therapeutics for KCNT1-associated epilepsy. It can also be used to identify existing drugs that could potentially be repurposed. With the increasing availability of experimental structures of potential drug targets (complemented by structural prediction), this approach has potential in initiating drug discovery and repurposing projects for other rare genetic disorders.
Clara Zourray, UCL Queen Square Institute of Neurology and UCL-GOSH, London
J Mills, UCL Queen Square Institute of Neurology; M Mainardi, IRCCS San Raffaele Scientific Institute, Milan, Italy; J Street, UCL Queen Square Institute of Neurology; G Mills, UCL Queen Square Institute of Neurology; J Bartkiewicz, UCL Queen Square Institute of Neurology; S Mihaylov, The Francis Crick Institute, J Sartorelli, UCL-GOSH; N O’Neill, UCL Queen Square Institute of Neurology; J Carpenter, UCL Queen Square Institute of Neurology; A Almacellas, UCL Queen Square Institute of Neurology, K Benriassa, UCL Queen Square Institute of Neurology; M Bari, UCL Queen Square Institute of Neurology; A Castiblanque, UCL Queen Square Institute of Neurology; A Papandreou, UCL-GOSH; A McTague, UCL-GOSH; S Schorge, UCL; S Sissodya, UCL Queen Square Institute of Neurology; S Ultanir, The Francis Crick Institute; M Thom, UCL Queen Square Institute of Neurology; M Kurian, UCL-GOSH; G Colasante, IRCCS San Raffaele Scientific Institute, Milan, Italy; S Barral, UCL-GOSH*; and G Lignani, UCL Queen Square Institute of Neurology*
Background: Dravet Syndrome (DS), caused by SCN1A haploinsufficiency, is a severe epilepsy syndrome characterized by early-onset seizures and cortical network hyperexcitability. While the role of SCN1A in mature inhibitory neurons is well-documented, its effects during early cortical development remain poorly understood.
Methods: We generated a human forebrain assembloid (hFA) model using iPSCs derived from two DS patients and their isogenic controls. In mature hFAs we used local field potential recordings to assess network excitability and transcriptomics to identify molecular changes. In early human cortical organoids we used a multi-omic approach combined with flow cytometry to identify changes in the cell cycle progression of cortical progenitors. To validate findings, we used a pulse-chase Brdu labelling assay in a mouse model of Scn1a haploinsufficiency.
Results: DS hFAs exhibited decreased latency to epileptiform activity and increased power spectral density upon proconvulsant exposure, mirroring patient phenotypes. Transcriptomic analyses revealed upregulation of cortical differentiation genes, such as SATB2, suggesting early changes in cortical development. Nav1.1 protein was found to be highly expressed during early corticogenesis, and we found that SCN1A haploinsufficiency induced a delay in the G2/M cell cycle transitions in cortical progenitors resulting in increased neurogenesis. Proteomic analyses implicated CDK1 dysregulation and kinetochore instability in mitotic delay. Finally, in vivo mouse studies confirmed prolonged G2 phase and increased neurogenesis in Scn1a+/- cortical progenitors.
Conclusions: SCN1A haploinsufficiency disrupts progenitor cell cycle dynamics, promoting premature neurogenesis and cortical network hyperexcitability. These findings highlight a novel and critical role for Nav1.1 in cortical progenitors during early corticogenesis and identifies new molecular pathways that could be targeted to mitigate DS developmental phenotypes.
Jonathan Horsley, Newcastle University
Gerard Hall, Newcastle University; Callum Simpson, Newcastle University; Csaba Kozma, Newcastle University; Rhys Thomas, Newcastle University; Yujiang Wang, Newcastle University; Jane de Tisi, University College London; Anna Miserocchi, University College London; Andrew McEvoy, University College London; Sjoerd Vos, The University of Western Australia; Gavin Winston, Queen’s University; John Duncan, University College London; Peter Taylor, Newcastle University
Background: Identifying focal brain abnormalities is essential for neurosurgical intervention. Current clinical approaches to identify structural abnormalities for surgical targeting in epilepsy do not use diffusion-weighted MRI. However, previous work has shown that diffusion abnormalities are present in epilepsy and may relate to the epileptogenic zone. Here, we investigate whether surgical resection of diffusion abnormalities relates to post-operative seizure freedom.
