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SPTAN1 encephalopathy: distinct phenotypes and genotypes

Tohyama J, Nakashima M, Nabatame S, Gaik-Siew C, Miyata R, Rener-Primec Z, et al.

J Hum Genet, 2015 Apr;60(4):167-73.
PMID: 25631096 DOI: 10.1038/jhg.2015.5

Recent progress in genetic analysis reveals that a significant proportion of cryptogenic epileptic encephalopathies are single-gene disorders. Mutations in numerous genes for early-onset epileptic encephalopathies have been rapidly identified, including in SPTAN1, which encodes α-II spectrin. The aim of this review is to delineate SPTAN1 encephalopathy as a distinct clinical syndrome. To date, a total of seven epileptic patients with four different in-frame SPTAN1 mutations have been identified. The major clinical features of SPTAN1 mutations include epileptic encephalopathy with hypsarrhythmia, no visual attention, acquired microcephaly, spastic quadriplegia and severe intellectual disability. Brainstem and cerebellar atrophy and cerebral hypomyelination, as observed by magnetic resonance imaging, are specific hallmarks of this condition. A milder variant is characterized by generalized epilepsy with pontocerebellar atrophy. Only in-frame SPTAN1 mutations in the last two spectrin repeats in the C-terminal region lead to dominant negative effects and these specific phenotypes. The last two spectrin repeats are required for α/β spectrin heterodimer associations and the mutations can alter heterodimer formation between the two spectrins. From these data we suggest that SPTAN1 encephalopathy is a distinct clinical syndrome owing to specific SPTAN1 mutations. It is important that this syndrome is recognized by pediatric neurologists to enable proper diagnostic work-up for patients.
Novel biallelic SZT2 mutations in 3 cases of early-onset epileptic encephalopathy

Tsuchida N, Nakashima M, Miyauchi A, Yoshitomi S, Kimizu T, Ganesan V, et al.

Clin Genet, 2018 02;93(2):266-274.
PMID: 28556953 DOI: 10.1111/cge.13061

The seizure threshold 2 (SZT2) gene encodes a large, highly conserved protein that is associated with epileptogenesis. In mice, Szt2 is abundantly expressed in the central nervous system. Recently, biallelic SZT2 mutations were found in 7 patients (from 5 families) presenting with epileptic encephalopathy with dysmorphic features and/or non-syndromic intellectual disabilities. In this study, we identified by whole-exome sequencing compound heterozygous SZT2 mutations in 3 patients with early-onset epileptic encephalopathies. Six novel SZT2 mutations were found, including 3 truncating, 1 splice site and 2 missense mutations. The splice-site mutation resulted in skipping of exon 20 and was associated with a premature stop codon. All individuals presented with seizures, severe developmental delay and intellectual disabilities with high variability. Brain MRIs revealed a characteristic thick and short corpus callosum or a persistent cavum septum pellucidum in each of the 2 cases. Interestingly, in the third case, born to consanguineous parents, had unexpected compound heterozygous missense mutations. She showed microcephaly despite the other case and previous ones presenting with macrocephaly, suggesting that SZT2 mutations might affect head size.
A novel homozygous truncating variant of NECAP1 in early infantile epileptic encephalopathy: the second case report of EIEE21

Mizuguchi T, Nakashima M, Moey LH, Ch'ng GS, Khoo TB, Mitsuhashi S, et al.

J Hum Genet, 2019 Apr;64(4):347-350.
PMID: 30626896 DOI: 10.1038/s10038-018-0556-2

