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Background: The tricarboxylic acid (TCA) cycle is a sequence of catabolic reactions within the mitochondrial matrix, and is a central pathway for cellular energy metabolism. Genetic defects affecting the TCA cycle are known to cause severe multisystem disorders., Methods: We performed whole exome sequencing of genomic DNA of a patient with progressive cerebellar and cerebral atrophy, hypotonia, ataxia, seizure disorder, developmental delay, ophthalmological abnormalities and hearing loss. We also performed biochemical studies using patient fibroblasts., Results: We identified new compound heterozygous mutations (c.1534G > A, p.Asp512Asn and c.1997G > C, p.Gly666Ala) in ACO2, which encodes aconitase 2, a component of the TCA cycle. In patient fibroblasts, the aconitase activity was reduced to 15% of that of the control, and the aconitase 2 level decreased to 36% of that of the control. As such a decrease in aconitase 2 in patient fibroblasts was partially restored by proteasome inhibition, mutant aconitase 2 was suggested to be relatively unstable and rapidly degraded after being synthesized. In addition, the activity of the father-derived variant of aconitase 2 (p.Gly666Ala), which had a mutation near the active center, was 55% of that of wild-type., Conclusion: The marked reduction of aconitase activity in patient fibroblasts was due to the combination of decreased aconitase 2 amount and activity due to mutations. Reduced aconitase activity directly suppresses the TCA cycle, resulting in mitochondrial dysfunction, which may lead to symptoms similar to those observed in mitochondrial diseases., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

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

Misato 1, mitochondrial distribution and morphology regulator (encoded by the MSTO1 gene), is involved in mitochondrial distribution and morphology. Recently, MSTO1 mutations have been shown to cause clinical manifestations suggestive of mitochondrial dysfunction, such as muscle weakness, short stature, motor developmental delay, and cerebellar atrophy. Both autosomal dominant and recessive modes of inheritance have been suggested. We performed whole-exome sequencing in two unrelated patients showing cerebellar atrophy, intellectual disability, and pigmentary retinopathy. Three novel mutations were identified: c.836 G > A (p.Arg279His), c.1099-1 G > A (p.Val367Trpfs*2), and c.79 C > T (p.Gln27*). Both patients had compound heterozygous mutations with a combination of protein-truncation mutation and missense mutation, the latter shared by them both. This survey of two patients with recessive and novel MSTO1 mutations provides additional clinical and genetic information on the pathogenicity of MSTO1 in humans.

Early-onset epileptic encephalopathies, including West syndrome (WS), are a group of neurological disorders characterized by developmental impairments and intractable seizures from early infancy. We have now identified biallelic CNPY3 variants in three individuals with WS; these include compound-heterozygous missense and frameshift variants in a family with two affected siblings (individuals 1 and 2) and a homozygous splicing variant in a consanguineous family (individual 3). All three individuals showed hippocampal malrotation. In individuals 1 and 2, electroencephalography (EEG) revealed characteristic fast waves and diffuse sharp- and slow-wave complexes. The fast waves were clinically associated with seizures. CNPY3 encodes a co-chaperone in the endoplasmic reticulum and regulates the subcellular distribution and responses of multiple Toll-like receptors. The amount of CNPY3 in lymphoblastoid cells derived from individuals 1 and 2 was severely lower than that in control cells. Cnpy3-knockout mice exhibited spastic or dystonic features under resting conditions and hyperactivity and anxiolytic behavior during the open field test. Also, their resting EEG showed enhanced activity in the fast beta frequency band (20-35 Hz), which could mimic the fast waves in individuals 1 and 2. These data suggest that CNPY3 and Cnpy3 perform essential roles in brain function in addition to known Toll-like receptor-dependent immune responses., (Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Purpose: Little is known regarding neuroimaging-genotype correlations in Joubert syndrome (JBTS). To elucidate one of these correlations, we investigated the neuroimaging findings of JBTS patients with C5orf42 mutations., Materials and Methods: Neuroimaging findings in five JBTS patients with C5orf42 mutations were retrospectively assessed with regard to the infratentorial and supratentorial structures on T1-magnetization prepared rapid gradient echo (MPRAGE), T2-weighted images, and color-coded fractional anisotropy (FA) maps; the findings were compared to those in four JBTS patients with mutations in other genes (including three with AHI1 and one with TMEM67 mutations)., Results: In C5orf42-mutant patients, the infratentorial magnetic resonance (MR) images showed normal or minimally thickened and minimally elongated superior cerebellar peduncles (SCP), normal or minimally deepened interpeduncular fossa (IF), and mild vermian hypoplasia (VH). However, in other patients, all had severe abnormalities in the SCP and IF, and moderate to marked VH. Supratentorial abnormalities were found in one individual in other JBTS. In JBTS with all mutations, color-coded FA maps showed the absence of decussation of the SCP (DSCP)., Conclusion: The morphological neuroimaging findings in C5orf42-mutant JBTS were distinctly mild and made diagnosis difficult. However, the absence of DSCP on color-coded FA maps may facilitate the diagnosis of JBTS., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

