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Biallelic variants in PUS3 have recently been recognized as a rare cause of neurodevelopmental disorders. Pseudouridine synthase-3 encoded by PUS3 is an enzyme important for modification of various RNAs, including transfer RNA (tRNA). Here we present the clinical and genetic features of 21 individuals with biallelic PUS3 variants: seven new and 14 previously reported individuals, where clinical features of two were updated. The clinical and genetic information were collected through collaborations or by literature search. All individuals were characterized by the local clinicians and the gene variants were identified by next generation sequencing (NGS) based methodologies. The clinical picture was dominated by global developmental delay, epilepsy, hypotonia and microcephaly. Gray sclera, which has previously been suggested to be a characteristic feature of PUS3-associated phenotypes, was reported in only seven individuals. The patients had some dysmorphic facial features, but a recognizable gestalt was not observed. In conclusion, homozygous and compound heterozygous PUS3 variants lead to a rare neurodevelopmental disorder. Further functional studies are necessary to understand involvement of PUS3 and tRNA biogenesis in normal and abnormal brain development., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Status epilepticus is a life-threatening medical condition which requires immediate diagnosis and treatment. In children, it may be a recurrent manifestation in the context of heterogeneous severe developmental genetic encephalopathies, as well as the first neurological manifestation. Mutations in several genes have been consistently associated with status epilepticus despite none of them can be considered as 'pure' Mendelian status epilepticus gene. Most genetic conditions featuring status epilepticus can be assigned to specific phenotypic subgroups, including cortical dysplasias, inborn errors of metabolism, mitochondrial diseases, or epileptic encephalopathies and childhood syndromes. Next generation sequencing (NGS) has increased the number of genes associated with, and improved the turnaround time for molecular diagnosis of, status epilepticus, allowing more timely and rationale management choices for specific conditions. Next generation sequencing might become part of the standard of care in the near future for a large subset of patients with status epilepticus, especially in early life. At present, trios whole exome sequencing, with a first analysis of point and copy number variants of an in silico panel containing 'status epilepticus' genes might represent best choice as it would allow a rapid screening. This article is part of the Special Issue "Proceedings of the 7th London-Innsbruck Colloquium on Status Epilepticus and Acute Seizures"., (Copyright © 2019. Published by Elsevier Inc.)

Objective: To delineate the epileptology, a key part of the SYNGAP1 phenotypic spectrum, in a large patient cohort., Methods: Patients were recruited via investigators' practices or social media. We included patients with (likely) pathogenic SYNGAP1 variants or chromosome 6p21.32 microdeletions incorporating SYNGAP1 . We analyzed patients' phenotypes using a standardized epilepsy questionnaire, medical records, EEG, MRI, and seizure videos., Results: We included 57 patients (53% male, median age 8 years) with SYNGAP1 mutations (n = 53) or microdeletions (n = 4). Of the 57 patients, 56 had epilepsy: generalized in 55, with focal seizures in 7 and infantile spasms in 1. Median seizure onset age was 2 years. A novel type of drop attack was identified comprising eyelid myoclonia evolving to a myoclonic-atonic (n = 5) or atonic (n = 8) seizure. Seizure types included eyelid myoclonia with absences (65%), myoclonic seizures (34%), atypical (20%) and typical (18%) absences, and atonic seizures (14%), triggered by eating in 25%. Developmental delay preceded seizure onset in 54 of 56 (96%) patients for whom early developmental history was available. Developmental plateauing or regression occurred with seizures in 56 in the context of a developmental and epileptic encephalopathy (DEE). Fifty-five of 57 patients had intellectual disability, which was moderate to severe in 50. Other common features included behavioral problems (73%); high pain threshold (72%); eating problems, including oral aversion (68%); hypotonia (67%); sleeping problems (62%); autism spectrum disorder (54%); and ataxia or gait abnormalities (51%)., Conclusions: SYNGAP1 mutations cause a generalized DEE with a distinctive syndrome combining epilepsy with eyelid myoclonia with absences and myoclonic-atonic seizures, as well as a predilection to seizures triggered by eating., (Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Background: Patients with Gaucher Disease (GD) exhibit three phenotypes, including type 1 (non-neuronopathic), type 2 (acute neuronopathic), and type 3 (subacute neuronopathic)., Aim: Identifying which GBA changes represent benign polymorphisms and which may result in disease-causing mutations is essential for diagnosis and genotype/phenotype correlations but is often challenging., Results: Here, we describe a patient with type 3 GD, presenting with drug-resistant epilepsy, who bears a set of GBA polymorphic variants including the novel c.363A > G (Gly82Gly) synonymous mutation. In silico predictions, mRNA and functional studies revealed that the new Gly82Gly mutation causes skipping of GBA exon 4, leading to a severe reduction of the wild type GBA mRNA. This is the first report of a synonymous change causing GD through loss of an exonic splicing enhancer sequence. The synonymous mutation is in trans with the Asn188Ser missense mutation, thus making the Asn188Ser responsible for the patient's phenotype and strengthening the association of Asn188Ser with the particular neurological phenotype of type 3 GD., Conclusion: We strengthen the association of Asn188Ser with the type 3 GD phenotype and progressive myoclonus epilepsy. Our data confirm that in silico predictions and mRNA analysis are mandatory in discriminating pathological mutations from the background of harmless polymorphisms, especially synonymous changes.

We describe a mother and son with focal epilepsy, mild cognitive impairment, and pachygyria, which was parieto-occipital in the mother and with remarkable posterior greater than anterior severity in the son. Overall clinical manifestations, although overlapping in type, were likewise slightly more severe in the son. Using targeted resequencing through a gene panel for malformations of cortical development, we identified the c.655 T > A [p.(Trp219Arg)] novel missense variant in the LIS1 gene, segregating in the proband and in his mother. Western Blot analysis, qPCR gene expression and RT-PCR disclosed no significant differences between proband, his parents, and controls. Epilepsy and mild cognitive impairment can be the only clinical presentation of constitutional LIS1 mutations, which can therefore be inherited if the associated phenotype implies limited or no reproductive disadvantage. Parents of patients harboring LIS1 mutations should be assessed for their mutation carrier status., (© 2018 Wiley Periodicals, Inc.)

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control neuronal excitability and their dysfunction has been linked to epileptogenesis but few individuals with neurological disorders related to variants altering HCN channels have been reported so far. In 2014, we described five individuals with epileptic encephalopathy due to de novo HCN1 variants. To delineate HCN1-related disorders and investigate genotype-phenotype correlations further, we assembled a cohort of 33 unpublished patients with novel pathogenic or likely pathogenic variants: 19 probands carrying 14 different de novo mutations and four families with dominantly inherited variants segregating with epilepsy in 14 individuals, but not penetrant in six additional individuals. Sporadic patients had epilepsy with median onset at age 7 months and in 36% the first seizure occurred during a febrile illness. Overall, considering familial and sporadic patients, the predominant phenotypes were mild, including genetic generalized epilepsies and genetic epilepsy with febrile seizures plus (GEFS+) spectrum. About 20% manifested neonatal/infantile onset otherwise unclassified epileptic encephalopathy. The study also included eight patients with variants of unknown significance: one adopted patient had two HCN1 variants, four probands had intellectual disability without seizures, and three individuals had missense variants inherited from an asymptomatic parent. Of the 18 novel pathogenic missense variants identified, 12 were associated with severe phenotypes and clustered within or close to transmembrane domains, while variants segregating with milder phenotypes were located outside transmembrane domains, in the intracellular N- and C-terminal parts of the channel. Five recurrent variants were associated with similar phenotypes. Using whole-cell patch-clamp, we showed that the impact of 12 selected variants ranged from complete loss-of-function to significant shifts in activation kinetics and/or voltage dependence. Functional analysis of three different substitutions altering Gly391 revealed that these variants had different consequences on channel biophysical properties. The Gly391Asp variant, associated with the most severe, neonatal phenotype, also had the most severe impact on channel function. Molecular dynamics simulation on channel structure showed that homotetramers were not conducting ions because the permeation path was blocked by cation(s) strongly complexed to the Asp residue, whereas heterotetramers showed an instantaneous current component possibly linked to deformation of the channel pore. In conclusion, our results considerably expand the clinical spectrum related to HCN1 variants to include common generalized epilepsy phenotypes and further illustrate how HCN1 has a pivotal function in brain development and control of neuronal excitability.

