Mutations in COA7 cause spinocerebellar ataxia with axonal neuropathy.

Several genes related to mitochondrial functions have been identified as causative genes of neuropathy or ataxia. Cytochrome c oxidase assembly factor 7 (COA7) may have a role in assembling mitochondrial respiratory chain complexes that function in oxidative phosphorylation. Here we identified four unrelated patients with recessive mutations in COA7 among a Japanese case series of 1396 patients with Charcot-Marie-Tooth disease (CMT) or other inherited peripheral neuropathies, including complex forms of CMT. We also found that all four patients had characteristic neurological features of peripheral neuropathy and ataxia with cerebellar atrophy, and some patients showed leukoencephalopathy or spinal cord atrophy on MRI scans. Validated mutations were located at highly conserved residues among different species and segregated with the disease in each family. Nerve conduction studies showed axonal sensorimotor neuropathy. Sural nerve biopsies showed chronic axonal degeneration with a marked loss of large and medium myelinated fibres. An immunohistochemical assay with an anti-COA7 antibody in the sural nerve from the control patient showed the positive expression of COA7 in the cytoplasm of Schwann cells. We also observed mildly elevated serum creatine kinase levels in all patients and the presence of a few ragged-red fibres and some cytochrome c oxidase-negative fibres in a muscle biopsy obtained from one patient, which was suggestive of subclinical mitochondrial myopathy. Mitochondrial respiratory chain enzyme assay in skin fibroblasts from the three patients showed a definitive decrease in complex I or complex IV. Immunocytochemical analysis of subcellular localization in HeLa cells indicated that mutant COA7 proteins as well as wild-type COA7 were localized in mitochondria, which suggests that mutant COA7 does not affect the mitochondrial recruitment and may affect the stability or localization of COA7 interaction partners in the mitochondria. In addition, Drosophila COA7 (dCOA7) knockdown models showed rough eye phenotype, reduced lifespan, impaired locomotive ability and shortened synaptic branches of motor neurons. Our results suggest that loss-of-function COA7 mutation is responsible for the phenotype of the presented patients, and this new entity of disease would be referred to as spinocerebellar ataxia with axonal neuropathy type 3.

Dominant mutations in ORAI1 cause tubular aggregate myopathy with hypocalcemia via constitutive activation of store-operated Ca²âº channels.

The store-operated Ca(2+) release-activated Ca(2+) (CRAC) channel is activated by diminished luminal Ca(2+) levels in the endoplasmic reticulum and sarcoplasmic reticulum (SR), and constitutes one of the major Ca(2+) entry pathways in various tissues. Tubular aggregates (TAs) are abnormal structures in the skeletal muscle, and although their mechanism of formation has not been clarified, altered Ca(2+) homeostasis related to a disordered SR is suggested to be one of the main contributing factors. TA myopathy is a hereditary muscle disorder that is pathologically characterized by the presence of TAs. Recently, dominant mutations in the STIM1 gene, encoding a Ca(2+) sensor that controls CRAC channels, have been identified to cause tubular aggregate myopathy (TAM). Here, we identified heterozygous missense mutations in the ORAI1 gene, encoding the CRAC channel itself, in three families affected by dominantly inherited TAM with hypocalcemia. Skeletal myotubes from an affected individual and HEK293 cells expressing mutated ORAI1 proteins displayed spontaneous extracellular Ca(2+) entry into cells without diminishment of luminal Ca(2+) or the association with STIM1. Our results indicate that STIM1-independent activation of CRAC channels induced by dominant mutations in ORAI1 cause altered Ca(2+) homeostasis, resulting in TAM with hypocalcemia.

Mutations in KLHL40 are a frequent cause of severe autosomal-recessive nemaline myopathy.

Nemaline myopathy (NEM) is a common congenital myopathy. At the very severe end of the NEM clinical spectrum are genetically unresolved cases of autosomal-recessive fetal akinesia sequence. We studied a multinational cohort of 143 severe-NEM-affected families lacking genetic diagnosis. We performed whole-exome sequencing of six families and targeted gene sequencing of additional families. We identified 19 mutations in KLHL40 (kelch-like family member 40) in 28 apparently unrelated NEM kindreds of various ethnicities. Accounting for up to 28% of the tested individuals in the Japanese cohort, KLHL40 mutations were found to be the most common cause of this severe form of NEM. Clinical features of affected individuals were severe and distinctive and included fetal akinesia or hypokinesia and contractures, fractures, respiratory failure, and swallowing difficulties at birth. Molecular modeling suggested that the missense substitutions would destabilize the protein. Protein studies showed that KLHL40 is a striated-muscle-specific protein that is absent in KLHL40-associated NEM skeletal muscle. In zebrafish, klhl40a and klhl40b expression is largely confined to the myotome and skeletal muscle, and knockdown of these isoforms results in disruption of muscle structure and loss of movement. We identified KLHL40 mutations as a frequent cause of severe autosomal-recessive NEM and showed that it plays a key role in muscle development and function. Screening of KLHL40 should be a priority in individuals who are affected by autosomal-recessive NEM and who present with prenatal symptoms and/or contractures and in all Japanese individuals with severe NEM.

