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.

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.

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.

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.

De novo mutations in SIK1 cause a spectrum of developmental epilepsies.

Developmental epilepsies are age-dependent seizure disorders for which genetic causes have been increasingly identified. Here we report six unrelated individuals with mutations in salt-inducible kinase 1 (SIK1) in a series of 101 persons with early myoclonic encephalopathy, Ohtahara syndrome, and infantile spasms. Individuals with SIK1 mutations had short survival in cases with neonatal epilepsy onset, and an autism plus developmental syndrome after infantile spasms in others. All six mutations occurred outside the kinase domain of SIK1 and each of the mutants displayed autophosphorylation and kinase activity toward HDAC5. Three mutations generated truncated forms of SIK1 that were resistant to degradation and also showed changes in sub-cellular localization compared to wild-type SIK1. We also report the human neuropathologic examination of SIK1-related developmental epilepsy, with normal neuronal morphology and lamination but abnormal SIK1 protein cellular localization. Therefore, these results expand the genetic etiologies of developmental epilepsies by demonstrating SIK1 mutations as a cause of severe developmental epilepsy.

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.

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.

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.

De novo mutations in the beta-tubulin gene TUBB2A cause simplified gyral patterning and infantile-onset epilepsy.

Tubulins, and microtubule polymers into which they incorporate, play critical mechanical roles in neuronal function during cell proliferation, neuronal migration, and postmigrational development: the three major overlapping events of mammalian cerebral cortex development. A number of neuronally expressed tubulin genes are associated with a spectrum of disorders affecting cerebral cortex formation. Such "tubulinopathies" include lissencephaly/pachygyria, polymicrogyria-like malformations, and simplified gyral patterns, in addition to characteristic extracortical features, such as corpus callosal, basal ganglia, and cerebellar abnormalities. Epilepsy is a common finding in these related disorders. Here we describe two unrelated individuals with infantile-onset epilepsy and abnormalities of brain morphology, harboring de novo variants that affect adjacent amino acids in a beta-tubulin gene TUBB2A. Located in a highly conserved loop, we demonstrate impaired tubulin and microtubule function resulting from each variant in vitro and by using in silico predictive modeling. We propose that the affected functional loop directly associates with the alpha-tubulin-bound guanosine triphosphate (GTP) molecule, impairing the intradimer interface and correct formation of the alpha/beta-tubulin heterodimer. This study associates mutations in TUBB2A with the spectrum of "tubulinopathy" phenotypes. As a consequence, genetic variations affecting all beta-tubulin genes expressed at high levels in the brain (TUBB2B, TUBB3, TUBB, TUBB4A, and TUBB2A) have been linked with malformations of cortical development.

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.

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.

Novel mutations in ATP1A3 associated with catastrophic early life epilepsy, episodic prolonged apnea, and postnatal microcephaly.

OBJECTIVE: Mutations of ATP1A3 have been associated with rapid onset dystonia-parkinsonism and more recently with alternating hemiplegia of childhood. Here we report one child with catastrophic early life epilepsy and shortened survival, and another with epilepsy, episodic prolonged apnea, postnatal microcephaly, and severe developmental disability. Novel heterozygous mutations (p.Gly358Val and p.Ile363Asn) were identified in ATP1A3 in these children. METHODS: Subjects underwent next-generation sequencing under a research protocol. Clinical data were collected retrospectively. The biochemical effects of the mutations on ATP1A3 protein function were investigated. Postmortem neuropathologic specimens from control and affected subjects were studied. RESULTS: The mutations localized to the P domain of the Na,K-ATPase α3 protein, and resulted in significant reduction of Na,K-ATPase activity in vitro. We demonstrate in both control human brain tissue and that from the subject with the p.Gly358Val mutation that ATP1A3 immunofluorescence is prominently associated with interneurons in the cortex, which may provide some insight into the pathogenesis of the disease. SIGNIFICANCE: The findings indicate these mutations cause severe phenotypes of ATP1A3-related disorder spectrum that include catastrophic early life epilepsy, episodic apnea, and postnatal microcephaly.

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".

Genetic disorders associated with postnatal microcephaly.