Background: Mucopolysaccharidosis-IVA (Morquio A disease) is a lysosomal disorder in which the abnormal accumulation of keratan sulfate and chondroitin-6-sulfate is consequent to mutations in the galactosamine-6-sulfatase (GALNS) gene. Since standard DNA sequencing analysis fails to detect about 16% of GALNS mutant alleles, gross DNA rearrangement screening and uniparental disomy evaluation are required to complete the molecular diagnosis. Despite this, the second pathogenic GALNS allele generally remains unidentified in ~ 5% of Morquio-A disease patients., Methods: In an attempt to bridge the residual gap between clinical and molecular diagnosis, we performed an mRNA-based evaluation of three Morquio-A disease patients in whom the second mutant GALNS allele had not been identified. We also performed sequence analysis of the entire GALNS gene in two patients., Results: Different aberrant GALNS mRNA transcripts were characterized in each patient. Analysis of these transcripts then allowed the identification, in one patient, of a disease-causing deep intronic GALNS mutation. The aberrant mRNA products identified in the other two individuals resulted in partial exon loss. Despite sequencing the entire GALNS gene region in these patients, the identity of a single underlying pathological lesion could not be unequivocally determined. We postulate that a combination of multiple variants, acting in cis, may synergise in terms of their impact on the splicing machinery., Conclusions: We have identified GALNS variants located within deep intronic regions that have the potential to impact splicing. These findings have prompted us to incorporate mRNA analysis into our diagnostic flow procedure for the molecular analysis of Morquio A disease.

Background: Allelic heterogeneity is an important feature of the GLA gene for which almost 900 known genetic variants have been discovered so far. Pathogenetic GLA variants cause alpha-galactosidase A (α-Gal A) enzyme deficiency leading to the X-linked lysosomal storage disorder Fabry disease (FD). Benign GLA intronic and exonic variants (e.g. pseudodeficient p.Asp313Tyr) have also been described. Some GLA missense variants, previously deemed to be pathogenetic (e.g. p.Glu66Gln and p.Arg118Cys), they have been reclassified as benign after re-evaluation by functional and population studies. Hence, the functional role of novel GLA variants should be investigated to assess their clinical relevance., Results: We identified six GLA variants in 4 males and 2 females who exhibited symptoms of FD: c.159C>G p.(Asn53Lys), c.400T>C p.(Tyr134His), c.680G>C (p.Arg227Pro), c.815A>T p.(Asn272Ile), c.907A>T p.(Ile303Phe) and c.1163_1165delTCC (p.Leu388del). We evaluated their impact on the α-Gal A protein by bioinformatic analysis and homology modelling, by analysis of the GLA mRNA, and by site-directed mutagenesis and in vitro expression studies. We also measured their responsiveness to the pharmacological chaperone DGJ., Conclusions: The six detected GLA variants cause deficient α-Gal A activity and impairment or loss of the protein wild-type structure. We found p.Asn53Lys and p.Ile303Phe variants to be susceptible to DGJ., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

V-type proton (H+) ATPase (v-ATPase) is a multi-subunit proton pump that regulates pH homeostasis in all eukaryotic cells; in neurons, v-ATPase plays additional and unique roles in synapse function. Through whole exome sequencing, we identified de novo heterozygous mutations (p.Pro27Arg, p.Asp100Tyr, p.Asp349Asn, p.Asp371Gly) in ATP6V1A, encoding the A subunit of v-ATPase, in four patients with developmental encephalopathy with epilepsy. Early manifestations, observed in all patients, were developmental delay and febrile seizures, evolving to encephalopathy with profound delay, hypotonic/dyskinetic quadriparesis and intractable multiple seizure types in two patients (p.Pro27Arg, p.Asp100Tyr), and to moderate delay with milder epilepsy in the other two (p.Asp349Asn, p.Asp371Gly). Modelling performed on the available prokaryotic and eukaryotic structures of v-ATPase predicted p.Pro27Arg to perturb subunit interaction, p.Asp100Tyr to cause steric hindrance and destabilize protein folding, p.Asp349Asn to affect the catalytic function and p.Asp371Gly to impair the rotation process, necessary for proton transport. We addressed the impact of p.Asp349Asn and p.Asp100Tyr mutations on ATP6V1A expression and function by analysing ATP6V1A-overexpressing HEK293T cells and patients' lymphoblasts. The p.Asp100Tyr mutant was characterized by reduced expression due to increased degradation. Conversely, no decrease in expression and clearance was observed for p.Asp349Asn. In HEK293T cells overexpressing either pathogenic or control variants, p.Asp349Asn significantly increased LysoTracker® fluorescence with no effects on EEA1 and LAMP1 expression. Conversely, p.Asp100Tyr decreased both LysoTracker® fluorescence and LAMP1 levels, leaving EEA1 expression unaffected. Both mutations decreased v-ATPase recruitment to autophagosomes, with no major impact on autophagy. Experiments performed on patients' lymphoblasts using the LysoSensor™ probe revealed lower pH of endocytic organelles for p.Asp349Asn and a reduced expression of LAMP1 with no effect on the pH for p.Asp100Tyr. These data demonstrate gain of function for p.Asp349Asn characterized by an increased proton pumping in intracellular organelles, and loss of function for p.Asp100Tyr with decreased expression of ATP6V1A and reduced levels of lysosomal markers. We expressed p.Asp349Asn and p.Asp100Tyr in rat hippocampal neurons and confirmed significant and opposite effects in lysosomal labelling. However, both mutations caused a similar defect in neurite elongation accompanied by loss of excitatory inputs, revealing that altered lysosomal homeostasis markedly affects neurite development and synaptic connectivity. This study provides evidence that de novo heterozygous ATP6V1A mutations cause a developmental encephalopathy with a pathomechanism that involves perturbations of lysosomal homeostasis and neuronal connectivity, uncovering a novel role for v-ATPase in neuronal development.

Mitochondrial diseases are a plethora of inherited neuromuscular disorders sharing defects in mitochondrial respiration, but largely different from one another for genetic basis and pathogenic mechanism. Whole exome sequencing was performed in a familiar trio (trio-WES) with a child affected by severe epileptic encephalopathy associated with respiratory complex I deficiency and mitochondrial DNA depletion in skeletal muscle. By trio-WES we identified biallelic mutations in SLC25A10, a nuclear gene encoding a member of the mitochondrial carrier family. Genetic and functional analyses conducted on patient fibroblasts showed that SLC25A10 mutations are associated with reduction in RNA quantity and aberrant RNA splicing, and to absence of SLC25A10 protein and its transporting function. The yeast SLC25A10 ortholog knockout strain showed defects in mitochondrial respiration and mitochondrial DNA content, similarly to what observed in the patient skeletal muscle, and growth susceptibility to oxidative stress. Albeit patient fibroblasts were depleted in the main antioxidant molecules NADPH and glutathione, transport assays demonstrated that SLC25A10 is unable to transport glutathione. Here, we report the first recessive mutations of SLC25A10 associated to an inherited severe mitochondrial neurodegenerative disorder. We propose that SLC25A10 loss-of-function causes pathological disarrangements in respiratory-demanding conditions and oxidative stress vulnerability., (© The Author(s) 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Trio exome sequencing has been successful in identifying genes with de novo mutations (DNMs) causing epileptic encephalopathy (EE) and other neurodevelopmental disorders. Here, we evaluate how well a case-control collapsing analysis recovers genes causing dominant forms of EE originally implicated by DNM analysis. We performed a genome-wide search for an enrichment of "qualifying variants" in protein-coding genes in 488 unrelated cases compared to 12,151 unrelated controls. These "qualifying variants" were selected to be extremely rare variants predicted to functionally impact the protein to enrich for likely pathogenic variants. Despite modest sample size, three known EE genes (KCNT1, SCN2A, and STXBP1) achieved genome-wide significance (p<2.68×10-6). In addition, six of the 10 most significantly associated genes are known EE genes, and the majority of the known EE genes (17 out of 25) originally implicated in trio sequencing are nominally significant (p<0.05), a proportion significantly higher than the expected (Fisher's exact p = 2.33×10-17). Our results indicate that a case-control collapsing analysis can identify several of the EE genes originally implicated in trio sequencing studies, and clearly show that additional genes would be implicated with larger sample sizes. The case-control analysis not only makes discovery easier and more economical in early onset disorders, particularly when large cohorts are available, but also supports the use of this approach to identify genes in diseases that present later in life when parents are not readily available.

