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Degeneration of the nigrostriatal system occurs in multiple system atrophy (MSA) and Parkinson's disease (PD) via distinct pathological mechanisms. Here, we investigated nigrostriatal degeneration in MSA and PD by combining a newly developed method for evaluating the regional accumulation of dopamine transporter single-photon emission computed tomography (DAT SPECT) and individual voxel-based morphometry adjusting covariates (iVAC). We recruited 17 MSA patients and 13 PD patients, and compared their clinical and imaging indices. All patients underwent DAT SPECT and head three-dimensional T1-weighted magnetic resonance imaging. We calculated the specific binding ratio (SBR) of the putamen and caudate separately using a region-based method, and evaluated the association between the SBR and iVAC Z-score. SBR values of the putamen and caudate were lower in the PD group than in the MSA group (p < 0.001 for both). The MSA and PD groups had lower SBR values in the putamen than in the caudate (p < 0.05 and p < 0.001, respectively). We found a negative correlation between the putamen SBR and iVAC Z-score in MSA (p < 0.001, r = -0.775), but such a correlation was not detected in PD. For the caudate, there was no correlation between the SBR and iVAC Z-score in MSA and PD. Our results indicate different mechanisms of reduced uptake of DATs in MSA and PD. The association between the striatal SBR and iVAC Z-score suggests parallel degeneration in the substantia nigra and striatum in MSA., (Copyright © 2024 Elsevier B.V. All rights reserved.)

KIF1A-related disorders (KRDs) encompass recessive and dominant variants with wide clinical variability. Recent genetic investigations have expanded the clinical phenotypes of heterozygous KIF1A variants. However, there have been a few long-term observational studies of patients with heterozygous KIF1A variants. A retrospective chart review of consecutive patients diagnosed with spastic paraplegia at Miyagi Children's Hospital from 2016 to 2020 identified six patients with heterozygous KIF1A variants. To understand the long-term changes in clinical symptoms, we examined these patients in terms of their characteristics, clinical symptoms, results of electrophysiological and neuroimaging studies, and genetic testing. The median follow-up period was 30 years (4-44 years). This long-term observational study showed that early developmental delay and equinus gait, or unsteady gait, are the first signs of disease onset, appearing with the commencement of independent walking. In addition, later age-related progression was observed in spastic paraplegia, and the appearance of axonal neuropathy and reduced visual acuity were characteristic features of the late disease phenotype. Brain imaging showed age-related progression of cerebellar atrophy and the appearance of hyperintensity of optic radiation on T2WI and FLAIR imaging. Long-term follow-up revealed a pattern of steady progression and a variety of clinical symptoms, including spastic paraplegia, peripheral neuropathy, reduced visual acuity, and some degree of cerebellar ataxia. Clinical variability between patients was observed to some extent, and therefore, further studies are required to determine the phenotype-genotype correlation., (© 2024 Wiley Periodicals LLC.)

