Bi-allelic Variants in RALGAPA1 Cause Profound Neurodevelopmental Disability, Muscular Hypotonia, Infantile Spasms, and Feeding Abnormalities.

Ral (Ras-like) GTPases play an important role in the control of cell migration and have been implicated in Ras-mediated tumorigenicity. Recently, variants in RALA were also described as a cause of intellectual disability and developmental delay, indicating the relevance of this pathway to neuropediatric diseases. Here, we report the identification of bi-allelic variants in RALGAPA1 (encoding Ral GTPase activating protein catalytic alpha subunit 1) in four unrelated individuals with profound neurodevelopmental disability, muscular hypotonia, feeding abnormalities, recurrent fever episodes, and infantile spasms . Dysplasia of corpus callosum with focal thinning of the posterior part and characteristic facial features appeared to be unifying findings. RalGAPA1 was absent in the fibroblasts derived from two affected individuals suggesting a loss-of-function effect of the RALGAPA1 variants. Consequently, RalA activity was increased in these cell lines, which is in keeping with the idea that RalGAPA1 deficiency causes a constitutive activation of RalA. Additionally, levels of RalGAPB, a scaffolding subunit of the RalGAP complex, were dramatically reduced, indicating a dysfunctional RalGAP complex. Moreover, RalGAPA1 deficiency clearly increased cell-surface levels of lipid raft components in detached fibroblasts, which might indicate that anchorage-dependence of cell growth signaling is disturbed. Our findings indicate that the dysregulation of the RalA pathway has an important impact on neuronal function and brain development. In light of the partially overlapping phenotype between RALA- and RALGAPA1-associated diseases, it appears likely that dysregulation of the RalA signaling pathway leads to a distinct group of genetic syndromes that we suggest could be named RALopathies.

Perampanel as precision therapy in rare genetic epilepsies.

OBJECTIVE: Perampanel, an antiseizure drug with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist properties, may have a targeted effect in genetic epilepsies with overwhelming glutamate receptor activation. Epilepsies with loss of γ-aminobutyric acid inhibition (e.g., SCN1A), overactive excitatory neurons (e.g., SCN2A, SCN8A), and variants in glutamate receptors (e.g., GRIN2A) hold special interest. We aimed to collect data from a large rare genetic epilepsy cohort treated with perampanel, to detect possible subgroups with high efficacy. METHODS: This multicenter project was based on the framework of NETRE (Network for Therapy in Rare Epilepsies), a web of pediatric neurologists treating rare epilepsies. Retrospective data from patients with genetic epilepsies treated with perampanel were collected. Outcome measures were responder rate (50% seizure reduction), and percentage of seizure reduction after 3 months of treatment. Subgroups of etiologies with high efficacy were identified. RESULTS: A total of 137 patients with 79 different etiologies, aged 2 months to 61 years (mean = 15.48 ± 9.9 years), were enrolled. The mean dosage was 6.45 ± 2.47 mg, and treatment period was 2.0 ± 1.78 years (1.5 months-8 years). Sixty-two patients (44.9%) were treated for >2 years. Ninety-eight patients (71%) were responders, and 93 (67.4%) chose to continue therapy. The mean reduction in seizure frequency was 56.61% ± 34.36%. Sixty patients (43.5%) sustained >75% reduction in seizure frequency, including 38 (27.5%) with >90% reduction in seizure frequency. The following genes showed high treatment efficacy: SCN1A, GNAO1, PIGA, PCDH19, SYNGAP1, POLG1, POLG2, and NEU1. Eleven of 17 (64.7%) patients with Dravet syndrome due to an SCN1A pathogenic variant were responders to perampanel treatment; 35.3% of them had >90% seizure reduction. Other etiologies remarkable for >90% reduction in seizures were GNAO1 and PIGA. Fourteen patients had a continuous spike and wave during sleep electroencephalographic pattern, and in six subjects perampanel reduced epileptiform activity. SIGNIFICANCE: Perampanel demonstrated high safety and efficacy in patients with rare genetic epilepsies, especially in SCN1A, GNAO1, PIGA, PCDH19, SYNGAP1, CDKL5, NEU1, and POLG, suggesting a targeted effect related to glutamate transmission.

Clinical, neuroimaging, and molecular spectrum of TECPR2-associated hereditary sensory and autonomic neuropathy with intellectual disability.