Mutations of genes within the phosphatidylinositol-3-kinase (PI3K)-AKT-MTOR pathway are well known causes of brain overgrowth (megalencephaly) as well as segmental cortical dysplasia (such as hemimegalencephaly, focal cortical dysplasia and polymicrogyria). Mutations of the AKT3 gene have been reported in a few individuals with brain malformations, to date. Therefore, our understanding regarding the clinical and molecular spectrum associated with mutations of this critical gene is limited, with no clear genotype-phenotype correlations. We sought to further delineate this spectrum, study levels of mosaicism and identify genotype-phenotype correlations of AKT3-related disorders. We performed targeted sequencing of AKT3 on individuals with these phenotypes by molecular inversion probes and/or Sanger sequencing to determine the type and level of mosaicism of mutations. We analysed all clinical and brain imaging data of mutation-positive individuals including neuropathological analysis in one instance. We performed ex vivo kinase assays on AKT3 engineered with the patient mutations and examined the phospholipid binding profile of pleckstrin homology domain localizing mutations. We identified 14 new individuals with AKT3 mutations with several phenotypes dependent on the type of mutation and level of mosaicism. Our comprehensive clinical characterization, and review of all previously published patients, broadly segregates individuals with AKT3 mutations into two groups: patients with highly asymmetric cortical dysplasia caused by the common p.E17K mutation, and patients with constitutional AKT3 mutations exhibiting more variable phenotypes including bilateral cortical malformations, polymicrogyria, periventricular nodular heterotopia and diffuse megalencephaly without cortical dysplasia. All mutations increased kinase activity, and pleckstrin homology domain mutants exhibited enhanced phospholipid binding. Overall, our study shows that activating mutations of the critical AKT3 gene are associated with a wide spectrum of brain involvement ranging from focal or segmental brain malformations (such as hemimegalencephaly and polymicrogyria) predominantly due to mosaic AKT3 mutations, to diffuse bilateral cortical malformations, megalencephaly and heterotopia due to constitutional AKT3 mutations. We also provide the first detailed neuropathological examination of a child with extreme megalencephaly due to a constitutional AKT3 mutation. This child has one of the largest documented paediatric brain sizes, to our knowledge. Finally, our data show that constitutional AKT3 mutations are associated with megalencephaly, with or without autism, similar to PTEN-related disorders. Recognition of this broad clinical and molecular spectrum of AKT3 mutations is important for providing early diagnosis and appropriate management of affected individuals, and will facilitate targeted design of future human clinical trials using PI3K-AKT pathway inhibitors., (© The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

We describe the clinical and whole genome sequencing (WGS) study of a non-consanguineous Italian family in which two siblings, a boy and a girl, manifesting a severe epileptic encephalopathy (EE) with skeletal abnormalities, carried novel SLC35A3 compound heterozygous mutations. Both siblings exhibited infantile spasms, associated with focal, and tonic vibratory seizures from early infancy. EEG recordings showed a suppression-burst (SB) pattern and multifocal paroxysmal activity in both. In addition both had quadriplegia, acquired microcephaly, and severe intellectual disability. General examination showed distal arthrogryposis predominant in the hands in both siblings and severe left dorso-lumbar convex scoliosis in one. WGS of the siblings-parents quartet identified novel compound heterozygous mutations in SLC35A3 in both children. SLC35A3 encodes the major Golgi uridine diphosphate N-acetylglucosamine transporter. With this study, we add SLC35A3 to the gene list of epilepsies. Neurological symptoms and skeletal abnormalities might result from impaired glycosylation of proteins involved in normal development and function of the central nervous system and skeletal apparatus., (© 2017 Wiley Periodicals, Inc.)

Objective: To explore the prognostic value of initial clinical and mutational findings in infants with SCN1A mutations., Methods: Combining sex, age/fever at first seizure, family history of epilepsy, EEG, and mutation type, we analyzed the accuracy of significant associations in predicting Dravet syndrome vs milder outcomes in 182 mutation carriers ascertained after seizure onset. To assess the diagnostic accuracy of all parameters, we calculated sensitivity, specificity, receiver operating characteristic (ROC) curves, diagnostic odds ratios, and positive and negative predictive values and the accuracy of combined information. We also included in the study demographic and mutational data of the healthy relatives of mutation carrier patients., Results: Ninety-seven individuals (48.5%) had Dravet syndrome, 49 (23.8%) had generalized/genetic epilepsy with febrile seizures plus, 30 (14.8%) had febrile seizures, 6 (3.5%) had focal epilepsy, and 18 (8.9%) were healthy relatives. The association study indicated that age at first seizure and frameshift mutations were associated with Dravet syndrome. The risk of Dravet syndrome was 85% in the 0- to 6-month group, 51% in the 6- to 12-month range, and 0% after the 12th month. ROC analysis identified onset within the sixth month as the diagnostic cutoff for progression to Dravet syndrome (sensitivity = 83.3%, specificity = 76.6%)., Conclusions: In individuals with SCN1A mutations, age at seizure onset appears to predict outcome better than mutation type. Because outcome is not predetermined by genetic factors only, early recognition and treatment that mitigates prolonged/repeated seizures in the first year of life might also limit the progression to epileptic encephalopathy., (© 2017 American Academy of Neurology.)

Targeted resequencing gene panels are used in the diagnostic setting to identify gene defects in epilepsy. We performed targeted resequencing using a 30-genes panel and a 95-genes panel in 349 patients with drug-resistant epilepsies beginning in the first years of life. We identified 71 pathogenic variants, 42 of which novel, in 30 genes, corresponding to 20.3% of the probands. In 66% of mutation positive patients, epilepsy onset occurred before the age of 6 months. The 95-genes panel allowed a genetic diagnosis in 22 (6.3%) patients that would have otherwise been missed using the 30-gene panel. About 50% of mutations were identified in genes coding for sodium and potassium channel components. SCN2A was the most frequently mutated gene followed by SCN1A, KCNQ2, STXBP1, SCN8A, CDKL5, and MECP2. Twenty-nine mutations were identified in 23 additional genes, most of them recently associated with epilepsy. Our data show that panels targeting about 100 genes represent the best cost-effective diagnostic option in pediatric drug-resistant epilepsies. They enable molecular diagnosis of atypical phenotypes, allowing to broaden phenotype-genotype correlations. Molecular diagnosis might influence patients' management and translate into better and specific treatment recommendations in some conditions., (© 2016 WILEY PERIODICALS, INC.)

Objective: To examine the role of mutations in GABRB3 encoding the β 3 subunit of the GABA A receptor in individual patients with epilepsy with regard to causality, the spectrum of genetic variants, their pathophysiology, and associated phenotypes., Methods: We performed massive parallel sequencing of GABRB3 in 416 patients with a range of epileptic encephalopathies and childhood-onset epilepsies and recruited additional patients with epilepsy with GABRB3 mutations from other research and diagnostic programs., Results: We identified 22 patients with heterozygous mutations in GABRB3, including 3 probands from multiplex families. The phenotypic spectrum of the mutation carriers ranged from simple febrile seizures, genetic epilepsies with febrile seizures plus, and epilepsy with myoclonic-atonic seizures to West syndrome and other types of severe, early-onset epileptic encephalopathies. Electrophysiologic analysis of 7 mutations in Xenopus laevis oocytes, using coexpression of wild-type or mutant β 3 , together with α 5 and γ 2s subunits and an automated 2-microelectrode voltage-clamp system, revealed reduced GABA-induced current amplitudes or GABA sensitivity for 5 of 7 mutations., Conclusions: Our results indicate that GABRB3 mutations are associated with a broad phenotypic spectrum of epilepsies and that reduced receptor function causing GABAergic disinhibition represents the relevant disease mechanism., (© 2017 American Academy of Neurology.)

Objective: To delineate phenotypic heterogeneity, we describe the clinical features of a cohort of patients with GABRA1 gene mutations., Methods: Patients with GABRA1 mutations were ascertained through an international collaboration. Clinical, EEG, and genetic data were collected. Functional analysis of 4 selected mutations was performed using the Xenopus laevis oocyte expression system., Results: The study included 16 novel probands and 3 additional family members with a disease-causing mutation in the GABRA1 gene. The phenotypic spectrum varied from unspecified epilepsy (1), juvenile myoclonic epilepsy (2), photosensitive idiopathic generalized epilepsy (1), and generalized epilepsy with febrile seizures plus (1) to severe epileptic encephalopathies (11). In the epileptic encephalopathy group, the patients had seizures beginning between the first day of life and 15 months, with a mean of 7 months. Predominant seizure types in all patients were tonic-clonic in 9 participants (56%) and myoclonic seizures in 5 (31%). EEG showed a generalized photoparoxysmal response in 6 patients (37%). Four selected mutations studied functionally revealed a loss of function, without a clear genotype-phenotype correlation., Conclusions: GABRA1 mutations make a significant contribution to the genetic etiology of both benign and severe epilepsy syndromes. Myoclonic and tonic-clonic seizures with pathologic response to photic stimulation are common and shared features in both mild and severe phenotypes., (© 2016 American Academy of Neurology.)