Introduction: Parkinson's disease (PD) presents with decreased heart rate variability (HRV) from its early stages. However, most of its evidence originates from HRV measurements in parasympathetic dominant states. In this study, we aimed to examine whether HRV in sympathetic dominant states during the head-up tilt table test (HUT) serves as a marker of autonomic dysfunction in PD and isolated REM sleep behavior disorder (iRBD)., Methods: We retrospectively assessed 102 patients with PD, 10 patients with iRBD, and 43 healthy controls. We then measured the coefficient of variation of RR intervals as an HRV parameter in sympathetic dominant states (CVRR-S) and parasympathetic dominant states (CVRR-P). Furthermore, we evaluated parameters of cardiac autonomic function, including HUT and the heart-to-mediastinum (H/M) ratio of cardiac metaiodobenzylguanidine scintigraphy., Results: Patients with iRBD and PD at Hoehn and Yahr stage I exhibited a significantly decreased CVRR-S compared to healthy controls (controls vs. iRBD vs. PD; 1.82 ± 0.64 % vs. 1.13 ± 0.41 % vs. 1.15 ± 0.51 %, p < 0.001), although no further deterioration was observed in PD at more severe Hoehn and Yahr stages. CVRR-S showed a significant correlation with the H/M ratio in PD (r = 0.51, p < 0.001). Additionally, receiver operating characteristic (ROC) analysis revealed a larger area under the ROC curve in CVRR-S compared to that in CVRR-P for discriminating PD or iRBD from healthy controls., Conclusion: HRV in sympathetic dominant states shows the potential to be a marker of autonomic dysfunction in iRBD and early-stage PD, aiding in early diagnosis and patient stratification., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purpose: Cerebellar hypoplasia and atrophy (CBHA) in children is an extremely heterogeneous group of disorders, but few comprehensive genetic studies have been reported. Comprehensive genetic analysis of CBHA patients may help differentiating atrophy and hypoplasia and potentially improve their prognostic aspects., Methods: Patients with CBHA in 176 families were genetically examined using exome sequencing. Patients with disease-causing variants were clinically evaluated., Results: Disease-causing variants were identified in 96 of the 176 families (54.5%). After excluding 6 families, 48 patients from 42 families were categorized as having syndromic associations with CBHA, whereas the remaining 51 patients from 48 families had isolated CBHA. In 51 patients, 26 aberrant genes were identified, of which, 20 (76.9%) caused disease in 1 family each. The most prevalent genes were CACNA1A, ITPR1, and KIF1A. Of the 26 aberrant genes, 21 and 1 were functionally annotated to atrophy and hypoplasia, respectively. CBHA+S was more clinically severe than CBHA-S. Notably, ARG1 and FOLR1 variants were identified in 2 families, leading to medical treatments., Conclusion: A wide genetic and clinical diversity of CBHA was revealed through exome sequencing in this cohort, which highlights the importance of comprehensive genetic analyses. Furthermore, molecular-based treatment was available for 2 families., Competing Interests: Conflict of Interest The authors declare no conflict of interest., (Copyright © 2022 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Vacuolar H + -ATPases (V-ATPases) transport protons across cellular membranes to acidify various organelles. ATP6V0A1 encodes the a1-subunit of the V0 domain of V-ATPases, which is strongly expressed in neurons. However, its role in brain development is unknown. Here we report four individuals with developmental and epileptic encephalopathy with ATP6V0A1 variants: two individuals with a de novo missense variant (R741Q) and the other two individuals with biallelic variants comprising one almost complete loss-of-function variant and one missense variant (A512P and N534D). Lysosomal acidification is significantly impaired in cell lines expressing three missense ATP6V0A1 mutants. Homozygous mutant mice harboring human R741Q (Atp6v0a1 R741Q ) and A512P (Atp6v0a1 A512P ) variants show embryonic lethality and early postnatal mortality, respectively, suggesting that R741Q affects V-ATPase function more severely. Lysosomal dysfunction resulting in cell death, accumulated autophagosomes and lysosomes, reduced mTORC1 signaling and synaptic connectivity, and lowered neurotransmitter contents of synaptic vesicles are observed in the brains of Atp6v0a1 A512P/A512P mice. These findings demonstrate the essential roles of ATP6V0A1/Atp6v0a1 in neuronal development in terms of integrity and connectivity of neurons in both humans and mice.

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.)

Although there are many known Mendelian genes linked to epileptic or developmental and epileptic encephalopathy (EE/DEE), its genetic architecture is not fully explained. Here, we address this incompleteness by analyzing exomes of 743 EE/DEE cases and 2366 controls. We observe that damaging ultra-rare variants (dURVs) unique to an individual are significantly overrepresented in EE/DEE, both in known EE/DEE genes and the other non-EE/DEE genes. Importantly, enrichment of dURVs in non-EE/DEE genes is significant, even in the subset of cases with diagnostic dURVs (P = 0.000215), suggesting oligogenic contribution of non-EE/DEE gene dURVs. Gene-based analysis identifies exome-wide significant (P = 2.04 × 10 -6 ) enrichment of damaging de novo mutations in NF1, a gene primarily linked to neurofibromatosis, in infantile spasm. Together with accumulating evidence for roles of oligogenic or modifier variants in severe neurodevelopmental disorders, our results highlight genetic complexity in EE/DEE, and indicate that EE/DEE is not an aggregate of simple Mendelian disorders.

