Generation of a human induced pluripotent stem cell line from a patient with GM3 synthase deficiency using self-replicating RNA vector.

GM3 synthase deficiency (GM3SD) is caused by biallelic variants in the ST3GAL5 gene. Early clinical features of GM3SD include infantile onset of severe irritability and feeding difficulties, early intractable seizures, growth failure, hypotonia, sensorineural hearing impairment. We describe the generation and characterization the human induced pluripotent stem cell (hiPSC) line derived from fibroblasts of a 13-year-old girl with GM3 synthase deficiency resulted compound heterozygous for two new variants in the ST3GAL5 gene, c.1166A > G (p.His389Arg) and the c.1024G > A (p.Gly342Ser). The generated hiPSC line shows a normal karyotype, expresses pluripotency markers, and is able to differentiate into the three germ layers.

Additional Results from Two Randomized, Placebo-Controlled Trials of Stiripentol in Dravet Syndrome Highlight a Rapid Antiseizure Efficacy with Longer Seizure-Free Periods.

INTRODUCTION: The efficacy of stiripentol in Dravet syndrome children was evidenced in two randomized, double-blind, placebo-controlled, phase 3 studies, namely STICLO France (October 1996-August 1998) and STICLO Italy (April 1999-October 2000), but data were not fully exploited at the time. METHODS: This post-hoc analysis used additional information, notably collected during the open-label extension (OLE) month, or reported by caregivers in individual diaries, to evaluate new outcomes. RESULTS: Overall, 64 patients were included (31 in the placebo group, 33 in the stiripentol group) of whom 34 (53.1%) were female. Patients' mean and median (25%; 75%) age were 9.2 years (range 3.0-20.7 years) and 8.7 years (6.0; 12.1) respectively. At the end of the double-blind treatment period, 72% of the patients in the stiripentol group had a ≥ 50% decrease in generalized tonic-clonic seizure (GTCS) frequency, versus 7% in the placebo group (P < 0.001), 56% had a profound (≥ 75%) decrease versus 3% in the placebo group (P < 0.001), and 38% were free of GTCS, but none in the placebo group (P < 0.001). The onset of stiripentol efficacy was rapid, significant from the fourth day of treatment onwards. The median longest period of consecutive days with no GTCS was 32 days in the stiripentol group compared to 8.5 days in the placebo group (P < 0.001). Further to the switch to the third month OLE, an 80.2% decrease in seizure frequency from baseline was observed in patients previously receiving placebo, while no change in efficacy was observed in those already on stiripentol. Adverse events were more frequent in the stiripentol group, with significantly more episodes of somnolence, anorexia, and weight decrease than in the placebo group. CONCLUSION: Altogether these new analyses of the STICLO data reinforce the evidence for a remarkable efficacy of stiripentol in Dravet syndrome, with a demonstrated rapid onset of action and sustained response, as also evidenced in further post-randomized trials.

Severe A(H1N1)pdm09 influenza acute encephalopathy outbreak in children in Tuscany, Italy, December 2023 to January 2024.

A severe outbreak of influenza A(H1N1pdm09) infection in seven children (median age: 52 months) occurred between December 2023 and January 2024 in Tuscany, Italy. Clinical presentation ranged from milder encephalopathy to acute necrotizing encephalopathy (ANE) with coma and multiorgan failure; one child died. This report raises awareness for clinicians to identify and treat early acute encephalopathy caused by H1N1 influenza and serves as a reminder of severe presentations of influenza in young children and the importance of vaccination.

Unilateral deep brain stimulation (DBS) of nucleus ventralis intermedius thalami (Vim) for the treatment of post-traumatic tremor in children: a multicentre experience.