Importance: Focal cortical dysplasia (FCD), hemimegalencephaly, and megalencephaly constitute a spectrum of malformations of cortical development with shared neuropathologic features. These disorders are associated with significant childhood morbidity and mortality., Objective: To identify the underlying molecular cause of FCD, hemimegalencephaly, and diffuse megalencephaly., Design, Setting, and Participants: Patients with FCD, hemimegalencephaly, or megalencephaly (mean age, 11.7 years; range, 2-32 years) were recruited from Pediatric Hospital A. Meyer, the University of Hong Kong, and Seattle Children's Research Institute from June 2012 to June 2014. Whole-exome sequencing (WES) was performed on 8 children with FCD or hemimegalencephaly using standard-depth (50-60X) sequencing in peripheral samples (blood, saliva, or skin) from the affected child and their parents and deep (150-180X) sequencing in affected brain tissue. Targeted sequencing and WES were used to screen 93 children with molecularly unexplained diffuse or focal brain overgrowth. Histopathologic and functional assays of phosphatidylinositol 3-kinase-AKT (serine/threonine kinase)-mammalian target of rapamycin (mTOR) pathway activity in resected brain tissue and cultured neurons were performed to validate mutations., Main Outcomes and Measures: Whole-exome sequencing and targeted sequencing identified variants associated with this spectrum of developmental brain disorders., Results: Low-level mosaic mutations of MTOR were identified in brain tissue in 4 children with FCD type 2a with alternative allele fractions ranging from 0.012 to 0.086. Intermediate-level mosaic mutation of MTOR (p.Thr1977Ile) was also identified in 3 unrelated children with diffuse megalencephaly and pigmentary mosaicism in skin. Finally, a constitutional de novo mutation of MTOR (p.Glu1799Lys) was identified in 3 unrelated children with diffuse megalencephaly and intellectual disability. Molecular and functional analysis in 2 children with FCD2a from whom multiple affected brain tissue samples were available revealed a mutation gradient with an epicenter in the most epileptogenic area. When expressed in cultured neurons, all MTOR mutations identified here drive constitutive activation of mTOR complex 1 and enlarged neuronal size., Conclusions and Relevance: In this study, mutations of MTOR were associated with a spectrum of brain overgrowth phenotypes extending from FCD type 2a to diffuse megalencephaly, distinguished by different mutations and levels of mosaicism. These mutations may be sufficient to cause cellular hypertrophy in cultured neurons and may provide a demonstration of the pattern of mosaicism in brain and substantiate the link between mosaic mutations of MTOR and pigmentary mosaicism in skin.

Purpose: PCDH19 mutations cause epilepsy and mental retardation limited to females (EFMR) or Dravet-like syndromes. Especially in the first years of life, epilepsy is known to be highly pharmacoresistant. The aim of our study was to evaluate the effectiveness of antiepileptic therapy in patients with PCDH19 mutations., Methods: We report a retrospective multicenter study of antiepileptic therapy in 58 female patients with PCDH19 mutations and epilepsy aged 2-27 years (mean age 10.6 years)., Results: The most effective drugs after 3 months were clobazam and bromide, with a responder rate of 68% and 67%, respectively, where response was defined as seizure reduction of at least 50%. Defining long-term response as the proportion of responders after 12 months of treatment with a given drug in relation to the number of patients treated for at least 3 months, the most effective drugs after 12 months were again bromide and clobazam, with a long-term response of 50% and 43%, respectively. Seventy-four percent of the patients became seizure-free for at least 3 months, 47% for at least one year., Significance: The most effective drugs in patients with PCDH19 mutations were bromide and clobazam. Although epilepsy in PCDH19 mutations is often pharmacoresistant, three quarters of the patients became seizure-free for at least for 3 months and half of them for at least one year. However, assessing the effectiveness of the drugs is difficult because a possible age-dependent spontaneous seizure remission must be considered., (Copyright © 2016 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

Infantile-onset skeletal myopathy Barth syndrome (OMIM #302060) is caused by mutations in the X-linked TAZ gene and hence usually manifests itself only in hemizygous males. Confirmatory testing is provided by mutational analysis of the TAZ gene and/or by biochemical dosage of the monolysocardiolipin/tetralinoleoyl cardiolipin ratio. Heterozygous females do not usually display a clinical phenotype but may undergo molecular genetic prenatal diagnosis during pregnancy. We characterized two novel and non-identical TAZ gene rearrangements in the offspring of a single female carrier of Barth syndrome. The hg19chrX:g.153634427&#95;153644361delinsKP&#95;123427.1 TAZ gene rearrangement was identified in her affected son, whereas the NM&#95;000116.3(TAZ)c.-72&#95;109+51del TAZ gene deletion was identified in a male foetus during a subsequent pregnancy. The unaffected mother was surprisingly found to harbour both variants in addition to a wild-type TAZ allele. A combination of breakpoint junction sequencing, linkage analysis and assessment of allelic dosage revealed that the two variants had originated independently from an apparently unstable/mutable TAZ maternal allele albeit via different mutational mechanisms. We conclude that molecular prenatal diagnosis in Barth syndrome families with probands carrying TAZ gene rearrangements should include investigation of the entire coding region of the TAZ gene. The identification of the breakpoint junctions of such gross gene rearrangements is important to ensure accurate ascertainment of carriership with a view to providing appropriate genetic counselling.

Background: Bilateral perisylvian polymicrogyria (BPP), the most common form of regional polymicrogyria, causes the congenital bilateral perisylvian syndrome, featuring oromotor dysfunction, cognitive impairment, and epilepsy. The causes of BPP are heterogeneous, but only a few genetic causes have been reported. The aim of this study was to identify additional genetic causes of BPP and characterise their frequency in this population., Methods: Children (aged ≤18 years) with polymicrogyria were enrolled into our research programme from July, 1980, to October, 2015, at two centres (Florence, Italy, and Seattle, WA, USA). We obtained samples (blood and saliva) throughout this period at both centres and did whole-exome sequencing on DNA from eight trios (two parents and one affected child) with BPP in 2014. After the identification of mosaic PIK3R2 mutations in two of these eight children, we performed targeted screening of PIK3R2 by two methods in a cohort of 118 children with BPP. First, we performed targeted sequencing of the entire PIK3R2 gene by single molecule molecular inversion probes (smMIPs) on 38 patients with BPP with normal to large head size. Second, we did amplicon sequencing of the recurrent PIK3R2 mutation (Gly373Arg) in 80 children with various types of polymicrogyria including BPP. One additional patient had clinical whole-exome sequencing done independently, and was included in this study because of the phenotypic similarity to our cohort., Findings: We identified a mosaic mutation (Gly373Arg) in a regulatory subunit of the PI3K-AKT-mTOR pathway, PIK3R2, in two children with BPP. Of the 38 patients with BPP and normal to large head size who underwent targeted next-generation sequencing by smMIPs, we identified constitutional and mosaic PIK3R2 mutations in 17 additional children. In parallel, one patient had the recurrent PIK3R2 mutation identified by clinical whole-exome sequencing. Seven of these 20 patients had BPP alone, and 13 had BPP in association with features of the megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH) syndrome. 19 patients had the same mutation (Gly373Arg), and one had a nearby missense mutation (Lys376Glu). Mutations were constitutional in 12 patients and mosaic in eight patients. In patients with mosaic mutations, we noted substantial variation in alternate (mutant) allele levels, ranging from ten (3%) of 377 reads to 39 (37%) of 106 reads, equivalent to 5-73% of cells analysed. Levels of mosaicism varied from undetectable to 37 (17%) of 216 reads in blood-derived DNA compared with 2030 (29%) of 6889 reads to 275 (43%) of 634 reads in saliva-derived DNA., Interpretation: Constitutional and mosaic mutations in the PIK3R2 gene are associated with developmental brain disorders ranging from BPP with a normal head size to the MPPH syndrome. The phenotypic variability and low-level mosaicism, which challenge conventional molecular methods, have important implications for genetic testing and counselling., Funding: US National Institutes of Health., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Protocadherin 19 (PCDH19) female limited epilepsy (PCDH19-FE; also known as epilepsy and mental retardation limited to females, EFMR; MIM300088) is an infantile onset epilepsy syndrome with or without intellectual disability (ID) and autism. We investigated transcriptomes of PCDH19-FE female and control primary skin fibroblasts, which are endowed to metabolize neurosteroid hormones. We identified a set of 94 significantly dysregulated genes in PCDH19-FE females. Intriguingly, 43 of the 94 genes (45.7%) showed gender-biased expression; enrichment of such genes was highly significant (P = 2.51E-47, two-tailed Fisher exact test). We further investigated the AKR1C1-3 genes, which encode crucial steroid hormone-metabolizing enzymes whose key products include allopregnanolone and estradiol. Both mRNA and protein levels of AKR1C3 were significantly decreased in PCDH19-FE patients. In agreement with this, the blood levels of allopregnanolone were also (P < 0.01) reduced. In conclusion, we show that the deficiency of neurosteroid allopregnanolone, one of the most potent GABA receptor modulators, may contribute to PCDH19-FE. Overall our findings provide evidence for a role of neurosteroids in epilepsy, ID and autism and create realistic opportunities for targeted therapeutic interventions., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Loss-of-function mutations of the FLNA gene cause a neuronal migration disorder defined as X-linked periventricular nodular heterotopia (PNH); gain-of-function mutations are associated with a group of X-linked skeletal dysplasias designed as otopalatodigital (OPD) spectrum. We describe a family in which a woman and her three daughters exhibited a complex phenotype combining PNH, epilepsy and Melnick-Needles syndrome (MNS), a skeletal disorder assigned to the OPD spectrum. All four individuals harboured a novel non-conservative missense mutation in FLNA exon 3., Methods: In all affected family members, we performed mutation analysis of the FLNA gene, RT-PCR, ultradeep sequencing analysis in FLNA cDNAs and western blot in lymphocyte cells to further characterise the mutation. We also assessed the effects on RT-PCR products of treatment of patients' lymphocytes with cycloheximide, a nonsense mediated mRNA decay (NMD) inhibitor., Results: We identified a novel c.622G>C change in FLNA exon 3, leading to the substitution of a highly conserved aminoacid (p.Gly208Arg). Gel electrophoresis and ultradeep sequencing revealed the missense mutation as well as retention of intron 3. Cycloheximide treatment demonstrated that the aberrant mRNA transcript-retaining intron 3 is subjected to NMD. Western blot analysis confirmed reduced FLNA levels in lymphocyte cells., Conclusions: The novel c.622G>C substitution leads to two aberrant FLNA transcripts, one of which carries the missense mutation, plus a longer transcript resulting from intron 3 retention. We propose that the exceptional co-occurrence of PNH and MNS, two otherwise mutually exclusive allelic phenotypes, is the consequence of a single mutational event resulting in co-occurring gain-of-function and loss-of-function effects., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Biotinidase deficiency (BD), which is caused by BTD genetic lesions, if untreated, can result in neurological and cutaneous manifestations. Biotin supplementation can improve or prevent symptoms. We herewith present a family, which we studied at biochemical and molecular level, after identifying the proband through a newborn screening programme. BTD gene molecular analysis showed the proband to be compound heterozygous for the c.1330G>C p.(Asp444His) mild known variant, and for the c.1475 C>T p.(Thr492Ile) new variant. Bioinformatic analysis allowed us to confirm the pathogenic role of the newly identified variant. The proband's father, who exhibited low biotinidase (BTD) enzyme activity, was homozygous for the mild variant, whereas the proband's mother, who exhibited borderline BTD values, the BTD mutation carrier status could not be detected. This is the first description of a patient with BD harbouring a variant whose origin is either de novo or the consequence of gonadal mosaicism. BTD molecular analysis and bioinformatic tools for the evaluation of pathogenicity of newly identified variants are necessary for diagnostic purposes (i.e., clarifying borderline enzyme assays and the carrier status of parents), and for genetic counselling., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objectives: To describe the antiepileptic drug (AED) treatment of patients with early infantile epileptic encephalopathy due to KCNQ2 mutations during the neonatal phase and the first year of life., Methods: We identified 15 patients and reviewed the electroclinical, neuroimaging, and AED treatment data., Results: Seizure onset was between 1 and 4 days of age with daily tonic asymmetric, focal and clonic seizures in nine patients and status epilepticus in the remaining six. Electroencephalography (EEG) showed multifocal epileptiform abnormalities in nine patients and a burst-suppression pattern in six. All patients were trialed with adequate daily doses of several AEDs before they reached seizure freedom. Six patients (40%) achieved seizure control within 2 weeks of carbamazepine (CBZ) administration and five (33%) were seizure-free with phenytoin (PHT). The last four patients (27%) were successfully treated with topiramate (TPM) (two patients), levetiracetam (LEV) (one), and a combination of LEV with TPM (one). Most patients reached seizure freedom within the first year of life and remained seizure-free thereafter. Twelve patients had moderate-to-severe developmental delay at follow-up. However, the two patients whose seizures ceased within a few days of onset showed only mild cognitive impairment., Significance: Our findings suggest that drugs acting on sodium channels including CBZ and PHT should be considered as first-line treatment in patients with KCNQ2 encephalopathy. Voltage-gated sodium and potassium channels co-localize at the neuronal membrane. Therefore, the efficacy of drugs acting as sodium-channel blockers could be linked to their modulating effect on both channels. The type of KCNQ2 mutation might influence AED response as well as developmental outcome. Early recognition of KCNQ2 encephalopathy followed by the most appropriate and effective treatment may be important for reducing the neurodevelopmental impairment associated with this disorder., (Wiley Periodicals, Inc. © 2015 International League Against Epilepsy.)

