Innovative LC-MS/MS method for therapeutic drug monitoring of fenfluramine and cannabidiol in the plasma of pediatric patients with epilepsy.

We present a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying fenfluramine (FFA), its active metabolite norfenfluramine (norFFA), and Epidyolex®, a pure cannabidiol (CBD) oral solution in plasma. Recently approved by the EMA for the adjunctive treatment of refractory seizures in patients with Dravet and Lennox-Gastaut syndromes aged above 2 years, FFA and CBD still do not have established therapeutic blood ranges, and thus need careful drug monitoring to manage potential pharmacokinetic and pharmacodynamic interactions. Our method, validated by ICH guidelines M10, utilizes a rapid extraction protocol from 100 µL of human plasma and a reversed-phase C-18 HPLC column, with deuterated internal standards. The Thermofisher Quantiva triple-quadrupole MS coupled with an Ultimate 3000 UHPLC allowed multiple reaction monitoring detection, ensuring precise analyte quantification. The assay exhibited linear responses across a broad spectrum of concentrations: ranging from 1.64 to 1000 ng/mL for both FFA and CBD, and from 0.82 to 500 ng/mL for norFFA. The method proves accurate and reproducible, free from matrix effect. Additionally, FFA stability in plasma at 4 °C and -20 °C for up to 7 days bolsters its clinical applicability. Plasma concentrations detected in patients samples, expressed as mean ± standard deviation, were 0.36 ± 0.09 ng/mL for FFA, 19.67 ± 1.22 ng/mL for norFFA. This method stands as a robust tool for therapeutic drug monitoring (TDM) of FFA and CBD, offering significant utility in assessing drug-drug interactions in co-treated patients, thus contributing to optimized patient care in complex therapeutic scenarios.

A de novo frameshift variant in MED13 gene in a patient with autism spectrum disorder and magnetic resonance imaging abnormalities mimicking tuberous sclerosis.

The mediator complex subunit 13 (MED13) gene is implicated in neurodevelopmental disorders including autism spectrum disorder (ASD), intellectual disability, and speech delay with varying severity and course. Additional, extra central nervous system, features include eye or vision problems, hypotonia, congenital heart abnormalities, and dysmorphisms. We describe a 7-year- and 4-month-old girl evaluated for ASD whose brain magnetic resonance imaging was suggestive of multiple cortical tubers. The exome sequencing (ES - trio analysis) uncovered a unique, de novo, frameshift variant in the MED13 gene (c.4880del, D1627Vfs*17), with a truncating effect on the protein. This case report thus expands the phenotypic spectrum of MED13-related disorders to include brain abnormalities.

Brain and spine malformations and neurodevelopmental disorders in a cohort of children with CAKUT.

BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) represent 20-30% of all birth defects and are often associated with extra-renal malformations. We investigated the frequency of brain/spine malformations and neurological features in children with CAKUT. METHODS: We reviewed the clinico-radiological and genetic data of 199 out of 1,165 children with CAKUT evaluated from 2006 to 2023 (99 males, mean age at MRI 6.4 years) who underwent brain and/or spine MRI. Patients were grouped according to the type of CAKUT (CAKUT-K involving the kidney and CAKUT-H involving the inferior urinary tract). Group comparisons were performed using χ2 and Fisher exact tests. RESULTS: Brain/spine malformations were observed in 101/199 subjects (50.7%), 8.6% (101/1165) of our CAKUT population, including midbrain-hindbrain anomalies (40/158, 25.3%), commissural malformations (36/158, 22.7%), malformation of cortical development (23/158, 14.5%), Chiari I anomaly (12/199, 6%), cranio-cervical junction malformations (12/199, 6%), vertebral defects (46/94, 48.9%), caudal regression syndrome (29/94, 30.8%), and other spinal dysraphisms (13/94, 13.8%). Brain/spine malformations were more frequent in the CAKUT-K group (62.4%, p < 0.001). Sixty-two subjects (62/199, 31.2%) had developmental delay/intellectual disability. Neurological examination was abnormal in 40/199 (20.1%). Seizures and/or electroencephalographic anomalies were reported in 28/199 (14%) and behavior problems in 19/199 subjects (9%). Developmental delay/intellectual disability was more frequent in kidney dysplasia (65.2%) and agenesis (40.7%) (p = 0.001). CONCLUSIONS: We report a relative high frequency of brain/spine malformations and neurodevelopmental disorders in children with CAKUT who underwent MRI examinations in a tertiary referral center, widening the spectrum of anomalies associated with this condition.