Methods: We investigated the association between surgical resection of diffusion abnormalities and post-operative seizure freedom in 200 individuals with drug-resistant focal epilepsy using dMRI. A cohort of 97 healthy controls provided a normative baseline for dMRI metrics, allowing calculation of voxel-wise z-scores to identify abnormal clusters in both grey and white matter.
Results: Surgical resections overlapping with the largest abnormal cluster significantly correlated with sustained seizure freedom at 12 months (83% vs 55%; ) and over five years (). Notably, resecting only a small proportion of the largest cluster was associated with better seizure outcomes than cases with no resection of this cluster (). Furthermore, sparing the largest cluster but resecting other large clusters still improved seizure freedom rates compared to no overlap ().
Conclusions: Mechanistically, our results suggest that abnormal clusters, identified using dMRI, are integral to the epileptogenic network, and even a partial removal of such an abnormal cluster is sufficient to achieve seizure freedom. The study highlights the potential of incorporating dMRI into pre-surgical planning to improve outcomes in focal epilepsy by reliably identifying and targeting diffusion abnormalities.
Anna Norton, University of Manchester, Manchester
Emily Pegg, University of Manchester; Sonia Kanom, Royal Manchester Children’s Hospital; Rhys Thomas, Newcastle University; with support from the Epilepsy Research Institute UK; Rebecca Bromley, University of Manchester
Background: While epilepsy has equal prevalence in males and females, women with epilepsy are disproportionately affected by the condition throughout their lifespan, with hormonal changes influencing and being influenced by seizure mechanisms and anti-seizure medications. This represents unique challenges for women with epilepsy and their clinicians alike; however, there remains a paucity of research in this area.
The aim of this project was to identify the priorities relating to epilepsy in women across the lifespan through further analysis of UK Epilepsy PSP priorities.
Methods: The UK Epilepsy PSP is a once-in-a-generation, national consensus that was undertaken to collate and rank the research priorities of the UK epilepsy community. Following the established James Lind Alliance (JLA) methodology, the priorities submitted in response to the first survey were categorised and grouped to inform a series of summary questions. These questions were evidence checked, and those deemed unanswered or partially answered were ranked by the UK epilepsy community, culminating in the Top Ten epilepsy research priorities.
Results: In total, 5418 priorities were submitted in response to the PSP first survey. Of these, 141 priorities were identified as relating to hormones and epilepsy in women, which informed Question 7 of the Top Ten: “How do hormonal changes in women throughout the lifespan impact epilepsy, and how can this impact be addressed?” These priorities were further reviewed and analysed to determine the sub themes identified by the UK epilepsy community, including the impact of hormonal changes on epilepsy in women during puberty, pregnancy and menopause.
Conclusions: The UK epilepsy community identified the life-long impact of hormones on epilepsy in women as a research priority; however, this is not represented currently by recently completed or ongoing research. The findings of this study, and the future research it will inform, aims to address this.
Jenna Carpenter, UCL Queen Square Institute of Neurology
F Hardjo, UCL Queen Square Institute of Neurology; X Yang, UCL Queen Square Institute of Neurology; N O’Neill, UCL Queen Square Institute of Neurology; A Almacellas Barbanoj, UCL Queen Square Institute of Neurology; N White, UCL Great Ormond Street Institute of Child Health; G Tanzi, San Raffaele Institute, Italy; F Moreira, UCL Queen Square Institute of Neurology, E Goss-Sampson, UCL Queen Square Institute of Neurology; T Savant, UCL Queen Square Institute of Neurology; O Goff, UCL Queen Square Institute of Neurology; G Turchiano, UCL Great Ormond Street Institute of Child Health; G Colasante, San Raffaele Institute, Italy; G Lignani, UCL Queen Square Institute of Neurology
Abstract not included
Anna Dimtsi, Newcastle University
Max Knops, Newcastle University; Isabella Davies, Newcastle University; Gavin Clowry, Newcastle University; Andrew Trevelyan, Newcastle University; Faye McLeod, Newcastle University
Background: Disease causing variants in Proline-Rich Transmembrane Protein 2 (PRRT2) are amongst the most common epilepsy risk genes associated with infantile seizures and movement disorders. PRRT2 is a synaptic protein with a putative role in negatively modulating exocytosis in neurons postnatally. However, neither its role in early human brain development, nor the underlying pathophysiological mechanisms associated with PRRT2 variants have been elucidated.