PURPOSE: Deep brain stimulation (DBS) of nucleus ventralis intermedius thalami (Vim) is a validated technique for the treatment of essential tremor (ET) in adults. Conversely, its use for post traumatic tremor (PTT) and in paediatric patients is still debated. We evaluated the efficacy of Vim-DBS for lesional tremor in three paediatric patients with drug-resistant post-traumatic unilateral tremor. METHODS: We retrospectively collected data regarding three patients with unilateral tremor due to severe head injury, with no MRI evidence of basal ganglia lesions. The three patients underwent stereotactic frame-based robot-assisted DBS of Vim contralateral to the tremor side. RESULTS: Mean follow-up was 48 months (range: 36-60 months). Tremor was reduced in all patients with a better control of voluntary movements and improvement of functional status (mean FIM scale improvement + 7 points). No surgical complications occurred. CONCLUSION: Unilateral contralateral DBS of Vim could be efficacious in post-traumatic tremor, even in paediatric patients and should be offered in PTT drug-resistant patients.

Clinical and molecular characterization of patients with YWHAG-related epilepsy.

OBJECTIVE: YWHAG variant alleles have been associated with a rare disease trait whose clinical synopsis includes an early onset epileptic encephalopathy with predominantly myoclonic seizures, developmental delay/intellectual disability, and facial dysmorphisms. Through description of a large cohort, which doubles the number of reported patients, we further delineate the spectrum of YWHAG-related epilepsy. METHODS: We included in this study 24 patients, 21 new and three previously described, with pathogenic/likely pathogenic variants in YWHAG. We extended the analysis of clinical, electroencephalographic, brain magnetic resonance imaging, and molecular genetic information to 24 previously published patients. RESULTS: The phenotypic spectrum of YWHAG-related disorders ranges from mild developmental delay to developmental and epileptic encephalopathy (DEE). Epilepsy onset is in the first 2 years of life. Seizure freedom can be achieved in half of the patients (13/24, 54%). Intellectual disability (23/24, 96%), behavioral disorders (18/24, 75%), neurological signs (13/24, 54%), and dysmorphisms (6/24, 25%) are common. A genotype-phenotype correlation emerged, as DEE is more represented in patients with missense variants located in the ligand-binding domain than in those with truncating or missense variants in other domains (90% vs. 19%, p < .001). SIGNIFICANCE: This study suggests that pathogenic YWHAG variants cause a wide range of clinical presentations with variable severity, ranging from mild developmental delay to DEE. In this allelic series, a genotype-phenotype correlation begins to emerge, potentially providing prognostic information for clinical management and genetic counseling.

Comparative efficacy and safety of stiripentol, cannabidiol and fenfluramine as first-line add-on therapies for seizures in Dravet syndrome: A network meta-analysis.

OBJECTIVES: Stiripentol, fenfluramine, and cannabidiol are licensed add-on therapies to treat seizures in Dravet Syndrome (DS). There are no direct or indirect comparisons assessing their full licensed dose regimens, across different jurisdictions, as first-line add-on therapies in DS. METHODS: We conducted a systematic review and frequentist network meta-analysis (NMA) of randomized controlled trial (RCT) data for licensed add-on DS therapies. We compared the proportions of patients experiencing: reductions from baseline in monthly convulsive seizure frequency (MCSF) of ≥50% (clinically meaningful), ≥75% (profound), and 100% (seizure-free); serious adverse events (SAEs); discontinuations due to AEs. RESULTS: We identified relevant data from two placebo-controlled RCTs for each drug. Stiripentol 50 mg/kg/day and fenfluramine 0.7 mg/kg/day had similar efficacy in achieving ≥50% (clinically meaningful) and ≥75% (profound) reductions from baseline in MCSF (absolute risk difference [RD] for stiripentol versus fenfluramine 1% [95% confidence interval: -20% to 22%; p = 0.93] and 6% [-15% to 27%; p = 0.59], respectively), and both were statistically superior (p < 0.05) to licensed dose regimens of cannabidiol (10 or 20 mg/kg/day, with/irrespective of clobazam) for these outcomes. Stiripentol was statistically superior in achieving seizure-free intervals compared to fenfluramine (RD = 26% [CI: 8% to 44%; p < 0.01]) and licensed dose regimens of cannabidiol. There were no significant differences in the proportions of patients experiencing SAEs. The risk of discontinuations due to AEs was lower for stiripentol, although the stiripentol trials were shorter. SIGNIFICANCE: This NMA of RCT data indicates stiripentol, as a first-line add-on therapy in DS, is at least as effective as fenfluramine and both are more effective than cannabidiol in reducing convulsive seizures. No significant difference in the incidence of SAEs between the three add-on agents was observed, but stiripentol may have a lower risk of discontinuations due to AEs. These results may inform clinical decision-making and the continued development of guidelines for the treatment of people with DS. PLAIN LANGUAGE SUMMARY: This study compared three drugs (stiripentol, fenfluramine, and cannabidiol) used alongside other medications for managing seizures in a severe type of epilepsy called DS. The study found that stiripentol and fenfluramine were similarly effective in reducing seizures and both were more effective than cannabidiol. Stiripentol was the best drug for stopping seizures completely based on the available clinical trial data. All three drugs had similar rates of serious side effects, but stiripentol had a lower chance of being stopped due to side effects. This information can help guide treatment choices for people with DS.