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.

Cerebellar atrophy is recognized in various types of childhood neurological disorders with clinical and genetic heterogeneity. Genetic analyses such as whole exome sequencing are useful for elucidating the genetic basis of these conditions. Pathological recessive mutations in Sep (O-phosphoserine) tRNA:Sec (selenocysteine) tRNA synthase (SEPSECS) have been reported in a total of 11 patients with pontocerebellar hypoplasia type 2, progressive cerebellocerebral atrophy or progressive encephalopathy, yet detailed clinical features are limited to only four patients. We identified two new families with progressive cerebellar atrophy, and by whole exome sequencing detected biallelic SEPSECS mutations: c.356A>G (p.Asn119Ser) and c.77delG (p.Arg26Profs*42) in family 1, and c.356A>G (p.Asn119Ser) and c.467G>A (p.Arg156Gln) in family 2. Their development was slightly delayed regardless of normal brain magnetic resonance imaging (MRI) in infancy. The progression of clinical symptoms in these families is evidently slower than in previously reported cases, and the cerebellar atrophy milder by brain MRI, indicating that SEPSECS mutations are also involved in milder late-onset cerebellar atrophy.

The voltage-gated Kv2.1 potassium channel encoded by KCNB1 produces the major delayed rectifier potassium current in pyramidal neurons. Recently, de novo heterozygous missense KCNB1 mutations have been identified in three patients with epileptic encephalopathy and a patient with neurodevelopmental disorder. However, the frequency of KCNB1 mutations in infantile epileptic patients and their effects on neuronal activity are yet unknown. We searched whole exome sequencing data of a total of 437 patients with infantile epilepsy, and found novel de novo heterozygous missense KCNB1 mutations in two patients showing psychomotor developmental delay and severe infantile generalized seizures with high-amplitude spike-and-wave electroencephalogram discharges. The mutation located in the channel voltage sensor (p.R306C) disrupted sensitivity and cooperativity of the sensor, while the mutation in the channel pore domain (p.G401R) selectively abolished endogenous Kv2 currents in transfected pyramidal neurons, indicating a dominant-negative effect. Both mutants inhibited repetitive neuronal firing through preventing production of deep interspike voltages. Thus KCNB1 mutations can be a rare genetic cause of infantile epilepsy, and insufficient firing of pyramidal neurons would disturb both development and stability of neuronal circuits, leading to the disease phenotypes.