Novel SYNGAP1 Variant in an Adult Individual Affected by Intellectual Disability and Epilepsy: A Cold Case Solved through Whole-Exome Sequencing.

Introduction: Nowadays, whole-exome sequencing (WES) analysis is an essential part in the diagnostic pathway of individuals with complex phenotypes when routine exams, such as array-CGH and gene panels, have proved inconclusive. However, data on the diagnostic rate of WES analysis in adult individuals, negative to first-tier tests, are lacking. This is because initiatives with the aim of diagnosing rare diseases focus mainly on pediatric unsolved cases. Case Presentation: We hereby present a 45-year-old woman with severe intellectual disability, previous psychomotor developmental delay, behavioral disorders, stereotypies, nonconvulsive epilepsy, and dysmorphisms. The proband first came to our attention when she was 4 years old (in 1982); since then, she has undergone several clinical and instrumental assessments, without reaching a genetic diagnosis. At last, through WES analysis, a novel de novo variant in SYNGAP1 was found. The clinical characteristics associated with SYNGAP1 are similar to those presented by the proband. Conclusion: The variant is predicted to be deleterious and is most probably the cause of the proband's phenotype. The perseverance of the clinicians and the family allowed us to reach a diagnosis in a woman with a more than 30-year history of clinical evaluations, instrumental assessments, and genetic tests. This diagnosis was of significant relevance in genetic counseling for family members and the proband herself.

Asynchronous combined central and peripheral demyelination (CCPD) in a girl with anti-MOG positivity: A case report and review of the literature.

The occurrence of combined central and peripheral demyelination (CCPD) is rare, data are limited to small case and cohort studies, mainly concerning adults. In few patients positivity to anti MOG antibody is reported, thus widening the spectrum of anti-MOG associated disorders (MOGAD). We describe a 7-year-old girl with optic neuritis followed 8 years later by peripheral demyelination, with fluctuating anti-MOG antibody positivity at cell-based assay. From the review of the literature, MOGAD-CCPD appear very rare in childhood, especially with asynchronous course. Clinicians should keep this possibility in mind to better define diagnosis in atypical demyelination syndromes, with therapeutical implications.

Acute pediatric encephalitis: etiology, course, and outcome of a 12-year single-center immunocompetent cohort.

BACKGROUND: Encephalitis is an uncommon but severe disorder due to an inflammation of the brain parenchyma, usually diagnosed on clinical, laboratory, electroencephalographic, and neuroradiological features. New causes of encephalitis have been reported in recent years, so diagnostic criteria have changed over time. We report on a single-center experience of a pediatric Hospital, the hub of its region, over 12 years (2008-2021), with the evaluation of all children managed for acute encephalitis. METHODS: We retrospectively reviewed clinical, laboratory, neuroradiological, and EEG data from the acute phase and outcome of all immunocompetent patients diagnosed with acute encephalitis. According to the newly proposed criteria for pediatric autoimmune encephalitis, we divided patients into infectious, definite autoimmune, probable autoimmune, and possible autoimmune, and performed a comparison between the different groups. RESULTS: 48 patients (26 females, mean age 4.4 years), 19 with infections, and 29 with autoimmune encephalitis, were included. Herpes simplex virus 1 encephalitis was the most frequently identified etiology followed by anti-NMDA receptor encephalitis. Movement disorders at onset and a longer hospital stay were observed more frequently in autoimmune compared to infectious encephalitis (p p < 0.001 and p = 0.001, respectively). Among the autoimmune group, children who started immunomodulatory treatment earlier (within 7 days from onset) had more frequent complete functional recovery (p = 0.002). CONCLUSIONS: Herpes virus and anti-NMDAR encephalitis are the most frequent etiologies within our cohort. Clinical onset and course are extremely variable. Since early immunomodulatory treatment was associated with a better functional outcome, our data confirm that a timely diagnostic classification in definite, probable, or possible autoimmune encephalitis can help the clinician in a successful therapeutic approach.