A WORLDWIDE ENIGMA STUDY ON EPILEPSY-RELATED GRAY AND WHITE MATTER COMPROMISE ACROSS THE ADULT LIFESPAN.

Objectives: Temporal lobe epilepsy (TLE) is commonly associated with mesiotemporal pathology and widespread alterations of grey and white matter structures. Evidence supports a progressive condition although the temporal evolution of TLE is poorly defined. This ENIGMA-Epilepsy study utilized multimodal magnetic resonance imaging (MRI) data to investigate structural alterations in TLE patients across the adult lifespan. We charted both grey and white matter changes and explored the covariance of age-related alterations in both compartments. Methods: We studied 769 TLE patients and 885 healthy controls across an age range of 17-73 years, from multiple international sites. To assess potentially non-linear lifespan changes in TLE, we harmonized data and combined median split assessments with cross-sectional sliding window analyses of grey and white matter age-related changes. Covariance analyses examined the coupling of grey and white matter lifespan curves. Results: In TLE, age was associated with a robust grey matter thickness/volume decline across a broad cortico-subcortical territory, extending beyond the mesiotemporal disease epicentre. White matter changes were also widespread across multiple tracts with peak effects in temporo-limbic fibers. While changes spanned the adult time window, changes accelerated in cortical thickness, subcortical volume, and fractional anisotropy (all decreased), and mean diffusivity (increased) after age 55 years. Covariance analyses revealed strong limbic associations between white matter tracts and subcortical structures with cortical regions. Conclusions: This study highlights the profound impact of TLE on lifespan changes in grey and white matter structures, with an acceleration of aging-related processes in later decades of life. Our findings motivate future longitudinal studies across the lifespan and emphasize the importance of prompt diagnosis as well as intervention in patients.

Patterns of subregional cerebellar atrophy across epilepsy syndromes: An ENIGMA-Epilepsy study.