KCNT1 mutations have been found in epilepsy of infancy with migrating focal seizures (EIMFS; also known as migrating partial seizures in infancy), autosomal dominant nocturnal frontal lobe epilepsy, and other types of early onset epileptic encephalopathies (EOEEs). We performed KCNT1-targeted next-generation sequencing (207 samples) and/or whole-exome sequencing (229 samples) in a total of 362 patients with Ohtahara syndrome, West syndrome, EIMFS, or unclassified EOEEs. We identified nine heterozygous KCNT1 mutations in 11 patients: nine of 18 EIMFS cases (50%) in whom migrating foci were observed, one of 180 West syndrome cases (0.56%), and one of 66 unclassified EOEE cases (1.52%). KCNT1 mutations occurred de novo in 10 patients, and one was transmitted from the patient's mother who carried a somatic mosaic mutation. The mutations accumulated in transmembrane segment 5 (2/9, 22.2%) and regulators of K(+) conductance domains (7/9, 77.8%). Five of nine mutations were recurrent. Onset ages ranged from the neonatal period (<1 month) in five patients (5/11, 45.5%) to 1-4 months in six patients (6/11, 54.5%). A generalized attenuation of background activity on electroencephalography was seen in six patients (6/11, 54.5%). Our study demonstrates that the phenotypic spectrum of de novo KCNT1 mutations is largely restricted to EIMFS., (Wiley Periodicals, Inc. © 2015 International League Against Epilepsy.)

Objective: Focal cortical dysplasia (FCD) type IIb is a cortical malformation characterized by cortical architectural abnormalities, dysmorphic neurons, and balloon cells. It has been suggested that FCDs are caused by somatic mutations in cells in the developing brain. Here, we explore the possible involvement of somatic mutations in FCD type IIb., Methods: We collected a total of 24 blood-brain paired samples with FCD, including 13 individuals with FCD type IIb, 5 with type IIa, and 6 with type I. We performed whole-exome sequencing using paired samples from 9 of the FCD type IIb subjects. Somatic MTOR mutations were identified and further investigated using all 24 paired samples by deep sequencing of the entire gene's coding region. Somatic MTOR mutations were confirmed by droplet digital polymerase chain reaction. The effect of MTOR mutations on mammalian target of rapamycin (mTOR) kinase signaling was evaluated by immunohistochemistry and Western blotting analyses of brain samples and by in vitro transfection experiments., Results: We identified four lesion-specific somatic MTOR mutations in 6 of 13 (46%) individuals with FCD type IIb showing mutant allele rates of 1.11% to 9.31%. Functional analyses showed that phosphorylation of ribosomal protein S6 in FCD type IIb brain tissues with MTOR mutations was clearly elevated, compared to control samples. Transfection of any of the four MTOR mutants into HEK293T cells led to elevated phosphorylation of 4EBP, the direct target of mTOR kinase., Interpretation: We found low-prevalence somatic mutations in MTOR in FCD type IIb, indicating that activating somatic mutations in MTOR cause FCD type IIb., (© 2015 American Neurological Association.)

Objective: To investigate the clinical spectrum caused by mutations in PIGA at Xp22.2, which is involved in the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor, among patients with early-onset epileptic encephalopathies (EOEEs)., Methods: Whole-exome sequencing was performed as a comprehensive genetic analysis for a cohort of 172 patients with EOEEs including early myoclonic encephalopathy, Ohtahara syndrome, and West syndrome, and PIGA mutations were carefully investigated., Results: We identified 4 PIGA mutations in probands showing early myoclonic encephalopathy, West syndrome, or unclassified EOEE. Flow cytometry of blood granulocytes from patients demonstrated reduced expression of GPI-anchored proteins. Expression of GPI-anchored proteins in PIGA-deficient JY5 cells was only partially or hardly restored by transient expression of PIGA mutants with a weak TATA box promoter, indicating a variable loss of PIGA activity. The phenotypic consequences of PIGA mutations can be classified into 2 types, severe and less severe, which correlate with the degree of PIGA activity reduction caused by the mutations. Severe forms involved myoclonus and asymmetrical suppression bursts on EEG, multiple anomalies with a dysmorphic face, and delayed myelination with restricted diffusion patterns in specific areas. The less severe form presented with intellectual disability and treatable seizures without facial dysmorphism., Conclusions: Our study confirmed that PIGA mutations are one genetic cause of EOEE, suggesting that GPI-anchor deficiencies may be an underlying cause of EOEE.