Bi-allelic TECPR2 variants have been associated with a complex syndrome with features of both a neurodevelopmental and neurodegenerative disorder. Here, we provide a comprehensive clinical description and variant interpretation framework for this genetic locus. Through international collaboration, we identified 17 individuals from 15 families with bi-allelic TECPR2-variants. We systemically reviewed clinical and molecular data from this cohort and 11 cases previously reported. Phenotypes were standardized using Human Phenotype Ontology terms. A cross-sectional analysis revealed global developmental delay/intellectual disability, muscular hypotonia, ataxia, hyporeflexia, respiratory infections, and central/nocturnal hypopnea as core manifestations. A review of brain magnetic resonance imaging scans demonstrated a thin corpus callosum in 52%. We evaluated 17 distinct variants. Missense variants in TECPR2 are predominantly located in the N- and C-terminal regions containing ß-propeller repeats. Despite constituting nearly half of disease-associated TECPR2 variants, classifying missense variants as (likely) pathogenic according to ACMG criteria remains challenging. We estimate a pathogenic variant carrier frequency of 1/1221 in the general and 1/155 in the Jewish Ashkenazi populations. Based on clinical, neuroimaging, and genetic data, we provide recommendations for variant reporting, clinical assessment, and surveillance/treatment of individuals with TECPR2-associated disorder. This sets the stage for future prospective natural history studies.

NEXMIF encephalopathy: an X-linked disorder with male and female phenotypic patterns.

PURPOSE: Pathogenic variants in the X-linked gene NEXMIF (previously KIAA2022) are associated with intellectual disability (ID), autism spectrum disorder, and epilepsy. We aimed to delineate the female and male phenotypic spectrum of NEXMIF encephalopathy. METHODS: Through an international collaboration, we analyzed the phenotypes and genotypes of 87 patients with NEXMIF encephalopathy. RESULTS: Sixty-three females and 24 males (46 new patients) with NEXMIF encephalopathy were studied, with 30 novel variants. Phenotypic features included developmental delay/ID in 86/87 (99%), seizures in 71/86 (83%) and multiple comorbidities. Generalized seizures predominated including myoclonic seizures and absence seizures (both 46/70, 66%), absence with eyelid myoclonia (17/70, 24%), and atonic seizures (30/70, 43%). Males had more severe developmental impairment; females had epilepsy more frequently, and varied from unaffected to severely affected. All NEXMIF pathogenic variants led to a premature stop codon or were deleterious structural variants. Most arose de novo, although X-linked segregation occurred for both sexes. Somatic mosaicism occurred in two males and a family with suspected parental mosaicism. CONCLUSION: NEXMIF encephalopathy is an X-linked, generalized developmental and epileptic encephalopathy characterized by myoclonic-atonic epilepsy overlapping with eyelid myoclonia with absence. Some patients have developmental encephalopathy without epilepsy. Males have more severe developmental impairment. NEXMIF encephalopathy arises due to loss-of-function variants.

Netrin-G2 dysfunction causes a Rett-like phenotype with areflexia.

We describe the underlying genetic cause of a novel Rett-like phenotype accompanied by areflexia in three methyl-CpG-binding protein 2-negative individuals from two unrelated families. Discovery analysis was performed using whole-exome sequencing followed by Sanger sequencing for validation and segregation. Functional studies using short-hairpin RNA for targeted gene knockdown were implemented by the transfection of mouse cultured primary hippocampal neurons and in vivo by in utero electroporation. All patients shared a common homozygous frameshift mutation (chr9:135073515, c.376dupT, p.(Ser126PhefsTer241)) in netrin-G2 (NTNG2, NM_032536.3) with predicted nonsense-mediated decay. The mutation fully segregated with the disease in both families. The knockdown of either NTNG2 or the related netrin-G family member NTNG1 resulted in severe neurodevelopmental defects of neuronal morphology and migration. While NTNG1 has previously been linked to a Rett syndrome (RTT)-like phenotype, this is the first description of a RTT-like phenotype caused by NTNG2 mutation. Netrin-G proteins have been shown to be required for proper axonal guidance during early brain development and involved in N-methyl- d-aspartate-mediated synaptic transmission. Our results demonstrating that knockdown of murine NTNG2 causes severe impairments of neuronal morphology and cortical migration are consistent with those of RTT animal models and the shared neurodevelopmental phenotypes between the individuals described here and typical RTT patients.

De Novo Mutations in PPP3CA Cause Severe Neurodevelopmental Disease with Seizures.