OBJECTIVE: The intricate neuroanatomical structure of the cerebellum is of longstanding interest in epilepsy, but has been poorly characterized within the current corticocentric models of this disease. We quantified cross-sectional regional cerebellar lobule volumes using structural magnetic resonance imaging in 1602 adults with epilepsy and 1022 healthy controls across 22 sites from the global ENIGMA-Epilepsy working group. METHODS: A state-of-the-art deep learning-based approach was employed that parcellates the cerebellum into 28 neuroanatomical subregions. Linear mixed models compared total and regional cerebellar volume in (1) all epilepsies, (2) temporal lobe epilepsy with hippocampal sclerosis (TLE-HS), (3) nonlesional temporal lobe epilepsy, (4) genetic generalized epilepsy, and (5) extratemporal focal epilepsy (ETLE). Relationships were examined for cerebellar volume versus age at seizure onset, duration of epilepsy, phenytoin treatment, and cerebral cortical thickness. RESULTS: Across all epilepsies, reduced total cerebellar volume was observed (d = .42). Maximum volume loss was observed in the corpus medullare (dmax = .49) and posterior lobe gray matter regions, including bilateral lobules VIIB (dmax = .47), crus I/II (dmax = .39), VIIIA (dmax = .45), and VIIIB (dmax = .40). Earlier age at seizure onset ( η ρ max 2 = .05) and longer epilepsy duration ( η ρ max 2 = .06) correlated with reduced volume in these regions. Findings were most pronounced in TLE-HS and ETLE, with distinct neuroanatomical profiles observed in the posterior lobe. Phenytoin treatment was associated with reduced posterior lobe volume. Cerebellum volume correlated with cerebral cortical thinning more strongly in the epilepsy cohort than in controls. SIGNIFICANCE: We provide robust evidence of deep cerebellar and posterior lobe subregional gray matter volume loss in patients with chronic epilepsy. Volume loss was maximal for posterior subregions implicated in nonmotor functions, relative to motor regions of both the anterior and posterior lobe. Associations between cerebral and cerebellar changes, and variability of neuroanatomical profiles across epilepsy syndromes argue for more precise incorporation of cerebellar subregional damage into neurobiological models of epilepsy.

Genotype-phenotype associations in 1018 individuals with SCN1A-related epilepsies.

OBJECTIVE: SCN1A variants are associated with epilepsy syndromes ranging from mild genetic epilepsy with febrile seizures plus (GEFS+) to severe Dravet syndrome (DS). Many variants are de novo, making early phenotype prediction difficult, and genotype-phenotype associations remain poorly understood. METHODS: We assessed data from a retrospective cohort of 1018 individuals with SCN1A-related epilepsies. We explored relationships between variant characteristics (position, in silico prediction scores: Combined Annotation Dependent Depletion (CADD), Rare Exome Variant Ensemble Learner (REVEL), SCN1A genetic score), seizure characteristics, and epilepsy phenotype. RESULTS: DS had earlier seizure onset than other GEFS+ phenotypes (5.3 vs. 12.0 months, p < .001). In silico variant scores were higher in DS versus GEFS+ (p < .001). Patients with missense variants in functionally important regions (conserved N-terminus, S4-S6) exhibited earlier seizure onset (6.0 vs. 7.0 months, p = .003) and were more likely to have DS (280/340); those with missense variants in nonconserved regions had later onset (10.0 vs. 7.0 months, p = .036) and were more likely to have GEFS+ (15/29, χ2 = 19.16, p < .001). A minority of protein-truncating variants were associated with GEFS+ (10/393) and more likely to be located in the proximal first and last exon coding regions than elsewhere in the gene (9.7% vs. 1.0%, p < .001). Carriers of the same missense variant exhibited less variability in age at seizure onset compared with carriers of different missense variants for both DS (1.9 vs. 2.9 months, p = .001) and GEFS+ (8.0 vs. 11.0 months, p = .043). Status epilepticus as presenting seizure type is a highly specific (95.2%) but nonsensitive (32.7%) feature of DS. SIGNIFICANCE: Understanding genotype-phenotype associations in SCN1A-related epilepsies is critical for early diagnosis and management. We demonstrate an earlier disease onset in patients with missense variants in important functional regions, the occurrence of GEFS+ truncating variants, and the value of in silico prediction scores. Status epilepticus as initial seizure type is a highly specific, but not sensitive, early feature of DS.

Morphometric network-based abnormalities correlate with psychiatric comorbidities and gene expression in PCDH19-related developmental and epileptic encephalopathy.

