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Aortic aneurysms are rare manifestations in children with tuberous sclerosis complex (TSC) with life threating implications. Although an association between TSC, aortic and other aneurysms has been recognized, mechanistic insights explaining the pathophysiology behind aneurysm development and genetic aberrations in TSC have so far been lacking. Here, we summarize existing knowledge on aneurysms in TSC and present a case of a 2-year-old boy with an infrarenal aortic aneurysm, successfully treated with open aortic reconstruction. Histologic examination of the excised aneurysm wall showed distortion of vessel wall structure with loss of elastin and a pathologic accumulation of smooth muscle cells. Until now, these pathologic features have puzzled researchers as proliferating smooth muscle cells would rather be expected to preserve vessel wall integrity. Recent reports exploring the biological consequences of the dysregulated intracellular signaling pathways in patients with TSC provide plausible explanations to this paradox, which may support the development of future therapeutic strategies.

TOSCA; Epilepsia; Registro TOSCA; Epilèpsia; Registre TOSCA; Epilepsy; Registry Background: Epilepsy is the most common neurological manifestation in individuals with tuberous sclerosis complex (TSC). However, real-world evidence on diagnosis and treatment patterns is limited. Here, we present data from TuberOus Sclerosis registry to increase disease Awareness (TOSCA) on changes in patterns of epilepsy diagnosis, treatments, and outcomes over time, and detailed epilepsy characteristics from the epilepsy substudy. Methods: TuberOus Sclerosis registry to increase disease Awareness (TOSCA) was a multicentre, international disease registry, consisting of a main study that collected data on overall diagnostic characteristics and associated clinical features, and six substudies focusing on specific TSC manifestations. The epilepsy substudy investigated detailed epilepsy characteristics and their correlation to genotype and intelligence quotient (IQ). Results: Epilepsy was reported in 85% of participants, more commonly in younger individuals (67.8% in 1970s to 91.8% in last decade), while rate of treatments was similar across ages (>93% for both infantile spasms and focal seizures, except prior to 1960). Vigabatrin (VGB) was the most commonly used antiepileptic drugs (AEDs). Individuals with infantile spasms showed a higher treatment response over time with lower usage of steroids. Individuals with focal seizures reported similar rates of drug resistance (32.5–43.3%). Use of vagus nerve stimulation (VNS), ketogenic diet, and surgery remained low. Discussion: The epilepsy substudy included 162 individuals from nine countries. At epilepsy onset, most individuals with infantile spasms (73.2%) and focal seizures (74.5%) received monotherapies. Vigabatrin was first-line treatment in 45% of individuals with infantile spasms. Changes in initial AEDs were commonly reported due to inadequate efficacy. TSC1 mutations were associated with less severe epilepsy phenotypes and more individuals with normal IQ. In individuals with TSC diagnosis before seizure onset, electroencephalogram (EEG) was performed prior to seizures in only 12.5 and 25% of subsequent infantile spasms and focal seizures, respectively. Conclusions: Our study confirms the high prevalence of epilepsy in TSC individuals and less severe phenotypes with TSC1 mutations. Vigabatrin improved the outcome of infantile spasms and should be used as first-line treatment. There is, however, still a need for improving therapies in focal seizures. Electroencephalogram follow-up prior to seizure-onset should be promoted for all infants with TSC in order to facilitate preventive or early treatment. The study was funded by Novartis Pharma AG. Novartis has contributed to study design, data analysis and the decision to publish. Novartis authors reviewed the draft for submission.

Recently, evidence from both animal studies and human cases has emerged that a dysbiosis in the gut may be associated with certain forms of epilepsy. The ketogenic diet is an alternative treatment of drug-resistant epilepsy, although its precise mechanism of action has been unclear. It has now been shown that the ketogenic diet changes the composition and function of the gut microbiome in epilepsy patients. Studies in mice have demonstrated that the gut microbiota was necessary for the therapeutic effect of the diet and a mechanism of action has been proposed, providing new potential strategies for treatment. Further studies are needed to confirm the clinical relevance of this discovery.Below, we will discuss the scientific evidence of the role of the microbiome in seizure disorders, the impact of the ketogenic diet on the intestinal microbiota as well as the interactions described between commonly used antiepileptic drugs and intestinal microbial communities. We also discuss the potential of modulators of the gut microbiota as possible future anti-seizure therapeutics. Keywords: Microbiota, Epilepsy, Ketogenic diet, Metagenomics, FMTs