MYT1L variant inherited by a mosaic father in a case of severe developmental and epileptic encephalopathy.

The MYT1L gene plays a critical role in brain development, promoting the differentiation and proliferation of cells, important for the formation of brain connections. MYT1L is also involved in regulating the development of the hypothalamus, which is a crucial actor in weight regulation. Genetic variants in the MYT1L are associated with a range of developmental disorders, including intellectual disability, autism spectrum disorder, facial dysmorphisms, and epilepsy. The specific role of MYT1L in epilepsy remains elusive and no patients with developmental and epileptic encephalopathy (DEE) have been described so far. In this study, we report a patient with DEE presenting with severe refractory epilepsy, obesity, and behavioral abnormalities. Exome sequencing led to the identification of the heterozygous variant NM_001303052.2: c.1717G>A, p.(Gly573Arg) (chr2-1910340-C-T; GRCh38.p14) in the MYT1L gene. This variant was found to be inherited by the father, who was a mosaic and did not suffer from any neuropsychiatric disorders. Our observations expand the molecular and phenotype spectrum of MYT1L-related disorders, suggesting that affected individuals may present with severe epileptic phenotype leading to neurocognitive deterioration. Furthermore, we show that mosaic parents may not display the disease phenotype, with relevant implications for genetic counseling.

Genotype-phenotype correlation in contactin-associated protein-like 2 (CNTNAP-2) developmental disorder.

Contactin-associated protein-like 2 (CNTNAP2) gene encodes for CASPR2, a presynaptic type 1 transmembrane protein, involved in cell-cell adhesion and synaptic interactions. Biallelic CNTNAP2 loss has been associated with "Pitt-Hopkins-like syndrome-1" (MIM#610042), while the pathogenic role of heterozygous variants remains controversial. We report 22 novel patients harboring mono- (n = 2) and bi-allelic (n = 20) CNTNAP2 variants and carried out a literature review to characterize the genotype-phenotype correlation. Patients (M:F 14:8) were aged between 3 and 19 years and affected by global developmental delay (GDD) (n = 21), moderate to profound intellectual disability (n = 17) and epilepsy (n = 21). Seizures mainly started in the first two years of life (median 22.5 months). Antiseizure medications were successful in controlling the seizures in about two-thirds of the patients. Autism spectrum disorder (ASD) and/or other neuropsychiatric comorbidities were present in nine patients (40.9%). Nonspecific midline brain anomalies were noted in most patients while focal signal abnormalities in the temporal lobes were noted in three subjects. Genotype-phenotype correlation was performed by also including 50 previously published patients (15 mono- and 35 bi-allelic variants). Overall, GDD (p < 0.0001), epilepsy (p < 0.0001), hyporeflexia (p = 0.012), ASD (p = 0.009), language impairment (p = 0.020) and severe cognitive impairment (p = 0.031) were significantly associated with the presence of biallelic versus monoallelic variants. We have defined the main features associated with biallelic CNTNAP2 variants, as severe cognitive impairment, epilepsy and behavioral abnormalities. We propose CASPR2-deficiency neurodevelopmental disorder as an exclusively recessive disease while the contribution of heterozygous variants is less likely to follow an autosomal dominant inheritance pattern.

Abrogation of MAP4K4 protein function causes congenital anomalies in humans and zebrafish.

We report 21 families displaying neurodevelopmental differences and multiple congenital anomalies while bearing a series of rare variants in mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4). MAP4K4 has been implicated in many signaling pathways including c-Jun N-terminal and RAS kinases and is currently under investigation as a druggable target for multiple disorders. Using several zebrafish models, we demonstrate that these human variants are either loss-of-function or dominant-negative alleles and show that decreasing Map4k4 activity causes developmental defects. Furthermore, MAP4K4 can restrain hyperactive RAS signaling in early embryonic stages. Together, our data demonstrate that MAP4K4 negatively regulates RAS signaling in the early embryo and that variants identified in affected humans abrogate its function, establishing MAP4K4 as a causal locus for individuals with syndromic neurodevelopmental differences.