Background: Rett syndrome (RTT) is a characteristic neurological disease presenting with regressive loss of neurodevelopmental milestones. Typical RTT is generally caused by abnormality of methyl-CpG binding protein 2 ( MECP2 ). Our objective to investigate the genetic landscape of MECP2 -negative typical/atypical RTT and RTT-like phenotypes using whole exome sequencing (WES)., Methods: We performed WES on 77 MECP2 -negative patients either with typical RTT (n=11), atypical RTT (n=22) or RTT-like phenotypes (n=44) incompatible with the RTT criteria., Results: Pathogenic or likely pathogenic single-nucleotide variants in 28 known genes were found in 39 of 77 (50.6%) patients. WES-based CNV analysis revealed pathogenic deletions involving six known genes (including MECP2 ) in 8 of 77 (10.4%) patients. Overall, diagnostic yield was 47 of 77 (61.0 %). Furthermore, strong candidate variants were found in four novel genes: a de novo variant in each of ATPase H + transporting V0 subunit A1 ( ATP6V0A1 ), ubiquitin-specific peptidase 8 ( USP8 ) and microtubule-associated serine/threonine kinase 3 ( MAST3 ), as well as biallelic variants in nuclear receptor corepressor 2 ( NCOR2 )., Conclusions: Our study provides a new landscape including additional genetic variants contributing to RTT-like phenotypes, highlighting the importance of comprehensive genetic analysis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

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.

We present a unique 11-year-old girl showing clinical features of Rett-related disorder with distinctive facial features and multiple congenital anomalies including ocular hypertelorism, arched eyebrows, a broad nose, dental anomalies, congenital heart disease, truncal obesity, and epilepsy. A novel de novo mutation in histone deacetylase 8 (HDAC8) (c.652G > T, p.Gly218Cys) was confirmed by whole exome sequencing and Sanger sequencing. X-chromosome inactivation analysis on DNA isolated from peripheral blood lymphocytes revealed a completely skewed pattern associated with an inactive maternal allele. Late clinical loss of acquired purposeful hand movements and psychomotor deterioration may be a feature of Rett-related disorder, while distinctive facial features and multiple congenital anomalies are reminiscent of Cornelia de Lange syndrome., (Copyright © 2018 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

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.)

Objective: α ( CAMK2A ) and β ( CAMK2B ) isoforms of Calcium/calmodulin-dependent protein kinase II (CaMKII) play a pivotal role in neuronal plasticity and in learning and memory processes in the brain. Here, we explore the possible involvement of α - and β -CaMKII variants in neurodevelopmental disorders., Methods: Whole-exome sequencing was performed for 976 individuals with intellectual disability, developmental delay, and epilepsy. The effect of CAMK2A and CAMK2B variants on CaMKII structure and firing of neurons was evaluated by computational structural analysis, immunoblotting, and electrophysiological analysis., Results: We identified a total of five de novo CAMK2A and CAMK2B variants in three and two individuals, respectively. Seizures were common to three individuals with CAMK2A variants. Using a minigene splicing assay, we demonstrated that a splice site variant caused skipping of exon 11 leading to an in-frame deletion of the regulatory segment of CaMKII α . By structural analysis, four missense variants are predicted to impair the interaction between the kinase domain and the regulatory segment responsible for the autoinhibition of its kinase activity. The Thr286/Thr287 phosphorylation as a result of release from autoinhibition was increased in three mutants when the mutants were stably expressed in Neuro-2a neuroblastoma cells. Expression of a CaMKII α mutant in primary hippocampal neurons significantly increased A-type K + currents, which facilitated spike repolarization of single action potentials., Interpretation: Our data highlight the importance of CaMKII α and CaMKII β and their autoinhibitory regulation in human brain function, and suggest the enhancement of A-type K + currents as a possible pathophysiological basis.