Exome sequencing has readily enabled the discovery of the genetic mutations responsible for a wide range of diseases. This success has been particularly remarkable in the severe epilepsies and other neurodevelopmental diseases for which rare, often de novo, mutations play a significant role in disease risk. Despite significant progress, the high genetic heterogeneity of these disorders often requires large sample sizes to identify a critical mass of individuals with disease-causing mutations in a single gene. By pooling genetic findings across multiple studies, we have identified six individuals with severe developmental delay (6/6), refractory seizures (5/6), and similar dysmorphic features (3/6), each harboring a de novo mutation in PPP3CA. PPP3CA encodes the alpha isoform of a subunit of calcineurin. Calcineurin encodes a calcium- and calmodulin-dependent serine/threonine protein phosphatase that plays a role in a wide range of biological processes, including being a key regulator of synaptic vesicle recycling at nerve terminals. Five individuals with de novo PPP3CA mutations were identified among 4,760 trio probands with neurodevelopmental diseases; this is highly unlikely to occur by chance (p = 1.2 × 10-8) given the size and mutability of the gene. Additionally, a sixth individual with a de novo mutation in PPP3CA was connected to this study through GeneMatcher. Based on these findings, we securely implicate PPP3CA in early-onset refractory epilepsy and further support the emerging role for synaptic dysregulation in epilepsy.

Biallelic Mutations in DNAJC12 Cause Hyperphenylalaninemia, Dystonia, and Intellectual Disability.

Phenylketonuria (PKU, phenylalanine hydroxylase deficiency), an inborn error of metabolism, can be detected through newborn screening for hyperphenylalaninemia (HPA). Most individuals with HPA harbor mutations in the gene encoding phenylalanine hydroxylase (PAH), and a small proportion (2%) exhibit tetrahydrobiopterin (BH4) deficiency with additional neurotransmitter (dopamine and serotonin) deficiency. Here we report six individuals from four unrelated families with HPA who exhibited progressive neurodevelopmental delay, dystonia, and a unique profile of neurotransmitter deficiencies without mutations in PAH or BH4 metabolism disorder-related genes. In these six affected individuals, whole-exome sequencing (WES) identified biallelic mutations in DNAJC12, which encodes a heat shock co-chaperone family member that interacts with phenylalanine, tyrosine, and tryptophan hydroxylases catalyzing the BH4-activated conversion of phenylalanine into tyrosine, tyrosine into L-dopa (the precursor of dopamine), and tryptophan into 5-hydroxytryptophan (the precursor of serotonin), respectively. DNAJC12 was undetectable in fibroblasts from the individuals with null mutations. PAH enzyme activity was reduced in the presence of DNAJC12 mutations. Early treatment with BH4 and/or neurotransmitter precursors had dramatic beneficial effects and resulted in the prevention of neurodevelopmental delay in the one individual treated before symptom onset. Thus, DNAJC12 deficiency is a preventable and treatable cause of intellectual disability that should be considered in the early differential diagnosis when screening results are positive for HPA. Sequencing of DNAJC12 may resolve any uncertainty and should be considered in all children with unresolved HPA.

Functional parameter measurements in children with ataxia telangiectasia.

AIM: To collect preliminary functional data on ataxia telangiectasia and create a disease specific scale: the Ataxia Telangiectasia Functional Scale (ATFS). METHOD: Retrospective information on patients with ataxia telangiectasia referred to the Assistive Technology Unit was included. Functional mobility scales (the Gross Motor Function Classification System [GMFCS] and the Functional Mobility Scale [FMS]-5, FMS-50, FMS-500) and activities of daily living [ADL] parameters were recorded. We created a 51-point ATFS, that consisted of three ambulation items adapted for ataxia telangiectasia in the frame of FMS (home, school, outdoors), five ADL items, and one schooling item. RESULTS: Twenty-seven participants (17 males, 10 females; mean age 10y 8mo [SD 5y 1mo], range 1y 9mo-25y 6mo), were enrolled; 168 measurements were recorded. Patients walked at a mean age of 1 year 4 months (SD 5y 4mo) and lost ambulatory capacity at 8 years 8 months (SD 2y 1mo). GMFCS level and FMS-5, FMS-50, FMS-500 assessments correlated with age (Spearman's correlations r=0.555, -0.617, -0.639, -0.662 respectively, p<0.01 for all), but plateaued after 12 years of age. ATFS mean score was 37.46 (SD 7.88) and increased with age (Spearman's correlation r=0.585, p<0.01). The scale showed three stages of disease progression. INTERPRETATION: In this pilot study we show longitudinal functional data of ambulation and ADL skills in ataxia telangiectasia, and created a framework for a functional scale. This functional scale closely approximated disease course, but further validation is required. WHAT THIS PAPER ADDS: The Gross Motor Function Classification System and the Functional Mobility Scale are ill-suited for ataxia telangiectasia assessments. Three functional mobility scales (home, school, outdoors) suited to ataxia telangiectasia were created. The Ataxia Telangiectasia Functional Scale (ATFS) combines mobility and items of activities of daily living. The ATFS closely approximates the three-stage progression of the disorder.