The phenotypic spectrum of epilepsy associated with periventricular nodular heterotopia.

BACKGROUND: Periventricular nodular heterotopia (PVNH) is a congenital brain malformation often associated with seizures. We aimed to clarify the spectrum of epilepsy phenotypes in PVNH and the significance of specific brain malformation patterns. METHODS: In this retrospective cohort study, we recruited people with PVNH and a history of seizures, and collected data via medical record review and a standardized questionnaire. RESULTS: One hundred individuals were included, aged 1 month to 61 years. Mean seizure onset age was 7.9 years. Ten patients had a self-limited epilepsy course and 35 more were pharmacoresponsive. Fifty-five had ongoing seizures, of whom 23 met criteria for drug resistance. Patients were subdivided as follows: isolated PVNH ("PVNH-Only") single nodule (18) or multiple nodules (21) and PVNH with additional brain malformations ("PVNH-Plus") single nodule (8) or multiple nodules (53). Of PVNH-Only single nodule, none had drug-resistant seizures. Amongst PVNH-Plus, 55% with multiple unilateral nodules were pharmacoresponsive, compared to only 21% with bilateral nodules. PVNH-Plus with bilateral nodules demonstrated the highest proportion of drug resistance (39%). A review of genetic testing results revealed eight patients with pathogenic or likely pathogenic single-gene variants, two of which were FLNA. Five had copy number variants, two of which were pathogenic. CONCLUSIONS: The spectrum of epilepsy phenotypes in PVNH is broad, and seizure patterns are variable; however, epilepsy course may be predicted to an extent by the pattern of malformation. Overall, drug-resistant epilepsy occurs in approximately one quarter of affected individuals. When identified, genetic etiologies are very heterogeneous.

GLUT1-DS Italian registry: past, present, and future: a useful tool for rare disorders.

BACKGROUND: GLUT1 deficiency syndrome is a rare, genetically determined neurological disorder for which Ketogenic Dietary Treatment represents the gold standard and lifelong treatment. Patient registries are powerful tools providing insights and real-world data on rare diseases. OBJECTIVE: To describe the implementation of a national web-based registry for GLUT1-DS. METHODS: This is a retrospective and prospective, multicenter, observational registry developed in collaboration with the Italian GLUT1-DS association and based on an innovative, flexible and configurable cloud computing technology platform, structured according to the most rigorous requirements for the management of patient's sensitive data. The Glut1 Registry collects baseline and follow-up data on the patient's demographics, history, symptoms, genotype, clinical, and instrumental evaluations and therapies. RESULTS: Five Centers in Italy joined the registry, and two more Centers are currently joining. In the first two years of running, data from 67 patients (40 females and 27 males) have been collected. Age at symptom onset was within the first year of life in most (40, 60%) patients. The diagnosis was formulated in infancy in almost half of the cases (34, 51%). Symptoms at onset were mainly paroxysmal (mostly epileptic seizure and paroxysmal ocular movement disorder) or mixed paroxysmal and fixed symptoms (mostly psychomotor delay). Most patients (53, 79%) are currently under Ketogenic dietary treatments. CONCLUSIONS: We describe the principles behind the design, development, and deployment of the web-based nationwide GLUT1-DS registry. It represents a stepping stone towards a more comprehensive understanding of the disease from onset to adulthood. It also represents a virtuous model from a technical, legal, and organizational point of view, thus representing a possible paradigmatic example for other rare disease registry implementation.

Extended Glasgow Outcome Scale to Evaluate the Functional Impairment of Patients With Subcortical Band Heterotopia: A Multicentric Cross-sectional Study.