A morphometric study to establish criteria for fetal and neonatal cerebellar hypoplasia: A special emphasis on trisomy 18.

Cerebellar hypoplasia (CH) is one of the congenital abnormalities of the central nervous system and is seen in several diseases and syndromes. This study was conducted in order to examine methods for evaluating CH in fetus and neonate because CH has been diagnosed without any morphometric criteria at autopsy. We sampled 140 autopsied cases including nineteen trisomy 18 (T18), four non-T18 with presumed CH, and 117 control cases without any brain malformation. Statistical significance was present in the cerebellar weight and weight ratio of cerebellum per total brain between T18 and the control. The exponential regression models (ERM) showed that cerebral weight, cerebellar weight, and weight ratio of cerebellum per total brain increased gradually relative to gestational age in both T18 and the control. However, cerebellar weight and weight ratio of cerebellum per total brain of T18 showed growth delay with clear distinction between the two groups. The non-T18 with presumed CH showed similar results. Body weight, total brain, and gestational age should be considered totally when evaluating fetal and neonatal cerebellar development. Furthermore, the ERM results may be useful to evaluate the cerebellar development of fetus and neonate at autopsy.

GLRB is the third major gene of effect in hyperekplexia.

Glycinergic neurotransmission is a major inhibitory influence in the CNS and its disruption triggers a paediatric and adult startle disorder, hyperekplexia. The postsynaptic α(1)-subunit (GLRA1) of the inhibitory glycine receptor (GlyR) and the cognate presynaptic glycine transporter (SLC6A5/GlyT2) are well-established genes of effect in hyperekplexia. Nevertheless, 52% of cases (117 from 232) remain gene negative and unexplained. Ligand-gated heteropentameric GlyRs form chloride ion channels that contain the α(1) and ß-subunits (GLRB) in a 2α(1):3ß configuration and they form the predominant population of GlyRs in the postnatal and adult human brain, brainstem and spinal cord. We screened GLRB through 117 GLRA1- and SLC6A5-negative hyperekplexia patients using a multiplex-polymerase chain reaction and Sanger sequencing approach. The screening identified recessive and dominant GLRB variants in 12 unrelated hyperekplexia probands. This primarily yielded homozygous null mutations, with nonsense (n = 3), small indel (n = 1), a large 95 kb deletion (n = 1), frameshifts (n = 1) and one recurrent splicing variant found in four cases. A further three cases were found with two homozygous and one dominant GLRB missense mutations. We provide strong evidence for the pathogenicity of GLRB mutations using splicing assays, deletion mapping, cell-surface biotinylation, expression studies and molecular modelling. This study describes the definitive assignment of GLRB as the third major gene for hyperekplexia and impacts on the genetic stratification and biological causation of this neonatal/paediatric disorder. Driven principally by consanguineous homozygosity of GLRB mutations, the study reveals long-term additive phenotypic outcomes for affected cases such as severe apnoea attacks, learning difficulties and developmental delay.

Compound heterozygous BRAT1 mutations cause familial Ohtahara syndrome with hypertonia and microcephaly.

Homozygous frameshift BRAT1 mutations were found in patients with lethal neonatal rigidity and multifocal seizure syndrome (MIM# 614498). Here, we report on two siblings with compound heterozygous mutations in BRAT1. They had intractable seizures from neonatal period, dysmorphic features and hypertonia. Progressive microcephaly was also observed. Initial electroencephalogram showed a suppression-burst pattern, leading to a diagnosis of Ohtahara syndrome. They both died from pneumonia at 1 year and 3 months, respectively. Whole-exome sequencing of one patient revealed a compound heterozygous BRAT1 mutations (c.176T>C (p.Leu59Pro) and c.962_963del (p.Leu321Profs*81)). We are unable to obtain DNA from another patient. The p.Leu59Pro mutation occurred at an evolutionarily conserved amino acid in a CIDE-N (N-terminal of an cell death-inducing DFF45-like effector) domain, which has a regulatory role in the DNA fragmentation pathway of apoptosis. Our results further support that mutations of BRAT1 could lead to epileptic encephalopathy.