Several genetic disorders are characterized by normal head size at birth, followed by deceleration in head growth resulting in postnatal microcephaly. Among these are classic disorders such as Angelman syndrome and MECP2-related disorder (formerly Rett syndrome), as well as more recently described clinical entities associated with mutations in CASK, CDKL5, CREBBP, and EP300 (Rubinstein-Taybi syndrome), FOXG1, SLC9A6 (Christianson syndrome), and TCF4 (Pitt-Hopkins syndrome). These disorders can be identified clinically by phenotyping across multiple neurodevelopmental and neurobehavioral realms, and enough data are available to recognize these postnatal microcephaly disorders as separate diagnostic entities in their own right. A second diagnostic grouping, comprised of Warburg MICRO syndrome, Cockayne syndrome, and Cerebral-oculo-facial skeletal syndrome, share similar features of somatic growth failure, ophthalmologic, and dysmorphologic features. Many postnatal microcephaly syndromes are caused by mutations in genes important in the regulation of gene expression in the developing forebrain and hindbrain, although important synaptic structural genes also play a role. This is an emerging group of disorders with a fascinating combination of brain malformations, specific epilepsies, movement disorders, and other complex neurobehavioral abnormalities.

Introduction: Brain malformations.

This issue of the American Journal of Medical Genetics Seminar Series Part C is dedicated to congenital brain malformations with a special focus on the molecular mechanisms underlying this fascinating, and often complex, group of developmental brain disorders. As with most genetic disorders, the past few years have witnessed a dramatic leap in our understanding of the molecular basis of these malformations that include both constitutional and post-zygotic (or mosaic) genetic aberrations. This is best exemplified by the recent identification of mutations within components of the PI3K-AKT-mTOR pathway in hemimegalencephaly and megalencephaly syndromes, and the rapidly increased identification of mutations within the tubulin family in a broad range of cortical and non-cortical brain malformations. These discoveries, particularly of the emerging "tubulinopathies" spectrum, have not only expanded our knowledge of these disorders but challenge our existing, and perhaps overly simplistic, classification of these malformations based on the primary neuronal stage at which the abnormality occurs. It is our hope that this series will facilitate a deeper understanding of these malformations beyond their clinical and neuroimaging features and syndromic associations to their molecular and pathway underpinnings. We believe this knowledge will most certainly be instrumental as we move into the era of delineating genotype-phenotype correlations and, ultimately, pathway-based therapies.

Mutations in CENPE define a novel kinetochore-centromeric mechanism for microcephalic primordial dwarfism.

Defects in centrosome, centrosomal-associated and spindle-associated proteins are the most frequent cause of primary microcephaly (PM) and microcephalic primordial dwarfism (MPD) syndromes in humans. Mitotic progression and segregation defects, microtubule spindle abnormalities and impaired DNA damage-induced G2-M cell cycle checkpoint proficiency have been documented in cell lines from these patients. This suggests that impaired mitotic entry, progression and exit strongly contribute to PM and MPD. Considering the vast protein networks involved in coordinating this cell cycle stage, the list of potential target genes that could underlie novel developmental disorders is large. One such complex network, with a direct microtubule-mediated physical connection to the centrosome, is the kinetochore. This centromeric-associated structure nucleates microtubule attachments onto mitotic chromosomes. Here, we described novel compound heterozygous variants in CENPE in two siblings who exhibit a profound MPD associated with developmental delay, simplified gyri and other isolated abnormalities. CENPE encodes centromere-associated protein E (CENP-E), a core kinetochore component functioning to mediate chromosome congression initially of misaligned chromosomes and in subsequent spindle microtubule capture during mitosis. Firstly, we present a comprehensive clinical description of these patients. Then, using patient cells we document abnormalities in spindle microtubule organization, mitotic progression and segregation, before modeling the cellular pathogenicity of these variants in an independent cell system. Our cellular analysis shows that a pathogenic defect in CENP-E, a kinetochore-core protein, largely phenocopies PCNT-mutated microcephalic osteodysplastic primordial dwarfism-type II patient cells. PCNT encodes a centrosome-associated protein. These results highlight a common underlying pathomechanism. Our findings provide the first evidence for a kinetochore-based route to MPD in humans.

The Developmental Brain Disorders Database (DBDB): a curated neurogenetics knowledge base with clinical and research applications.

