Clinical and functional consequences of GRIA variants in patients with neurological diseases.

AMPA receptors are members of the glutamate receptor family and mediate a fast component of excitatory synaptic transmission at virtually all central synapses. Thus, their functional characteristics are a critical determinant of brain function. We evaluate intolerance of each GRIA gene to genetic variation using 3DMTR and report here the functional consequences of 52 missense variants in GRIA1-4 identified in patients with various neurological disorders. These variants produce changes in agonist EC50, response time course, desensitization, and/or receptor surface expression. We predict that these functional and localization changes will have important consequences for circuit function, and therefore likely contribute to the patients' clinical phenotype. We evaluated the sensitivity of variant receptors to AMPAR-selective modulators including FDA-approved drugs to explore potential targeted therapeutic options.

Mendelian etiologies identified with whole exome sequencing in cerebral palsy.

OBJECTIVES: Cerebral palsy (CP) is the most common childhood motor disability, yet its link to single-gene disorders is under-characterized. To explore the genetic landscape of CP, we conducted whole exome sequencing (WES) in a cohort of patients with CP. METHODS: We performed comprehensive phenotyping and WES on a prospective cohort of individuals with cryptogenic CP (who meet criteria for CP; have no risk factors), non-cryptogenic CP (who meet criteria for CP; have at least one risk factor), and CP masqueraders (who could be diagnosed with CP, but have regression/progressive symptoms). We characterized motor phenotypes, ascertained medical comorbidities, and classified brain MRIs. We analyzed WES data using an institutional pipeline. RESULTS: We included 50 probands in this analysis (20 females, 30 males). Twenty-four had cryptogenic CP, 20 had non-cryptogenic CP, five had CP masquerader classification, and one had unknown classification. Hypotonic-ataxic subtype showed a difference in prevalence across the classification groups (p = 0.01). Twenty-six percent of participants (13/50) had a pathogenic/likely pathogenic variant in 13 unique genes (ECHS1, SATB2, ZMYM2, ADAT3, COL4A1, THOC2, SLC16A2, SPAST, POLR2A, GNAO1, PDHX, ACADM, ATL1), including one patient with two genetic disorders (ACADM, PDHX) and two patients with a SPAST-related disorder. The CP masquerader category had the highest diagnostic yield (n = 3/5, 60%), followed by the cryptogenic CP category (n = 7/24, 29%). Fifteen percent of patients with non-cryptogenic CP (n = 3/20) had a Mendelian disorder on WES. INTERPRETATION: WES demonstrated a significant prevalence of Mendelian disorders in individuals clinically diagnosed with CP, including in individuals with known CP risk factors.

A diagnostic confidence scheme for CLN3 disease.

Over the past 20 years, diagnostic testing for genetic diseases has evolved, leading to variable diagnostic certainty for individuals included in long-term natural history studies. Using genotype and phenotype data from an ongoing natural history study of CLN3 disease, we developed a hierarchical diagnostic confidence scheme with three major classes: Definite, Probable, or Possible CLN3 disease. An additional level, CLN3 Disease PLUS, includes individuals with CLN3 disease plus an additional disorder with a separate etiology that substantially affects the phenotype. Within the Definite and Probable CLN3 disease classes, we further divided individuals into subclasses based on phenotype. After assigning participants to classes, we performed a blinded reclassification to assess the reliability of this scheme. A total of 134 individuals with suspected CLN3 disease were classified: 100 as Definite, 21 as Probable, and 7 as Possible. Six individuals were classified as CLN3-PLUS. Phenotypes included the classical juvenile-onset syndromic phenotype, a "vision loss only" phenotype, and an atypical syndromic phenotype. Some individuals were too young to fully classify phenotype. Test-retest reliability showed 96% agreement. We created a reliable diagnostic confidence scheme for CLN3 disease that has excellent face validity. This scheme has implications for clinical research in CLN3 and other rare genetic neurodegenerative disorders.

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.

BioVR: a platform for virtual reality assisted biological data integration and visualization.

