De novo variants in RHOBTB2, an atypical Rho GTPase gene,cause epileptic encephalopathy

By whole exome sequencing, we identified three de novo RHOBTB2 variants in three patients with epileptic encephalopathies (EEs). Interestingly, all three patients showed acute encephalopathy (febrile status epilepticus), with magnetic resonance imaging revealing hemisphere swelling or reduced diffusion in various brain regions. RHOBTB2 encodes Rho‐related BTB domain‐containing protein 2, an atypical Rho GTPase that is a substrate‐specific adaptor or itself is a substrate for the Cullin‐3 (CUL3)‐based ubiquitin ligase complex. Transient expression experiments in Neuro‐2a cells revealed that mutant RHOBTB2 was more abundant than wild‐type RHOBTB2. Coexpression of CUL3 with RHOBTB2 decreased the level of wild‐type RHOBTB2 but not the level of any of the three mutants, indicating impaired CUL3 complex‐dependent degradation of the three mutants. These data indicate that RHOBTB2 variants are a rare genetic cause of EEs, in which acute encephalopathy might be a characteristic feature, and that precise regulation of RHOBTB2 levels is essential for normal brain function.     

De novo variants in CELF2 that disrupt the nuclear localization signal cause developmental and epileptic encephalopathy

We report heterozygous CELF2 (NM_006561.3) variants in five unrelated individuals: Individuals 1–4 exhibited developmental and epileptic encephalopathy (DEE) and Individual 5 had intellectual disability and autistic features. CELF2 encodes a nucleocytoplasmic shuttling RNA‐binding protein that has multiple roles in RNA processing and is involved in the embryonic development of the central nervous system and heart. Whole‐exome sequencing identified the following CELF2 variants: two missense variants [c.1558C>T:p.(Pro520Ser) in unrelated Individuals 1 and 2, and c.1516C>G:p.(Arg506Gly) in Individual 3], one frameshift variant in Individual 4 that removed the last amino acid of CELF2 c.1562dup:p.(Tyr521Ter), possibly resulting in escape from nonsense‐mediated mRNA decay (NMD), and one canonical splice site variant, c.272‐1G>C in Individual 5, also probably leading to NMD. The identified variants in Individuals 1, 2, 4, and 5 were de novo, while the variant in Individual 3 was inherited from her mosaic mother. Notably, all identified variants, except for c.272‐1G>C, were clustered within 20 amino acid residues of the C‐terminus, which might be a nuclear localization signal. We demonstrated the extranuclear mislocalization of mutant CELF2 protein in cells transfected with mutant CELF2 complementary DNA plasmids. Our findings indicate that CELF2 variants that disrupt its nuclear localization are associated with DEE.     

De novo KCNA1 variants in the PVP motif cause infantile epileptic encephalopathy and cognitive impairment similar to recurrent KCNA2 variants

Derangements in voltage‐gated potassium channel function are responsible for a range of paroxysmal neurologic disorders. Pathogenic variants in the KCNA1 gene, which encodes the voltage‐gated potassium channel Kv1.1, are responsible for Episodic Ataxia Type 1 (EA1). Patients with EA1 have an increased incidence of epilepsy, but KCNA1 variants have not been described in epileptic encephalopathy. Here, we describe four patients with infantile‐onset epilepsy and cognitive impairment who harbor de novo KCNA1 variants located within the Kv‐specific Pro‐Val‐Pro (PVP) motif which is essential for channel gating. The first two patients have KCNA1 variants resulting in (p.Pro405Ser) and (p.Pro405Leu), respectively, and a set of identical twins has a variant affecting a nearby residue (p.Pro403Ser). Notably, recurrent de novo variants in the paralogous PVP motif of KCNA2 have previously been shown to abolish channel function and also cause early‐onset epileptic encephalopathy. Importantly, this report extends the range of phenotypes associated with KCNA1 variants to include epileptic encephalopathy when the PVP motif is involved.     