Methods: We have established a unique model system, which allows the study of the intact, developing, human cortical network at the prenatal stage, and how it is affected by altered gene function. The Human Developmental Biology Resource (www.hdbr.org) in Newcastle provides ethically approved samples of human foetal cerebral cortical tissue (12–18 post conception weeks; pcw), from which we can prepare living organotypic cultures.
Results: We observed high PRRT2 expression in the developing human cortex between 12-17 pcw, with PRRT2 present mainly at glutamatergic, but also GABAergic synapses. Additionally, PRRT2 immunoreactive puncta more than doubled between 0 and 27 days in vitro in human cortical slice cultures, at both glutamatergic and GABAergic pre-synaptic sites. We next investigated the effects of manipulating PRRT2 expression using a short hairpin RNA interference approach, as most gene variants lead to a loss of function. We established a clone that successfully diminishes PRRT2 levels and results in a decrease in the pre-synaptic marker vGlut1 and an increase in the post-synaptic marker Homer1 at glutamatergic sites. No differences were found at GABAergic sites.
Conclusions: We show that PRRT2 is highly expressed in the human developing cortex and that loss of PRRT2 alters early glutamatergic synaptic networks which are important for neocortical maturation. Our model provides crucial insights into potential disease-causing mechanisms.
Ceri Pickering, Imperial College London
Faye McLeod, Newcastle University; Adriana Bakoulina, Imperial College London; Angeliki Pantziarou, Imperial College London; Alicia Seet, Imperial College London; Alishba Saleemi, Imperial College London; Nicholas D. Mazarakis, Imperial College London
Background: Cyclin Dependant Kinase-Like 5 (CDKL5) Deficiency Disorder (CDD) is a rare developmental epileptic encephalopathy, typically caused by loss of function variants in the gene encoding the X-linked serine-threonine kinase CDKL5. Currently there is no effective cure. Interestingly, alterations in astrocyte function have been linked to the pathophysiology of epilepsy, which might also occur in CDD. We hypothesise that astrocytic dysfunction contributes to the phenotype of CDD, knowledge of which could pave the way for novel therapeutic strategies.
Methods: Induced pluripotent stem cells harbouring a CDKL5 loss-of-function mutation (and isogenic controls) were derived from CDD patient fibroblasts and differentiated into astrocytes (iAstros). Mature iAstros, cultured in maturation medium for 18-45 days, were profiled using RNA sequencing, proteomics and functional assays. Human adult and foetal (17-18 post-conception weeks) cortical organotypic brain slices were transduced with an adeno-associated virus encoding a short-hairpin RNA to knock down CDKL5 (shAAV), or encoding a scrambled control (ScrAAV). Slice cultures were analysed 10-20 days post transduction via extracellular field potential recordings and proteomics.
Results: Analysis of CDD iAstros revealed transcriptomic and proteomic dysregulation in astrocyte-specific genes and proteins relevant to epilepsy and development. Furthermore, CDKL5null iAstros demonstrated altered cytokine and chemokine release in response to stimuli. CDKL5 expression was successfully knocked down in human adult and foetal cortical organotypic slice cultures transduced with shAAV, which resulted in increased spontaneous network activity and reduced phosphorylation of a known CDKL5 substrate in foetal samples. Moreover, slices transduced with shAAV replicated dysregulation of the same astrocytic proteins as CDD iAstros.
Conclusions: We provide evidence that a loss of astrocytic CDKL5 causes transcriptomic, proteomic and functional dysregulation in these cells. Additionally, we have successfully generated two novel human in vitro models of CDD – providing a platform to interrogate cell-type specific and network consequences of CDKL5 deficiency.
Kathryn J Bush, Newcastle University
B Fonferko-Shadrach, Swansea University; HA Strafford, Swansea University; AS Lacey, Swansea University; A Kingston, Newcastle University; RH Thomas, Newcastle University; SE Ramsay, Newcastle University; WO Pickrell; Swansea University and Swansea Bay University Health Board
Background: Population-level linked routinely collected datasets such as SAIL Databank in Wales, represent a unique opportunity for epilepsy epidemiology. However, validation studies are required to ensure the code algorithms used are accurate. There are no previous validation studies of epilepsy cases identified in United Kingdom (UK) inpatient hospital data, or examining epilepsy cause/types.