TOSCA; Everolimus; Estudi de seguretat post-autorització TOSCA; Everolimus; Estudio de seguridad posterior a la autorización TOSCA; Everolimus; Post-authorization safety study This non-interventional post-authorisation safety study (PASS) assessed the long-term safety of everolimus in patients with tuberous sclerosis complex (TSC) who participated in the TuberOus SClerosis registry to increase disease Awareness (TOSCA) clinical study and received everolimus for the licensed indications in the European Union. The rate of adverse events (AEs), AEs that led to dose adjustments or treatment discontinuation, AEs of potential clinical interest, treatment-related AEs (TRAEs), serious AEs (SAEs), and deaths were documented. One hundred seventy-nine patients were included in the first 5 years of observation; 118 of 179 patients had an AE of any grade, with the most common AEs being stomatitis (7.8%) and headache (7.3%). AEs caused dose adjustments in 56 patients (31.3%) and treatment discontinuation in nine patients (5%). AEs appeared to be more frequent and severe in children. On Tanner staging, all patients displayed signs of age-appropriate sexual maturation. Twenty-two of 106 female (20.8%) patients had menstrual cycle disorders. The most frequent TRAEs were stomatitis (6.7%) and aphthous mouth ulcer (5.6%). SAEs were reported in 54 patients (30.2%); the most frequent SAE was pneumonia (>3% patients; grade 2, 1.1%, and grade 3, 2.8%). Three deaths were reported, all in patients who had discontinued everolimus for more than 28 days, and none were thought to be related to everolimus according to the treating physicians. The PASS sub-study reflects the safety and tolerability of everolimus in the management of TSC in real-world routine clinical practice.

Background The low carbohydrate, high fat ketogenic diet can be an effective anticonvulsant treatment in some pediatric patients with pharmacoresistant epilepsy. Its mechanism(s) of action, however, remain uncertain. Direct sampling of cerebrospinal fluid before and during metabolic therapy may reveal key changes associated with differential clinical outcomes. We characterized the relationship between seizure responsiveness and changes in lipid and carbohydrate metabolites. Methods We performed metabolomic analysis of cerebrospinal fluid samples taken before and during ketogenic diet treatment in patients with optimal response (100% seizure remission) and patients with no response (no seizure improvement) to search for differential diet effects in hallmark metabolic compounds in these two groups. Optimal responders and non-responders were similar in age range and included males and females. Seizure types and the etiologies or syndromes of epilepsy varied but did not appear to differ systematically between responders and non-responders. Results Analysis showed a strong effect of ketogenic diet treatment on the cerebrospinal fluid metabolome. Longitudinal and between-subjects analyses revealed that many lipids and carbohydrates were changed significantly by ketogenic diet, with changes typically being of larger magnitude in responders. Notably, responders had more robust changes in glucose and the ketone bodies β-hydroxybutyrate and acetoacetate than non-responders; conversely, non-responders had significant increases in fructose and sorbose, which did not occur in responders. Conclusions The data suggest that a differential and stronger metabolic response to the ketogenic diet may predict a better anticonvulsant response, and such variability is likely due to inherent biological factors of individual patients. Strategies to boost the metabolic response may be beneficial.

Renal angiomyolipomas are one of the most common renal manifestations in patients with tuberous sclerosis complex (TSC), with potentially life-threatening complications and a poor prognosis. Despite the considerable progress in understanding TSC-associated renal angiomyolipomas, there are no large scale real-world data. The aim of our present study was to describe in detail the prevalence and outcome of renal angiomyolipomas in patients with TSC, enrolled into the TuberOus SClerosis registry to increase disease Awareness (TOSCA) from 170 sites across 31 countries worldwide. We also sought to evaluate the relationship of TSC-associated renal angiomyolipomas with age, gender and genotype. The potential risk factors for renal angiomyolipoma-related bleeding and chronic kidney disease (CKD) were studied in patients who participated in the TOSCA renal angiomyolipoma substudy. Of the 2,211 eligible patients, 1,062 (48%) reported a history of renal angiomyolipomas. The median age of TSC diagnosis for the all subjects (n = 2,211) was 1 year. The median age of diagnosis of renal angiomyolipoma in the 1,062 patients was 13 years. Renal angiomyolipomas were significantly more prevalent in female patients (p < 0.0001). Rates of angiomyolipomas >3 cm (p = 0.0119), growing lesions (p = 0.0439), and interventions for angiomyolipomas (p = 0.0058) were also higher in females than males. Pre-emptive intervention for renal angiomyolipomas with embolisation, surgery, or mammalian target of rapamycin (mTOR) inhibitor may have abolished the gender difference in impaired renal function, hypertension, and other complications. The rate of interventions for angiomyolipomas was less common in children than in adults, but interventions were reported in all age groups. In the substudy of 76 patients the complication rate was too low to be useful in predicting risk for more severe CKD. In addition, in this substudy no patient had a renal hemorrhage after commencing on an mTOR inhibitor. Our findings confirmed that renal angiomyolipomas in subjects with TSC1 mutations develop on average at the later age, are relatively smaller in size and less likely to be growing; however, by age 40 years, no difference was observed in the percentage of patients with TSC1 and TSC2 mutations needing intervention. The peak of appearance of new renal angiomyolipomas was observed in patients aged between 18 and 40 years, but, given that angiomyolipomas can occur later, lifelong surveillance is necessary. We found that pre-emptive intervention was dramatically successful in altering the outcome compared to historical controls; with high pre-emptive intervention rates but low rates of bleeding and other complications. This validates the policy of surveillance and pre-emptive intervention recommended by clinical guidelines.