BACKGROUND: Functional characterization of single nucleotide variants (SNVs) involves two steps, the first step is to convert DNA to protein and the second step is to visualize protein sequences with their structures. As massively parallel sequencing has emerged as a leading technology in genomics, resulting in a significant increase in data volume, direct visualization of SNVs together with associated protein sequences/structures in a new user interface (UI) would be a more effective way to assess their potential effects on protein function. RESULTS: We have developed BioVR, an easy-to-use interactive, virtual reality (VR)-assisted platform for integrated visual analysis of DNA/RNA/protein sequences and protein structures using Unity3D and the C# programming language. It utilizes the cutting-edge Oculus Rift, and Leap Motion hand detection, resulting in intuitive navigation and exploration of various types of biological data. Using Gria2 and its associated gene product as an example, we present this proof-of-concept software to integrate protein and nucleic acid data. For any amino acid or nucleotide of interest in the Gria2 sequence, it can be quickly linked to its corresponding location on Gria2 protein structure and visualized within VR. CONCLUSIONS: Using innovative 3D techniques, we provide a VR-based platform for visualization of DNA/RNA sequences and protein structures in aggregate, which can be extended to view omics data.

Association of Severe Hydrocephalus With Congenital Zika Syndrome.

Importance: Hydrocephalus is a treatable but potentially fatal complication that has not been previously described in congenital Zika syndrome (CZS). Objective: To describe the clinical features and imaging findings in 24 patients with congenital Zika syndrome (CZS) who developed hydrocephalus. Design, Setting, and Participants: This case series included patients with hydrocephalus who were born in October and November 2015 and followed up until mid-2017 in the 2 largest national referral centers for CZS in Brazil. The participants included consecutively enrolled children with a clinical and laboratorial diagnosis of CZS who developed clinical and/or image findings suggestive of hydrocephalus and who were confirmed to experience increased intracranial hypertension during ventriculoperitoneal shunt procedures. Main Outcomes and Measures: To retrospectively describe clinical and image findings in these 24 patients. Results: This multicenter cohort included 308 patients with CZS; 24 consecutive children were enrolled in this study. These children were aged between 3 to 18 months, and 13 of 24 (54%) were female. All patients presented with at least 1 positive test result for anti-Zika antibodies in cerebrospinal fluid or serum and had classic signs of CZS. At the time of hydrocephalus diagnosis, only 14 of 24 patients (58%) had symptoms and signs suggestive of hydrocephalus (mainly worsening seizures, vomiting, irritability, and/or sudden increase of head circumference percentile). Two of 24 patients (8%) had no symptoms suggestive of hydrocephalus but were found to have reduced brain volume on repeated imaging. Cerebellar or brainstem hypoplasia on baseline imaging were found in 18 of 23 patients (78%). At the second computed tomographic scan, all patients showed a marked increase of ventricular volume, compatible with communicating hydrocephalus, and reduction of brain tissue that was visibly worse than on baseline imaging for the 23 patients with repeated scans. Conclusions and Relevance: We present evidence that hydrocephalus is a complication of CZS in at least a proportion of patients. The clinical spectrum of this condition continues to evolve, but given that presenting signs and symptoms of hydrocephalus can be challenging to recognize in CZS, we provisionally recommend that high suspicion and appropriate monitoring for hydrocephalus should be part of the standard care of patients with CZS.

Phenotypes, genotypes, and the management of paroxysmal movement disorders.

As a consequence of the genomic revolution, a large number of publications describing paroxysmal movement disorders have been published in the last few years, shedding light on their molecular pathology. Routine gene testing is not necessary to guide treatment for typical forms of paroxysmal kinesigenic dyskinesia (PKD), paroxysmal nonkinesigenic dyskinesia (PNKD), and episodic ataxia type 1 or 2. It can, however, be helpful in the management of atypical or complex cases, especially for genetic counselling, treatment strategies, and the offer of preimplantation genetic diagnosis. Antiepileptic drugs remain the treatment of choice for PKD and episodic ataxia type 1, benzodiazepines are often useful for PNKD, and episodic ataxia type 2 benefits from acetazolamide regardless of the genetic etiology. WHAT THE PAPER ADDS: A growing number of genes have been associated with classic and newly described paroxysmal movement disorders. Paroxysmal movement disorders share common mechanisms and clinical features with other neurological paroxysmal phenomena including epilepsy and migraine.

Genetic Diagnostics for Neurologists.