MEDICIONES DE PARÁMETROS FUNCIONALES EN NIÑOS CON ATAXIA TELANGIECTASIA: OBJETIVO: Recopilar datos funcionales preliminares sobre la ataxia telangiectasia y crear una escala específica de la enfermedad: la escala funcional de la ataxia telangiectasia (ATFS). MÉTODO: Se incluyó información retrospectiva sobre pacientes con ataxia telangiectasia remitidos a la Unidad de Tecnología Asistiva. Se registraron las escalas de movilidad funcional (el sistema de clasificación de la función motora gruesa [GMFCS] y la escala de movilidad funcional [FMS-5, FMS-50, FMS-500]) y los parámetros de las actividades de la vida diaria [ADL]. Creamos un ATFS de 51 puntos, que constaba de tres elementos de asistencia a la deambulación adaptados para la ataxia telangiectasia según los requerimientos de FMS (hogar, escuela, exteriores), cinco elementos de ADL y un elemento de escolarización. RESULTADOS: Se reclutaron 27 participantes (17 varones, 10 mujeres; edad media 10a 8m[DE 5a 1m], rango 1a 9m- 25a 6m); se registraron 168 mediciones. Los pacientes caminaron a una edad promedio de 1 año y 4 meses (DE 5 y 4 meses) y perdieron la capacidad ambulatoria a los 8 años y 8 meses (DE 2 y 1 meses). Las evaluaciones GMFCS y FMS-5, FMS-50, FMS-500 se correlacionaron con la edad (correlaciones de Spearman r = 0,555, -0,617, -0,639, -0,662 respectivamente, p <0,01 para todos), pero se estancaron después de los 12 años de edad. La puntuación media de ATFS fue 37,46 (SD 7,88) y aumentó con la edad (correlación de Spearman r = 0,585, p <0,01). La escala mostró tres etapas de progresión de la enfermedad. INTERPRETACIÓN: En este estudio piloto, mostramos datos funcionales longitudinales de ambulación y habilidades de ADL en la ataxia telangiectasia, y creamos un marco para una escala funcional. Esta escala funcional se aproximó mucho al curso de la enfermedad, pero se requiere validación adicional.

MEDIDAS DE PARÂMETROS FUNCIONAIS EM CRIANÇAS COM ATAXIA TELANGIECTASIA: OBJETIVO: Coletar dados funcionais prelimiares sobre ataxia telangiectasia e criar uma escala específica para a doença: a Escala Funcional de Ataxia Telangiectasia (EFAT). MÉTODO: Informações retrospectivas sobre pacientes com ataxia telangiectasia encaminhados para a Unidade de Tecnologia Assistiva foram incluídas. Escalas de mobilidade funcional (o Sistema de Classificação da Função Motora grossa [GMFCS] e a Escala de Mobilidade Funcional [FMS]-5, FMS-50, FMS-500) e parâmetros de atividades da vida diária [AVD] foram registrados. Uma EFAT de 51 pontos foi criada, consistindo de três itens de deambulação adaptados para ataxia telangiectasia na estrutura da FMF (casa, escola, comunidade), cinco itens de AVDs, e um item sobre escolaridade. RESULTADOS: Vinte e sete participantes (17 do sexo masculino, 10 nomes do sexo feminino; média de idade 10a 8m [DP 5a 1m], variação 1a 9m-25a 6m), foram recrutados; 168 medidas foram registradas. Os pacientes deambularam com uma média de (DP 5a 4m) e perderam a capacidade deambulatória com 8a 8m (DP 2a 1m). As avaliações de GMFCS e FMS-5, FMS-50, FMS-500 se correlacionara mcom a idade (correlações de r=0,555, -0,617, -0,639, -0,662 respectivamen, p<0,01 para todos), mas atingiram platô após a idade de 12 anos. O escore médio da EFAT foi 37,46 (DP 7,88) e houve aumento com a idade (correlação de Spearman r=0,585, p<0,01). A escala mostrou três estágios de progressão da doença. INTERPRETAÇÃO: Neste estudo piloto, mostramos dados funcionais longitudinais sobre a deambulação e AVDs em ataxia telangiectasia, e criamos uma estrutura para uma escala fncional. Esta escala funcional se assemelhou com o curso da doença, porém maiores estudos para validação são necessaries.