Morquio A syndrome (MPS IVA) is a systemic lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), encoded by the GALNS gene. We studied 37 MPS IV A patients and defined genotype-phenotype correlations based on clinical data, biochemical assays, molecular analyses, and in silico structural analyses of associated mutations. We found that standard sequencing procedures, albeit identifying 14 novel small GALNS genetic lesions, failed to characterize the second disease-causing mutation in the 16% of the patients' cohort. To address this drawback and uncover potential gross GALNS rearrangements, we developed molecular procedures (CNV [copy-number variation] assays, QF-PCRs [quantitative fluorescent-PCRs]), endorsed by CGH-arrays. Using this approach, we characterized two new large deletions and their corresponding breakpoints. Both deletions were heterozygous and included the first exon of the PIEZO1 gene, which is associated with dehydrated hereditary stomatocitosis, an autosomal-dominant syndrome. In addition, we characterized the new GALNS intronic lesion c.245-11C>G causing m-RNA defects, although identified outside the GT/AG splice pair. We estimated the occurrence of the disease in the Italian population to be approximately 1:300,000 live births and defined a molecular testing algorithm designed to help diagnosing MPS IVA and foreseeing disease progression., (© 2014 WILEY PERIODICALS, INC.)

Objective: SCN8A encodes the sodium channel voltage-gated α8-subunit (Nav1.6). SCN8A mutations have recently been associated with epilepsy and neurodevelopmental disorders. We aimed to delineate the phenotype associated with SCN8A mutations., Methods: We used high-throughput sequence analysis of the SCN8A gene in 683 patients with a range of epileptic encephalopathies. In addition, we ascertained cases with SCN8A mutations from other centers. A detailed clinical history was obtained together with a review of EEG and imaging data., Results: Seventeen patients with de novo heterozygous mutations of SCN8A were studied. Seizure onset occurred at a mean age of 5 months (range: 1 day to 18 months); in general, seizures were not triggered by fever. Fifteen of 17 patients had multiple seizure types including focal, tonic, clonic, myoclonic and absence seizures, and epileptic spasms; seizures were refractory to antiepileptic therapy. Development was normal in 12 patients and slowed after seizure onset, often with regression; 5 patients had delayed development from birth. All patients developed intellectual disability, ranging from mild to severe. Motor manifestations were prominent including hypotonia, dystonia, hyperreflexia, and ataxia. EEG findings comprised moderate to severe background slowing with focal or multifocal epileptiform discharges., Conclusion: SCN8A encephalopathy presents in infancy with multiple seizure types including focal seizures and spasms in some cases. Outcome is often poor and includes hypotonia and movement disorders. The majority of mutations arise de novo, although we observed a single case of somatic mosaicism in an unaffected parent., (© 2015 American Academy of Neurology.)

We report a three-generation, clinically heterogeneous family in which we identify a novel inherited splicing mutation of the SCN1A gene. Thirteen subjects were submitted to genetic analysis, clinical and instrumental examination, and neuropsychological assessment. In eight subjects, a heterozygous c.2946+5G>A donor splice site alteration in the SCN1A gene was found. Half of them had never had a seizure and showed normal EEG and cognitive profile, whereas the other half had a history of seizures and variable neuropsychological impairments ranging from moderate cognitive disabilities to mild visual-motor impairments. Different clinical phenotypes were identified, including generalized epilepsy with febrile seizure plus (GEFS+), Dravet syndrome, and partial epilepsy with febrile seizure plus (PEFS+). Remarkable clinical heterogeneity can be found among family members carrying the same SCN1A gene mutation. Variable involvement of visual-motor abilities might represent a neuropsychological feature which needs to be further explored in other familial cases., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: Mutations involving the cyclin-dependent kinase-like 5 (CDKL5) gene cause an early onset epileptic encephalopathy (EE) with severe neurologic impairment and a skewed 12:1 female-to-male ratio. To date, 18 mutations have been described in boys. We analyzed our cohort of boys with early onset EE to assess the diagnostic yield of our molecular approach., Methods: We studied 74 boys who presented early onset severe seizures, including infantile spasms and developmental delay, in the setting of EE, using Sanger sequencing, next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA)., Results: We identified alterations involving CDKL5 in four boys (5.4%) using NGS in one and MLPA in three. Three of four mutations were indicative of somatic mosaicism., Significance: CDKL5 gene mutations accounted for 5.4% of boys with early onset EE. Somatic mosaic mutations might be even more represented than germline mutations, probably because their less deleterious effect enhances viability of the male embryo. The molecular approach used for CDKL5 screening remarkably influences the diagnostic yield in boys. Diagnosis is optimized by Sanger sequencing combined with array-based methods or MLPA; alternatively, NGS targeted resequencing designed to also detect copy number alterations, may be performed., (Wiley Periodicals, Inc. © 2014 International League Against Epilepsy.)