SCN8A heterozygous variants are associated with anoxic‐epileptic seizures

Anoxic‐epileptic seizures (AES) are rare outcomes of common childhood reflex anoxic syncope that trigger a true epileptic seizure. The term AES was coined by Stephenson in 1983, to differentiate these events from convulsive syncopes and the more common reflex anoxic syncopes. A genetic susceptibility for AES has been postulated; but, its molecular basis has up to now been elusive. We report here two illustrative cases and show the association of de novo SCN8A variants and AES. One of them had focal or generalized seizures and autonomic symptoms triggered by orthostatism; the second had breath‐holding spells triggered by pain or exercise leading to tonic–clonic seizures; both had repeatedly normal EEGs and a family history of reflex syncope. The data of three additional AES patients further suggest, for the first time, a link between SCN8A pathogenic variants and AES. The neurodevelopment of four patients was abnormal. Four of the five SCN8A mutations observed here were previously described in patients with seizure disorders. Seizures responded particularly well to sodium channel blockers. Our observation enriches the spectrum of seizures linked with SCN8A pathogenic variants.     

De novo SCN1A mutations are a major cause of severe myoclonic epilepsy of infancy

Severe myoclonic epilepsy of infancy (SMEI or Dravet syndrome) is a rare disorder occurring in young children often without a family history of a similar disorder. The earliest disease manifestations are usually fever-associated seizures. Later in life, patients display different types of afebrile seizures including myoclonic seizures. Arrest of psychomotor development occurs in the second year of life and most patients become ataxic. Patients are resistant to antiepileptic drug therapy. Recently, we described de novo mutations of the neuronal sodium channel alpha-subunit gene SCN1A in seven isolated SMEI patients. To investigate the contribution of SCN1A mutations to the etiology of SMEI, we examined nine additional SMEI patients. We observed eight coding and one noncoding mutation. In contrast to our previous study, most mutations are missense mutations clustering in the S4-S6 region of SCN1A. These findings demonstrate that de novo mutations in SCN1A are a major cause of isolated SMEI.     

De novo variants are a common cause of genetic hearing loss

PurposeDe novo variants (DNVs) are a well-recognized cause of genetic disorders. The contribution of DNVs to hearing loss (HL) is poorly characterized. We aimed to evaluate the rate of DNVs in HL-associated genes and assess their contribution to HL.MethodsTargeted genomic enrichment and massively parallel sequencing were used for molecular testing of all exons and flanking intronic sequences of known HL-associated genes, with no exclusions on the basis of type of HL or clinical features. Segregation analysis was performed, and previous reports of DNVs in PubMed and ClinVar were reviewed to characterize the rate, distribution, and spectrum of DNVs in HL.ResultsDNVs were detected in 10% (24/238) of trios for whom segregation analysis was performed. Overall, DNVs were causative in at least ～1% of probands for whom a genetic diagnosis was resolved, with marked variability based on inheritance mode and phenotype. DNVs of MITF were most common (21% of DNVs), followed by GATA3 (13%), STRC (13%), and ACTG1 (8%). Review of reported DNVs revealed gene-specific variability in contribution of DNV to the mutational spectrum of HL-associated genes.ConclusionDNVs are a relatively common cause of genetic HL and must be considered in all cases of sporadic HL.     

De novo missense variants in RAC3 cause a novel neurodevelopmental syndrome

PurposeRAC3 is an underexamined member of the Rho GTPase gene family that is expressed in the developing brain and linked to key cellular functions. De novo missense variants in the homolog RAC1 were recently associated with developmental disorders. In the RAC subfamily, transforming missense changes at certain shared residues have been observed in human cancers and previously characterized in experimental studies. The purpose of this study was to determine whether constitutional dysregulation of RAC3 is associated with human disease.MethodsWe discovered a RAC3 variant in the index case using genome sequencing, and searched for additional variants using international data-sharing initiatives. Functional effects of the variants were assessed using a multifaceted approach generalizable to most clinical laboratory settings.ResultsWe rapidly identified five individuals with de novo monoallelic missense variants in RAC3, including one recurrent change. Every participant had severe intellectual disability and brain malformations. In silico protein modeling, and prior in vivo and in situ experiments, supported a transforming effect for each of the three different RAC3 variants. All variants were observed in databases of somatic variation in cancer.ConclusionsMissense variants in RAC3 cause a novel brain disorder, likely through a mechanism of constitutive protein activation.     