We report the second case of early infantile epileptic encephalopathy (EIEE) arising from a homozygous truncating variant of NECAP1. The boy developed infantile-onset tonic-clonic and tonic seizures, then spasms in clusters. His electroencephalogram (EEG) showed a burst suppression pattern, leading to the diagnosis of Ohtahara syndrome. Whole-exome sequencing revealed the canonical splice-site variant (c.301 + 1 G > A) in NECAP1. In rodents, Necap1 protein is enriched in neuronal clathrin-coated vesicles and modulates synaptic vesicle recycling. cDNA analysis confirmed abnormal splicing that produced early truncating mRNA. There has been only one previous report of a mutation in NECAP1 in a family with EIEE; this was a nonsense mutation (p.R48*) that was cited as EIEE21. Decreased mRNA levels and the loss of the WXXF motif in both the families suggests that loss of NECAP1 function is a common pathomechanism for EIEE21. This study provided additional support that synaptic vesicle recycling plays a key role in epileptogenesis.
De novo variants in CUL3 are associated with global developmental delays with or without infantile spasms

Nakashima M, Kato M, Matsukura M, Kira R, Ngu LH, Lichtenbelt KD, et al.

J Hum Genet, 2020 Sep;65(9):727-734.
PMID: 32341456 DOI: 10.1038/s10038-020-0758-2

The ubiquitin-proteasome system is the principal system for protein degradation mediated by ubiquitination and is involved in various cellular processes. Cullin-RING ligases (CRL) are one class of E3 ubiquitin ligases that mediate polyubiquitination of specific target proteins, leading to decomposition of the substrate. Cullin 3 (CUL3) is a member of the Cullin family proteins, which act as scaffolds of CRL. Here we describe three cases of global developmental delays, with or without epilepsy, who had de novo CUL3 variants. One missense variant c.854T>C, p.(Val285Ala) and two frameshift variants c.137delG, p.(Arg46Leufs*32) and c.1239del, p.(Asp413Glufs*42) were identified by whole-exome sequencing. The Val285 residue located in the Cullin N-terminal domain and p.Val285Ala CUL3 mutant showed significantly weaker interactions to the BTB domain proteins than wild-type CUL3. Our findings suggest that de novo CUL3 variants may cause structural instability of the CRL complex and impairment of the ubiquitin-proteasome system, leading to diverse neuropsychiatric disorders.
Fulltext PLPBP mutations cause variable phenotypes of developmental and epileptic encephalopathy

Shiraku H, Nakashima M, Takeshita S, Khoo CS, Haniffa M, Ch'ng GS, et al.

Epilepsia Open, 2018 Dec;3(4):495-502.
PMID: 30525118 DOI: 10.1002/epi4.12272

Objective: Vitamin B6-dependent epilepsies are treatable disorders caused by variants in several genes, such as ALDH7A1,PNPO, and others. Recently, biallelic variants in PLPBP, formerly known as PROSC, were identified as a novel cause of vitamin B6-dependent epilepsies. Our objective was to further delineate the phenotype of PLPBP mutation.
Methods: We identified 4 unrelated patients harboring a total of 4 variants in PLPBP, including 3 novel variants, in a cohort of 700 patients with developmental and epileptic encephalopathies. Clinical information in each case was collected.
Results: Each patient had a different clinical course of epilepsy, with seizure onset from the first day of life to 3 months of age. Generalized tonic-clonic seizures were commonly noted. Myoclonic seizures or focal seizures were also observed in 2 patients. Interictal electroencephalography showed variable findings, such as suppression burst, focal or multifocal discharges, and diffuse slow activity. Unlike previous reports, all the patients had some degree of intellectual disability, although some of them had received early treatment with vitamin B6, suggesting that different mutation types influence the severity and outcome of the seizures.
Significance: PLPBP variants should be regarded as among the causative genes of developmental and epileptic encephalopathy, even when it occurs after the neonatal period. Early diagnosis and proper treatment with pyridoxine or pyridoxal phosphate is essential to improve the neurologic prognosis in neonates or young children with poorly controlled seizures.
Identification of de novo CSNK2A1 and CSNK2B variants in cases of global developmental delay with seizures

Nakashima M, Tohyama J, Nakagawa E, Watanabe Y, Siew CG, Kwong CS, et al.