Background: Although there is increasing recognition of the role of somatic mutations in genetic disorders, the prevalence of somatic mutations in neurodevelopmental disease and the optimal techniques to detect somatic mosaicism have not been systematically evaluated., Methods: Using a customized panel of known and candidate genes associated with brain malformations, we applied targeted high-coverage sequencing (depth, ≥200×) to leukocyte-derived DNA samples from 158 persons with brain malformations, including the double-cortex syndrome (subcortical band heterotopia, 30 persons), polymicrogyria with megalencephaly (20), periventricular nodular heterotopia (61), and pachygyria (47). We validated candidate mutations with the use of Sanger sequencing and, for variants present at unequal read depths, subcloning followed by colony sequencing., Results: Validated, causal mutations were found in 27 persons (17%; range, 10 to 30% for each phenotype). Mutations were somatic in 8 of the 27 (30%), predominantly in persons with the double-cortex syndrome (in whom we found mutations in DCX and LIS1), persons with periventricular nodular heterotopia (FLNA), and persons with pachygyria (TUBB2B). Of the somatic mutations we detected, 5 (63%) were undetectable with the use of traditional Sanger sequencing but were validated through subcloning and subsequent sequencing of the subcloned DNA. We found potentially causal mutations in the candidate genes DYNC1H1, KIF5C, and other kinesin genes in persons with pachygyria., Conclusions: Targeted sequencing was found to be useful for detecting somatic mutations in patients with brain malformations. High-coverage sequencing panels provide an important complement to whole-exome and whole-genome sequencing in the evaluation of somatic mutations in neuropsychiatric disease. (Funded by the National Institute of Neurological Disorders and Stroke and others.).

Background: X-linked Ornithine Transcarbamylase deficiency (OTCD) is often unrecognized in adults, as clinical manifestations are non-specific, often episodic and unmasked by precipitants, and laboratory findings can be normal outside the acute phase. It may thus be associated with significant mortality if not promptly recognized and treated. The aim of this study was to provide clues for recognition of OTCD in adults and analyze the environmental factors that, interacting with OTC gene mutations, might have triggered acute clinical manifestations., Methods: We carried out a clinical, biochemical and molecular study on five unrelated adult patients (one female and four males) with late onset OTCD, who presented to the Emergency Department (ED) with initial fatal encephalopathy. The molecular study consisted of OTC gene sequencing in the probands and family members and in silico characterization of the newly detected mutations., Results: We identified two new, c.119G>T (p.Arg40Leu) and c.314G>A (p.Gly105Glu), and three known OTC mutations. Both new mutations were predicted to cause a structural destabilization, correlating with late onset OTCD. We also identified, among the family members, 8 heterozygous females and 2 hemizygous asymptomatic males. Patients' histories revealed potential environmental triggering factors, including steroid treatment, chemotherapy, diet changes and hormone therapy for in vitro fertilization., Conclusions: This report raises awareness of the ED medical staff in considering OTCD in the differential diagnosis of sudden neurological and behavioural disorders associated with hyperammonemia at any age and in both genders. It also widens the knowledge about combined effect of genetic and environmental factors in determining the phenotypic expression of OTCD.

Malformations of cortical development are common causes of developmental delay and epilepsy. Some patients have early, severe neurological impairment, but others have epilepsy or unexpected deficits that are detectable only by screening. The rapid evolution of molecular biology, genetics, and imaging has resulted in a substantial increase in knowledge about the development of the cerebral cortex and the number and types of malformations reported. Genetic studies have identified several genes that might disrupt each of the main stages of cell proliferation and specification, neuronal migration, and late cortical organisation. Many of these malformations are caused by de-novo dominant or X-linked mutations occurring in sporadic cases. Genetic testing needs accurate assessment of imaging features, and familial distribution, if any, and can be straightforward in some disorders but requires a complex diagnostic algorithm in others. Because of substantial genotypic and phenotypic heterogeneity for most of these genes, a comprehensive analysis of clinical, imaging, and genetic data is needed to properly define these disorders. Exome sequencing and high-field MRI are rapidly modifying the classification of these disorders., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Purine nucleoside phosphorylase (PNP) deficiency is a rare form of autosomal recessive combined primary immunodeficiency caused by a enzyme defect leading to the accumulation of inosine, 2'-deoxy-inosine (dIno), guanosine, and 2'-deoxy-guanosine (dGuo) in all cells, especially lymphocytes. Treatments are available and curative for PNP deficiency, but their efficacy depends on the early approach. PNP-combined immunodeficiency complies with the criteria for inclusion in a newborn screening program., Objective: This study evaluate whether mass spectrometry can identify metabolite abnormalities in dried blood spots (DBSs) from affected patients, with the final goal of individuating the disease at birth during routine newborn screening., Methods: DBS samples from 9 patients with genetically confirmed PNP-combined immunodeficiency, 10,000 DBS samples from healthy newborns, and 240 DBSs from healthy donors of different age ranges were examined. Inosine, dIno, guanosine, and dGuo were tested by using tandem mass spectrometry (TMS). T-cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) levels were evaluated by using quantitative RT-PCR only for the 2 patients (patients 8 and 9) whose neonatal DBSs were available., Results: Mean levels of guanosine, inosine, dGuo, and dIno were 4.4, 133.3, 3.6, and 3.8 μmol/L, respectively, in affected patients. No indeterminate or false-positive results were found. In patient 8 TREC levels were borderline and KREC levels were abnormal; in patient 9 TRECs were undetectable, whereas KREC levels were normal., Conclusion: TMS is a valid method for diagnosis of PNP deficiency on DBSs of affected patients at a negligible cost. TMS identifies newborns with PNP deficiency, whereas TREC or KREC measurement alone can fail., (Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Objective: Autosomal dominant cortical myoclonus and epilepsy (ADCME) is characterized by distal, fairly rhythmic myoclonus and epilepsy with variable severity. We have previously mapped the disease locus on chromosome 2p11.1-q12.2 by genome-wide linkage analysis. Additional pedigrees affected by similar forms of epilepsy have been associated with chromosomes 8q, 5p, and 3q, but none of the causing genes has been identified. We aim to identify the mutant gene responsible for this form of epilepsy., Methods: Genes included in the ADCME critical region were directly sequenced. Coimmunoprecipitation, immunofluorescent, and electrophysiologic approaches to transfected human cells have been utilized for testing the functional significance of the identified mutation., Results: Here we show that mutation in the α2 -adrenergic receptor subtype B (α2B -AR) is associated with ADCME by identifying a novel in-frame insertion/deletion in 2 Italian families. The mutation alters several conserved residues of the third intracellular loop, hampering neither the α2B -AR plasma membrane localization nor the arrestin-mediated internalization capacity, but altering the binding with the scaffolding protein spinophilin upon neurotransmitter activation. Spinophilin, in turn, regulates interaction of G protein coupled receptors with regulator of G protein signaling proteins. Accordingly, the mutant α2B -AR increases the epinephrine-stimulated calcium signaling., Interpretation: The identified mutation is responsible for ADCME, as the loss of α2B -AR/spinophilin interaction causes a gain of function effect. This work implicates for the first time the α-adrenergic system in human epilepsy and opens new ways of understanding the molecular pathway of epileptogenesis, widening the spectrum of possible therapeutic targets., (© 2014 Child Neurology Society/American Neurological Association.)

Objectives: To determine the frequency of KCNQ2 mutations in patients with neonatal epileptic encephalopathy (NEE), and to expand the phenotypic spectrum of KCNQ2 epileptic encephalopathy., Methods: Eighty-four patients with unexplained NEE were screened for KCNQ2 mutations using classic Sanger sequencing. Clinical data of 6 additional patients with KCNQ2 mutations detected by gene panel were collected. Detailed phenotyping was performed with particular attention to seizure frequency, cognitive outcome, and video-EEG., Results: In the cohort, we identified 9 different heterozygous de novo KCNQ2 missense mutations in 11 of 84 patients (13%). Two of 6 missense mutations detected by gene panel were recurrent and present in patients of the cohort. Seizures at onset typically consisted of tonic posturing often associated with focal clonic jerking, and were accompanied by apnea with desaturation. One patient diagnosed by gene panel had seizure onset at the age of 5 months. Based on seizure frequency at onset and cognitive outcome, we delineated 3 clinical subgroups, expanding the spectrum of KCNQ2 encephalopathy to patients with moderate intellectual disability and/or infrequent seizures at onset. Recurrent mutations lead to relatively homogenous phenotypes. One patient responded favorably to retigabine; 5 patients had a good response to carbamazepine. In 6 patients, seizures with bradycardia were recorded. One patient died of probable sudden unexpected death in epilepsy., Conclusion: KCNQ2 mutations cause approximately 13% of unexplained NEE. Patients present with a wide spectrum of severity and, although rare, infantile epilepsy onset is possible.

Background: Mutations in the CTSA gene, that encodes the protective protein/cathepsin A or PPCA, lead to the secondary deficiency of β-galactosidase (GLB1) and neuraminidase 1 (NEU1), causing the lysosomal storage disorder galactosialidosis (GS). Few clinical cases of GS have been reported in the literature, the majority of them belonging to the juvenile/adult group of patients., Methods: The correct nomenclature of mutations for this gene is discussed through the analysis of the three PPCA/CTSA isoforms available in the GenBank database. Phenotype-genotype correlation has been assessed by computational analysis and review of previously reported single amino acid substitutions., Results: We report the clinical and mutational analyses of four cases with the rare infantile form of GS. We identified three novel nucleotide changes, two of them resulting in the missense mutations, c.347A>G (p.His116Arg), c.775T>C (p.Cys259Arg), and the third, c.1216C>T, resulting in the p.Gln406* stop codon, a type of mutation identified for the first time in GS. An Italian founder effect of the c.114delG mutation can be suggested according to the origin of the only three patients carrying this mutation reported here and in the literature., Conclusions: In early reports mutations nomenclature was selected according to all CTSA isoforms (three different isoforms), thus generating a lot of confusion. In order to assist physicians in the interpretation of detected mutations, we mark the correct nomenclature for CTSA mutations. The complexity of pathology caused by the multifunctions of CTSA, and the very low numbers of mutations (only 23 overall) in relation to the length of the CTSA gene are discussed.In addition, the in silico functional predictions of all reported missense mutations allowed us to closely predict the early infantile, late infantile and juvenile phenotypes, also disclosing different degrees of severity in the juvenile phenotype.