We report an 11-month-old boy with acetazolamide-responsive epileptic apnea and inherited glycosylphosphatidylinositol (GPI)-anchor deficiency who presented with decreased serum alkaline phosphatase associated with compound PIGT mutations. The patient exhibited congenital anomalies, severe intellectual disability, and seizures, including epileptic apnea with epileptiform discharges from bilateral temporal areas. Brain magnetic resonance imaging revealed delayed myelination and progressive atrophy of the brainstem, cerebellum, and cerebrum. Whole-exome sequencing revealed compound heterozygous mutations in PIGT (c.250G>T, p.Glu84X and c.1096G>T, p.Gly366Trp), which encodes a subunit of the GPI transamidase complex. Flow cytometry revealed decreased expression of CD16 (a GPI anchor protein) on granulocytes, supporting the putative pathogenicity of the mutations. Phenobarbital, clonazepam, and potassium bromide decreased the frequency of tonic seizure and acetazolamide decreased epileptic apnea. To our knowledge, this is the first reported case of intractable seizures accompanied by epileptic apnea associated with GPI anchor deficiency and a compound PIGT mutation., (Copyright © 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Cerebral, ocular, dental, auricular, skeletal (CODAS) syndrome is a rare autosomal recessive multisystem disorder caused by mutations in LONP1. It is characterized by intellectual disability, cataracts, delayed tooth eruption, malformed auricles and skeletal abnormalities. We performed whole-exome sequencing on a 12-year-old Japanese male with severe intellectual disability, congenital bilateral cataracts, spasticity, hypotonia with motor regression and progressive cerebellar atrophy with hyperintensity of the cerebellar cortex on T2-weighted images. We detected compound heterozygous mutation in LONP1. One allele contained a paternally inherited frameshift mutation (p.Ser100Glnfs*46). The other allele contained a maternally inherited missense mutation (p.Arg786Trp), which was predicted to be pathogenic by web-based prediction tools. The two mutations were not found in Exome Variant Server or our 575 in-house control exomes. Some features were not consistent with CODAS syndrome but overlapped with Marinesco-Sjögren syndrome, a multisystem disorder caused by a mutation in SIL1. An atypical mutation site may result in atypical presentation of the LONP1 mutation.

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.)

Crystallization of probucol (PBL) from both solution and glassy solid state was investigated. In the crystallization study from solution, six solvents and three methods, i.e., evaporation, addition of a poor solvent, and cooling on ice, were used to obtain various crystal forms. In addition to common two crystal forms (forms I and II), two further forms (forms III and cyclohexane-solvate) were found in this study, and their thermodynamic relationships were determined. Forms I and II are likely to be enantiotropically related with thermodynamic transition temperature below 5°C. Isothermal crystallization studies revealed that PBL glass initially crystallized into form III between 25 and 50°C, and then transformed to form I. The isothermal crystallization appears to be a powerful option to find uncommon crystal forms. The crystallization of PBL was identified to be pressure controlled, thus the physical stability of PBL glass is higher than that of typical compounds., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Aromatic l-amino acid decarboxylase (AADC) deficiency is an autosomal recessive disorder, caused by defects in the DDC gene. AADC catalyzes the synthesis of the neurotransmitters dopamine and serotonin from l-dopa and 5-HT respectively. Most patients are bed ridden for life, with little response to treatment. We now report one female patient who improved her motor and cognitive function after being prescribed a MAO-B inhibitor., Case: A five years old female presented with the typical clinical features of AADC deficiency. She was floppy, with no head control, had intermittent limb dystonia, and an upward deviation of the eyes (oculogyric crisis). This patient possessed compound heterozygous mutations in DDC (p.Trp105Cys, p.Pro129Ser), with a CSF draw indicating abnormal patterns of biogenic amine metabolites, compatible with AADC deficiency., Results: After her diagnosis at 3years of age, medication with levodopa and vitamin B6 failed to show any efficacy. Subsequent administration with a MAO-B inhibitor improved her psychomotor functions to the extent that at 5years of age she could walk several meters with support., Conclusion: Our analyses of chemical findings, together with in silico structure predictions, lead us to hypothesize that this patient retained some AADC activity. In these cases, accurate diagnosis and early treatment should improve patient outcome., (Copyright © 2016 The Japanese Society of Child Neurology. Published by 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.)

Three new sesquiterpenoids-vetiverianines A (1), B (2), and C (3)-and a known eudesmane sesquiterpenoid (4) were isolated from the roots of Vetiveria zizanioides. Vetiverianine A (1) has a unique carbon framework comprising a rigid tricyclic ring system. Vetiverianines B (2) and C (3) are new eremophilane sesquiterpenoids. The structures of sesquiterpenoids 1-3, including the absolute configurations, were determined by NMR spectroscopic, X-ray crystallography, and vibrational circular dichroism data analysis. Vetiverianine C (3) exhibited weak cytotoxic activity against HL-60 cells.

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&#95;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&#95;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&#95;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.