Methods: We validated multiple code algorithms identifying adults with epilepsy and epilepsy types/cause, against data extracted via Natural Language Processing (NLP) from Swansea Bay University Health Board Neurology clinic letters between 2014-2021. Cases with linkage to SAIL primary care data were included and comprised the validation cohort. True positive epilepsy cases were defined as > 2 letters including an epilepsy diagnosis and true negatives as > 2 letters with no diagnosis of epilepsy or seizures.
Results: 3009 individuals had NLP data from letters indicating an epilepsy diagnosis and 3866 individuals an alternate diagnosis. The optimal algorithm using primary care and hospital data combined was: 2 anti-seizure medication codes in 6 months, and an epilepsy code within +/-12 months. This identified epilepsy cases with a sensitivity of (2687/3009) 89.3%, specificity 98.8% and PPV 98.3%. 76 (2.8%) cases were only identified with the addition of hospital data. Hospital data alone identified 2263 (75.2%) cases.
463/2687 (17.2%) validated cases had an epilepsy syndrome specified in their letter(s), 198/463 (42.8%) had a syndrome code. 288/2687(10.7%) had a specified cause and a code for this pathology was identified in the following proportions: genetic syndromes (100%), infective pathologies (76.2%), stroke/cerebrovascular disease (84.2%), tumours (94.7%) and brain injuries (64.9%).
Conclusions: Primary care and hospital data can be combined to accurately identify adults with epilepsy and their underlying cause in UK data. Epilepsy syndromes are coded less frequently than acquired pathologies. These algorithms should now be applied to population-level data to identify the types and causes of epilepsy and estimate the proportion of preventable epilepsies.
Julie Werenberg Dreier, National Centre for Register-Based Research, Aarhus University, Denmark
Betina B. Trabjerg, Aarhus University; Kasper Lolk, Aarhus University; Oleguer Plana-Ripoll, Aarhus University and Aarhus University Hospital; Jakob Christensen, Aarhus University Hospital
Background: We quantify the loss of working years for people with epilepsy compared to the general population and consider variation by aetiology, psychiatric comorbidity, sex and age.
Methods: This population-based cohort study included all individuals aged 18–65 years living in Denmark from 1995-2018. Using nationwide registers since 1977, we identified people with epilepsy and obtained information on the main source of income or employment for each year during follow-up from 1995 to 2020. The main outcome was number of working years lost in people with epilepsy compared to the general population of same sex and age, capturing both working life lost due to permanent (death, disability pension, early retirement) and temporary (unemployment, sick leave) factors.
Results: The study comprised 5,466,140 individuals, including 74,980 (1.4%) with epilepsy (median age at onset=23.9 years, IQR=11.6-41.9 years). In people with epilepsy, the number of working years was on average reduced by 6.6 (95% CI:6.5-6.7) years compared with the general population, largely due to disability pension (4.8 years, 95% CI:4.7-4.9) and premature death (1.6 years, 95% CI:1.6-1.7). Loss of working life was more pronounced in those with a presumed underlying aetiology (9.0 years (95% CI:8.9-9.2) versus 5.4 years (95% CI:5.2-5.5) in those with unknown aetiology), in those with early onset of epilepsy (e.g., 11.5 years (95% CI:11.3-11.7) among those with onset <20 years), and in men (7.2 years (95% CI:7.1-7.3) versus 5.9 (95% CI:5.8-6.0) years in women). Furthermore, loss of working life was substantial in people with epilepsy and psychiatric comorbidity, particularly in those with comorbid intellectual disability (27.7 years), autism (24.6 years), ADHD (21.2 years), and schizophrenia (18.6 years).
Conclusions: Epilepsy was associated with significant loss of working life resulting from both disability and premature death, indicating that more efforts are needed to help people with epilepsy secure and retain employment.
