Valentina De Giorgis

Improving molecular diagnosis in epilepsy by a dedicated high-throughput sequencing platform

We analyzed by next-generation sequencing (NGS) 67 epilepsy genes in 19 patients with different types of either isolated or syndromic epileptic disorders and in 15 controls to investigate whether a quick and cheap molecular diagnosis could be provided. The average number of nonsynonymous and splice site mutations per subject was similar in the two cohorts indicating that, even with relatively small targeted platforms, finding the disease gene is not an univocal process. Our diagnostic yield was 47% with nine cases in which we identified a very likely causative mutation. In most of them no interpretation would have been possible in absence of detailed phenotype and familial information. Seven out of 19 patients had a phenotype suggesting the involvement of a specific gene. Disease-causing mutations were found in six of these cases. Among the remaining patients, we could find a probably causative mutation only in three. None of the genes affected in the latter cases had been suspected a priori. Our protocol requires 8–10 weeks including the investigation of the parents with a cost per patient comparable to sequencing of 1–2 medium-to-large-sized genes by conventional techniques. The platform we used, although providing much less information than whole-exome or whole-genome sequencing, has the advantage that can also be run on ‘benchtop’ sequencers combining rapid turnaround times with higher manageability.

Post-Operative Benefits of Animal-Assisted Therapy in Pediatric Surgery: A Randomised Study

Background Interest in animal-assisted therapy has been fuelled by studies supporting the many health benefits. The purpose of this study was to better understand the impact of an animal-assisted therapy program on children response to stress and pain in the immediate post-surgical period. Patients and Methods Forty children (3–17 years) were enrolled in the randomised open-label, controlled, pilot study. Patients were randomly assigned to the animal-assisted therapy-group (n = 20, who underwent a 20 min session with an animal-assisted therapy dog, after surgery) or the standard-group (n = 20, standard postoperative care). The study variables were determined in each patient, independently of the assigned group, by a researcher unblinded to the patient’s group. The outcomes of the study were to define the neurological, cardiovascular and endocrinological impact of animal-assisted therapy in response to stress and pain. Electroencephalogram activity, heart rate, blood pressure, oxygen saturation, cerebral prefrontal oxygenation, salivary cortisol levels and the faces pain scale were considered as outcome measures. Results After entrance of the dog faster electroencephalogram diffuse beta-activity (> 14 Hz) was reported in all children of the animal-assisted therapy group; in the standard-group no beta-activity was recorded (100% vs 0%, p<0.001). During observation, some differences in the time profile between groups were observed for heart rate (test for interaction p = 0.018), oxygen saturation (test for interaction p = 0.06) and cerebral oxygenation (test for interaction p = 0.09). Systolic and diastolic blood pressure were influenced by animal-assisted therapy, though a higher variability in diastolic pressure was observed. Salivary cortisol levels did not show different behaviours over time between groups (p=0.70). Lower pain perception was noted in the animal-assisted group in comparison with the standard-group (p = 0.01). Conclusion Animal-assisted therapy facilitated rapid recovery in vigilance and activity after anaesthesia, modified pain perception and induced emotional prefrontal responses. An adaptative cardiovascular response was also present. Trial Registration ClinicalTrials.gov NCT02284100

The ketogenic diet for Dravet syndrome and other epileptic encephalopathies: an Italian consensus

Ketogenic diet is a nonpharmacologic treatment for childhood epilepsy not amenable to drugs. At the present time, two works based on national research, one in Germany and one in the United States provide international guidelines to ensure a correct management of the ketogenic diet. Our Italian collaborative study group was set up in order to formulate a consensus statement regarding the clinical management of the ketogenic diet, patient selection, pre–ketogenic diet, counseling, setting and enforcement of dietary induction of ketosis, follow‐up management, and eventual discontinuation of the diet.

Glucose transporter type 1 deficiency: Ketogenic diet in three patients with atypical phenotype

Glucose transporter type I deficiency syndrome (GLUT-1 DS) is an inborn error of glucose transport characterized by seizures, developmental delay, spasticity, acquired microcephaly and ataxia. Diagnosis is based on the finding of low cerebrospinal fluid glucose, in the absence of hypoglycemia, and identification of GLUT-1 gene mutation on chromosome 1. The classic phenotype is a severe form of early onset epileptic encephalopathy, but patient with different clinical presentation have been reported expanding the clinical spectrum. In particular, many patients show a prominent movement disorder other than epilepsy. It is known that this disease represents a treatable condition and ketogenic diet (KD) is the elective treatment in GLUT-1 DS patients. We report on KD in three unrelated Italian GLUT-1 DS female patients, diagnosed in early adulthood, all presenting with an atypical phenotype. Preliminary results seem to demonstrate efficacy of KD on paroxysmal movement disorder while positive effect on cognitive impairment result less evident.