A new neolignan glycoside (1) and four known aromatic compounds (2-5) were isolated. from the roots of Vetiveria zizanioides. The structure of compound 1 was determined based on spectroscopic analysis and hydrolysis. The structure of known flavonoid glycoside 3 was confirmed by X-ray crystallography. Compound 5 showed weak cytotoxic activity against HL-60 cells with an IC₅₀ value of 13.1 ± 0.04 μM.

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.

Objective: GABRA1 mutations have been identified in patients with familial juvenile myoclonic epilepsy, sporadic childhood absence epilepsy, and idiopathic familial generalized epilepsy. In addition, de novo GABRA1 mutations were recently reported in a patient with infantile spasms and four patients with Dravet syndrome. Those reports suggest that GABRA1 mutations are associated with infantile epilepsy including early onset epileptic encephalopathies. In this study, we searched for GABRA1 mutations in patients with infantile epilepsy to investigate the phenotypic spectrum of GABRA1 mutations., Methods: In total, 526 and 145 patients with infantile epilepsy were analyzed by whole-exome sequencing and GABRA1-targeted resequencing, respectively., Results: We identified five de novo missense GABRA1 mutations in six unrelated patients. A p.R112Q mutation in the long extracellular N-terminus was identified in a patient with infantile epilepsy; p.P260L, p.M263T, and p.M263I in transmembrane spanning domain 1 (TM1) were identified in three unrelated patients with West syndrome and a patient with Ohtahara syndrome, respectively; and p.V287L in TM2 was identified in a patient with unclassified early onset epileptic encephalopathy. Four of these mutations have not been observed previously., Significance: Our study suggests that de novo GABRA1 mutations can cause early onset epileptic encephalopathies, including Ohtahara syndrome and West syndrome., (Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.)

Purpose: The purpose of this study was to report transumbilical arterial embolization of a large dural arteriovenous fistula (AVF) in a low-birth-weight neonate with congestive heart failure (CHF)., Case Presentation: A female neonate was delivered by cesarean section at 31 weeks of gestation. Her birth weight was 1538 g and Apgar scores were 6 at both 1 and 5 min. Because of dyspnea and retracted respiration immediately after birth, she required mechanical ventilation. Ultrasound revealed right cardiac overload and a large cystic mass at the posterior brain. Magnetic resonance imaging on day 1 showed a large dural AVF (dural sinus malformation with arteriovenous (AV) shunts) at the torcular herophili. Umbilical artery and vein catheterization were performed on the same day for neurointervention. CHF prompted emergency embolization on day 8. The transfemoral arterial route could not be used because of its small size and compromised femoral artery blood flow. Transumbilical arterial embolization shrank the AV shunts markedly, resulting in clinical improvement, thus requiring no further intervention. Follow-up angiography at 4 months confirmed no residual AVF. Her growth and development were normal at the last follow-up at age 4 years., Conclusion: This patient apparently was the lowest birth weight neonate with a large AVF successfully treated by embolization, which is usually performed through the transfemoral arterial route. The transumbilical arterial route is an alternative for neonates with birth weight <2000 g and very small femoral arteries.

Recently, de novo KIF1A mutations were identified in patients with intellectual disability, spasticity and cerebellar atrophy and/or optic nerve atrophy. In this study, we analyzed a total of 62 families, including 68 patients with genetically unsolved childhood cerebellar atrophy, by whole-exome sequencing (WES). We identified five de novo missense KIF1A mutations, including only one previously reported mutation (p.Arg316Trp). All the mutations are located in the motor domain of KIF1A. In all patients, initial symptom onset was during the infantile period, and included developmental delay in three patients and gait disturbance in two. Thereafter, they showed gait disturbances, exaggerated deep tendon reflexes, cerebellar symptoms and cerebellar atrophy on brain magnetic resonance imaging. Four patients showed lower limb spasticity, upper limb clumsiness and visual disturbances. Nerve conduction study revealed peripheral neuropathy in three patients. This study further delineates clinical features of de novo KIF1A mutations. Genetic testing of KIF1A should be considered in children with developmental delay, cerebellar atrophy and pyramidal features.

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.)