PURPOSE OF REVIEW: This article puts advances in the field of neurogenetics into context and provides a quick review of the broad concepts necessary for current practice in neurology. RECENT FINDINGS: The exponential growth of genetic testing is due to its increased speed and decreasing cost, and it is now a routine part of the clinical care for a number of neurologic patients. In addition, phenotypic pleiotropy (mutations in the same gene causing very disparate phenotypes) and genetic heterogeneity (the same clinical phenotype resulting from mutations in different genes) are now known to exist in a number of conditions, adding an additional layer of complexity for genetic testing in these disorders. SUMMARY: Although the growing complexity of technical knowledge in the ordering and interpretation of genetic tests makes it necessary for neurologists to consult medical geneticists, limitations in the availability of such professionals often means neurologists will be on the front line dealing with suspected or confirmed neurogenetic conditions. The growing availability of broad genetic testing through chromosomal microarray and next-generation sequencing and the expanded phenotypic spectrum of many conditions has implications for genetic counseling and medical management. This article discusses the various forms of genetic variability and how to test for each of them. It also provides an update on the most common forms of neurologic presentations of genetic disease and a review of testing strategies.

Expanding the neurodevelopmental phenotype of PURA syndrome.

PURA syndrome is a recently described developmental encephalopathy presenting with neonatal hypotonia, feeding difficulties, global developmental delay, severe intellectual disability, and frequent apnea and epilepsy. We describe 18 new individuals with heterozygous sequence variations in PURA. A neuromotor disorder starting with neonatal hyptonia, but ultimately allowing delayed progression to walking, was present in nearly all individuals. Congenital apnea was present in 56% during infancy, but all cases in this cohort resolved during the first year of life. Feeding difficulties were frequently reported, with gastrostomy tube placement required in 28%. Epilepsy was present in 50% of the subjects, including infantile spasms and Lennox-Gastaut syndrome. Skeletal complications were found in 39%. Disorders of gastrointestinal motility and nystagmus were also recurrent features. Autism was diagnosed in one individual, potentially expanding the neurodevelopmental phenotype associated with this syndrome. However, we did not find additional PURA sequence variations in a cohort of 120 subjects with autism. We also present the first neuropathologic studies of PURA syndrome, and describe chronic inflammatory changes around the arterioles within the deep white matter. We did not find significant correlations between mutational class and severity, nor between location of the sequence variation in PUR repeat domains. Further studies are required in larger cohorts of subjects with PURA syndrome to clarify these genotype-phenotype associations.

CEDNIK: Phenotypic and Molecular Characterization of an Additional Patient and Review of the Literature.

Synaptosomal-associated protein 29 (SNAP29) is a t-SNARE protein that is implicated in intracellular vesicle fusion. Mutations in the SNAP29 gene have been associated with cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma syndrome (CEDNIK). In patients with 22q11.2 deletion syndrome, mutations in SNAP29 on the nondeleted chromosome are linked to similar ichthyotic and neurological phenotypes. Here, the authors report a patient with cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma syndrome who presented with global developmental delay, polymicrogyria, dysgenesis of the corpus callosum, optic nerve dysplasia, gaze apraxia, and dysmorphic features. He has developed ichthyosis and palmoplantar keratoderma as he has grown. Exome sequencing identified a homozygous nonsense mutation in SNAP29 gene designated as c.85C>T (p.Arg29X). The authors compare the findings in the proband with previously reported cases. The previously unreported mutation in this patient and his phenotype add to the characterization of cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma syndrome and the accumulating scientific evidence that implicates synaptic protein dysfunction in various neuroectodermal conditions.

India Allele Finder: a web-based annotation tool for identifying common alleles in next-generation sequencing data of Indian origin.

OBJECTIVE: We built India Allele Finder, an online searchable database and command line tool, that gives researchers access to variant frequencies of Indian Telugu individuals, using publicly available fastq data from the 1000 Genomes Project. Access to appropriate population-based genomic variant annotation can accelerate the interpretation of genomic sequencing data. In particular, exome analysis of individuals of Indian descent will identify population variants not reflected in European exomes, complicating genomic analysis for such individuals. RESULTS: India Allele Finder offers improved ease-of-use to investigators seeking to identify and annotate sequencing data from Indian populations. We describe the use of India Allele Finder to identify common population variants in a disease quartet whole exome dataset, reducing the number of candidate single nucleotide variants from 84 to 7. India Allele Finder is freely available to investigators to annotate genomic sequencing data from Indian populations. Use of India Allele Finder allows efficient identification of population variants in genomic sequencing data, and is an example of a population-specific annotation tool that simplifies analysis and encourages international collaboration in genomics research.