The changing face of dietary therapy for epilepsy

AbstractKetogenic diet is an established and effective non-pharmacologic treatment for drug-resistant epilepsy. Ketogenic diet represents the treatment of choice for GLUT-1 deficiency syndrome and pyruvate dehydrogenase complex deficiency. Infantile spasms, Dravet syndrome and myoclonic-astatic epilepsy are epilepsy syndromes for which ketogenic diet should be considered early in the therapeutic pathway. Recently, clinical indications for ketogenic diet have been increasing, as there is emerging evidence regarding safety and effectiveness. Specifically, ketogenic diet response has been investigated in refractory status epilepticus and encephalopathy with status epilepticus during sleep. New targets in neuropharmacology, such as mitochondrial permeability transition, are being studied and might lead to using it effectively in other neurological diseases. But, inefficient connectivity and impaired ketogenic diet proposal limit ideal availability of this therapeutic option. Ketogenic diet in Italy is not yet considered as standard of care, not even as a therapeutic option for many child neurologists and epileptologists. Conclusions: The aim of this review is to revisit ketogenic diet effectiveness and safety in order to highlight its importance in drug-resistant epilepsy and other neurological disorders.What is Known:• Ketogenic diet efficacy is now described in large case series, with adequate diet compliance and side effects control.• Ketogenic diet is far from being attempted as a first line therapy. Its availability varies worldwide.What is New:• New pharmacological targets such as mitochondrial permeability transition and new epileptic syndromes and etiologies responding to the diet such as refractory status epilepticus are being pointed out.• Ketogenic diet can function at its best when used as a tailor-made therapy. Fine tuning is crucial.

Short-term effects of ketogenic diet on anthropometric parameters, body fat distribution, and inflammatory cytokine production in GLUT1 deficiency syndrome

OBJECTIVE The aim of this study was to evaluate the effects of a 12-wk ketogenic diet (KD) on inflammatory status, adipose tissue activity biomarkers, and abdominal visceral (VAT) and subcutaneous fat (SAT) in children affected by glucose transporter 1 deficiency syndrome GLUT1 DS. METHODS We carried out a short-term longitudinal study on 10 children (mean age: 8.4 y, range 3.3-12 y, 5 girls, 5 boys) to determine fasting serum proinflammatory cytokines (high sensitivity C-reactive protein, tumor necrosis factor-α interleukin-6), adipocyte-derived chemokines (leptin and adiponectin), lipid profile, homeostatic model assessment-insulin resistance (HOMA-IR), quantitative insulin sensitivity index (QUICKI), anthropometric measurements, and VAT and SAT (by ultrasonography). RESULTS Children showed no significant changes in inflammatory and adipose tissue activity biomarkers, blood glucose, lipid profile, anthropometric measurements, VAT, and SAT. Fasting insulin decreased (6 ± 3.2 μU/mL versus 3 ± 2 μU/mL; P = 0.001), and both HOMA-IR and QUICKI indexes were significantly modified (1.2 ± 0.6 versus 0.6 ± 0.4; P = 0.002; 0.38 ± 0.03 versus 0.44 ± 0.05; P = 0.002, respectively). CONCLUSIONS Only HOMA-IR and QUICKI indexes changed after 12 wk on a KD, suggesting that over a short period of time KD does not affect inflammatory cytokines production and abdominal fat distribution despite being a high-fat diet. Long-term studies are needed to provide answers concerning adaptive metabolic changes during KD.