Protocadherin-19 (PCDH19) developmental and epileptic encephalopathy causes an early-onset epilepsy syndrome with limbic seizures, typically occurring in clusters and variably associated with intellectual disability and a range of psychiatric disorders including hyperactive, obsessive-compulsive and autistic features. Previous quantitative neuroimaging studies revealed abnormal cortical areas in the limbic formation (parahippocampal and fusiform gyri) and underlying white-matter fibers. In this study, we adopted morphometric, network-based and multivariate statistical methods to examine the cortex and substructure of the hippocampus and amygdala in a cohort of 20 PCDH19-mutated patients and evaluated the relation between structural patterns and clinical variables at individual level. We also correlated morphometric alterations with known patterns of PCDH19 expression levels. We found patients to exhibit high-significant reductions of cortical surface area at a whole-brain level (left/right pvalue = 0.045/0.084), and particularly in the regions of the limbic network (left/right parahippocampal gyri pvalue = 0.230/0.016; left/right entorhinal gyri pvalue = 0.002/0.327), and bilateral atrophy of several subunits of the amygdala and hippocampus, particularly in the CA regions (head of the left CA3 pvalue = 0.002; body of the right CA3 pvalue = 0.004), and differences in the shape of hippocampal structures. More severe psychiatric comorbidities correlated with more significant altered patterns, with the entorhinal gyrus (pvalue = 0.013) and body of hippocampus (pvalue = 0.048) being more severely affected. Morphometric alterations correlated significantly with the known expression patterns of PCDH19 (rvalue = -0.26, pspin = 0.092). PCDH19 encephalopathy represents a model of genetically determined neural network based neuropsychiatric disease in which quantitative MRI-based findings correlate with the severity of clinical manifestations and had have a potential predictive value if analyzed early.

Outcome of Epilepsy Surgery in MRI-Negative Patients Without Histopathologic Abnormalities in the Resected Tissue.

BACKGROUND AND OBJECTIVE: Patients with presumed nonlesional focal epilepsy-based on either MRI or histopathologic findings-have a lower success rate of epilepsy surgery compared with lesional patients. In this study, we aimed to characterize a large group of patients with focal epilepsy who underwent epilepsy surgery despite a normal MRI and had no lesion on histopathology. Determinants of their postoperative seizure outcomes were further studied. METHODS: We designed an observational multicenter cohort study of MRI-negative and histopathology-negative patients who were derived from the European Epilepsy Brain Bank and underwent epilepsy surgery between 2000 and 2012 in 34 epilepsy surgery centers within Europe. We collected data on clinical characteristics, presurgical assessment, including genetic testing, surgery characteristics, postoperative outcome, and treatment regimen. RESULTS: Of the 217 included patients, 40% were seizure-free (Engel I) 2 years after surgery and one-third of patients remained seizure-free after 5 years. Temporal lobe surgery (adjusted odds ratio [AOR]: 2.62; 95% CI 1.19-5.76), shorter epilepsy duration (AOR for duration: 0.94; 95% CI 0.89-0.99), and completely normal histopathologic findings-versus nonspecific reactive gliosis-(AOR: 4.69; 95% CI 1.79-11.27) were significantly associated with favorable seizure outcome at 2 years after surgery. Of patients who underwent invasive monitoring, only 35% reached seizure freedom at 2 years. Patients with parietal lobe resections had lowest seizure freedom rates (12.5%). Among temporal lobe surgery patients, there was a trend toward favorable outcome if hippocampectomy was part of the resection strategy (OR: 2.94; 95% CI 0.98-8.80). Genetic testing was only sporadically performed. DISCUSSION: This study shows that seizure freedom can be reached in 40% of nonlesional patients with both normal MRI and histopathology findings. In particular, nonlesional temporal lobe epilepsy should be regarded as a relatively favorable group, with almost half of patients achieving seizure freedom at 2 years after surgery-even more if the hippocampus is resected-compared with only 1 in 5 nonlesional patients who underwent extratemporal surgery. Patients with an electroclinically identified focus, who are nonlesional, will be a promising group for advanced molecular-genetic analysis of brain tissue specimens to identify new brain somatic epilepsy genes or epilepsy-associated molecular pathways.