ObjectivesTo describe the process and results of the updated Swedish practice guidelines for monotherapy in epilepsy.Materials and MethodsThe Swedish Medical Products Agency led the process together with medical experts. Evidence rating in accordance with the International League Against Epilepsy (ILAE) template was linked to the Cochrane group's GRADE system. Evidence from recently published trials and meta‐analyses was added. A national expert panel participated in the project and contributed their clinical experience.ResultsIn seizures with focal onset, carbamazepine, lamotrigine, or levetiracetam is recommended for children and adults (ILAE level A‐C for adults/Cochrane level strong for children and adults). Oxcarbazepine is an alternative for children, although its level A evidence, in a single class I trial, could relate to poor phenytoin tolerability. Eslicarbazepine acetate, lacosamide, and zonisamide are alternatives for adults and gabapentin for the elderly (ILAE level A). Carbamazepine is not a first choice for the elderly due to its high potential for interactions. In generalized epilepsy with tonic‐clonic seizures (GTC), lamotrigine, levetiracetam, and sodium valproate are recommended for children and adults (ILAE level C‐D/Cochrane level moderate‐strong) although sodium valproate is contraindicated in girls and women of childbearing age unless special considerations are met. Ethosuximide is the first choice in absence epilepsy without GTC (ILAE level A).ConclusionsLamotrigine and levetiracetam can be used as first choice for focal seizures and generalized epilepsy with GTC, suitable in all age‐groups and for both men and women. Recommendations for GTC seizures have lower evidence than those for focal seizures. (Less)

Background Renal angiomyolipoma occurs at a high frequency in patients with tuberous sclerosis complex (TSC) and is associated with potentially life-threatening complications. Despite this frequency and severity, there are no large population-based cohort studies. Here we present baseline and follow-up data of the international TuberOus SClerosis registry to increase disease Awareness (TOSCA) with an aim to provide detailed clinical characteristics of renal angiomyolipoma among patients with TSC. Methods Patients of any age with a documented clinic visit for TSC within 12 months or who were newly diagnosed with TSC before participation in the registry were eligible. Data specific to renal angiomyolipoma included physical tumour characteristics (multiple, bilateral, lesion size and growing lesions), clinical signs and symptoms, and management. The effects of age, gender and genotype on the prevalence of renal angiomyolipoma were also evaluated. Results Renal angiomyolipoma was reported in 51.8% of patients at baseline, with higher frequency in female patients (57.8% versus 42.2%). The median age at diagnosis was 12 years. Prevalence of angiomyolipoma was higher in patients with TSC2 compared with TSC1 mutations (59.2% versus 33.3%, P 3 cm in 34.3% of patients. Most patients were asymptomatic (82%). Frequently reported angiomyolipoma-related symptoms included bleeding, pain, elevated blood pressure and impaired renal function. Embolization and mammalian target of rapamycin inhibitors were the two most common treatment modalities. Conclusions The TOSCA registry highlights the burden of renal angiomyolipoma in patients with TSC and shows that renal manifestations are initially asymptomatic and are influenced by gender and genotype. Furthermore, the occurrence of significant problems from angiomyolipoma in a minority of younger patients suggests that surveillance should begin in infancy or at initial diagnosis.

Ketogenic diet is an effective treatment which has the potential to achieve a significant seizure reduction in drug-resistant epilepsy. The mechanism behind this effect is unclear, but one hypothesis is that the mechanism is anti-inflammatory. In this prospective study on pediatric patients we compared levels of cytokines and chemokines in the cerebrospinal fluid before and after three months on treatment to evaluate a possible anti-inflammatory effect. We analyzed 34 cytokines and chemokines in the cerebrospinal fluid of pediatric patients (n = 21) with refractory epilepsy by a multiplex assay. Beta-hydroxybutyric acid was measured in blood and cerebrospinal fluid. Seizure frequency in relation to diet treatment was assessed. For 9 different cytokines (CCL 7, CCL 21, CCL 22, CCL 25, CCL 27, IL-2, IL-10, CX3CL1 and MIF), a significant decrease ranging from 7 to 27% was seen after three months as compared to levels before the diet. In contrast, no cytokine displayed a significant increase during diet. A seizure reduction ≥ 50 % was seen in 15/21 patients (71 %) but no significant differences in cytokine decreases were found between responders and non-responders during treatment. A non-significant trend towards higher initial pre-treatment levels of cytokines was seen in responders, which were reduced following treatment. The levels of betahydroxybutyric acid were not related to seizure response. We conclude that while it is not possible to state a primary anti-inflammatory effect by dietary treatment from these data, an unequivocal immunological effect is seen and may be a part of the mechanism of ketogenic dietary treatment.