MECR Mutations Cause Childhood-Onset Dystonia and Optic Atrophy, a Mitochondrial Fatty Acid Synthesis Disorder.

Mitochondrial fatty acid synthesis (mtFAS) is an evolutionarily conserved pathway essential for the function of the respiratory chain and several mitochondrial enzyme complexes. We report here a unique neurometabolic human disorder caused by defective mtFAS. Seven individuals from five unrelated families presented with childhood-onset dystonia, optic atrophy, and basal ganglia signal abnormalities on MRI. All affected individuals were found to harbor recessive mutations in MECR encoding the mitochondrial trans-2-enoyl-coenzyme A-reductase involved in human mtFAS. All six mutations are extremely rare in the general population, segregate with the disease in the families, and are predicted to be deleterious. The nonsense c.855T>G (p.Tyr285∗), c.247_250del (p.Asn83Hisfs∗4), and splice site c.830+2_830+3insT mutations lead to C-terminal truncation variants of MECR. The missense c.695G>A (p.Gly232Glu), c.854A>G (p.Tyr285Cys), and c.772C>T (p.Arg258Trp) mutations involve conserved amino acid residues, are located within the cofactor binding domain, and are predicted by structural analysis to have a destabilizing effect. Yeast modeling and complementation studies validated the pathogenicity of the MECR mutations. Fibroblast cell lines from affected individuals displayed reduced levels of both MECR and lipoylated proteins as well as defective respiration. These results suggest that mutations in MECR cause a distinct human disorder of the mtFAS pathway. The observation of decreased lipoylation raises the possibility of a potential therapeutic strategy.

Cerebral and portal vein thrombosis, macrocephaly and atypical absence seizures in Glycosylphosphatidyl inositol deficiency due to a PIGM promoter mutation.

Defects of the glycosylphosphatidylinositol (GPI) biosynthesis pathway constitute an emerging subgroup of congenital disorders of glycosylation with heterogeneous phenotypes. A mutation in the promoter of PIGM, resulting in a syndrome with portal vein thrombosis and persistent absence seizures, was previously described in three patients. We now report four additional patients in two unrelated families, with further clinical, biochemical and molecular delineation of this unique entity. We also describe the first prenatal diagnosis of PIGM deficiency, allowing characterization of the natural history of the disease from birth. The patients described herein expand the phenotypic spectrum of PIGM deficiency to include macrocephaly and infantile-onset cerebrovascular thrombotic events. Finally, we offer insights regarding targeted treatment of this rare disorder with sodium phenylbutyrate.

Loss of Protocadherin-12 Leads to Diencephalic-Mesencephalic Junction Dysplasia Syndrome.

OBJECTIVE: To identify causes of the autosomal-recessive malformation, diencephalic-mesencephalic junction dysplasia (DMJD) syndrome. METHODS: Eight families with DMJD were studied by whole-exome or targeted sequencing, with detailed clinical and radiological characterization. Patient-derived induced pluripotent stem cells were derived into neural precursor and endothelial cells to study gene expression. RESULTS: All patients showed biallelic mutations in the nonclustered protocadherin-12 (PCDH12) gene. The characteristic clinical presentation included progressive microcephaly, craniofacial dysmorphism, psychomotor disability, epilepsy, and axial hypotonia with variable appendicular spasticity. Brain imaging showed brainstem malformations and with frequent thinned corpus callosum with punctate brain calcifications, reflecting expression of PCDH12 in neural and endothelial cells. These cells showed lack of PCDH12 expression and impaired neurite outgrowth. INTERPRETATION: DMJD patients have biallelic mutations in PCDH12 and lack of protein expression. These patients present with characteristic microcephaly and abnormalities of white matter tracts. Such pathogenic variants predict a poor outcome as a result of brainstem malformation and evidence of white matter tract defects, and should be added to the phenotypic spectrum associated with PCDH12-related conditions. Ann Neurol 2018;84:646-655.