Leat-associated seizures the possible role of EAAT2, pyruvate carboxylase and glutamine synthetase.

BACKGROUND: Drug-resistant epilepsy is a common condition in patients with brain neoplasms. The pathogenesis of tumor-associated seizures is poorly understood. Among the possible pathogenetic mechanisms, the increase in glutamate concentration has been proposed. Glutamate transporters, glutamine synthetase and pyruvate carboxylase are involved in maintaining the physiological concentration of glutamate in the intersynaptic spaces. In our previous research on angiocentric gliomas, we demonstrated that all tumors lacked the expression of the main glutamate transporter EAAT2, while the expression of glutamine synthetase and pyruvate carboxylase was mostly preserved. METHODS: In the present study, we evaluated the immunohistochemical expression of EAAT2, glutamine synthetase and pyruvate carboxylase in a heterogeneous series of 25 long-term epilepsy-associated tumors (10 dysembryoplastic neuroepithelial tumors, 7 gangliogliomas, 3 subependymal giant cell astrocytomas, 3 rosette forming glioneuronal tumors, 1 diffuse astrocytoma MYB- or MYBL1-altered and 1 angiocentric glioma). In order to evaluate the incidence of variants in the SLC1A2 gene, encoding EAAT2, in a large number of central nervous system tumors we also queried the PedcBioPortal. RESULTS: EAAT2 protein expression was lost in 9 tumors (36 %: 3 dysembryoplastic neuroepithelial tumors, 1 ganglioglioma, 3 subependymal giant cell astrocytomas, 1 diffuse astrocytoma MYB- or MYBL1-altered and 1 angiocentric glioma). Glutamine synthetase protein expression was completely lost in 2 tumors (8 %; 1 ganglioglioma and 1 diffuse astrocytoma MYB- or MYBL1-altered). All tumors of our series but rosette forming glioneuronal tumors (in which neurocytic cells were negative) were diffusely positive for pyruvate carboxylase. Consultation of the PedcBioPortal revealed that of 2307 pediatric brain tumors of different histotype and grade, 20 (< 1%) had variants in the SLC1A2 gene. Among the SLC1A2-mutated tumors, there were no angiocentric gliomas or other LEATs CONCLUSIONS: In conclusion, unlike angiocentric gliomas where the EAAT2 loss is typical and constant, the current study shows the loss of EAAT2 expression only in a fraction of the LEATs. In these cases, we may hypothesize some possible epileptogenic role of the EAAT2 loss. The retained expression of pyruvate carboxylase may contribute to determining a pathological glutamate excess unopposed by glutamine synthetase that resulted expressed to a variable extent in the majority of the tumors. Furthermore, we can assume that the EAAT2 loss in brain tumors in general and in LEATs in particular is more conceivably epigenetic.

Bilateral temporal lobe dysplasia and seizure onset associated with biallelic CNTNAP2 variants.

Biallelic CNTNAP2 variants have been associated with Pitt-Hopkins-like syndrome. We describe six novel and one previously reported patients from six independent families and review the literature including 64 patients carrying biallelic CNTNAP2 variants. Initial reports highlighted intractable focal seizures and the failure of epilepsy surgery in children, but subsequent reports did not expand on this aspect. In all our patients (n = 7), brain MRI showed bilateral temporal gray/white matter blurring with white matter high signal intensity, more obvious on the T2-FLAIR sequences, consistent with bilateral temporal lobe dysplasia. All patients had focal seizures with temporal lobe onset and semiology, which were recorded on EEG in five, showing bilateral independent temporal onset in four. Epilepsy was responsive to anti-seizure medications in two patients (2/7, 28.5%), and pharmaco-resistant in five (5/7, 71.5%). Splice-site variants identified in five patients (5/7, 71.5%) were the most common mutational finding. Our observation expands the phenotypic and genetic spectrum of biallelic CNTNAP2 alterations focusing on the neuroimaging features and provides evidence for an elective bilateral anatomoelectroclinical involvement of the temporal lobes in the associated epilepsy, with relevant implications on clinical management.