Diagnostic utility of whole exome sequencing in patients showing cerebellar and/or vermis atrophy in childhood.

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.

Clinical spectrum of early onset epileptic encephalopathies caused by KCNQ2 mutation.

PURPOSE: KCNQ2 mutations have been found in patients with benign familial neonatal seizures, myokymia, or early onset epileptic encephalopathy (EOEE). In this study, we aimed to delineate the clinical spectrum of EOEE associated with KCNQ2 mutation. METHODS: A total of 239 patients with EOEE, including 51 cases with Ohtahara syndrome and 104 cases with West syndrome, were analyzed by high-resolution melting (HRM) analysis or whole-exome sequencing. Detailed clinical information including electroencephalography (EEG) and brain magnetic resonance imaging (MRI) were collected from patients with KCNQ2 mutation. KEY FINDINGS: A total of nine de novo and one inherited mutations were identified (two mutations occurred recurrently). The initial seizures, which were mainly tonic seizures, occurred in the early neonatal period in all 12 patients. A suppression-burst pattern on EEG was found in most. Only three patients showed hypsarrhythmia on EEG; eight patients became seizure free when treated with carbamazepine, zonisamide, phenytoin, topiramate, or valproic acid. Although the seizures were relatively well controlled, moderate-to-profound intellectual disability was found in all except one patient who died at 3 months. SIGNIFICANCE: De novo KCNQ2 mutations are involved in EOEE, most of which cases were diagnosed as Ohtahara syndrome. These cases showed distinct features with early neonatal onset, tonic seizures, a suppression-burst EEG pattern, infrequent evolution to West syndrome, and good response to sodium channel blockers, but poor developmental prognosis. Genetic testing for KCNQ2 should be considered for patients with EOEE.

A child with three episodes of reversible splenial lesion.

In this study, we report the case of an 8-year-old girl who had three episodes of reversible splenial lesion of the corpus callosum (SCC) in 2 years. Vomiting, hypoglycemia, and fever were followed by altered consciousness and diminished muscle tone. In each episode, the clinical manifestations and abnormalities detected during magnetic resonance imaging resolved in 2 weeks. Transient alteration of vision and spike discharges revealed by interictal electroencephalogram implied the SCC lesions were related to epileptic activities. At follow-up, the patient had not presented with SCC lesions or altered consciousness for more than 4 years after undergoing carbamazepine treatment. Our case is the first report of a patient who presented with three episodes of reversible splenial lesion.

Contiguous deletion of SLC6A8 and BAP31 in a patient with severe dystonia and sensorineural deafness.

We report here a 6-year-old boy exhibiting severe dystonia, profound intellectual and developmental disability with liver disease, and sensorineural deafness. A deficient creatine peak in brain (1)H-MR spectroscopy and high ratio of creatine/creatinine concentration in his urine lead us to suspect a creatine transporter (solute carrier family 6, member 8; SLC6A8) deficiency, which was confirmed by the inability to take up creatine into fibroblasts. We found a large ~19 kb deletion encompassing exons 5-13 of SLC6A8 and exons 5-8 of the B-cell receptor-associated protein (BAP31) gene. This case is the first report in which the SLC6A8 and BAP31 genes are both deleted. The phenotype of BAP31 mutations has been reported only as a part of Xq28 deletion syndrome or contiguous ATP-binding cassette, sub-family D, member 1 (ABCD1)/DXS1375E (BAP31) deletion syndrome [MIM ID #300475], where liver dysfunction and sensorineural deafness have been suggested to be attributed to the loss of function of BAP31. Our case supports the idea that the loss of BAP31 is related to liver dysfunction and hearing loss.

[Clinical characteristics of acute encephalopathies associated with influenza H1N1-2009 in children].

We report 12 cases of acute encephalopathy associated with influenza H1N1-2009 treated according to Japanese guideline (2009). In all 12 cases, electroencephalogram presented diffuse or localized high-amplitude slow waves. Brain CT and MRI showed abnormalities in 4 and 6 cases, respectively. We used hypothermia therapy for 5 patients. One patient showed impairment in short term memory, while the rest of the patients showed no sequelae. These 12 cases presented here suggest the early recognition and therapy according to the newly proposed guideline may reduce severe sequelae and mortality by acute encephalopathy associated with influenza H1N1-2009.