In vivo activities of absorption enhancers coencapsulated with poorly absorptive drugs in the same enteric-coated particles were evaluated. Lopinavir [a substrate of cytochrome P450 3A (CYP3A)] and ritonavir (an inhibitor of CYP3A-mediatd metabolism) were used as a model drug and a model absorption enhancer, respectively. Lopinavir and ritonavir were encapsulated into enteric-coated particles as amorphous forms using coaxial electrospray deposition. The electrospray treatment resulted in dramatic improvement of dissolution profiles of both compounds, probably because of complete amorphization and superior dispersion efficiency of the particles. Poor absorption of lopinavir in rats was observed after oral administration of enteric-coated particles containing lopinavir alone. When the particles were coadministered with enteric-coated particles containing ritonavir alone, lopinavir absorption was boosted. The boosting effect was further enhanced when ritonavir was coencapsulated with lopinavir into the same enteric-coated particles. A significant increase in area under the plasma concentration-time curve reflected an extension of mean residence time rather than an elevation of Cmax . Lopinavir absorption was improved presumably because lopinavir was always accompanied by a practical amount of ritonavir required for the boosting during the gastrointestinal transit of the particles. Not only did the electrospray coencapsulation technique improve drug absorption, but also increased trough concentration that might result in the reduction of the number of doses., (© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Defects in the mitochondrial translation apparatus can impair energy production in affected tissues and organs. Most components of this apparatus are encoded by nuclear genes, including GFM2, which encodes a mitochondrial ribosome recycling factor. A few patients with mutations in some of these genes have been reported to date. Here, we present two female siblings with arthrogryposis multiplex congenita, optic atrophy and severe mental retardation. The younger sister had a progressive cerebellar atrophy and bilateral neuropathological findings in the brainstem. Although her cerebrospinal fluid (CSF) levels of lactate and pyruvate were not increased, brain magnetic resonance spectroscopy showed a lactate peak. Additionally, her CSF lactate/pyruvate and serum beta-hydroxybutyrate/acetoacetate ratios were high, and levels of oxidative phosphorylation in skin fibroblasts were reduced. We therefore diagnosed Leigh syndrome. Genomic investigation confirmed the presence of compound heterozygous GFM2 mutations (c.206+4A>G and c.2029-1G>A) in both siblings, causing aberrant splicing with premature stop codons (p.Gly50Glufs*4 and p.Ala677Leufs*2, respectively). These findings suggest that GFM2 mutations could be causative of a phenotype of Leigh syndrome with arthrogryposis multiplex congenita.

Rett syndrome (RTT) is a neurodevelopmental disorder predominantly affecting females. Females with the MECP2 mutations exhibit a broad spectrum of clinical manifestations ranging from classical Rett syndrome to asymptomatic carriers. Mutations of genes encoding cyclin-dependent kinase-like 5 (CDKL5) and forkhead box G1 (FOXG1) are also found in early onset RTT variants. Here, we present the first report of a female patient with RTT-like phenotype caused by SHANK3 (SH3 and multiple ankylin repeat domain 3) mutation, indicating that the clinical spectrum of SHANK3 mutations may extend to RTT-like phenotype in addition to (severe) developmental delay, absence of expressive speech, autistic behaviors and intellectual disability., (© 2015 Wiley Periodicals, Inc.)

Acute virus-associated encephalopathy induces seizures. Serum N-terminal pro-B-type natriuretic peptide (NTproBNP) levels are elevated following febrile and afebrile seizures. However, the role of NTproBNP in acute virus-associated encephalopathy pathology is unknown. We enrolled 10 patients with acute virus-associated encephalopathy and convulsions (E group: 7 boys, 3 girls; median age, 3.10 ± 1.92 years) and 130 patients with febrile seizure (FS group: 80 boys, 50 girls; median age, 3.23 ± 2.44 years). The E group had significantly higher NTproBNP levels (345 ± 141 pg/mL) compared with the FS group (166 ± 228 pg/mL) (P < .0005). Furthermore, subjects with prolonged seizure within the E group had significantly higher NTproBNP levels (303 ± 107 pg/mL) compared with subjects with prolonged seizure within the FS group (134 ± 100 pg/mL) (P < .005). Our findings suggest that serum NTproBNP levels are increased during the acute phase of acute virus-associated encephalopathy associated with convulsion., (© The Author(s) 2014.)