Christine Embury, Young Epilepsy
Zelekha Seedat, Young Epilepsy; Tim Tierney, University College London; Kelly St. Pier, Young Epilepsy; Caroline Scott, Young Epilepsy; Gareth Barnes, University College London; Matthew Walker, University College London; Umesh Vivekananda, University College London; J. Helen Cross, University College London and Great Ormond Street Hospital
Background: Current epilepsy investigations such as EEG and cryogenic MEG scans fall short in precisely mapping the underlying source of brain activity in paediatric populations. New technologies are emerging to make mapping epileptogenic activity more precise and allow for movement, where the sensors are not fixed but are housed in smaller and variably-sized child-friendly helmets. To this end, we demonstrate the use of OPM-MEG, a wearable, near room temperature version of MEG, in mapping epileptogenic activity and eloquent cortex in children with epilepsy. The technique is passive and non-invasive, and does not require adhesives, rather relying on sized helmets (similar to a bike helmet) to fit the size and shape of the child’s head.
Methods: We scanned 11 children (ages 5-15) with the OPM-MEG during rest and while performing tasks. Our OPM-MEG array consisted of 64 QuSpin dual axis sensors (128 channels) housed in child-sized helmets within a light MuRoom coupled with active shielding (Cerca Magnetics Ltd., Nottingham, England, UK). For motor cortex mapping, participants were visually cued to perform a rapid right index finger abduction, completing 50 trials. We beamformed motor responses and mapped equivalent current dipoles for detected spike activity in BESA Research 7.1 (BESA GmbH, Gräfelfing, Germany).
Results: We are able to map epileptogenic activity in those with clear spikes during the recording period and further map motor responses to contralateral motor cortex in all participants.
Conclusions: New technologies such as OPM-MEG may revolutionise the diagnostic and treatment pathways for children with epilepsy. Notable advantages of the technique include enhanced tolerability particularly in children with sensory sensitivities, adaptability in array definition and sizing, and the ability to move more naturally while recording. OPM-MEG shows great promise in delivering precise mapping of epileptic activity and nearby eloquent cortices, to enhance treatment precision and patient experience for children with epilepsy.
Professor John R Terry, University of Birmingham & Neuronostics Ltd
Isabella Marinelli, University of Birmingham; Jamie J. Walker, University of Exeter; Udaya Seneviratne, Monash University; Wendyl D’Souza, University of Melbourne; Mark J. Cook, University of Melbourne; Clare Anderson, University of Birmingham; Andrew P. Bagshaw, University of Birmingham; Stafford L. Lightman, University of Bristol; Wessel Woldman, University of Birmingham & Neuronostics Ltd.
Background: Classically, seizures are assumed to occur at random. However, recent research has uncovered underlying rhythms both in seizures and in key signatures of epilepsy—so-called interictal epileptiform discharges—with timescales that vary from hours and days through to months. Many people with epilepsy identify precipitants of their seizures, the most common of which include stress, sleep deprivation and fatigue.
Methods: To quantify the impact of potential physiological factors on the occurrence of interictal epileptiform discharges, 24-hour EEG recordings from a cohort of 107 people with idiopathic generalized epilepsy were analysed using a mathematical model that incorporated the time-varying effect of physiological factors on the level of excitability in brain regions.
Results: Two subgroups with distinct distributions of epileptiform discharges were found: one with highest incidence during sleep and the other during day-time. By calibrating the forcing term of the mathematical model using independently collected human cortisol (the primary stress-responsive hormone characterised by circadian and ultradian patterns of secretion) data and sleep-staged EEG from healthy human participants it was shown that either the dynamics of cortisol or sleep stage transition, or a combination of both, could explain most of the observed distributions of epileptiform discharges.
Conclusions: These findings provide conceptual evidence for the existence of underlying physiological drivers of rhythms of epileptiform discharges. These findings should motivate future research to explore these mechanisms in carefully designed experiments using animal models or people with epilepsy.