BACKGROUND: Subcortical band heterotopia (SBH) is a rare malformation of the cortical development characterized by a heterotopic band of gray matter between cortex and ventricles. The clinical presentation typically includes intellectual disability and epilepsy. PURPOSE: To evaluate if the Extended Glasgow Outcome Scale-pediatric version (EGOS-ped) is a feasible tool for evaluating the functional disability of patients with (SBH). METHOD: Cross-sectional multicenter study of a cohort of 49 patients with SBH (female n = 30, 61%), recruited from 23 Italian centers. RESULTS: Thirty-nine of 49 (80%) cases showed high functional disability at EGOS-ped assessment. In the poor result subgroup (EGOS-ped >3) motor deficit, language impairment, and lower intelligence quotient were more frequent (P < 0.001, P = 0.02, and P = 0.01, respectively); the age at epilepsy onset was remarkably lower (P < 0.001); and the prevalence of epileptic encephalopathy (West syndrome or Lennox-Gastaut-like encephalopathy) was higher (P = 0.04). The thickness and the extension of the heterotopic band were associated with EGOS-ped score (P < 0.01 and P = 0.02). Pachygyria was found exclusively among patients with poor outcome (P < 0.01). CONCLUSIONS: The EGOS-ped proved to be a reliable tool for stratifying the functional disability of patients with SBH. According to this score, patients could be dichotomized: group 1 (80%) is characterized by a poor overall functionality with early epilepsy onset, thick heterotopic band, and pachygyria, whereas group 2 (20%) is characterized by a good overall functionality with later epilepsy onset and thinner heterotopic band.

Ketamine as advanced second-line treatment in benzodiazepine-refractory convulsive status epilepticus in children.

Status epilepticus (SE) is one of the most common neurological emergencies in children. To date, there is no definitive evidence to guide treatment of SE refractory to benzodiazepines. The main objectives of treatment protocols are to expedite therapeutic decisions and to use fast- and short-acting medications without significant adverse effects. Protocols differ among institutions, and most frequently valproate, phenytoin, and levetiracetam are used as second-line treatment. After failure of first- and second-line medications, admission to the intensive care unit and continuous infusion of anesthetics are usually indicated. Ketamine is a noncompetitive N-methyl-D-aspartate receptor antagonist that has been safely used for the treatment of refractory SE in adults and children. In animal models of SE, ketamine demonstrated antiepileptic and neuroprotective properties and synergistic effects with other antiseizure medications. We reviewed the literature to demonstrate the potential role of ketamine as an advanced second-line agent in the treatment of SE. Pharmacological targets, pathophysiology of SE, and the receptor trafficking hypothesis are reviewed and presented. The pharmacology of ketamine is outlined with related properties, advantages, and side effects. We summarize the most recent and relevant publications on experimental and clinical studies on ketamine in SE. Key expert opinion is also reported. Considering the current knowledge on SE pathophysiology, early sequential polytherapy should include ketamine for its wide range of positive assets. Future research and clinical trials on SE pharmacotherapy should focus on the role of ketamine as second-line medication.

Evaluating the central vein sign in paediatric-onset multiple sclerosis: A case series study.

The central vein sign (CVS) has been proposed as a biomarker of multiple sclerosis (MS). In adult-onset MS (AOMS), 40%-threshold of CVS positive (+) lesions demonstrated high accuracy for MS diagnosis. However, CVS+ lesions' performance has not been characterized in paediatric-onset (POMS) yet. We compared the CVS contribution to MS diagnosis in 10 POMS and 12 disease-duration-matched AOMS patients. Three POMS patients did not meet the 40%-threshold, while all AOMS patients were correctly diagnosed as having MS. The high proportion of periventricular confluent lesions, excluded from the CVS assessment, seemed to impair CVS sensitivity in POMS diagnosis.

Early Immunotherapy and Longer Corticosteroid Treatment Are Associated With Lower Risk of Relapsing Disease Course in Pediatric MOGAD.