Introduction: The TuberOus SClerosis registry to increase disease Awareness (TOSCA) is an international disease registry designed to provide insights into the clinical characteristics of patients with Tuberous Sclerosis Complex (TSC). The aims of this study were to identify issues that arose during the design, execution, and publication phases of TOSCA, and to reflect on lessons learnt that may guide future registries in rare and complex diseases. Methods: A questionnaire was designed to identify the strengths, weaknesses, and issues that arose at any stage of development and implementation of the TOSCA registry. The questionnaire contained 225 questions distributed in 7 sections (identification of issues during registry planning, during the operation of the registry, during data analysis, during the publication of the results, other issues, assessment of lessons learnt, and additional comments), and was sent by e-mail to 511 people involved in the registry, including 28 members of the Scientific Advisory Board (SAB), 162 principal investigators (PIs), and 321 employees of the sponsor belonging to the medical department or that were clinical research associate (CRA). Questionnaires received within the 2 months from the initial mailing were included in the analysis. Results: A total of 53 (10.4%) questionnaires were received (64.3% for SAB members, 12.3% for PIs and 4.7% for employees of the sponsor), and the overall completeness rate for closed questions was 87.6%. The most common issues identified were the limited duration of the registry (38%) and issues related to handling of missing data (32%). In addition, 25% of the respondents commented that biases might have compromised the validity of the results. More than 80% of the respondents reported that the registry improved the knowledge on the natural history and manifestations of TSC, increased disease awareness and helped to identify relevant information for clinical research in TSC. Conclusions: This analysis shows the importance of registries as a powerful tool to increase disease awareness, to produce real-world evidence, and to generate questions for future research. However, there is a need to implement strategies to ensure patient retention and long-term sustainability of patient registries, to improve data quality, and to reduce biases.

The gut microbiota has been linked to various neurological disorders via the gut–brain axis. Diet influences the composition of the gut microbiota. The ketogenic diet (KD) is a high-fat, adequate-protein, low-carbohydrate diet established for treatment of therapy-resistant epilepsy in children. Its efficacy in reducing seizures has been confirmed, but the mechanisms remain elusive. The diet has also shown positive effects in a wide range of other diseases, including Alzheimer’s, depression, autism, cancer, and type 2 diabetes. We collected fecal samples from 12 children with therapy-resistant epilepsy before starting KD and after 3 months on the diet. Parents did not start KD and served as diet controls. Applying shotgun metagenomic DNA sequencing, both taxonomic and functional profiles were established. Here we report that alpha diversity is not changed significantly during the diet, but differences in both taxonomic and functional composition are detected. Relative abundance of bifidobacteria as well as E. rectale and Dialister is significantly diminished during the intervention. An increase in relative abundance of E. coli is observed on KD. Functional analysis revealed changes in 29 SEED subsystems including the reduction of seven pathways involved in carbohydrate metabolism. Decomposition of these shifts indicates that bifidobacteria and Escherichia are important contributors to the observed functional shifts. As relative abundance of health-promoting, fiber-consuming bacteria becomes less abundant during KD, we raise concern about the effects of the diet on the gut microbiota and overall health. Further studies need to investigate whether these changes are necessary for the therapeutic effect of KD., Gut–brain axis: The ketogenic diet alters functional gut microbiota The ketogenic diet changes both the relative abundance of gut microbiota and their metabolic activities. The diet forces a shift from carbohydrates to ketones as a primary energy source and has demonstrated efficacy in reducing epileptic seizures in children. After animal models implicated gut microbiota in this amelioration, Stefanie Prast-Nielsen, of Sweden’s Karolinska Institutet, and her team sequenced microbiotic DNA of fecal samples from 12 children with epilepsy before and after 3 months on a ketogenic diet. Changes included reductions in the numbers of Bifidobacterium and an increase in Escherichia coli. Carbohydrate metabolism significantly changed after 3 months on the diet. Some reductions raise questions about the diet’s potential impact on gut and overall health. More studies are also needed to discern the mechanistic impact of these changes on seizure activity.

Brain malformations are a major cause of therapy-refractory epilepsy as well as neurological and developmental disabilities in children. This study examined the frequency and the nature of copy number variations among children with structural brain malformations and refractory epilepsy. The medical records of all children born between 1990 and 2009 in the epilepsy registry at the Astrid Lindgren's Children's Hospital were reviewed and 86 patients with refractory epilepsy and various brain malformations were identified. Array-CGH analysis was performed in 76 of the patients. Pathogenic copy number variations were detected in seven children (9.2%). In addition, rearrangements of unclear significance, but possibly pathogenic, were detected in 11 (14.5%) individuals. In 37 (48.7%) patients likely benign, but previously unreported, copy number variants were detected. Thus, a large proportion of our patients had at least one rare copy number variant. Our results suggest that array-CGH should be considered as a first line genetic test for children with cerebral malformations and refractory epilepsy unless there is a strong evidence for a specific monogenic syndrome.