Dr Amy McTague, UCL Great Ormond Institute of Child Health and Great Ormond Street Hospital, London
A D’Gama, Boston Children’s Hospital, Boston; V Chau, Hospital for Sick Children, Toronto, and University of Toronto; S Mulhern, Murdoch Children’s Research Institute, Melbourne, and University of Melbourne; E Scotchman, North Thames Genomic Laboratory Hub, Great Ormond Street NHS Foundation Trust; M Lachgar-Ruiz, North Thames Genomic Laboratory Hub, Great Ormond Street NHS Foundation Trust ; T Higginbotham, Genome Diagnostics, The Hospital for Sick Children, Toronto ; J Nguyen, SickKids Research Institute, Toronto; K Bell, Bioinformatics, MCRI, Melbourne; A Griffiths, Neurosciences, MCRI, Melbourne; B Hanson, North Thames Genomic Laboratory Hub, Great Ormond Street NHS Foundation Trust; P Lombard, North Thames Genomic Laboratory Hub, Great Ormond Street NHS Foundation Trust; C Magro, North Thames Genomic Laboratory Hub, Great Ormond Street NHS Foundation Trust; CR Marshall, Genome Diagnostics, The Hospital for Sick Children, Toronto ; S Nesbitt, Genome Diagnostics, The Hospital for Sick Children, Toronto; B Trost, Molecular Medicine, The Hospital for Sick Children, Toronto ; J Coleman, Neurosciences, MCRI, Melbourne; M Coleman, Neurosciences, MCRI, Melbourne; W Shao, Harvard Medical School, Boston; J Sidhu, SickKids Research Institute, Toronto ; B Paternoster, Genome Diagnostics, The Hospital for Sick Children, Toronto ; S Nesbitt, Genome Diagnostics, The Hospital for Sick Children, Toronto; G Rose, Genome Diagnostics, The Hospital for Sick Children, Toronto ; B Sheidly, Boston Children’s Hospital, Boston; Gene-STEPS Study Group; Z Stark, Murdoch Children’s Research Institute, Melbourne; NJ Chandler, Genome Diagnostics, The Hospital for Sick Children, Toronto ; L Chitty, Genome Diagnostics, The Hospital for Sick Children, Toronto ; IE Scheffer, University of Melbourne, Melbourne; Austin Health, and Florey Institute of Neuroscience and Mental Health, Melbourne; JH Cross, UCL Great Ormond Institute of Child Health and Great Ormond Street Hospital; A Poduri, Harvard Medical School and Epilepsy Genetics Programme, Boston Children’s Hospital; NINDs ; G Costain, SickKids Research Institute, Toronto; K Howell, Neurosciences, MCRI, Melbourne and Royal Children’s Hospital, Melbourne
Background: Accurate and timely genetic diagnosis in infantile-onset epilepsies is vital as it can dictate epilepsy management and determine prognosis. In Gene-STEPS, a prospective international multi-centre study, we set out to examine diagnostic yield and impact of rapid trio genome sequencing (GS) with advanced interpretation strategies on outcomes for patients with epilepsy under 12
Methods: We recruited infants <12 months old at seizure onset within 6 weeks of presentation. Clinical data was collected and rapid trio GS performed in clinically accredited laboratories at each site. For participants without genetic diagnoses after clinical GS (n>170), we applied advanced genomic strategies including 1) re-evaluation using an agnostic approach, 2) manual review for a second variant in cases with a single pathogenic variant in an autosomal recessive gene, 3) reanalysis of all coding SNVs and indels, including in a consensus epilepsy gene list (n=1,045), 4) assessment of mosaic, structural, and mitochondrial variants, and 5) targeted RNA-sequencing.
Results: We recruited over 400 infants with a sustained diagnostic yield of 45% for primary analysis. 93 distinct genes/genomic regions were identified with treatment and/or surveillance implications for 99/179 (55%). The top 5 genes (PRRT2, KCNQ2, SCN1A, SCN2A, SCN8A) accounted for 35% of diagnoses, all with management implications. Re-analysis established 15 further diagnoses including a complex structural variant and a deep intronic variant in DNM1 confirmed with RNA sequencing to cause aberrant splicing.
Conclusions: Gene-STEPS is the first prospective study of rapid GS in a cohort of patients with epilepsy, demonstrating high diagnostic yield and feasibility across varied healthcare systems. Reanalysis improved yield from short-read GS in patients with infantile onset epilepsy, with significant impacts for patient management and reproductive counselling. This study deepens our understanding of the genetic landscape of early onset epilepsy and supports rapid GS with regular re-analysis for infants with new-onset epilepsy. Ongoing research will examine developmental and epilepsy outcomes and impacts on the patient and family’s experience.
Ilaria Belluomo, Imperial College London
Eleonora Lugarà, University College London; Grace Luff, University College London; Joao Pedro Rodrigues Pizarro, University College London Hospitals; Khrystyna Forbes, University College London Hospitals; Matthew Walker, University College London; George B Hanna, Imperial College London
Background: About 20-30% of patients are misdiagnosed with epilepsy. Inaccurate diagnosis can result in unnecessary treatments with potential adverse effects and social issues. Together with diagnosis, another unmet requirement for the management of epilepsy is the predictability of seizure onset, posing daily life challenges for people experiencing recurring seizures. The identification of specific biomarkers before, during and after seizures would allow the development of diagnostic and predictive tests.