Expanding the genotype–phenotype correlation of de novo heterozygous missense variants in YWHAG as a cause of developmental and epileptic encephalopathy

Developmental and Epileptic encephalopathies (DEE) describe heterogeneous epilepsy syndromes, characterized by early‐onset, refractory seizures and developmental delay (DD). Several DEE associated genes have been reported. With increased access to whole exome sequencing (WES), new candidate genes are being identified although there are fewer large cohort papers describing the clinical phenotype in such patients. We describe 6 unreported individuals and provide updated information on an additional previously reported individual with heterozygous de novo missense variants in YWHAG. We describe a syndromal phenotype, report 5 novel, and a recurrent p.Arg132Cys YWHAG variant and compare developmental trajectory and treatment strategies in this cohort. We provide further evidence of causality in YWHAG variants. WES was performed in five patients via Deciphering Developmental Disorders Study and the remaining two were identified via Genematcher and AnnEX databases. De novo variants identified from exome data were validated using Sanger sequencing. Seven out of seven patients in the cohort have de novo, heterozygous missense variants in YWHAG including 2/7 patients with a recurrent c.394C > T, p.Arg132Cys variant; 1/7 has a second, pathogenic variant in STAG1. Characteristic features included: early‐onset seizures, predominantly generalized tonic–clonic and absence type (7/7) with good response to standard anti‐epileptic medications; moderate DD; Intellectual Disability (ID) (5/7) and Autism Spectrum Disorder (3/7). De novo YWHAG missense variants cause EE, characterized by early‐onset epilepsy, ID and DD, supporting the hypothesis that YWHAG loss‐of‐function causes a neurological phenotype. Although the exact mechanism of disease resulting from alterations in YWHAG is not fully known, it is possible that haploinsufficiency of YWHAG in developing cerebral cortex may lead to abnormal neuronal migration resulting in DEE.     

De novo GRIN1 mutations: An emerging cause of severe early infantile encephalopathy

De novo GRIN1 mutations have recently been shown to cause severe intellectual disability, hypotonia, hyperkinetic and stereotyped movements, and epilepsy. We report two new cases of severe early onset encephalopathy associated with hyperkinetic and oculogyric-like movements, caused by mutations in the GRIN1 gene; both were identified by whole exome sequencing. One of the patients harbored the novel mutation p.Ser688Tyr and the other patient harbored the p.Gly827Arg mutation, which was previously reported in three patients. In silico studies suggested that the p.Se688Tyr mutation results in disruption of NMDA ligand binding and the p.Gly827Arg mutation results in disrupted gating of the ion channel. Our study highlights the importance of GRIN1 mutations in the etiology of isolated cases of early onset encephalopathy, and the valuable role of whole exome sequencing in identifying these mutations.     

De novo variants in CNOT9 cause a neurodevelopmental disorder with or without epilepsy

PurposeThe study aimed to clinically and molecularly characterize the neurodevelopmental disorder associated with heterozygous de novo variants in CNOT9.MethodsIndividuals were clinically examined. Variants were identified using exome or genome sequencing. These variants were evaluated using in silico predictions, and their functional relevance was further assessed by molecular models and research in the literature. The variants have been classified according to the criteria of the American College of Medical Genetics.ResultsWe report on 7 individuals carrying de novo missense variants in CNOT9, p.(Arg46Gly), p.(Pro131Leu), and p.(Arg227His), and, recurrent in 4 unrelated individuals, p.(Arg292Trp). All affected persons have developmental delay/intellectual disability, with 5 of them showing seizures. Other symptoms include muscular hypotonia, facial dysmorphism, and behavioral abnormalities. Molecular modeling predicted that the variants are damaging and would lead to reduced protein stability or impaired recognition of interaction partners. Functional analyses in previous studies showed a pathogenic effect of p.(Pro131Leu) and p.(Arg227His).ConclusionWe propose CNOT9 as a novel gene for neurodevelopmental disorder and epilepsy.     