Background This Scandinavian collaborative retrospective study of children treated with ketogenic diet (KD) highlights indications and effectiveness over two years follow-up. Methods Five centres specialised in KD collected data retrospectively on 315 patients started on KD from 1999 to 2009. Twenty-five patients who stopped the diet within four weeks because of compliance-problems and minor side-effects were excluded. Seizure-type(s), seizure-frequency, anti-epileptic drugs and other treatments, mental retardation, autism-spectrum disorder and motor-dysfunction were identified and treatment-response was evaluated. Results An intention-to-treat analysis was used. Responders (>50% seizure-frequency reduction) at 6, 12 and 24 months were 50%, 46% and 28% respectively, seizure-free were 16%, 13% and 10%. Still on the diet were 80%, 64% and 41% after 6, 12 and 24 months. No child had an increased seizure-frequency. The best seizure outcome was seen in the group with not-daily seizures at baseline ( n = 22), where 45%, 41% and 32% became seizure-free at 6, 12 and 24 months A significant improvement in seizure-frequency was seen in atonic seizures at three months and secondary generalised seizures at three and six months. Side-effects were noted in 29 subjects; most could be treated and only two stopped due to hyperlipidaemia and two due to kidney-stones. In 167 patients treated with potassium-citrate, one developed kidney-stones, compared with six of 123 without potassium-citrate treatment (relative risk = 8.1). Conclusions As the first study of implementing KD in children in the Scandinavian countries, our survey of 290 children showed that KD is effective and well tolerated, even in such severe patients with therapy-resistant epilepsy, more than daily seizures and intellectual disability in the majority of patients. Long-term efficacy of KD was comparable or even better than reported in newer AEDs. Addition of potassium citrate reduced risk of kidney-stones. Our data indicate that the response might be predicted by seizure-frequency before initiation of the diet but not by age, seizure-type or aetiology.

The ketogenic diet (KD) is a non-pharmacological treatment of medically refractory epilepsy in children. Its mechanisms of action are still unclear but monoamine neurotransmitters have been proposed to be involved. Norepinephrine, dopamine, and serotonin are known to modulate seizure susceptibility in many animal models. We examined whether the concentrations of norepinephrine, dopamine, and serotonin metabolites were affected by the KD in children with pharmacoresistant epilepsy. The metabolites of norepinephrine, HMPG, of dopamine, HVA, and of serotonin, 5-HIAA, were analyzed in cerebrospinal fluid (CSF) before and 3 months after starting the KD. Twenty-six children (mean age 5.9 years) participated. Twenty-one children had generalized epilepsy and five partial. CSF was sampled by lumbar puncture. Seizure frequency before and during the diet was determined. Highly significant changes were found for HVA (p=0.0002) and 5-HIAA (p=0.004), which were both decreased during the KD compared to before diet. The levels of HMPG were unchanged. However, no differences were found between response groups. Valproate medication affected the levels of HMPG during diet with decreased levels in children on valproate and increased in those not on valproate (p=0.04). Our study indicates that the KD significantly alters the levels of metabolites of dopamine and serotonin but with a stable ratio HVA/5-HIAA in the CSF of children with refractory epilepsy, which finding may be of importance for the mechanism of action.

Purpose: To examine the influence of the ketogenicdiet (KD) on linear growth and insulin-likegrowth factor I (IGF-I) levels in children withpharmacotherapy-resistantepilepsy.Methods: A prospective study was designed toevaluate growth, serum IGF-I levels, bloodb-hydroxybutyric acid (b-OHB), and seizurefrequency before and during KD in 22 children(median age 5.5 years). Growth was assessed bymeasurements of weight, height, body mass index(BMI), and height velocity. Standard deviationscores (SDS) were calculated for all measuredparameters as well as for serum IGF-I to eliminatethe influence of age- and sex-related differencesamongpatients.Results: Fourteen of the 22 patients responded tothe KD. Weight, height, BMI, and height velocitydecreased significantly during the KD. We foundthat the KD had profound influence on growth andIGF-I levels. No correlation was found betweenseizure response and growth alterations. Heightvelocity correlated negatively with b-OHB duringthe KD. The slope of the regression of heightvelocity against IGF-I decreased significantly dur-ingtheKD.Conclusions: Height velocity was most affected inthose with pronounced ketosis, which impliesthat, in clinical practice, the level of ketosisshould be related to outcomes in seizure responseand growth. Our data indicate that growth dis-turbances and the decreased sensitivity of growthto similar IGF-I levels during KD are indepen-dent of seizure reduction. The metabolic statusinduced by KD may be the mechanism underlyingboth alterations of linear growth and seizurereduction.KEY WORDS: Growth, IGF-I, Ketogenic diet,Pharmacoresistantepilepsy.Theketogenicdiet(KD),introducedbyWilderin1921,isusedinthetreatmentofchildrenwithpharmacotherapy-resistant epilepsy (Wilder, 1921). Despite its long historyof clinical use, it is still not clear how the diet affects thebrain and what the mechanisms are that underlie itsseizure-suppressive action (Cheng et al., 2003a; Bough R Liuet al., 2003). Since an increasing number of patients aretreated with the KD, it is important to evaluate its effectsongrowthandnutritionalstatus.

The ketogenic diet (KD) is an established, effective nonpharmacologic treatment for intractable childhood epilepsy. The KD is provided differently throughout the world, with occasionally significant variations in its administration. There exists a need for more standardized protocols and management recommendations for clinical and research use. In December 2006, The Charlie Foundation commissioned a panel comprised of 26 pediatric epileptologists and dietitians from nine countries with particular expertise using the KD. This group was created in order to create a consensus statement regarding the clinical management of the KD. Subsequently endorsed by the Practice Committee of the Child Neurology Society, this resultant manuscript addresses issues such as patient selection, pre-KD counseling and evaluation, specific dietary therapy selection, implementation, supplementation, follow-up management, adverse event monitoring, and eventual KD discontinuation. This paper highlights recommendations based on best evidence, including areas of agreement and controversy, unanswered questions, and future research.