PLXNA1 developmental encephalopathy with syndromic features: A case report and review of the literature.

Developmental encephalopathies constitute a broad and genetically heterogeneous spectrum of disorders associated with global developmental delay, intellectual disability, frequent epilepsy, and other neurofunctional abnormalities. Here, we report a male presenting with infantile onset epilepsy and syndromic features resembling Dubowitz syndrome identified to have a de novo PLXNA1 variant by whole exome sequencing. This constitutes the second report of PLXNA1 sequence variation associated with early onset epilepsy, and the first to expand on the clinical features of this emerging disorder. This reports suggests that nonsynonymous de novo sequence variations in PLXNA1 are associated with a novel human phenotype characterized by intractable early onset epilepsy, intellectual disability, and syndromic features.

Developing a novel epileptic discharge localization algorithm for electroencephalogram infantile spasms during hypsarrhythmia.

Infantile spasms (ISS) is a devastating epileptic syndrome that affects children under the age of 1 year. The diagnosis of ISS is based on the semiology of the seizure and the electroencephalogram (EEG) background characterized by hypsarrhythmia (HYPS). However, even skilled electrophysiologists may interpret the EEG of children with ISS differently, and commercial software or existing epilepsy detection algorithms are not helpful. Since EEG is a key factor in the diagnosis of ISS, misinterpretation could result in serious consequences including inappropriate treatment. In this paper, we developed a novel algorithm to localize the relevant electrical abnormality known as epileptic discharges (or spikes) to provide a quantitative assessment of ISS in HYPS. The proposed algorithm extracts novel time-frequency features from the EEG signals and localizes the epileptic discharges associated with ISS in HYPS using a support vector machine classifier. We evaluated the proposed method on an EEG dataset with ISS subjects and obtained an average true positive and false negative of 98 and 7%, respectively, which was a significant improvement compared to the results obtained using the clinically available software. The proposed automated method provides a quantitative assessment of ISS in HYPS, which could significantly enhance our knowledge in therapy management of ISS.

Genetics and genotype-phenotype correlations in early onset epileptic encephalopathy with burst suppression.

OBJECTIVE: We sought to identify genetic causes of early onset epileptic encephalopathies with burst suppression (Ohtahara syndrome and early myoclonic encephalopathy) and evaluate genotype-phenotype correlations. METHODS: We enrolled 33 patients with a referral diagnosis of Ohtahara syndrome or early myoclonic encephalopathy without malformations of cortical development. We performed detailed phenotypic assessment including seizure presentation, electroencephalography, and magnetic resonance imaging. We confirmed burst suppression in 28 of 33 patients. Research-based exome sequencing was performed for patients without a previously identified molecular diagnosis from clinical evaluation or a research-based epilepsy gene panel. RESULTS: In 17 of 28 (61%) patients with confirmed early burst suppression, we identified variants predicted to be pathogenic in KCNQ2 (n = 10), STXBP1 (n = 2), SCN2A (n = 2), PNPO (n = 1), PIGA (n = 1), and SEPSECS (n = 1). In 3 of 5 (60%) patients without confirmed early burst suppression, we identified variants predicted to be pathogenic in STXBP1 (n = 2) and SCN2A (n = 1). The patient with the homozygous PNPO variant had a low cerebrospinal fluid pyridoxal-5-phosphate level. Otherwise, no early laboratory or clinical features distinguished the cases associated with pathogenic variants in specific genes from each other or from those with no prior genetic cause identified. INTERPRETATION: We characterize the genetic landscape of epileptic encephalopathy with burst suppression, without brain malformations, and demonstrate feasibility of genetic diagnosis with clinically available testing in >60% of our cohort, with KCNQ2 implicated in one-third. This electroclinical syndrome is associated with pathogenic variation in SEPSECS. Ann Neurol 2017;81:419-429.

Epilepsy-causing sequence variations in SIK1 disrupt synaptic activity response gene expression and affect neuronal morphology.