The face of Glut1-DS patients: A 3D craniofacial morphometric analysis

Glut1 deficiency syndrome (Glut1‐DS) is a neurological and metabolic disorder caused by impaired transport of glucose across the blood brain barrier (BBB). Mutations on the SCL2A1 gene encoding the glucose transporter protein in the BBB cause the syndrome, which encompasses epilepsy, movement disorders, and mental delay. Such variability of symptoms presents an obstacle to early diagnosis. The patients seem to share some craniofacial features, and identification and quantification of these could help in prompt diagnosis and clinical management. We performed a three‐dimensional morphometric analysis of the faces of 11 female Glut1‐DS patients using a stereophotogrammetric system. Data were analyzed using both inter‐landmark distances and Principal Component Analysis. Compared with data collected from age‐, sex‐, and ethnicity‐matched control subjects, common and homogenous facial features were identified among patients, which were mainly located in the mandible and the eyes. Glut1‐DS patients had a more anterior chin; their mandibular body was longer but the rami were shorter, with a reduced gonial angle; they had smaller and down‐slanted eyes with a reduced intercanthal distance. This study highlights the importance of morphometric analysis for defining the facial anatomical characteristics of the syndrome better, potentially helping clinicians to diagnose Glut1‐DS. Improved knowledge of the facial anatomy of these patients can provide insights into their facial and cerebral embryological development, perhaps further clarifying the molecular basis of the syndrome. Clin. Anat. 30:644–652, 2017.

Biochemical phenotyping unravels novel metabolic abnormalities and potential biomarkers associated with treatment of GLUT1 deficiency with ketogenic diet

Global metabolomic profiling offers novel opportunities for the discovery of biomarkers and for the elucidation of pathogenic mechanisms that might lead to the development of novel therapies. GLUT1 deficiency syndrome (GLUT1-DS) is an inborn error of metabolism due to reduced function of glucose transporter type 1. Clinical presentation of GLUT1-DS is heterogeneous and the disorder mirrors patients with epilepsy, movement disorders, or any paroxysmal events or unexplained neurological manifestation triggered by exercise or fasting. The diagnostic biochemical hallmark of the disease is a reduced cerebrospinal fluid (CSF)/blood glucose ratio and the only available treatment is ketogenic diet. This study aimed at advancing our understanding of the biochemical perturbations in GLUT1-DS pathogenesis through biochemical phenotyping and the treatment of GLUT1-DS with a ketogenic diet. Metabolomic analysis of three CSF samples from GLUT1-DS patients not on ketogenic diet was feasible inasmuch as CSF sampling was used for diagnosis before to start with ketogenic diet. The analysis of plasma and urine samples obtained from GLUT1-DS patients treated with a ketogenic diet showed alterations in lipid and amino acid profiles. While subtle, these were consistent findings across the patients with GLUT1-DS on ketogenic diet, suggesting impacts on mitochondrial physiology. Moreover, low levels of free carnitine were present suggesting its consumption in GLUT1-DS on ketogenic diet. 3-hydroxybutyrate, 3-hydroxybutyrylcarnitine, 3-methyladipate, and N-acetylglycine were identified as potential biomarkers of GLUT1-DS on ketogenic diet. This is the first study to identify CSF, plasma, and urine metabolites associated with GLUT1-DS, as well as biochemical changes impacted by a ketogenic diet. Potential biomarkers and metabolic insights deserve further investigation.

Brain correlates of spike and wave discharges in GLUT1 deficiency syndrome

Purpose To provide imaging biomarkers of generalized spike-and-wave discharges (GSWD) in patients with GLUT1 deficiency syndrome (GLUT1DS). Methods Eighteen GLUT1DS patients with pathogenetic mutation in SLC2A1 gene were studied by means of Video-EEG simultaneously recorded with functional MRI (VideoEEG-fMRI). A control group of sex and age-matched patients affected by Genetic Generalized Epilepsy (GGE) with GSWD were investigated with the same protocol. Within and between groups comparison was performed as appropriated. For GLUT1DS, correlations analyses between the contrast of interest and the main clinical measurements were provided. Results EEG during fMRI revealed interictal GSWD in 10 GLUT1DS patients. Group-level analysis showed BOLD signal increases at the premotor cortex and putamen. With respect to GGE, GLUT1DS patients demonstrated increased neuronal activity in the putamen, precuneus, cingulate cortex, SMA and paracentral lobule. Whole-brain correlation analyses disclosed a linear relationship between the GSWD-related BOLD changes and the levels of glycorrhachia at diagnosis over the sensory-motor cortex and superior parietal lobuli. Conclusion The BOLD dynamics related to GSWD in GLUT1DS are substantially different from typical GGE showing the former an increased activity in the premotor-striatal network and a decrease in the thalamus. The revealed hemodynamic maps might represent imaging biomarkers of GLUT1DS, being potentially useful for a precocious diagnosis of this genetic disorder.