The brain aspartate-glutamate carrier (AGC1) is specifically expressed in neurons, where it transports aspartate from the mitochondria to the cytosol, and plays a role in transfer of nicotinamide adenine dinucleotide (NADH)-reducing equivalents into the mitochondria as a part of the malate-aspartate shuttle. Deficient function of AGC1 underlies an inborn error of metabolism that presents with severe hypotonia, arrested psychomotor development, and seizures from a few months of age. In AGC1 deficiency, there is secondary hypomyelination due to lack of N-acetylaspartate (NAA), which is normally generated by acetylation of aspartate in the neuron and required for fatty acid synthesis by the adjacent oligodendrocyte. Based on experiences from AGC2 deficiency, we predicted that reduced glycolysis should compensate for the metabolic defect and allow resumed myelination in AGC1 deficiency. Carbohydrate restriction was therefore initiated in a patient with AGC1 deficiency at 6 years of age by introducing a ketogenic diet. The response was dramatic, clinically as well as radiologically. Psychomotor development showed clear improvement, and magnetic resonance imaging (MRI) indicated resumed myelination. This is the first successful treatment of secondary hypomyelination reported. Because AGC1 is driven by the proton gradient generated by the neuronal mitochondrial respiratory chain, the results have potential relevance for secondary hypomyelination in general.

The ketogenic diet (KD) is an established treatment for medically refractory pediatric epilepsy. Its anticonvulsant mechanism is still unclear. We examined the influence of the KD on the CSF levels of excitatory and inhibitory amino acids in 26 children (mean age 6.1 years) with refractory epilepsy. Seventeen amino acids were determined before and at a mean of 4 months after the start of the KD. Seizures were quantified. Highly significant changes were found in eight amino acids: increases in GABA, taurine, serine, and glycine and decreases in asparagine, alanine, tyrosine and phenylalanine. However, aspartate, glutamate, arginine, threonine, citrulline, leucine, isoleucine and valine/methionine remained unchanged. A significant correlation with seizure response was found for threonine (P=0.016). The GABA levels were higher in responders (>50% seizure reduction) than in nonresponders during the diet (P=0.041). In the very good responders (>90% seizure reduction), the GABA levels were significantly higher at baseline as well as during the diet. Age differences were found with significantly larger decreases in glutamate and increases in GABA in connection with the diet in younger children. Our results indicate that the KD significantly alters the levels of several CSF amino acids that may be involved in its mechanism of action and the increase in GABA is of particular interest.

The influence of age and comedication on the dose-to-level ratio of topiramate was examined in 91 children with epilepsy treated with topiramate. The topiramate dosing and plasma concentrations, as well as those of their concomitant antiepileptic drugs were examined retrospectively. The dose-to-level ratio was used as a measure of clearance and was calculated as the weight-normalized topiramate dose (mg/kg/day) divided by the steady-state trough plasma drug level in the child. The children were classified in age groups and treatment groups; topiramate was administered with an enzyme inducer (n = 32), with a nonenzyme inducer (n = 49), or as monotherapy (n = 10). The topiramate clearance in children aged 0-8 years compared with those aged 9-17 years was more than twofold higher if treated with an enzyme-inducing antiepileptic drug and 1.5-fold higher if treated with a nonenzyme inducer. Children receiving enzyme inducers had a more than twofold higher clearance compared with those who did not. Within all age groups, significant differences in topiramate clearance were observed between those receiving enzyme inducers and those receiving nonenzyme inducers or monotherapy. Thus younger age and concomitant enzyme inducers, both acting independently, significantly increased the clearance of topiramate in children. This effect has to be considered to optimize treatment in the individual patient.

Background Tuberous sclerosis complex (TSC) is a rare, multisystem, genetic disorder with an estimated prevalence between 1/6800 and 1/15000. Although recent years have seen huge progress in understanding the pathophysiology and in the management of TSC, several questions remain unanswered. A disease registry could be an effective tool to gain more insights into TSC and thus help in the development of improved management strategies. Methods TuberOus SClerosis registry to increase disease Awareness (TOSCA) is a multicentre, international disease registry to assess manifestations, interventions, and outcomes in patients with TSC. Patients of any age diagnosed with TSC, having a documented visit for TSC within the preceding 12 months, or newly diagnosed individuals are eligible. Objectives include mapping the course of TSC manifestations and their effects on prognosis, identifying patients with rare symptoms and co-morbidities, recording interventions and their outcomes, contributing to creation of an evidence-base for disease assessment and therapy, informing further research on TSC, and evaluating the quality of life of patients with TSC. The registry includes a ‘core’ section and subsections or ‘petals’. The ‘core’ section is designed to record general information on patients’ background collected at baseline and updated annually. Subsections will be developed over time to record additional data related to specific disease manifestations and will be updated annually. The registry aimed to enrol approximately 2000 patients from about 250 sites in 31 countries. The initial enrolment period was of 24 months. A follow-up observation period of up to 5 years is planned. Results A pre-planned administrative analysis of ‘core’ data from the first 100 patients was performed to evaluate the feasibility of the registry. Results showed a high degree of accuracy of the data collection procedure. Annual interim analyses are scheduled. Results of first interim analysis will be presented subsequent to data availability in 2014. Implications The results of TOSCA will assist in filling the gaps in understanding the natural history of TSC and help in planning better management and surveillance strategies. This large-scale international registry to study TSC could serve as a model to encourage planning of similar registries for other rare diseases.