Developmental epileptic encephalopathy in DLG4-related synaptopathy.

OBJECTIVE: The postsynaptic density protein of excitatory neurons PSD-95 is encoded by discs large MAGUK scaffold protein 4 (DLG4), de novo pathogenic variants of which lead to DLG4-related synaptopathy. The major clinical features are developmental delay, intellectual disability (ID), hypotonia, sleep disturbances, movement disorders, and epilepsy. Even though epilepsy is present in 50% of the individuals, it has not been investigated in detail. We describe here the phenotypic spectrum of epilepsy and associated comorbidities in patients with DLG4-related synaptopathy. METHODS: We included 35 individuals with a DLG4 variant and epilepsy as part of a multicenter study. The DLG4 variants were detected by the referring laboratories. The degree of ID, hypotonia, developmental delay, and motor disturbances were evaluated by the referring clinician. Data on awake and sleep electroencephalography (EEG) and/or video-polygraphy and brain magnetic resonance imaging were collected. Antiseizure medication response was retrospectively assessed by the referring clinician. RESULTS: A large variety of seizure types was reported, although focal seizures were the most common. Encephalopathy related to status epilepticus during slow-wave sleep (ESES)/developmental epileptic encephalopathy with spike-wave activation during sleep (DEE-SWAS) was diagnosed in >25% of the individuals. All but one individual presented with neurodevelopmental delay. Regression in verbal and/or motor domains was observed in all individuals who suffered from ESES/DEE-SWAS, as well as some who did not. We could not identify a clear genotype-phenotype relationship even between individuals with the same DLG4 variants. SIGNIFICANCE: Our study shows that a subgroup of individuals with DLG4-related synaptopathy have DEE, and approximately one fourth of them have ESES/DEE-SWAS. Our study confirms DEE as part of the DLG4-related phenotypic spectrum. Occurrence of ESES/DEE-SWAS in DLG4-related synaptopathy requires proper investigation with sleep EEG.

Structural and non-coding variants increase the diagnostic yield of clinical whole genome sequencing for rare diseases.

BACKGROUND: Whole genome sequencing is increasingly being used for the diagnosis of patients with rare diseases. However, the diagnostic yields of many studies, particularly those conducted in a healthcare setting, are often disappointingly low, at 25-30%. This is in part because although entire genomes are sequenced, analysis is often confined to in silico gene panels or coding regions of the genome. METHODS: We undertook WGS on a cohort of 122 unrelated rare disease patients and their relatives (300 genomes) who had been pre-screened by gene panels or arrays. Patients were recruited from a broad spectrum of clinical specialties. We applied a bioinformatics pipeline that would allow comprehensive analysis of all variant types. We combined established bioinformatics tools for phenotypic and genomic analysis with our novel algorithms (SVRare, ALTSPLICE and GREEN-DB) to detect and annotate structural, splice site and non-coding variants. RESULTS: Our diagnostic yield was 43/122 cases (35%), although 47/122 cases (39%) were considered solved when considering novel candidate genes with supporting functional data into account. Structural, splice site and deep intronic variants contributed to 20/47 (43%) of our solved cases. Five genes that are novel, or were novel at the time of discovery, were identified, whilst a further three genes are putative novel disease genes with evidence of causality. We identified variants of uncertain significance in a further fourteen candidate genes. The phenotypic spectrum associated with RMND1 was expanded to include polymicrogyria. Two patients with secondary findings in FBN1 and KCNQ1 were confirmed to have previously unidentified Marfan and long QT syndromes, respectively, and were referred for further clinical interventions. Clinical diagnoses were changed in six patients and treatment adjustments made for eight individuals, which for five patients was considered life-saving. CONCLUSIONS: Genome sequencing is increasingly being considered as a first-line genetic test in routine clinical settings and can make a substantial contribution to rapidly identifying a causal aetiology for many patients, shortening their diagnostic odyssey. We have demonstrated that structural, splice site and intronic variants make a significant contribution to diagnostic yield and that comprehensive analysis of the entire genome is essential to maximise the value of clinical genome sequencing.