Congenital disorders of glycosylation type IIb with MOGS mutations cause early infantile epileptic encephalopathy, dysmorphic features, and hepatic dysfunction.

AIM: MOGS mutations cause congenital disorders of glycosylation type IIb (CDG-IIb or GCS1-CDG). The specific manifestations caused by the mutations in this gene remain unknown. We aimed to describe the clinical features of CDG- IIb and the effectiveness of urinary oligosaccharide analysis in the diagnosis of CDG- IIb. METHODS: Patient 1 was analyzed with whole-exome sequencing (WES) to identify the causative gene of intractable epilepsy and severe developmental delay. After detecting MOGS mutation in patient 1, we analyzed patients 2 and 3 who were siblings and had clinical features similar to those in patient 1. Urinary oligosaccharide analysis was performed to confirm CDG- IIb diagnosis in patient 1. The clinical features of these patients were analyzed and compared with those in eight published cases. RESULTS: Our three patients presented with early infantile epileptic encephalopathy, generalized hypotonia, hepatic dysfunction and dysmorphic features. In two cases, compound heterozygous mutations in MOGS were identified by WES. Isolation and characterization of the urinary oligosaccharide was performed in one of these cases to confirm the diagnosis of CDG-IIb. Although the isoelectric focusing of transferrin (IEF-T) of serum in this patient was normal, urinary excretion of Hex4 corresponding to Glc3Man was observed by mass spectrometry. CONCLUSION: This report provides clinical manifestations of CDG-IIb with MOGS mutation. CDG-IIb shows a normal IEF profile of serum transferrin and cannot be detected by structural analysis of the patient's glycoproteins. Characterization of urinary oligosaccharides should be considered to detect this disorder.

Dysbindin, syncoilin, and beta-synemin mRNA levels in dystrophic muscles.

Progressive muscular dystrophies are genetic diseases with various modes of transmission. Duchenne muscular dystrophy (DMD) is caused by the defect of dystrophin, and Fukuyama congenital muscular dystrophy (FCMD) is caused by an abnormal fukutin gene leading to the glycosylation defect of alpha-dystroglycan. Dystrobrevin is one member of the dystrophin glycoprotein complex and its binding partners include dysbindin, syncoilin, and beta-synemin (desmuslin). Dysbindin is reported to be upregulated at the protein level in mdx mouse muscles, and syncoilin protein is also reported to be upregulated in biopsied muscles with neuromuscular disorders. In the present study we measured mRNA levels of dysbindin, syncoilin, and beta-synemin in biopsied muscles with DMD and FCMD. Upregulation of human dysbindin mRNA was observed in DMD muscles in comparison with normal muscles (p < .05). The differences in human syncoilin and beta-synemin mRNA ratios between DMD and normal muscles were not statistically significant, although upregulation tendency of human syncoilin mRNA was noted in DMD muscles (.05 < p < .1). Furthermore, the differences of human dysbindin, syncoilin, and beta-synemin mRNA ratios between FCMD and normal muscles were not statistically significant. These data provide insight into the pathophysiology of these muscular dystrophies.

Aggregate formation analysis of GFAPR416W found in one case of Alexander disease.

Alexander disease (AxD) is a neurodegenerative disease in astrocytes caused by a mutation in the gene encoding glial fibrillary acidic protein, GFAP. We herein present the case of a 12-year-old girl who showed intermittent exotropia at 3 years of age and central precocious puberty at 7 years of age. The periventricular and medulla oblongata showed high signal intensity on T2-weighted magnetic resonance imaging. The patient was diagnosed with AxD after direct sequencing revealing a de novo recurrent mutation, c.1246C>T (p.R416W) in GFAP. The transient expression of GFAPR416W in cells resulted in the significant formation of aggregates, which recapitulated the hallmark of AxD. We firstly utilized In Cell analyzer to prove the tendency of aggregate formation by mutants of GFAP.

Changes in the amounts of myelin lipids and molecular species of plasmalogen PE in the brain of an autopsy case with D-bifunctional protein deficiency.