Background Zonisamide is a new generation antiepileptic drug (AED) widely used in children with refractory epilepsy, although until recently, it was used to a large extent as off-label or unlicensed medication due to the lack of evidence-based studies. Children have a different pharmacokinetic profile than adults and an adult dose regimen cannot be directly translated into pediatric use. Patients and Methods In this retrospective noninterventional study of the medical records of 75 children with pharmacoresistant epilepsy, the pharmacokinetics, efficacy and safety of zonisamide were examined. The dose-to-concentration ratio, the daily weight-normalized dose of zonisamide divided by its plasma concentration, was used as a measure of clearance. In addition, data on the efficacy of zonisamide to reduce seizures and reported adverse events were extracted from the medical records and analyzed. Results Young children (range, 0–4 years) had a significantly increased zonisamide clearance compared with older ones (range, 5–17 years) and those with enzyme-inducing comedication (carbamazepine, phenobarbital, or phenytoin) had increased clearance compared with those on nonenzyme inducers; the increases were 1.7-fold and 1.8-fold, respectively. No significant difference in clearance was found between female and male subjects. The clearances of concomitant AEDs were not affected by zonisamide administration. The overall efficacy of zonisamide for reducing seizure frequency ≥50% was 35% and the most frequent adverse event was fatigue, reported in 23% of the patients. Conclusion Patients with enzyme-inducing comedication or of young age (range, 0–4 years) might need higher weight-normalized doses to achieve the same plasma levels as in patients with no enzyme-inducing comedication or patients of older age. Zonisamide was not found to influence the pharmacokinetics of concomitant AEDs. The shortage of pharmacokinetic studies of zonisamide in children highlights the need for research of this kind.

Summary: Purpose: To evaluate the effect of low-dose clonazepam (CZP) on the amount of epileptiform activity in children with focal and generalized epilepsy. Methods: In a single-blind pilot study, followed by a double-blind, placebo-controlled, randomized, crossover study, 15 children with epilepsy were evaluated by using 24-h long-term EEG recordings during baseline days and days after injections of placebo and CZP. The drug was given as a single i.m. injection of 0.02 mg/kg BW. Blood samples were obtained regularly for analysis of plasma concentrations of CZP. The number of epileptiform discharges was determined during corresponding periods with the individual child in the same state of alertness, the same real time of day, and with concomitant antiepileptic drugs (AEDs) unchanged. Results: In the double-blind study, low-dose CZP produced a highly significant (p = 0.0015) decrease in the amount of epileptiform activity (mean, -69% vs. placebo, -2%) obtained during periods when median plasma concentrations ranged from 18 to

Objective : To evaluate the impact of the time factor on the amount of epileptiform activity in long-term EEG recordings in children with epilepsy. Methods : Ten children with epilepsy of different types underwent three 24h EEG examinations during two consecutive days and with a month's interval. The number of epileptiform discharges during selected corresponding periods of time was counted. Results : The number of epileptiform discharges on three repeated examination days showed no significant difference (ANOVA P=0.88) as intraindividual increases and decreases on different days counterbalanced each other within the group. However the standard deviations of the relative changes were larger between recordings with a month's interval compared to those for consecutive days (86% and 33%). The mean magnitude of change was 55% between days separated by a month compared to 24% on consecutive days. The difference was non-significant but showed a trend towards larger changes with a longer interval (P=0.07). Conclusions : The variability of epileptiform activity was larger when the interval between recordings was 1 month compared to consecutive days. The magnitude of the relative changes between intervals of 1 and 30 days showed a trend towards a difference although not statistically significant. When evaluating repeated long-term EEGs in relation to therapy in children, these variations should be considered.

Summary FGF21 is a critical metabolic regulator, pivotal for fasting adaptation and directly regulated by PPARα in rodents. However, the physiological role of FGF21 in man is not yet defined and was investigated in our study. Serum FGF21 varied 250-fold among 76 healthy individuals and did not relate to age, gender, body mass index (BMI), serum lipids, or plasma glucose. FGF21 levels had no diurnal variation and were unrelated to bile acid or cholesterol synthesis. Ketosis induced by a 2 day fast or feeding a ketogenic diet (KD) did not influence FGF21 levels, whereas a 74% increase occurred after 7 days of fasting. Hypertriglyceridemic nondiabetic patients had 2-fold elevated FGF21 levels, which were further increased by 28% during fenofibrate treatment. FGF21 circulates in human plasma and increases by extreme fasting and PPARα activation. The wide interindividual variation and the induction of ketogenesis independent of FGF21 levels indicate that the physiological role of FGF21 in humans may differ from that in mice.