Background: Barth syndrome (BS) is an X-linked infantile-onset cardioskeletal disease characterized by cardiomyopathy, hypotonia, growth delay, neutropenia and 3-methylglutaconic aciduria. It is caused by mutations in the TAZ gene encoding tafazzin, a protein involved in the metabolism of cardiolipin, a mitochondrial-specific phospholipid involved in mitochondrial energy production., Methods: Clinical, biochemical and molecular characterization of a group of six male patients suspected of having BS. Three patients presented early with severe metabolic decompensation including respiratory distress, oxygen desaturation and cardiomyopathy and died within the first year of life. The remaining three patients had cardiomyopathy, hypotonia and growth delay and are still alive. Cardiomyopathy was detected during pregnancy through a routine check-up in one patient. All patients exhibited 3-methylglutaconic aciduria and neutropenia, when tested and five of them also had lactic acidosis., Results: We confirmed the diagnosis of BS with sequence analysis of the TAZ gene, and found five new mutations, c.641A>G p.His214Arg, c.284dupG (p.Thr96Aspfs*37), c.678&#95;691del14 (p.Tyr227Trpfs*79), g.8009&#95;16445del8437 and g.[9777&#95;9814del38; 9911-?&#95;14402del] and the known nonsense mutation c.367C>T (p.Arg123Term). The two gross rearrangements ablated TAZ exons 6 to 11 and probably originated by non-allelic homologous recombination and by Serial Replication Slippage (SRS), respectively. The identification of the breakpoints boundaries of the gross deletions allowed the direct detection of heterozygosity in carrier females., Conclusions: Lactic acidosis associated with 3-methylglutaconic aciduria is highly suggestive of BS, whilst the severity of the metabolic decompensation at disease onset should be considered for prognostic purposes. Mutation analysis of the TAZ gene is necessary for confirming the clinical and biochemical diagnosis in probands in order to identify heterozygous carriers and supporting prenatal diagnosis and genetic counseling.

A homozygous SCN1B mutation was previously identified in a patient with early onset epileptic encephalopathy (EOEE) described as Dravet syndrome (DS) despite a more severe phenotype than DS. We investigated whether SCN1B mutations are a common cause of DS. Patients with DS who did not have a SCN1A sequencing mutation or copy number variation were studied. Genomic DNA was Sanger sequenced for mutations in the 6 exons of SCN1B. In 54 patients with DS recruited from four centres, no SCN1B mutations were identified. SCN1B mutation is not a common cause of DS., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Purpose: Mutations of the protocadherin19 gene (PCDH19) cause a female-related epilepsy of variable severity, with or without mental retardation and autistic features. Despite the increasing number of patients and mutations reported, the epilepsy phenotype associated with PCDH19 mutations is still unclear. We analyzed seizure semiology through ictal video-electroencephalography (EEG) recordings in a large series of patients., Methods: We studied 35 patients with PCDH19 gene-related epilepsy and analyzed clinical history and ictal video-EEG recordings obtained in 34 of them., Key Findings: Clusters of focal febrile and afebrile seizures had occurred in 34 patients, at a mean age of 10 months. The predominant and more consistent ictal sign was fearful screaming, occurring in 24 patients (70.5%); it was present since epilepsy onset in 12 and appeared later on, during the course in the remaining 12 patients. In infancy, fearful screaming mainly appeared within the context of seizures with prominent hypomotor semiology, whereas during follow-up it was associated with prominent early motor manifestations. In 16 patients, seizures were video-EEG recorded both at onset and during follow-up: in five patients (31%) seizure semiology remained identical, in 7 (44%) semiology varied and in four patients it was unclear whether ictal semiology changed with age. Three patients (9%) had both focal and generalized seizures, the latter consisting of absences and myoclonus. Ictal EEG during focal seizures showed a prominent involvement of the frontotemporal regions (22 patients). About 45% of patients had an alternating EEG pattern, with the ictal discharge migrating from one hemisphere to the contralateral during the same ictal event. Status epilepticus occurred in 30% of patients. Cognitive impairment occurred in 70%, ranging from mild (42%) to moderate (54%) and severe (4%); autistic features occurred in 28.5%. Direct sequencing detected 33 different heterozygous candidate mutations, 8 of which were novel. Mutations were missense substitutions (48.5%), premature termination (10 frameshift, 4 nonsense, and 2 splice-site mutations; 48.5%), and one in-frame deletion. Thirty candidate mutations (91%) were de novo. No specific genotype-phenotype correlation could be established, as missense and truncating mutations were associated with phenotypes of comparable severity., Significance: Most patients with PCDH19 mutations exhibit a distinctive electroclinical pattern of focal seizures with affective symptoms, suggesting an epileptogenic dysfunction involving the frontotemporal limbic system. Awareness of this distinctive phenotype will likely enhance recognition of this disorder., (Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.)

Objective: Benign familial infantile epilepsy (BFIE) is an autosomal dominant epilepsy syndrome characterized by afebrile seizures beginning at about 6 months of age. Mutations in PRRT2, encoding the proline-rich transmembrane protein 2 gene, have recently been identified in the majority of families with BFIE and the associated syndrome of infantile convulsions and choreoathetosis (ICCA). We asked whether the phenotypic spectrum of PRRT2 was broader than initially recognized by studying patients with sporadic benign infantile seizures and non-BFIE familial infantile seizures for PRRT2 mutations., Methods: Forty-four probands with infantile-onset seizures, infantile convulsions with mild gastroenteritis, and benign neonatal seizures underwent detailed phenotyping and PRRT2 sequencing. The familial segregation of mutations identified in probands was studied., Results: The PRRT2 mutation c.649-650insC (p.R217fsX224) was identified in 11 probands. Nine probands had a family history of BFIE or ICCA. Two probands had no family history of infantile seizures or paroxysmal kinesigenic dyskinesia and had de novo PRRT2 mutations. Febrile seizures with or without afebrile seizures were observed in 2 families with PRRT2 mutations., Conclusions: PRRT2 mutations are present in >80% of BFIE and >90% ICCA families, but are not a common cause of other forms of infantile epilepsy. De novo mutations of PRRT2 can cause sporadic benign infantile seizures. Seizures with fever may occur in BFIE such that it may be difficult to distinguish BFIE from febrile seizures and febrile seizures plus in small families.

The purpose of the study is to explore the causative role of TUBB2B gene mutations in patients with different malformations of cortical development. We collected and evaluated clinical and MRI data of a cohort of 128 consecutive patients (61 females and 67 males) in whom brain MRI had detected a spectrum of malformations of cortical development including polymicrogyria or pachygyria, who were mutation-negative to other possible causative genes. Mutation analysis of the TUBB2B gene was performed. We identified three new TUBB2B mutations in three unrelated patients (3 out of 128; 2.3%) with a diffuse and rather symmetrical cortical abnormality, including diffuse polymicrogyria in two and bilateral regional pachygyria in one. One patient harbored a p.Asp417Asn amino-acid substitution in the C-terminal domain of the protein; one patient a p.Asn256Ser amino-acid substitution in the intermediate domain and one patient a p.Leu117Pro amino-acid substitution in the N-terminal domain. The localization of each mutation within the secondary structure of the β2-tubulin polypeptide suggests that these mutations might alter the proper functions of microtubules. The phenotypic spectrum associated with TUBB2B mutations is wider than previously reported and includes diffuse, symmetric malformations of cortical development.

Malformations of cerebral cortical development include a wide range of developmental disorders that are common causes of neurodevelopmental delay and epilepsy. In addition, study of these disorders contributes greatly to the understanding of normal brain development and its perturbations. The rapid recent evolution of molecular biology, genetics and imaging has resulted in an explosive increase in our knowledge of cerebral cortex development and in the number and types of malformations of cortical development that have been reported. These advances continue to modify our perception of these malformations. This review addresses recent changes in our perception of these disorders and proposes a modified classification based upon updates in our knowledge of cerebral cortical development.