Defects of the human glycosylphosphatidylinositol (GPI) anchor biosynthetic pathway show a broad range of clinical phenotypes. A homozygous mutation in PIGN, a member of genes involved in the GPI anchor-synthesis pathway, was previously reported to cause dysmorphic features, multiple congenital anomalies, severe neurological impairment, and seizure in a consanguineous family. Here, we report two affected siblings with compound heterozygous PIGN mutations [c.808T >C (p.Ser270Pro) and c.963G >A] showing congenital anomalies, developmental delay, hypotonia, epilepsy, and progressive cerebellar atrophy. The c.808C >T mutation altered an evolutionarily conserved amino acid residue (Ser270), while reverse transcription-PCR and sequencing demonstrated that c.963G >A led to aberrant splicing, in which two mutant transcripts with premature stop codons (p.Ala322Valfs*24 and p.Glu308Glyfs*2) were generated. Expression of GPI-anchored proteins such as CD16 and CD24 on granulocytes from affected siblings was significantly decreased, and expression of the GPI-anchored protein CD59 in PIGN-knockout human embryonic kidney 293 cells was partially or hardly restored by transient expression of p.Ser270Pro and p.Glu308Glyfs*2 mutants, respectively, suggesting severe and complete loss of PIGN activity. Our findings confirm that developmental delay, hypotonia, and epilepsy combined with congenital anomalies are common phenotypes of PIGN mutations and add progressive cerebellar atrophy to this clinical spectrum.

Rett syndrome (RTT) is a neurodevelopmental disorder mostly caused by MECP2 mutations. We identified a de novo WDR45 mutation, which caused a subtype of neurodegeneration with brain iron accumulation, in a patient showing clinically typical RTT. The mutation (c.830+1G>A) led to aberrant splicing in lymphoblastoid cells. Sequential brain magnetic resonance imaging demonstrated that iron deposition in the globus pallidus and the substantia nigra was observed as early as at 11 years of age. Because the patient showed four of the main RTT diagnostic criteria, WDR45 should be investigated in patients with RTT without MECP2 mutations.

Cerebellar and/or vermis atrophy is recognized in various types of childhood disorders with clinical and genetic heterogeneity. Although careful evaluation of clinical features and neuroimaging can lead to correct diagnosis of disorders, their diagnosis is sometimes difficult because clinical features can overlap with each other. In this study, we performed family-based whole exome sequencing of 23 families including 25 patients with cerebellar and/or vermis atrophy in childhood, who were unable to be diagnosed solely by clinical examination. Pathological mutations of seven genes were found in ten patients from nine families (9/23, 39.1 %): compound heterozygous mutations in FOLR1, C5orf42, POLG, TPP1, PEX16, and de novo mutations in CACNA1A, and ITPR1. Patient 1A with FOLR1 mutations showed extremely low concentration of 5-methyltetrahydrofolate in the cerebrospinal fluid and serum, and Patient 6 with TPP1 mutations demonstrated markedly lowered tripeptidyl peptidase 1 activity in leukocytes. Furthermore, Patient 8 with PEX16 mutations presented a mild increase of very long chain fatty acids in the serum as supportive data for genetic diagnosis. The main clinical features of these ten patients were nonspecific and mixed, and included developmental delay, intellectual disability, ataxia, hypotonia, and epilepsy. Brain MRI revealed both cerebellar and vermis atrophy in eight patients (8/10, 80 %), vermis atrophy/hypoplasia in two patients (2/10, 20 %), and brainstem atrophy in one patient (1/10, 10 %). Our data clearly demonstrate the utility of whole exome sequencing for genetic diagnosis of childhood cerebellar and/or vermis atrophy.

Autosomal recessive cerebellar ataxias and autosomal recessive hereditary spastic paraplegias are clinically and genetically heterogeneous disorders with diverse neurological and non-neurological features. We herein describe a Japanese patient with a slowly progressive form of ataxia and spastic paraplegia. Using whole exome sequencing, we identified a novel homozygous frameshift mutation in SPG7, encoding paraplegin, in this patient. This is the first report of an SPG7 mutation in the Japanese population. For disorders previously undetected in a particular population, or unrecognized/atypical phenotypes, exome sequencing may facilitate molecular diagnosis.