BACKGROUND AND OBJECTIVES: We sought to identify early factors associated with relapse and outcome in paediatric-onset myelin oligodendrocyte glycoprotein antibody-associated disorders (MOGAD). METHODS: In a multicenter retrospective cohort of pediatric MOGAD (≤18 years), onset features and treatment were compared in patients with monophasic vs relapsing disease (including cases with follow-up ≥12 months after onset or relapse at any time) and in patients with final Expanded Disability Status Scale (EDSS) 0 vs ≥1 at last follow-up (including cases with follow-up >3 months after last event or EDSS0 at any time). Multivariable logistic regression models were used to evaluate factors associated with relapsing disease course and EDSS ≥ 1 at final follow-up. RESULTS: Seventy-five children were included (median onset age 7 years; median 30 months of follow-up). Presentation with acute disseminated encephalomyelitis was more frequent in children aged 8 years or younger (66.7%, 28/42) than in older patients (30.3%, 10/33) (p = 0.002), whereas presentation with optic neuritis was more common in children older than 8 years (57.6%, 19/33) than in younger patients (21.4%, 9/42) (p = 0.001). 40.0% (26/65) of patients relapsed. Time to first relapse was longer in children aged 8 years or younger than in older patients (median 18 vs 4 months) (p = 0.013). Factors at first event independently associated with lower risk of relapsing disease course were immunotherapy <7 days from onset (6.7-fold reduced odds of relapsing course, OR 0.15, 95% CI 0.03-0.61, p = 0.009), corticosteroid treatment for ≥5 weeks (6.7-fold reduced odds of relapse, OR 0.15, 95% CI 0.03-0.80, p = 0.026), and abnormal optic nerves on onset MRI (12.5-fold reduced odds of relapse, OR 0.08, 95% CI 0.01-0.50, p = 0.007). 21.1% (15/71) had EDSS ≥ 1 at final follow-up. Patients with a relapsing course had a higher proportion of final EDSS ≥ 1 (37.5%, 9/24) than children with monophasic disease (12.8%, 5/39) (p = 0.022, univariate analysis). Each 1-point increment in worst EDSS at onset was independently associated with 6.7-fold increased odds of final EDSS ≥ 1 (OR 6.65, 95% CI 1.33-33.26, p = 0.021). DISCUSSION: At first attack of pediatric MOGAD, early immunotherapy, longer duration of corticosteroid treatment, and abnormal optic nerves on MRI seem associated with lower risk of relapse, whereas higher disease severity is associated with greater risk of final disability (EDSS ≥ 1).

Brivaracetam add-on treatment in pediatric patients with severe drug-resistant epilepsy: Italian real-world evidence.

PURPOSE: To report the efficacy and tolerability of brivaracetam (BRV) in add-on therapy in pediatric patients with severe drug-resistant epilepsy. Prognostic factors of clinical outcome were also analyzed. METHODS: This Italian multicenter retrospective observational study was conducted on 45 pediatric patients with severe drug-resistant epilepsy, treated with BRV for at least 1 month and with a follow-up >6 months. Demographic, clinical, and treatment variables were assessed at T0 (baseline, BRV introduction) and T1 (6 months after BRV introduction). The response was defined as ≥50% seizure frequency reduction; responders and non-responders were then compared to assess potential prognostic factors. RESULTS: Forty-five patients (M = 28, mean age 12.4+/-4.4 years) were enrolled (focal epilepsy=14; generalized epilepsy=2; epileptic encephalopathy=29). At T1, 19/45 patients (42.2%) were responders (≥50% seizure frequency reduction), with 4 patients (8.9%) achieving a ≥ 75% seizure reduction and 2 patients (4.4%) becoming seizure free. Epilepsy onset at >12 months of age (p = 0.001), disease duration ≤6 years (p = 0.036), and lower seizure frequency at baseline (p = 0.008) were the prognostic factors significantly associated with a better prognosis. No significant difference emerged for demographics, epilepsy types/etiology, intellectual disability, or therapy variables. At T1, 21 patients (46.6%) discontinued BRV, mainly due to lack of efficacy (13 subjects; 28.9%) and adverse events in 8 patients (17.8%). CONCLUSION: Brivaracetam was an effective and tolerated treatment in pediatric patients with severe drug-resistant epilepsy, especially when the seizure onset was at >12 months of age, the epilepsy duration ≤6 years, and the seizure frequency before BRV treatment was low. Further and controlled studies are needed.

Electroencephalographic findings in ATRX syndrome: A new case series and review of literature.