Background: SCN1A is the most clinically relevant epilepsy gene, most mutations causing Dravet syndrome (also known as severe myoclonic epilepsy of infancy or SMEI). We evaluated clinical differences, if any, between young patients with and without a SCN1A mutations and a definite clinical diagnosis of Dravet syndrome., Methods: Twenty-five patients with a diagnosis of Dravet Syndrome (7 males, 18 females; mean age at inclusion: 10.3; median: 9±7; range: 18 months-30 years) were retrospectively studied. A clinical and genetic study focusing on SCN1A was performed, using DHPLC, gene sequencing and MLPA to detect genomic deletions/duplications. A formal cognitive and behavioral assessment was available for all patients., Results: Analysis revealed SCN1A mutations comprising missense, truncating mutations and genomic deletions/duplications in eighteen patients and no mutation in seven. The phenotype of mutation positive patients was characterized by a higher number of seizures/month in the first year of life, an earlier seizure onset and a higher frequency of episodes of status epilepticus. The cognitive and behavioral profile was slightly worst in mutation positive patients., Conclusions: These findings confirm that SCN1A gene mutations are strongly associated to a more severe phenotype in patients with Dravet syndrome., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objective: The idiopathic generalized epilepsies (IGE) are the most common genetically determined epilepsies. However, the underlying genes are largely unknown. We screened the SLC2A1 gene, encoding the glucose transporter type 1 (GLUT1), for mutations in a group of 95 European patients with familial IGE., Methods: The affected individuals were examined clinically by EEG and brain imaging. The coding regions of SLC2A1 were sequenced in the index cases of all families. Wild-type and mutant transporters were expressed and functionally characterized in Xenopus laevis oocytes., Results: We detected a novel nonsynonymous SLC2A1 mutation (c.694C>T, p.R232C) in one IGE family. Nine family members were affected mainly by absence epilepsies with a variable age at onset, from early childhood to adulthood. Childhood absence epilepsy in one individual evolved into juvenile myoclonic epilepsy. Eight affected and 4 unaffected individuals carried the mutation, revealing a reduced penetrance of 67%. The detected mutation was not found in 846 normal control subjects. Functional analysis revealed a reduced maximum uptake velocity for glucose, whereas the affinity to glucose and the protein expression were not different in wild-type and mutant transporters., Conclusion: Our study shows that GLUT1 defects are a rare cause of classic IGE. SLC2A1 screening should be considered in IGE featuring absence epilepsies with onset from early childhood to adult life, because this diagnosis may have important implications for treatment and genetic counseling.

Objective: To determine if a significant proportion of patients with myoclonic-astatic epilepsy (MAE) have glucose transporter 1 (GLUT1) deficiency., Design: Genetic analysis., Setting: Ambulatory and hospitalized care., Patients: Eighty-four unrelated probands with MAE were phenotyped and SLC2A1 was sequenced and analyzed by multiplex ligation-dependent probe amplification. Any identified mutations were then screened in controls., Main Outcome Measure: Any SLC2A1 mutations., Results: Four of 84 probands with MAE had a mutation of SLC2A1 on sequencing. Multiplex ligation-dependent probe amplification analysis did not reveal any genomic rearrangements in 75 of the remaining cases; 5 could not be tested. Two patients with MAE with SLC2A1 mutations also developed paroxysmal exertional dyskinesia in childhood., Conclusions: Five percent of our patients with MAE had SLC2A1 mutations, suggesting that patients with MAE should be tested for GLUT1 deficiency. Diagnosis of GLUT1 deficiency is a strong indication for early use of the ketogenic diet, which may substantially improve outcome of this severe disorder.

Dravet syndrome (DS), otherwise known as severe myoclonic epilepsy of infancy (SMEI), is an epileptic encephalopathy presenting in the first year of life. DS has a genetic etiology: between 70% and 80% of patients carry sodium channel α1 subunit gene (SCN1A) abnormalities, and truncating mutations account for about 40% and have a significant correlation with an earlier age of seizures onset. The remaining SCN1A mutations comprise splice-site and missense mutations, most of which fall into the pore-forming region of the sodium channel. Mutations are randomly distributed across the SCN1A protein. Most mutations are de novo, but familial SCN1A mutations also occur. Somatic mosaic mutations have also been reported in some patients and might explain the phenotypical variability seen in some familial cases. SCN1A exons deletions or chromosomal rearrangements involving SCN1A and contiguous genes are also detectable in about 2-3% of patients. A small percentage of female patients with a DS-like phenotype might carry PCDH19 mutations. Rare mutations have been identified in the GABARG2 and SCN1B genes. The etiology of about 20% of DS patients remains unknown, and additional genes are likely to be implicated., (Wiley Periodicals, Inc. © 2011 International League Against Epilepsy.)

Fabry Disease (FD) is an X-linked multisystemic lysosomal disorder caused by mutations of alpha-galactosidase (GLA) gene. Only a few of the 450 genetic lesions identified so far have been characterised by in vitro expression studies. Thus the significance of newly identified GLA nucleotide variants in FD patients which lead to alpha-galactosidase (GAL-A) amino acid substitutions or intronic changes can be uncertain. We identified three GLA mutations, c.155G>A (p.C52Y), c.548G>C (p.G183A), c.647A>G (p.Y216C) in as many individuals (two male; one female) and performed in vitro expression studies and Western blot analysis in order to clarify their functional effects. Reduced GAL-A activity and normal or partially reduced mutant proteins were present in all overexpressed mutant systems in which three-dimensional structural analysis showed that the active site was not directly involved. We hypothesize that the three new mutations affect the GAL-A protein, leading to conformational FD. When mutant proteins overexpressed in COS-1 cells and in patients' lymphocytes were tested in the presence of the 1-deoxygalactonojirimicin (DGJ) chaperone, the p.G183A and p.Y216C systems showed increased GAL-A enzyme activities and protein stabilisation while p.C52Y was not responsive. We underline that genetic, biochemical and functional studies are helpful in clarifying the consequences of the missense genetic lesions detected in FD. ERT is the elective therapy for Fabry patients, but it is not always possible to issue the enzyme's active form in all involved organs. Our study endorses the hypothesis that an active site-specific chemical chaperone, which could be administered orally, might be effective in treating GAL-A conformational defects., (Copyright 2009 Elsevier B.V. All rights reserved.)

Objective: We aimed to determine the type, frequency, and size of microchromosomal copy number variations (CNVs) affecting the neuronal sodium channel α 1 subunit gene (SCN1A) in Dravet syndrome (DS), other epileptic encephalopathies, and generalized epilepsy with febrile seizures plus (GEFS+)., Methods: Multiplex ligation-dependent probe amplification (MLPA) was applied to detect SCN1A CNVs among 289 cases (126 DS, 97 GEFS+, and 66 with other phenotypes). CNVs extending beyond SCN1A were further characterized by comparative genome hybridization (array CGH)., Results: Novel SCN1A CNVs were found in 12.5% of DS patients where sequence-based mutations had been excluded. We identified the first partial SCN1A duplications in two siblings with typical DS and in a patient with early-onset symptomatic generalized epilepsy. In addition, a patient with DS had a partial SCN1A amplification of 5-6 copies. The remaining CNVs abnormalities were four partial and nine whole SCN1A deletions involving contiguous genes. Two CNVs (a partial SCN1A deletion and a duplication) were inherited from a parent, in whom there was mosaicism. Array CGH showed intragenic deletions of 90 kb and larger, with the largest of 9.3 Mb deleting 49 contiguous genes and extending beyond SCN1A., Discussion: Duplication and amplification involving SCN1A are now added to molecular mechanisms of DS patients. Our findings showed that 12.5% of DS patients who are mutation negative have MLPA-detected SCN1A CNVs with an overall frequency of about 2-3%. MLPA is the established second-line testing strategy to reliably detect all CNVs of SCN1A from the megabase range down to one exon. Large CNVs extending outside SCN1A and involving contiguous genes can be precisely characterized by array CGH.

Purpose: Bilateral frontoparietal polymicrogyria (BFPP) has been reported in sporadic patients and in recessive pedigrees. Eleven mutations in GPR56, a gene encoding an evolutionarily dynamic G-protein-coupled receptor, have been identified in 29 patients from 18 families. The clinical features of BFPP include severe mental retardation, motor and language impairment, and epilepsy. No detailed description of the epilepsy is available for the patients reported to date. We report three consanguineous families in which four affected individuals with BFPP and GPR56 mutations had Lennox-Gastaut syndrome., Methods: Family studies, brain magnetic resonance imaging (MRI), electroencephalography (EEG)-video recordings, and mutation analysis., Results: In Family 1, with one affected proband, we found an R565W change in the second extracellular loop of GPR56, involving a highly conserved aminoacidic residue. In Family 2, with one affected proband, we found an R79X change affecting the protein N-terminus and predicted to cause a premature truncation with loss of the G-protein-coupled receptor proteolytic site. In family 3, with two affected siblings, we found an R33P substitution in the protein N-terminus, involving a highly conserved aminoacidic residue. Epilepsy, present in all four patients, had started between ages 1 and 8 years, with infantile spasms in one patient and with de novo Lennox-Gastaut syndrome in the remaining three. All patients had Lennox-Gastaut syndrome when last observed, at ages 13 to 32 years., Discussion: Several genes, when mutated, can cause malformations of cortical development that have been associated with the Lennox-Gastaut syndrome. BFPP caused by GPR56 mutations represents an additional, although rare, genetically determined cause of Lennox-Gastaut syndrome.