Changes in the molecular species of lipids associated with peroxisomal d-bifunctional protein (d-BP) deficiency were investigated in cerebral tissues to elucidate the pathological mechanisms underlying this disorder. Total phospholipids in the gray and white matters of the patient's brain were decreased to approximately 73% and 50% of control levels, respectively, and profound declines in myelin lipids, i.e. galactosyl ceramide and sulfatides, indicated dysmyelination in our patient with d-BP deficiency. Although the total ganglioside amounts in the gray and white matter of this patient's brain were also decreased to 61% and 37% of control levels and GM1 in the white matter was 20% of the control level, the relative amounts of GM2 in both the gray and the white matter of this patient's brain were increased in comparison to those in the control, indicating altered metabolism of gangliosides. In addition, among molecular species of phospholipids, plasmalogen-type and polyunsaturated fatty acid-containing phosphatidylethanolamine were characteristically decreased in the patient's gray matter. These alterations in the molecular species of brain lipids may affect sensitivity to oxidative stress and the membrane fluidity of neural cells, thereby producing the brain pathology of d-BP deficiency.

Early onset West syndrome with cerebral hypomyelination and reduced cerebral white matter.

Numerous numbers of pre-, peri- and postnatal damages cause West syndrome in early infancy, however, etiology in many cases are not still elucidated despite intensive biochemical and neuroradiologic investigations. We described four patients having early onset epileptic encephalopathy with severe hypomyelination and reduction in cerebral white matter. The clinical symptoms of these patients are impaired visual attention, acquired microcephaly, spastic tetraplegia, profound psychomotor delay and infantile spasms since early infancy. All patients had striking hypomyelination of cerebrum, reduced volume of white matter and cortical atrophy on MRI. Serial MRI investigations in three patients showed absence of myelination of the white matter. On EEG, one patient revealed suppression-burst and other three had hypsarrhythmia. Despite having intractable seizures, no patient showed deterioration of neurological development. The group of these findings is mimicking to clinical manifestations of 3-phosphoglycerate dehydrogenase deficiency, and has some overlap with progressive encephalopathy with edema, hypsarrhythmia, and optic atrophy (PEHO) like syndrome, however it is not compatible with these two conditions. The findings observed in our patients can be regarded as a new clinical condition associated with early onset West syndrome.

Peripheral nerve pathology at fixed stage in spinal muscular atrophy with respiratory distress type 1.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is characterized by severe respiratory failure due to diaphragmatic paralysis and distal muscular weakness in early infancy. After an initial decline in respiratory state and motor function until 1-2years of age, residual capabilities reach a plateau. We report the peripheral neuropathological findings of a patient with SMARD1 at 1year and 1month of age, when his muscle strength and respiratory symptoms had deteriorated and then stabilized for several months. Peripheral nerve biopsy revealed severely progressed axonal degeneration. This finding suggests the rapid progression of peripheral axonal neuropathy in SMARD1 that leads to its characteristic clinical course of respiratory failure and paralysis in the early infantile period.

Japanese Leigh syndrome case treated with EPI-743.

BACKGROUND: Leigh syndrome is a mitochondrial disease caused by respiratory chain deficiency, and there are no proven effective therapies. EPI-743 is a potent cellular oxidative stress protectant and results of clinical trials for mitochondrial diseases are accumulating. CASE: At 5months, a girl presented with the scarce eye movement and diminished muscle tone. She was diagnosed with Leigh encephalopathy from blood and cerebrospinal fluid lactate elevation and MRI findings. Sequence analysis for mitochondrial DNA revealed a T10158C mutation in the mitochondrial encoded ND3 gene in complex I. RESULTS: At 8months, succinate was prescribed expected to restore the electron transport chain system. After that her condition got worse and succinate was discontinued. Subsequent administration of EPI-743 improved her eye movement, fine motor movements of the extremities, and bowel movement. She is now 5years old. Although brain atrophy has progressed, she has still respiratory free time. CONCLUSION: Our patient showed visible improvement with EPI-743 treatment and the only patient surviving after 4years. There is a possibility that EPI-743 is modifying the natural course of the syndrome.

Patients with a sodium channel alpha 1 gene mutation show wide phenotypic variation.

We investigated the roles of mutations in voltage-gated sodium channel alpha 1 subunit gene (SCN1A) in epilepsies and psychiatric disorders. The SCN1A gene was screened for mutations in three unrelated Japanese families with generalized epilepsy with febrile seizure plus (GEFS+), febrile seizure with myoclonic seizures, or intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTC). In the family with GEFS+, one individual was affected with panic disorder and seizures, and another individual was diagnosed with Asperger syndrome and seizures. The novel mutation V1366I was found in all probands and patients with psychiatric disorders of the three families. These results suggest that SCN1A mutations may confer susceptibility to psychiatric disorders in addition to variable epileptic seizures. Unidentified modifiers may play critical roles in determining the ultimate phenotype of patients with sodium channel mutations.