Deleterious variants in TRAK1 disrupt mitochondrial movement and cause fatal encephalopathy.

Cellular distribution and dynamics of mitochondria are regulated by several motor proteins and a microtubule network. In neurons, mitochondrial trafficking is crucial because of high energy needs and calcium ion buffering along axons to synapses during neurotransmission. The trafficking kinesin proteins (TRAKs) are well characterized for their role in lysosomal and mitochondrial trafficking in cells, especially neurons. Using whole exome sequencing, we identified homozygous truncating variants in TRAK1 (NM_001042646:c.287-2A > C), in six lethal encephalopathic patients from three unrelated families. The pathogenic variant results in aberrant splicing and significantly reduced gene expression at the RNA and protein levels. In comparison with normal cells, TRAK1-deficient fibroblasts showed irregular mitochondrial distribution, altered mitochondrial motility, reduced mitochondrial membrane potential, and diminished mitochondrial respiration. This study confirms the role of TRAK1 in mitochondrial dynamics and constitutes the first report of this gene in association with a severe neurodevelopmental disorder.

Liver Disease in Pediatric Patients With Ataxia Telangiectasia: A Novel Report.

OBJECTIVE: Ataxia telangiectasia (A-T) is a rare genetic multiorgan disease. Although gastrointestinal involvement is known, hepatic involvement in A-T has not been investigated. We aimed to study the hepatic involvement in a large cohort of patients with A-T. METHODS: A retrospective review of patients, studied from January 1986 to January 2015 at a National A-T Center. Clinical data including demographic, genetic, laboratory, nutritional, radiographic, and histological data were retrieved. RESULTS: Fifty-three patients, 27 (49%) boys, age 14.6 ±â€Š5.2 years (range 5.9-26.1 years), were included. Twenty-three patients (43.4%), age 9.9 ±â€Š5.1 years, had consistently abnormal liver enzymes. The mean enzyme levels were alanine aminotransferase 76.8 ±â€Š73.8 IU/L, aspartate aminotransferase 70 ±â€Š50 IU/L, alkaline phosphatase 331 ±â€Š134 IU/L, and gamma glutamyl transferase 114.7 ±â€Š8 IU/L. Evaluation of other etiology of liver disease was negative. Ultrasonography revealed fatty liver in 9 of them (39%). Liver biopsy was performed in 2 patients, revealing mild-to-moderate steatosis in both, and fibrosis in 1 patient. Progression to advanced liver disease occurred in 2 of 23 (9%) patients within 2 to 5 years. Dyslipidemia was significantly associated with abnormal liver enzymes: 3 of 30 (10%) patients without abnormal liver enzymes versus 10 of 23 (45.5%) patients with abnormal liver enzymes, respectively (P < 0.05, Fisher exact test). No correlation was found between hepatic involvement and HbA1C, sex, presence of malignancy, or type of mutation. CONCLUSIONS: Abnormal liver enzymes and fatty liver are common in patients with A-T and may progress to advanced liver disease at a young age. These findings are novel and implicate that patients with A-T with abnormal liver enzymes should be evaluated for the presence of liver disease.

Whole-exome sequencing in undiagnosed genetic diseases: interpreting 119 trios.

PURPOSE: Despite the recognized clinical value of exome-based diagnostics, methods for comprehensive genomic interpretation remain immature. Diagnoses are based on known or presumed pathogenic variants in genes already associated with a similar phenotype. Here, we extend this paradigm by evaluating novel bioinformatics approaches to aid identification of new gene-disease associations. METHODS: We analyzed 119 trios to identify both diagnostic genotypes in known genes and candidate genotypes in novel genes. We considered qualifying genotypes based on their population frequency and in silico predicted effects we also characterized the patterns of genotypes enriched among this collection of patients. RESULTS: We obtained a genetic diagnosis for 29 (24%) of our patients. We showed that patients carried an excess of damaging de novo mutations in intolerant genes, particularly those shown to be essential in mice (P = 3.4 × 10(-8)). This enrichment is only partially explained by mutations found in known disease-causing genes. CONCLUSION: This work indicates that the application of appropriate bioinformatics analyses to clinical sequence data can also help implicate novel disease genes and suggest expanded phenotypes for known disease genes. These analyses further suggest that some cases resolved by whole-exome sequencing will have direct therapeutic implications.