SIK1 syndrome is a newly described developmental epilepsy disorder caused by heterozygous mutations in the salt-inducible kinase SIK1. To better understand the pathophysiology of SIK1 syndrome, we studied the effects of SIK1 pathogenic sequence variations in human neurons. Primary human fetal cortical neurons were transfected with a lentiviral vector to overexpress wild-type and mutant SIK1 protein. We evaluated the transcriptional activity of known downstream gene targets in neurons expressing mutant SIK1 compared with wild type. We then assayed neuronal morphology by measuring neurite length, number and branching. Truncating SIK1 sequence variations were associated with abnormal MEF2C transcriptional activity and decreased MEF2C protein levels. Epilepsy-causing SIK1 sequence variations were associated with significantly decreased expression of ARC (activity-regulated cytoskeletal-associated) and other synaptic activity response element genes. Assay of mRNA levels for other MEF2C target genes NR4A1 (Nur77) and NRG1, found significantly, decreased the expression of these genes as well. The missense p.(Pro287Thr) SIK1 sequence variation was associated with abnormal neuronal morphology, with significant decreases in mean neurite length, mean number of neurites and a significant increase in proximal branches compared with wild type. Epilepsy-causing SIK1 sequence variations resulted in abnormalities in the MEF2C-ARC pathway of neuronal development and synapse activity response. This work provides the first insights into the mechanisms of pathogenesis in SIK1 syndrome, and extends the ARX-MEF2C pathway in the pathogenesis of developmental epilepsy.

Phenotype Differentiation of FOXG1 and MECP2 Disorders: A New Method for Characterization of Developmental Encephalopathies.

OBJECTIVE: To differentiate developmental encephalopathies by creating a novel quantitative phenotyping tool. STUDY DESIGN: We created the Developmental Encephalopathy Inventory (DEI) to differentiate disorders with complex multisystem neurodevelopmental symptoms. We then used the DEI to study the phenotype features of 20 subjects with FOXG1 disorder and 11 subjects with MECP2 disorder. RESULTS: The DEI identified core domains of fine motor and expressive language that were severely impaired in both disorders. Individuals with FOXG1 disorder were overall more severely impaired. Subjects with FOXG1 disorder were less able to walk, had worse fine motor skills, more disability in receptive language and reciprocity, and had more disordered sleep than did subjects with MECP2 disorder (P <.05). Covariance, cluster, and principal component analysis confirmed a relationship between impaired awareness, reciprocity, and language in both disorders. In addition, abnormal ambulation was a first principal component for FOXG1 but not for MECP2 disorder, suggesting that impaired ambulation is a strong differentiating factor clinically between the 2 disorders. CONCLUSIONS: We have developed a novel quantitative developmental assessment tool for developmental encephalopathies and propose this tool as a method to identify and illustrate core common and differential domains of disability in these complex disorders. These findings demonstrate clear phenotype differences between FOXG1 and MECP2 disorders.

Familial recurrences of FOXG1-related disorder: Evidence for mosaicism.

FOXG1-related disorders are caused by heterozygous mutations in FOXG1 and result in a spectrum of neurodevelopmental phenotypes including postnatal microcephaly, intellectual disability with absent speech, epilepsy, chorea, and corpus callosum abnormalities. The recurrence risk for de novo mutations in FOXG1-related disorders is assumed to be low. Here, we describe three unrelated sets of full siblings with mutations in FOXG1 (c.515_577del63, c.460dupG, and c.572T > G), representing familial recurrence of the disorder. In one family, we have documented maternal somatic mosaicism for the FOXG1 mutation, and all of the families presumably represent parental gonadal (or germline) mosaicism. To our knowledge, mosaicism has not been previously reported in FOXG1-related disorders. Therefore, this report provides evidence that germline mosaicism for FOXG1 mutations is a likely explanation for familial recurrence and should be considered during recurrence risk counseling for families of children with FOXG1-related disorders.

Autism spectrum disorder and epilepsy: Disorders with a shared biology.

There is an increasing recognition of clinical overlap in patients presenting with epilepsy and autism spectrum disorder (ASD), and a great deal of new information regarding the genetic causes of both disorders is available. Several biological pathways appear to be involved in both disease processes, including gene transcription regulation, cellular growth, synaptic channel function, and maintenance of synaptic structure. We review several genetic disorders where ASD and epilepsy frequently co-occur, and we discuss the screening tools available for practicing neurologists and epileptologists to help determine which patients should be referred for formal ASD diagnostic evaluation. Finally, we make recommendations regarding the workflow of genetic diagnostic testing available for children with both ASD and epilepsy. This article is part of a Special Issue entitled "Autism and Epilepsy".