Acute chorea in a child receiving second dose of human papilloma virus vaccine

To the Editor, Since a program of immunization with human papilloma virus (HPV) quadrivalent vaccine Gardasil has began, adverse events’ surveillance has started. To date, the most important neurologic adverse events reported are Guillain–Barr e syndrome (1), demyelinizating diseases (2, 3) and cases of acute disseminated encephalo myelitis (ADEM) (4, 5). An 11-yr-old girl was admitted in our Child Neurology and Psychiatry Unit presenting generalized choreic movements involving arms, legs and trunk. These were associated with facial grimaces, ataxic gait and dysarthria. The girl is the secondborn sibling of healthy non-consanguineous parents. At the age of 14, her brother started suffering from symptomatic epilepsy as a complication of a surgery he had undergone to remove a fibrillar grade 1 astrocytoma. The girl had always been in good health since she developed the movement disorder. Her symptoms appeared rapidly and persisted for days. We performed accurate anamnestic interview. The patient was born after a full-term pregnancy, by vaginal delivery. No pre-perinatal problems were referred. She had received vaccines following Italian schedule and she had never reported adverse events. The patient received first dose of HPV vaccine Gardasil on February 25th, 2013. Second Gardasil dose was administered on April 8th, 2013. After 1 month, she developed acute chorea and came to our Unit. To exclude common causes of chorea in young children, we performed specific blood examinations. Complete blood count, liver enzymes and renal functioning were normal. Considering the hypothesis of rheumatic disease, we dosed antistreptolysin titer and found a value not sufficiently high to justify a rheumatic chorea according to our experience (363 IU/ml). Streptococcus pyogenes was not detected in the patient’s throat swab specimen. As in rheumatic chorea antistreptolysin titer can also be within normal limits, we performed electrocardiogram and echocardiography and excluded rheumatic carditis. Inflammatory indexes (Erythrocyte sedimentation rate, C reactive protein) as well as serum and 24 h urinary copper and serum ceruloplasmin were normal. We performed a complete autoimmune screening: immunoglobulins subpopulations, complement C3 and C4 fragments, antinuclear antibodies (ANA), extractable nuclear antigens (ENA), antineutrophils cytoplasm antibodies (ANCA), antibeta 2 microglobulin antibodies, antitireoperoxidase antibodies, anti-endomisium antibodies, anticardiolipin antibodies, antiphospolipids antibodies (PLP), lupus anticoagulant. All indexes were in the range except for antiperoxidase antibodies 43 U/l (normal range inferior to 35). We underline that thyroid functioning resulted normal and no familiar history of thyropathy was reported. Immunoglobulins class E were elevated (1265 IU/ml); we ascribed this data to allergic diathesis (the girl suffers from allergic rhinitis). Brain magnetic resonance imaging showed normal findings. Finally, we studied a sample of cerebrospinal fluid and obtained normal results. No oligoclonal bands were signaled. We hypothesized an autoimmune reaction after second dose of Gardasil vaccine (administered 1 month before) and decided to start therapy with oral methylprednisolone 2 mg/kg/die for 4 wk. This was then gradually reduced and completely stopped. The girl started improving after 2 days of therapy and obtained a complete resolution of symptoms within 1 wk. Follow-up visits were carried out monthly after discharge proving a complete recovery. In this article, we discuss the case of a completely healthy girl before acute choreic signs raising 1 month after the second immunization with Gardasil. We excluded rheumatic disease, other autoimmune disease (i.e., Lupus erythematosus systemicus), and other inflammatory processes in central nervous system and copper metabolism disorders. There is a strong temporary association between the administration of vaccine and the appearance of symptoms. As in other cases previously reported (4), symptoms tend to occur after booster vaccination rather than after first application and this is our case. Finally, we believe that prompt answer to steroid treatment could be another element, suggesting that an autoimmune post-vaccine mechanism of action could have caused symptoms. An autoimmune pathogenetic mechanism – that is molecular mimicry – has been postulated for this vaccine’s adverse events (6). In particular, it has been observed that Gardasil recombinant proteins and components other that proteins (i.e., Aluminum) have a high antigenicity. Noteworthy, the population eligible for the wide immunization program – girls in pubertal age – is per se predisposed to develop autoimmune reactions. We describe a further case of neurologic complication after Gardasil administration, underlying that surveillance on adverse effect of the vaccine is still needed.