J Hum Genet, 2019 Apr;64(4):313-322.
PMID: 30655572 DOI: 10.1038/s10038-018-0559-z

Casein kinase 2 (CK2) is a serine threonine kinase ubiquitously expressed in eukaryotic cells and involved in various cellular processes. In recent studies, de novo variants in CSNK2A1 and CSNK2B, which encode the subunits of CK2, have been identified in individuals with intellectual disability syndrome. In this study, we describe four patients with neurodevelopmental disorders possessing de novo variants in CSNK2A1 or CSNK2B. Using whole-exome sequencing, we detected two de novo variants in CSNK2A1 in two unrelated Japanese patients, a novel variant c.571C>T, p.(Arg191*) and a recurrent variant c.593A>G, p.(Lys198Arg), and two novel de novo variants in CSNK2B in Japanese and Malaysian patients, c.494A>G, p.(His165Arg) and c.533_534insGT, p.(Pro179Tyrfs*49), respectively. All four patients showed mild to profound intellectual disabilities, developmental delays, and various types of seizures. This and previous studies have found a total of 20 CSNK2A1 variants in 28 individuals with syndromic intellectual disability. The hotspot variant c.593A>G, p.(Lys198Arg) was found in eight of 28 patients. Meanwhile, only five CSNK2B variants were identified in five individuals with neurodevelopmental disorders. We reviewed the previous literature to verify the phenotypic spectrum of CSNK2A1- and CSNK2B-related syndromes.
Detection of copy number variations in epilepsy using exome data

Tsuchida N, Nakashima M, Kato M, Heyman E, Inui T, Haginoya K, et al.

Clin Genet, 2018 03;93(3):577-587.
PMID: 28940419 DOI: 10.1111/cge.13144

Epilepsies are common neurological disorders and genetic factors contribute to their pathogenesis. Copy number variations (CNVs) are increasingly recognized as an important etiology of many human diseases including epilepsy. Whole-exome sequencing (WES) is becoming a standard tool for detecting pathogenic mutations and has recently been applied to detecting CNVs. Here, we analyzed 294 families with epilepsy using WES, and focused on 168 families with no causative single nucleotide variants in known epilepsy-associated genes to further validate CNVs using 2 different CNV detection tools using WES data. We confirmed 18 pathogenic CNVs, and 2 deletions and 2 duplications at chr15q11.2 of clinically unknown significance. Of note, we were able to identify small CNVs less than 10 kb in size, which might be difficult to detect by conventional microarray. We revealed 2 cases with pathogenic CNVs that one of the 2 CNV detection tools failed to find, suggesting that using different CNV tools is recommended to increase diagnostic yield. Considering a relatively high discovery rate of CNVs (18 out of 168 families, 10.7%) and successful detection of CNV with <10 kb in size, CNV detection by WES may be able to surrogate, or at least complement, conventional microarray analysis.
GRIN2D variants in three cases of developmental and epileptic encephalopathy

Tsuchida N, Hamada K, Shiina M, Kato M, Kobayashi Y, Tohyama J, et al.

Clin Genet, 2018 12;94(6):538-547.
PMID: 30280376 DOI: 10.1111/cge.13454

N-methyl-d-aspartate (NMDA) receptors are glutamate-activated ion channels that are widely distributed in the central nervous system and essential for brain development and function. Dysfunction of NMDA receptors has been associated with various neurodevelopmental disorders. Recently, a de novo recurrent GRIN2D missense variant was found in two unrelated patients with developmental and epileptic encephalopathy. In this study, we identified by whole exome sequencing novel heterozygous GRIN2D missense variants in three unrelated patients with severe developmental delay and intractable epilepsy. All altered residues were highly conserved across vertebrates and among the four GluN2 subunits. Structural consideration indicated that all three variants are probably to impair GluN2D function, either by affecting intersubunit interaction or altering channel gating activity. We assessed the clinical features of our three cases and compared them to those of the two previously reported GRIN2D variant cases, and found that they all show similar clinical features. This study provides further evidence of GRIN2D variants being causal for epilepsy. Genetic diagnosis for GluN2-related disorders may be clinically useful when considering drug therapy targeting NMDA receptors.
Fulltext De Novo Truncating Variants in the Last Exon of SEMA6B Cause Progressive Myoclonic Epilepsy

Hamanaka K, Imagawa E, Koshimizu E, Miyatake S, Tohyama J, Yamagata T, et al.