Short Stature and Distinct Growth Characteristics in Angelman Syndrome.

Objectives - Angelman syndrome (AS) is a rare, genetic, neurodevelopmental disorder characterized by severe impairments in speech, cognition and motor skills accompanied by unique behaviors, distinct facial features and high prevalence of epilepsy and sleep problems. Despite some reports of short stature among AS patients, this feature is not included in the clinical criteria defined in 2005. We investigated growth patterns among AS patients with respect to mutation type, growth periods, family history and endocrine abnormalities. Methods - Data was collected from patients' medical files in AS national clinic. Mutation subtypes were divided to deletion and non-deletion. Four growth periods were defined: preschool, childhood, peak-height velocity, and final-height. Results - The cohort included 88 individuals (46 males), with 54 (61.4%) carrying deletion subtype. A median of 3 observations per individual , produced 280 data points. Final-height-SDS was significantly lower compared to general population (-1.23±1.26, p<0.001), and in deletion group vs. non-deletion (-1.67±1.3 vs. -0.65±0.96, p=0.03). Final-height-SDS was significantly lower compared to height-SDS in preschool period (-1.32 vs -0.47, p=0.007). Patient's final-height-SDS was significantly lower than the parents' (∆final-height-SDS=0.94±0.99, p=0.002). IGF1-SDS was significantly decreased compared to general population (-0.55±1.61, p=0.04), with lower values among deletion group (-0.70±1.44, p=0.01) Conclusions - AS patients demonstrate specific growth pattern with deceleration during childhood and adolescence resulting in significantly decreased final height compared to normal population, and even lower among deletion subgroup, which could be attributed to reduced IGF1 levels. We propose adding short stature to the clinical criteria and developing adjusted growth curves for AS population.

Gaboxadol in angelman syndrome: A double-blind, parallel-group, randomized placebo-controlled phase 3 study.

PURPOSE: To evaluate efficacy and safety of gaboxadol for treatment of children with Angelman syndrome (AS). METHOD: In this international, double-blind, phase 3 trial, we randomized children 4-12 years old with a molecular diagnosis of AS and a Clinical Global Impression (CGI)-severity score ≥3 to either daily administration of weight-based gaboxadol or matching placebo for 12 weeks. The primary endpoint was the CGI-Improvement-AS (CGI-I-AS) score at week 12. Secondary endpoints included the proportion of participants with CGI-I-AS response of ≤3 (i.e., at least "minimal improvement") and ≤2 (i.e., at least "much improvement") at week 12. Safety and tolerability were monitored throughout the study. Weight based dosing of study drug ranged from 0.125 mg/kg to 0.24 mg/kg depending on weight range. RESULTS: Between August 2019 and November 2020, 104 participants were enrolled: participants 4-12 years old were randomly (1:1) assigned to gaboxadol (n = 47) or placebo (n = 50), and 7 other participants 2─3 years old who received gaboxadol and were assessed for safety only. All gaboxadol-treated participants and 48 of 50 placebo-treated participants completed treatment. There was no significant difference in CGI-I-AS between groups: at week 12, mean CGI-I-AS score was 3.3 (SD, 1.00) and 3.2 (SD, 1.05) in the gaboxadol and placebo groups, respectively, yielding a least squares mean difference of zero (p = 0.83). There were no between-group significant differences with respect to CGI-I-AS responses. Gaboxadol was well tolerated in all age groups of this study. CONCLUSIONS: There was no significant difference in CGI-I-AS between gaboxadol and placebo after 12 weeks of study treatment in pediatric AS participants. CLINICALTRIALS: GOV: NCT04106557.

A defect in mitochondrial fatty acid synthesis impairs iron metabolism and causes elevated ceramide levels.

In most eukaryotic cells, fatty acid synthesis (FAS) occurs in the cytoplasm and in mitochondria. However, the relative contribution of mitochondrial FAS (mtFAS) to the cellular lipidome is not well defined. Here we show that loss of function of Drosophila mitochondrial enoyl coenzyme A reductase (Mecr), which is the enzyme required for the last step of mtFAS, causes lethality, while neuronal loss of Mecr leads to progressive neurodegeneration. We observe a defect in Fe-S cluster biogenesis and increased iron levels in flies lacking mecr, leading to elevated ceramide levels. Reducing the levels of either iron or ceramide suppresses the neurodegenerative phenotypes, indicating an interplay between ceramide and iron metabolism. Mutations in human MECR cause pediatric-onset neurodegeneration, and we show that human-derived fibroblasts display similar elevated ceramide levels and impaired iron homeostasis. In summary, this study identifies a role of mecr/MECR in ceramide and iron metabolism, providing a mechanistic link between mtFAS and neurodegeneration.