Alpha-thalassemia X-linked intellectual disability syndrome (ATRX) is a rare genetic condition caused by mutations in the ATRX gene characterized by distinctive dysmorphic features, alpha thalassemia, mild-to-profound intellectual disability, and epilepsy, reported in nearly 30% of the patients. To date, different types of seizures are reported in patients with ATRX syndrome including either clonic, tonic, myoclonic seizures or myoclonic absences. However, an accurate analysis of electroencephalographic features is lacking in literature. We report on the epileptic and electroencephalographic phenotype of seven unpublished patients with ATRX syndrome, highlighting the presence of a peculiar EEG pattern characterized by diffuse background slowing with superimposed low voltage fast activity. Likewise, we also review the available literature on this topic.

Variant-specific changes in RAC3 function disrupt corticogenesis in neurodevelopmental phenotypes.

Variants in RAC3, encoding a small GTPase RAC3 which is critical for the regulation of actin cytoskeleton and intracellular signal transduction, are associated with a rare neurodevelopmental disorder with structural brain anomalies and facial dysmorphism. We investigated a cohort of 10 unrelated participants presenting with global psychomotor delay, hypotonia, behavioural disturbances, stereotyped movements, dysmorphic features, seizures and musculoskeletal abnormalities. MRI of brain revealed a complex pattern of variable brain malformations, including callosal abnormalities, white matter thinning, grey matter heterotopia, polymicrogyria/dysgyria, brainstem anomalies and cerebellar dysplasia. These patients harboured eight distinct de novo RAC3 variants, including six novel variants (NM_005052.3): c.34G > C p.G12R, c.179G > A p.G60D, c.186_188delGGA p.E62del, c.187G > A p.D63N, c.191A > G p.Y64C and c.348G > C p.K116N. We then examined the pathophysiological significance of these novel and previously reported pathogenic variants p.P29L, p.P34R, p.A59G, p.Q61L and p.E62K. In vitro analyses revealed that all tested RAC3 variants were biochemically and biologically active to variable extent, and exhibited a spectrum of different affinities to downstream effectors including p21-activated kinase 1. We then focused on the four variants p.Q61L, p.E62del, p.D63N and p.Y64C in the Switch II region, which is essential for the biochemical activity of small GTPases and also a variation hot spot common to other Rho family genes, RAC1 and CDC42. Acute expression of the four variants in embryonic mouse brain using in utero electroporation caused defects in cortical neuron morphology and migration ending up with cluster formation during corticogenesis. Notably, defective migration by p.E62del, p.D63N and p.Y64C were rescued by a dominant negative version of p21-activated kinase 1. Our results indicate that RAC3 variants result in morphological and functional defects in cortical neurons during brain development through variant-specific mechanisms, eventually leading to heterogeneous neurodevelopmental phenotypes.

Acute Neurological Presentation in Children With SARS-CoV-2 Infection.

Background: In the pediatric population, the knowledge of the acute presentation of SARS-CoV-2 infection is mainly limited to small series and case reports, particularly when dealing with neurological symptoms. We describe a large cohort of children with acute SARS-CoV-2 infection, focusing on the neurological manifestations and investigating correlations between disease severity and population demographics. Methods: Patients aged 0-18 years with a positive molecular swab were recruited between April 2020 and March 2021 from a tertiary Italian pediatric centre. Clinical data, imaging, and laboratory test results were retrieved from our local dataset and statistically analyzed. Results: A total of 237 patients with a median age of 3.2 years were eligible; thirty-two (13.5%) presented with neurological symptoms, including headache (65.6%), altered awareness (18.8%), ageusia/anosmia (12.5%), seizures (6.3%), and vertigo (6.3%), combined in 7 (21.9%) cases. Respiratory (59.5%) and gastrointestinal (25.3%) symptoms were the most common among the 205 (86.5%) patients without neurological involvement. Neurological symptoms did not significantly influence the severity of the triage access codes. Moreover, pre-existing medical conditions were not higher in the group with neurological manifestations. Overall, fifty-nine patients (25%, 14/59 with neurological symptoms) required treatment, being antibiotics, systemic steroids, and heparin those most prescribed. Conclusion: Our study supports the overall benign course of the SARS-CoV-2 infection in children. Neurological manifestations, except for headache, remain a rare presenting symptom, and disease severity seems unrelated to pre-existing medical conditions.