Am J Hum Genet, 2020 04 02;106(4):549-558.
PMID: 32169168 DOI: 10.1016/j.ajhg.2020.02.011

De novo variants (DNVs) cause many genetic diseases. When DNVs are examined in the whole coding regions of genes in next-generation sequencing analyses, pathogenic DNVs often cluster in a specific region. One such region is the last exon and the last 50 bp of the penultimate exon, where truncating DNVs cause escape from nonsense-mediated mRNA decay [NMD(-) region]. Such variants can have dominant-negative or gain-of-function effects. Here, we first developed a resource of rates of truncating DNVs in NMD(-) regions under the null model of DNVs. Utilizing this resource, we performed enrichment analysis of truncating DNVs in NMD(-) regions in 346 developmental and epileptic encephalopathy (DEE) trios. We observed statistically significant enrichment of truncating DNVs in semaphorin 6B (SEMA6B) (p value: 2.8 × 10-8; exome-wide threshold: 2.5 × 10-6). The initial analysis of the 346 individuals and additional screening of 1,406 and 4,293 independent individuals affected by DEE and developmental disorders collectively identified four truncating DNVs in the SEMA6B NMD(-) region in five individuals who came from unrelated families (p value: 1.9 × 10-13) and consistently showed progressive myoclonic epilepsy. RNA analysis of lymphoblastoid cells established from an affected individual showed that the mutant allele escaped NMD, indicating stable production of the truncated protein. Importantly, heterozygous truncating variants in the NMD(+) region of SEMA6B are observed in general populations, and SEMA6B is most likely loss-of-function tolerant. Zebrafish expressing truncating variants in the NMD(-) region of SEMA6B orthologs displayed defective development of brain neurons and enhanced pentylenetetrazole-induced seizure behavior. In summary, we show that truncating DNVs in the final exon of SEMA6B cause progressive myoclonic epilepsy.
Fulltext Impaired neuronal KCC2 function by biallelic SLC12A5 mutations in migrating focal seizures and severe developmental delay

Saitsu H, Watanabe M, Akita T, Ohba C, Sugai K, Ong WP, et al.

Sci Rep, 2016 07 20;6:30072.
PMID: 27436767 DOI: 10.1038/srep30072

Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the early-onset epileptic syndromes characterized by migrating polymorphous focal seizures. Whole exome sequencing (WES) in ten sporadic and one familial case of EIMFS revealed compound heterozygous SLC12A5 (encoding the neuronal K(+)-Cl(-) co-transporter KCC2) mutations in two families: c.279 + 1G > C causing skipping of exon 3 in the transcript (p.E50_Q93del) and c.572 C >T (p.A191V) in individuals 1 and 2, and c.967T > C (p.S323P) and c.1243 A > G (p.M415V) in individual 3. Another patient (individual 4) with migrating multifocal seizures and compound heterozygous mutations [c.953G > C (p.W318S) and c.2242_2244del (p.S748del)] was identified by searching WES data from 526 patients and SLC12A5-targeted resequencing data from 141 patients with infantile epilepsy. Gramicidin-perforated patch-clamp analysis demonstrated strongly suppressed Cl(-) extrusion function of E50_Q93del and M415V mutants, with mildly impaired function of A191V and S323P mutants. Cell surface expression levels of these KCC2 mutants were similar to wildtype KCC2. Heterologous expression of two KCC2 mutants, mimicking the patient status, produced a significantly greater intracellular Cl(-) level than with wildtype KCC2, but less than without KCC2. These data clearly demonstrated that partially disrupted neuronal Cl(-) extrusion, mediated by two types of differentially impaired KCC2 mutant in an individual, causes EIMFS.
De novo hotspot variants in CYFIP2 cause early-onset epileptic encephalopathy

Nakashima M, Kato M, Aoto K, Shiina M, Belal H, Mukaida S, et al.