The number of single genes associated with neurodevelopmental disorders has increased dramatically over the past decade. The identification of causative genes for these disorders is important to clinical outcome as it allows for accurate assessment of prognosis, genetic counseling, delineation of natural history, inclusion in clinical trials, and in some cases determines therapy. Clinicians face the challenge of correctly identifying neurodevelopmental phenotypes, recognizing syndromes, and prioritizing the best candidate genes for testing. However, there is no central repository of definitions for many phenotypes, leading to errors of diagnosis. Additionally, there is no system of levels of evidence linking genes to phenotypes, making it difficult for clinicians to know which genes are most strongly associated with a given condition. We have developed the Developmental Brain Disorders Database (DBDB: https://www.dbdb.urmc.rochester.edu/home), a publicly available, online-curated repository of genes, phenotypes, and syndromes associated with neurodevelopmental disorders. DBDB contains the first referenced ontology of developmental brain phenotypes, and uses a novel system of levels of evidence for gene-phenotype associations. It is intended to assist clinicians in arriving at the correct diagnosis, select the most appropriate genetic test for that phenotype, and improve the care of patients with developmental brain disorders. For researchers interested in the discovery of novel genes for developmental brain disorders, DBDB provides a well-curated source of important genes against which research sequencing results can be compared. Finally, DBDB allows novel observations about the landscape of the neurogenetics knowledge base.

Epilepsy and outcome in FOXG1-related disorders.

OBJECTIVE: FOXG1-related disorders are associated with severe intellectual disability, absent speech with autistic features, and epilepsy. Children with deletions or intragenic mutations of FOXG1 also have postnatal microcephaly, morphologic abnormalities of the corpus callosum, and choreiform movements. Duplications of 14q12 often present with infantile spasms, and have subsequent intellectual disability with autistic features. Long-term epilepsy outcome and response to treatment have not been studied systematically in a well-described cohort of subjects with FOXG1-related disorders. We report on the epilepsy features and developmental outcome of 23 new subjects with deletions or intragenic mutations of FOXG1, and 7 subjects with duplications. METHODS: Subjects had either chromosomal microarray or FOXG1 gene sequencing performed as part of routine clinical care. Development and epilepsy follow-up data were collected from medical records from treating neurologists and through telephone parental interviews using standardized questionnaires. RESULTS: Epilepsy was diagnosed in 87% of the subjects with FOXG1-related disorders. The mean age of epilepsy diagnosis in FOXG1 duplications was significantly younger than those with deletions/intragenic mutations (p = 0.0002). All of the duplication FOXG1 children with infantile spasms responded to hormonal therapy, and only one required long-term antiepileptic therapy. In contrast, more children with deletions/intragenic mutations required antiepileptic drugs on follow-up (p < 0.0005). All subjects with FOXG1-related disorders had neurodevelopmental disabilities after 3 years of age, regardless of the epilepsy type or intractability of seizures. All had impaired verbal language and social contact, and three duplication subjects were formally diagnosed with autism. Subjects with deletion/intragenic mutations, however, had significantly worse ambulation (p = 0.04) and functional hand use (p < 0.0005). SIGNIFICANCE: Epilepsy and developmental outcome characteristics allow clinicians to distinguish among the FOXG1-related disorders. Further genotype-phenotype studies of FOXG1 may help to elucidate why children develop different forms of developmental epilepsy.

MEF2C Haploinsufficiency features consistent hyperkinesis, variable epilepsy, and has a role in dorsal and ventral neuronal developmental pathways.

MEF2C haploinsufficiency syndrome is an emerging neurodevelopmental disorder associated with intellectual disability, autistic features, epilepsy, and abnormal movements. We report 16 new patients with MEF2C haploinsufficiency, including the oldest reported patient with MEF2C deletion at 5q14.3. We detail the neurobehavioral phenotype, epilepsy, and abnormal movements, and compare our subjects with those previously reported in the literature. We also investigate Mef2c expression in the developing mouse forebrain. A spectrum of neurofunctional deficits emerges, with hyperkinesis a consistent finding. Epilepsy varied from absent to severe, and included intractable myoclonic seizures and infantile spasms. Subjects with partial MEF2C deletion were statistically less likely to have epilepsy. Finally, we confirm that Mef2c is present both in dorsal primary neuroblasts and ventral gamma-aminobutyric acid(GABA)ergic interneurons in the forebrain of the developing mouse. Given interactions with several key neurodevelopmental genes such as ARX, FMR1, MECP2, and TBR1, it appears that MEF2C plays a role in several developmental stages of both dorsal and ventral neuronal cell types.