Many children with epilepsy do not satisfactorily respond to conventional pharmacological therapy, but to the ketogenic diet, a high-fat, low-carbohydrate diet. This diet increases the concentrations of ketone bodies and polyunsaturated fatty acids (PUFAs) in cerebrospinal fluid (CSF) and plasma. However, its anticonvulsant mechanism is not known.To investigate the mechanism by which the diet protects against seizures, we studied the effects of several PUFAs (docosahexaenoic acid, eicosapentaenoic acid, and linoleic acid), ketone bodies (beta-hydroxybuturic acid and acetoacetic acid), and CSF from patients on the ketogenic diet on the voltage-gated Shaker K channel expressed in Xenopus oocytes.We found that PUFAs at concentrations down to 21microM clearly increased the K current by shifting the conductance versus voltage curve in negative direction along the voltage axis. CSF from patients on the ketogenic diet has similar but smaller effects. In contrast, high concentrations (1-5mM) of ketone bodies did not affect the K current. Computer simulations showed that the observed shifts for clinically relevant concentrations of PUFAs, and CSF from patients could effectively impair repetitive firing.These data suggest that the ketogenic diet could prevent epileptic seizures by PUFA-induced openings of voltage-gated K channels.

The ketogenic diet (KD) is used to treat medically refractory epilepsy in children. Alterations of fatty acid (FA) levels may reflect one mechanism of action. We examined the influence of the KD on FA levels and seizure control. The levels of 17 FAs in plasma phospholipids were determined before and 1, 3, 6, and 12 months after initiation of the KD in 25 children (mean age 6.3 years) with intractable epilepsy. Fluid omega-3 FA was supplemented in the diet after one month. Highly significant changes of the levels of several FAs were found. Linoleic acid (LA) and eicosapentaenoic acid (EPA) increased, whereas arachidonic acid (AA) and Mead acid (20:3 n-9) decreased. Docosahexaenoic acid (DHA) increased insignificantly. However, no correlation of changes in FA levels with seizure response was found. The ratio of omega-6 to omega-3 gradually decreased from 7.0 before to 4.9 at 12 months after starting the diet, presumably a cardiovascular benefit. The composition of the KD differs as to FA content and type between different treating centers but, still, the efficacy reports are very similar. This study demonstrates the possibility of composing the KD in such a way that the FA profile is kept within a normal range, which may reduce cardiovascular risks.

The acute effects of low-dose clonazepam on seizure frequency in children with epilepsy was evaluated. In an open study, 19 children with epilepsy (15 generalized and four partial) were examined during hospitalization with recordings of seizures by trained personnel. Seizures were counted during two 24-hour periods: before and after a single intramuscular injection of clonazepam 0.01-0.04 mg/kg body weight. Plasma concentrations of clonazepam were determined. The median number of seizures in all children on control days was 22 (range = 1-180) and, on days after low-dose clonazepam, the median was 6 (range = 0-73). The relative changes demonstrated a median of -70% (range from -100% to + 43%). A significant reduction of seizures (P = 0.0031) at median maximal plasma levels of clonazepam of 23 nmol/L (range = 11-41 nmol/L) was found. Thus in this study of the acute effects of a low-dose level of clonazepam on seizure frequency a significant reduction was found at plasma levels below those usually recommended. Inhibition of seizure activity seems to be achieved already at low plasma levels of clonazepam. These results suggest to start treatment at low doses of clonazepam and evaluate the individual effect carefully during dose escalation aiming at lowest possible dose with therapeutic effect.

1FC2.6 DOK7 congenital myasthenic syndrome in childhood: early diagnostic clues S.A. Robb1 *, J.C. McHugh2, M.C. Pitt2, C.A. Sewry1, S. Finlayson3, A.Y. Manzur1, C. De Vile4, S. Jayawant5, J. Palace3, D. Beeson6, F. Muntoni1. 1Dubowitz Neuromuscular Centre, Institute of child Health and Great Ormond Street Hospital, United Kingdom, 2Department of Clinical Neurophysiology, Institute of Child Health and Great Ormond Street Hospital, London, United Kingdom, 3Department of Neurology, John Radcliffe Hospital, Oxford, United Kingdom, 4Department of Neurology, Institute of Child Health and Great Ormond Street Hospital, London, United Kingdom, 5Department of Paediatric Neurology, Oxford Childrens’ Hospital, Oxford, United Kingdom, 6Neurosciences Group, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom

In an attempt to investigate whether benzodiazepines at low dosage have a significant effect in reducing spasticity among children with cerebral palsy, we carried out a double-blind, placebo-controlled, cross-over study. Twelve children with either spastic diplegia or hemiplegia participated in this study. The mean age was 14 years. The restraint of passive knee movements was determined with a dynamic dynamometer and spastic stretch reflexes were measured as EMG activity in muscles stretched. Clonazepam was given at low dosage (0.02 mg/kg body weight). In each child measurements of passive restraint were made on 2 different days immediately before and 3 h after an i.m. injection of either clonazepam or placebo in randomized order. Clonazepam significantly reduced spastic restraint (P < 0.001) compared to non-significant reduction with placebo. The mean plasma concentration of clonazepam at time of spasticity evaluation was 21 mmol/l which is in the low dose range, far below conventional doses. The study thus shows a positive effect of low dose clonazepam in reducing spasticity in children when given as a single dose.