The Long-Term Effectiveness and Safety of Cannabidiol-Enriched Oil in Children With Drug-Resistant Epilepsy.

BACKGROUND: Several retrospective studies on pediatric epilepsy reported positive effects of cannabidiol-enriched artisanal cannabis oil and pure cannabidiol oil on seizure reduction. METHODS: This is a retrospective study of children and adolescents with refractory epilepsy caused by various etiologies who were treated with artisanal cannabis oil during January 2014 to June 2019, with at least one year follow-up. RESULTS: Of 114 patients, 84 (73.3%) reported some improvement in seizure frequency at some point during treatment. Fifty-one (59%) of the 86 patients who continued treatment for at least one year showed >50% improvement in seizure frequency. Seizure etiology, seizure type, and patients' age and sex were not found to be associated with the response to cannabidiol-enriched cannabis oil. Side effects were minor, and positive effects beyond seizure reduction were noted. CONCLUSIONS: Artisanal cannabidiol-enriched cannabis may be an effective and safe long-term treatment for refractory epilepsy.

Felbamate for pediatric epilepsy-should we keep on using it as the last resort?

Introduction: Concerns regarding felbamate adverse effects restrict its widespread use in children with drug-resistant epilepsy. We aimed to examine the efficacy and safety of felbamate in those children and identify the ones who may benefit most from its use. Methods: We retrospectively reviewed the medical files of all patients who were treated with felbamate in a tertiary pediatric epilepsy clinic between 2009-2021. Drug efficacy was determined at the first 3 months of treatment and thereafter. Therapeutic response and adverse reactions were monitored throughout the course of treatment. Results: Our study included 75 children (age 8.9 ± 3.7 years), of whom 53 were treated with felbamate for seizures, 16 for electrical status epilepticus during sleep and 6 for both. The median follow-up time was 16 months (range 1-129 months). The most common cause for epilepsy was genetic (29%). The median number of previous anti-seizure medications was six [4-8]. A therapeutic response ≥50% was documented in 37 (51%) patients, and a complete response in 9 (12%). Nineteen patients (25%) sustained adverse reactions, including three cases of elevated liver enzymes and one case of neutropenia with normal bone marrow aspiration. In all cases, treatment could be continued. All children with intractable epilepsy following herpes encephalitis showed a response to felbamate. Conclusion: Felbamate is an efficacious and safe anti-seizure medication in the pediatric population.

Clinical impact of exome sequencing in the setting of a general pediatric ward for hospitalized children with suspected genetic disorders.

Background: Genetic conditions contribute a significant portion of disease etiologies in children admitted to general pediatric wards worldwide. While exome sequencing (ES) has improved clinical diagnosis and management over a variety of pediatric subspecialties, it is not yet routinely used by general pediatric hospitalists. We aim to investigate the impact of exome sequencing in sequencing-naive children suspected of having monogenic disorders while receiving inpatient care. Methods: We prospectively employed exome sequencing in children admitted to the general pediatric inpatient service at a large tertiary medical center in Israel. Genetic analysis was triggered by general and/or subspecialist pediatricians who were part of the primary inpatient team. We determined the diagnostic yield among children who were referred for exome sequencing and observed the effects of genetic diagnosis on medical care. Results: A total of fifty probands were evaluated and exome sequenced during the study period. The most common phenotypes included were neurodevelopmental (56%), gastrointestinal (34%), and congenital cardiac anomalies (24%). A molecular diagnosis was reached in 38% of patients. Among seven patients (37%), the molecular genetic diagnosis influenced subsequent clinical management already during admission or shortly following discharge. Conclusion: We identified a significant fraction of genetic etiologies among undiagnosed children admitted to the general pediatric ward. Our results support that early application of exome sequencing may be maximized by pediatric hospitalists' high index of suspicion for an underlying genetic etiology, prompting an in-house genetic evaluation. This framework should include a multidisciplinary co-management approach of the primary care team working alongside with subspecialties, geneticists and bioinformaticians.