Methods: Profiling exhaled breath volatile organic compounds (VOCs) offers an attractive new strategy for the development of non-invasive tests. VOCs reflect systemic biochemical processes underlying physio-pathological states. The potential role of volatiles in epilepsy is already known: dogs possess the ability to predict oncoming seizures. Research has demonstrated that dog’s seizure perception does not depend on emotion, posture or motion, as they can differentiate samples from subjects experiencing a seizure and those engaging in physical exercise, suggesting the existence of an odour cue linked to seizures.
Results: In the first part of this study – VIBES, Volatile non-Invasive Biomarkers of Epileptic Seizures – we recruited 133 patients at the telemetry unit of the National Hospital for Neurology and Neurosurgery and Chalfont Hospital. We collected breath before, and at three time points after seizures, to evaluate the VOC fluctuations related to the type of seizures and their seizure predictive potential. VOC-based tests are well-accepted by patients, making possible to perform them in emergency settings. The preliminary data collection has been completed, and the analysis is ongoing. We expect to identify a set of VOCs in breath that distinguish epileptic and non-epileptic seizures, as well as biomarkers associated with preictal states.
Conclusions: This study seek to uncover key VOCs associated with epilepsy and preictal states, which may offer novel insights into seizure pathophysiology and support the development of breath-based diagnostic and predictive tools.
Xiyu Feng, UCL Great Ormond Street Institute of Child Health, London
Rory Piper, UCL Great Ormond Street Institute of Child Health and Great Ormond Street Hospital; Freya Prentice, UCL Great Ormond Street Institute of Child Health; Maria H. Eriksson, UCL Great Ormond Street Institute of Child Health and The Hospital for Sick Children, Toronto, Canada; Martin Tisdall, UCL Great Ormond Street Institute of Child Health and Great Ormond Street Hospital; Jonathan Clayden, UCL Great Ormond Street Institute of Child Health ; Torsten Baldeweg, UCL Great Ormond Street Institute of Child Health
Background: The thalamus plays a key role in propagating epileptic activity and a target for neuromodulation in epilepsy treatment. Functional and structural rearrangements of brain networks may mediate the large-scale spread of epileptic activity. In adults with temporal lobe epilepsy (TLE), there is evidence of thalamic atrophy and abnormal functional connectivity but less is known about these changes in paediatric TLE. This study provides a multimodal characterisation of thalamic sub-nuclei in paediatric TLE, examining both volumetric and functional connectivity.
Methods: Structural and functional MRI were retrospectively analysed for 86 children with unilateral TLE (ages 6–19) and 72 controls (ages 6–20). We examined four thalamic nuclei groups (anterior, lateral, medial, posterior/pulvinar) in each hemisphere for 1) volume, 2) seed-to-voxel functional connectivity, and 3) functional connectivity-based hubness, which indicate whether a region is highly connected or isolated within a whole-brain network. Age- and sex-adjusted measures were compared between groups using t-tests to examine their linked to TLE phenotypes and surgical outcomes.
Results: We observed thalamic volume reduction ipsilateral to the seizure focus, especially in the pulvinar nuclei, which co-occurred with reduced functional hubness. Seed-to-voxel analyses revealed increased functional connectivity between the ipsilateral thalamus and bilateral midbrain structures (subthalamic nucleus, red nucleus, substantia nigra). Subgroup analysis linked greater ipsilateral thalamic atrophy with hippocampal sclerosis and altered bilateral hubness with focal-to-bilateral tonic-clonic seizures. Notably, greater atrophy and hubness of the ipsilateral thalamus was associated with seizure recurrence after temporal lobe surgery.
Conclusions: This study offers a detailed examination of thalamic alterations in paediatric TLE, emphasizing ipsilateral atrophy and abnormal bilateral functional connectivity. Thalamic subfields exhibited differential sensitivity to TLE phenotypes, reflecting their specific roles in the disorder. Combined functional and structural imaging markers may provide insights into brain network reorganization underlying disease severity in paediatric TLE.