Confirming the recessive inheritance of SCN1B mutations in developmental epileptic encephalopathy

Dominant SCN1B mutations are known to cause several epilepsy syndromes in humans. Only 2 epilepsy patients to date have been reported to have recessive mutations in SCN1B as the likely cause of their phenotype. Here, we confirm the recessive inheritance of 2 novel SCN1B mutations in 5 children from 3 families with developmental epileptic encephalopathy. The recessive inheritance and early death in these patients is consistent with the Dravet‐like phenotype observed in Scn1b^?/? mice. The ‘negative’ clinical exome in one of these families highlights the need to consider recessive mutations in the interpretation of variants in typically dominant genes.     

De novo 5q14.3 translocation 121.5-kb upstream of MEF2C in a patient with severe intellectual disability and early-onset epileptic encephalopathy

Recent studies have shown that haploinsufficiency of MEF2C causes severe intellectual disability, epilepsy, hypotonia, and cerebral malformations. We report on a female patient with severe intellectual disability, early-onset epileptic encephalopathy, and hypoplastic corpus callosum, possessing a de novo balanced translocation, t(5;15)(q13.3;q26.1). The patient showed upward gazing and tonic seizure of lower extremities followed by generalized clonic seizures at 4 months of age. Electroencephalogram showed hypsarrhythmia when asleep. By using fluorescent in situ hybridization (FISH), southern hybridization and inverse PCR, the translocation breakpoints were determined at the nucleotide level. The 5q14.3 breakpoint was localized 121.5-kb upstream of MEF2C. The 15q26.2 breakpoint was mapped 119-kb downstream of LOC91948 non-coding RNA. We speculate that the translocation may disrupt the proper regulation of MEF2C expression in the developing brain, resulting in severe intellectual disability and early-onset epileptic encephalopathy.     

Dominant negative effects of SCN5A missense variants

PurposeUp to 30% of patients with Brugada syndrome (BrS) carry loss-of-function (LoF) variants in the cardiac sodium channel gene SCN5A encoding for the protein Na_V1.5. Recent studies suggested that Na_V1.5 can dimerize, and some variants exert dominant negative effects. In this study, we sought to explore the generality of missense variant Na_V1.5 dominant negative effects and their clinical severity.MethodsWe identified 35 LoF variants (<10% of wild type [WT] peak current) and 15 partial LoF variants (10%-50% of WT peak current) that we assessed for dominant negative effects. SCN5A variants were studied in HEK293T cells, alone or in heterozygous coexpression with WT SCN5A using automated patch clamp. To assess the clinical risk, we compared the prevalence of dominant negative vs putative haploinsufficient (frameshift, splice, or nonsense) variants in a BrS consortium and the Genome Aggregation Database population database.ResultsIn heterozygous expression with WT, 32 of 35 LoF and 6 of 15 partial LoF variants showed reduction to <75% of WT-alone peak current, showing a dominant negative effect. Individuals with dominant negative LoF variants had an elevated disease burden compared with the individuals with putative haploinsufficient variants (2.7-fold enrichment in BrS cases, P = .019).ConclusionMost SCN5A missense LoF variants exert a dominant negative effect. This class of variant confers an especially high burden of BrS.     

De novo variants in sporadic cases of childhood onset schizophrenia

Childhood-onset schizophrenia (COS), defined by the onset of illness before age 13 years, is a rare severe neurodevelopmental disorder of unknown etiology. Recently, sequencing studies have identified rare, potentially causative de novo variants in sporadic cases of adult-onset schizophrenia and autism. In this study, we performed exome sequencing of 17 COS trios in order to test whether de novo variants could contribute to this disease. We identified 20 de novo variants in 17 COS probands, which is consistent with the de novo mutation rate reported in the adult form of the disease. Interestingly, the missense de novo variants in COS have a high likelihood for pathogenicity and were enriched for genes that are less tolerant to variants. Among the genes found disrupted in our study, SEZ6, RYR2, GPR153, GTF2IRD1, TTBK1 and ITGA6 have been previously linked to neuronal function or to psychiatric disorders, and thus may be considered as COS candidate genes.     

De novo nonsense mutations in the sodium channel gene,SCN2A,in sporadic intractable epilepsy

De novo mutations of voltage‐gated sodium channel αII gene SCN2A in intractable epilepsies Ogiwara et al. (2009) Neurology 73(13): 1046–1053