Objective: Recently, de novo mutations in GRIN1 have been identified in patients with nonsyndromic intellectual disability and epileptic encephalopathy. Whole exome sequencing (WES) analysis of patients with genetically unsolved epileptic encephalopathies identified four patients with GRIN1 mutations, allowing us to investigate the phenotypic spectrum of GRIN1 mutations., Methods: Eighty-eight patients with unclassified early onset epileptic encephalopathies (EOEEs) with an age of onset <1 year were analyzed by WES. The effect of mutations on N-methyl-D-aspartate (NMDA) receptors was examined by mapping altered amino acids onto three-dimensional models., Results: We identified four de novo missense GRIN1 mutations in 4 of 88 patients with unclassified EOEEs. In these four patients, initial symptoms appeared within 3 months of birth, including hyperkinetic movements in two patients (2/4, 50%), and seizures in two patients (2/4, 50%). Involuntary movements, severe developmental delay, and intellectual disability were recognized in all four patients. In addition, abnormal eye movements resembling oculogyric crises and stereotypic hand movements were observed in two and three patients, respectively. All the four patients exhibited only nonspecific focal and diffuse epileptiform abnormality, and never showed suppression-burst or hypsarrhythmia during infancy. A de novo mosaic mutation (c.1923G>A) with a mutant allele frequency of 16% (in DNA of blood leukocytes) was detected in one patient. Three mutations were located in the transmembrane domain (3/4, 75%), and one in the extracellular loop near transmembrane helix 1. All the mutations were predicted to impair the function of the NMDA receptor., Significance: Clinical features of de novo GRIN1 mutations include infantile involuntary movements, seizures, and hand stereotypies, suggesting that GRIN1 mutations cause encephalopathy resulting in seizures and movement disorders., (Wiley Periodicals, Inc. © 2015 International League Against Epilepsy.)

Mutations in the inositol 1,4,5-triphosphate receptor type 1 gene (ITPR1) have been identified in families with early-onset spinocerebellar ataxia type 29 (SCA29) and late-onset SCA15, but have not been found in sporadic infantile-onset cerebellar ataxia. We examined if mutations of ITPR1 are also involved in sporadic infantile-onset SCA. Sixty patients with childhood-onset cerebellar atrophy of unknown etiology and their families were examined by whole-exome sequencing. We found de novo heterozygous ITPR1 missense mutations in four unrelated patients with sporadic infantile-onset, nonprogressive cerebellar ataxia. Patients displayed nystagmus, tremor, and hypotonia from very early infancy. Nonprogressive ataxia, motor delay, and mild cognitive deficits were common clinical findings. Brain magnetic resonance imaging revealed slowly progressive cerebellar atrophy. ITPR1 missense mutations cause infantile-onset cerebellar ataxia. ITPR1-related SCA includes sporadic infantile-onset cerebellar ataxia as well as SCA15 and SCA29.

Whole-exome sequencing (WES) is becoming a standard tool for detecting nucleotide changes, and determining whether WES data can be used for the detection of copy-number variations (CNVs) is of interest. To date, several algorithms have been developed for such analyses, although verification is needed to establish if they fit well for the appropriate purpose, depending on the characteristics of each algorithm. Here, we performed WES CNV analysis using the eXome Hidden Markov Model (XHMM). We validated its performance using 27 rare CNVs previously identified by microarray as positive controls, finding that the detection rate was 59%, or higher (89%) with three or more targets. XHMM can be effectively used, especially for the detection of >200 kb CNVs. XHMM may be useful for deletion breakpoint detection. Next, we applied XHMM to genetically unsolved patients, demonstrating successful identification of pathogenic CNVs: 1.5-1.9-Mb deletions involving NSD1 in patients with unknown overgrowth syndrome leading to the diagnosis of Sotos syndrome, and 6.4-Mb duplication involving MECP2 in affected brothers with late-onset spasm and progressive cerebral/cerebellar atrophy confirming the clinical suspect of MECP2 duplication syndrome. The possibility of an 'exome-first' approach for clinical genetic investigation may be considered to save the cost of multiple investigations.