Patterns of subregional cerebellar atrophy across epilepsy syndromes: An ENIGMA-Epilepsy study.

Objective: The intricate neuroanatomical structure of the cerebellum is of longstanding interest in epilepsy, but has been poorly characterized within the current cortico-centric models of this disease. We quantified cross-sectional regional cerebellar lobule volumes using structural MRI in 1,602 adults with epilepsy and 1,022 healthy controls across twenty-two sites from the global ENIGMA-Epilepsy working group. Methods: A state-of-the-art deep learning-based approach was employed that parcellates the cerebellum into 28 neuroanatomical subregions. Linear mixed models compared total and regional cerebellar volume in i) all epilepsies; ii) temporal lobe epilepsy with hippocampal sclerosis (TLE-HS); iii) non-lesional temporal lobe epilepsy (TLE-NL); iv) genetic generalised epilepsy; and (v) extra-temporal focal epilepsy (ETLE). Relationships were examined for cerebellar volume versus age at seizure onset, duration of epilepsy, phenytoin treatment, and cerebral cortical thickness. Results: Across all epilepsies, reduced total cerebellar volume was observed (d=0.42). Maximum volume loss was observed in the corpus medullare (dmax=0.49) and posterior lobe grey matter regions, including bilateral lobules VIIB (dmax= 0.47), Crus I/II (dmax= 0.39), VIIIA (dmax=0.45) and VIIIB (dmax=0.40). Earlier age at seizure onset (ηρ2max=0.05) and longer epilepsy duration (ηρ2max=0.06) correlated with reduced volume in these regions. Findings were most pronounced in TLE-HS and ETLE with distinct neuroanatomical profiles observed in the posterior lobe. Phenytoin treatment was associated with reduced posterior lobe volume. Cerebellum volume correlated with cerebral cortical thinning more strongly in the epilepsy cohort than in controls. Significance: We provide robust evidence of deep cerebellar and posterior lobe subregional grey matter volume loss in patients with chronic epilepsy. Volume loss was maximal for posterior subregions implicated in non-motor functions, relative to motor regions of both the anterior and posterior lobe. Associations between cerebral and cerebellar changes, and variability of neuroanatomical profiles across epilepsy syndromes argue for more precise incorporation of cerebellum subregions into neurobiological models of epilepsy.

Steps to Improve Precision Medicine in Epilepsy.

Precision medicine is an old concept, but it is not widely applied across human health conditions as yet. Numerous attempts have been made to apply precision medicine in epilepsy, this has been based on a better understanding of aetiological mechanisms and deconstructing disease into multiple biological subsets. The scope of precision medicine is to provide effective strategies for treating individual patients with specific agent(s) that are likely to work best based on the causal biological make-up. We provide an overview of the main applications of precision medicine in epilepsy, including the current limitations and pitfalls, and propose potential strategies for implementation and to achieve a higher rate of success in patient care. Such strategies include establishing a definition of precision medicine and its outcomes; learning from past experiences, from failures and from other fields (e.g. oncology); using appropriate precision medicine strategies (e.g. drug repurposing versus traditional drug discovery process); and using adequate methods to assess efficacy (e.g. randomised controlled trials versus alternative trial designs). Although the progress of diagnostic techniques now allows comprehensive characterisation of each individual epilepsy condition from a molecular, biological, structural and clinical perspective, there remain challenges in the integration of individual data in clinical practice to achieve effective applications of precision medicine in this domain.