Ann Neurol, 2018 04;83(4):794-806.
PMID: 29534297 DOI: 10.1002/ana.25208

OBJECTIVE: The cytoplasmic fragile X mental retardation 1 interacting proteins 2 (CYFIP2) is a component of the WASP-family verprolin-homologous protein (WAVE) regulatory complex, which is involved in actin dynamics. An obvious association of CYFIP2 variants with human neurological disorders has never been reported. Here, we identified de novo hotspot CYFIP2 variants in neurodevelopmental disorders and explore the possible involvement of the CYFIP2 mutants in the WAVE signaling pathway.
METHODS: We performed trio-based whole-exome sequencing (WES) in 210 families and case-only WES in 489 individuals with epileptic encephalopathies. The functional effect of CYFIP2 variants on WAVE signaling was evaluated by computational structural analysis and in vitro transfection experiments.
RESULTS: We identified three de novo CYFIP2 variants at the Arg87 residue in 4 unrelated individuals with early-onset epileptic encephalopathy. Structural analysis indicated that the Arg87 residue is buried at an interface between CYFIP2 and WAVE1, and the Arg87 variant may disrupt hydrogen bonding, leading to structural instability and aberrant activation of the WAVE regulatory complex. All mutant CYFIP2 showed comparatively weaker interactions to the VCA domain than wild-type CYFIP2. Immunofluorescence revealed that ectopic speckled accumulation of actin and CYFIP2 was significantly increased in cells transfected with mutant CYFIP2.
INTERPRETATION: Our findings suggest that de novo Arg87 variants in CYFIP2 have gain-of-function effects on the WAVE signaling pathway and are associated with severe neurological disorders. Ann Neurol 2018;83:794-806.
Fulltext Biallelic TBCD Mutations Cause Early-Onset Neurodegenerative Encephalopathy

Miyake N, Fukai R, Ohba C, Chihara T, Miura M, Shimizu H, et al.

Am J Hum Genet, 2016 Oct 06;99(4):950-961.
PMID: 27666374 DOI: 10.1016/j.ajhg.2016.08.005

We describe four families with affected siblings showing unique clinical features: early-onset (before 1 year of age) progressive diffuse brain atrophy with regression, postnatal microcephaly, postnatal growth retardation, muscle weakness/atrophy, and respiratory failure. By whole-exome sequencing, we identified biallelic TBCD mutations in eight affected individuals from the four families. TBCD encodes TBCD (tubulin folding co-factor D), which is one of five tubulin-specific chaperones playing a pivotal role in microtubule assembly in all cells. A total of seven mutations were found: five missense mutations, one nonsense, and one splice site mutation resulting in a frameshift. In vitro cell experiments revealed the impaired binding between most mutant TBCD proteins and ARL2, TBCE, and β-tubulin. The in vivo experiments using olfactory projection neurons in Drosophila melanogaster indicated that the TBCD mutations caused loss of function. The wide range of clinical severity seen in this neurodegenerative encephalopathy may result from the residual function of mutant TBCD proteins. Furthermore, the autopsied brain from one deceased individual showed characteristic neurodegenerative findings: cactus and somatic sprout formations in the residual Purkinje cells in the cerebellum, which are also seen in some diseases associated with mitochondrial impairment. Defects of microtubule formation caused by TBCD mutations may underlie the pathomechanism of this neurodegenerative encephalopathy.
De novo variants in CELF2 that disrupt the nuclear localization signal cause developmental and epileptic encephalopathy

Itai T, Hamanaka K, Sasaki K, Wagner M, Kotzaeridou U, Brösse I, et al.

Hum Mutat, 2021 01;42(1):66-76.
PMID: 33131106 DOI: 10.1002/humu.24130

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.

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