Microdevices designed for practical environmental pollution monitoring need to detect specific pollutants such as dioxins. Bisphenol A (BPA) has been widely used as a monomer for the synthesis of polycarbonate and epoxy resins. However, the recent discovery of its high potential ability to disrupt human endocrine systems has made the development of smart systems and microdevices for its detection and removal necessary. Molecule-responsive microsized hydrogels with β-cycrodextrin (β-CD) as ligands are prepared by photopolymerization using a fluorescence microscope. The molecule-responsive micro-hydrogels show ultra-quick shrinkage in response to target BPA. Furthermore, the flow rate of a microchannel is autonomously regulated by the molecule-responsive shrinking of their hydrogels as smart microvalves., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A boy with spastic paraplegia type 2 (SPG2) due to a novel splice site mutation of PLP1 presented with progressive spasticity of lower limbs, which was first observed during late infancy, when he gained the ability to walk with support. His speech was slow and he had dysarthria. The patient showed mildly delayed intellectual development. Subtotal dysmyelination in the central nervous system was revealed, which was especially prominent in structures known to be myelinated during earlier period, whereas structures that are myelinated later were better myelinated. These findings on the brain magnetic resonance imaging were unusual for subjects with PLP1 mutations. Peaks I and II of the auditory brainstem response (ABR) were normally provoked, but peaks III-V were not clearly demarcated, similarly to the findings in Pelizaeus-Merzbacher disease. These findings of brain MRI and ABR may be characteristic for a subtype of SPG2 patients., (Copyright © 2014 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Objective: De novo SCN8A mutations have been reported in patients with epileptic encephalopathy. Herein we report seven patients with de novo heterozygous SCN8A mutations, which were found in our comprehensive genetic analysis (target capture or whole-exome sequencing) for early onset epileptic encephalopathies (EOEEs)., Methods: A total of 163 patients with EOEEs without mutations in known genes, including 6 with malignant migrating partial seizures in infancy (MMPSI), and 60 with unclassified EOEEs, were analyzed by target capture (28 samples) or whole-exome sequencing (135 samples)., Results: We identified de novo SCN8A mutations in 7 patients: 6 of 60 unclassified EOEEs (10.0%), and one of 6 MMPSI cases (16.7%). The mutations were scattered through the entire gene: four mutations were located in linker regions, two in the fourth transmembrane segments, and one in the C-terminal domain. The type of the initial seizures was variable including generalized tonic-clonic, atypical absence, partial, apneic attack, febrile convulsion, and loss of tone and consciousness. Onset of seizures was during the neonatal period in two patients, and between 3 and 7 months of age in five patients. Brain magnetic resonance imaging (MRI) showed cerebellar and cerebral atrophy in one and six patients, respectively. All patients with SCN8A missense mutations showed initially uncontrollable seizures by any drugs, but eventually one was seizure-free and three were controlled at the last examination. All patients showed developmental delay or regression in infancy, resulting in severe intellectual disability., Significance: Our data reveal that SCN8A mutations can cause variable phenotypes, most of which can be diagnosed as unclassified EOEEs, and rarely as MMPSI. Together with previous reports, our study further indicates that genetic testing of SCN8A should be considered in children with unclassified severe epilepsy., (Wiley Periodicals, Inc. © 2014 International League Against Epilepsy.)

Under light stress, the reaction center-binding protein D1 of PSII is photo-oxidatively damaged and removed from PSII complexes by proteases located in the chloroplast. A protease considered to be responsible for degradation of the damaged D1 protein is the metalloprotease FtsH. We showed previously that the active hexameric FtsH protease is abundant at the grana margin and the grana end membranes, and this homo-complex removes the photodamaged D1 protein in the grana. Here, we showed a change in the distribution of FtsH in spinach thylakoids during excessive illumination by transmission electron microscopy (TEM) and immunogold labeling of FtsH. The change in distribution of the protease was accompanied by structural changes to the thylakoids, which we detected using spinach leaves by TEM after chemical fixation of the samples. Quantitative analyses showed several characteristic changes in the structure of the thylakoids, including shrinkage of the grana, outward bending of the marginal portions of the thylakoids and an increase in the height of the grana stacks under excessive illumination. The increase in the height of the grana stacks may include swelling of the thylakoids and an increase in the partition gaps between the thylakoids. These data strongly suggest that excessive illumination induces partial unstacking of the thylakoids, which enables FtsH to access easily the photodamaged D1 protein. Finally three-dimensional tomography of the grana was recorded to observe the effect of light stress on the overall structure of the thylakoids., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.