Ryosuke Miyamoto

A mutation in the low voltage-gated calcium channel CACNA1G alters the physiological properties of the channel, causing spinocerebellar ataxia

BackgroundSpinocerebellar ataxia (SCA) is a genetically heterogeneous disease. To date, 36 dominantly inherited loci have been reported, and 31 causative genes have been identified.ResultsIn this study, we analyzed a Japanese family with autosomal dominant SCA using linkage analysis and exome sequencing, and identified CACNA1G, which encodes the calcium channel CaV3.1, as a new causative gene. The same mutation was also found in another family with SCA. Although most patients exhibited the pure form of cerebellar ataxia, two patients showed prominent resting tremor in addition to ataxia. CaV3.1 is classified as a low-threshold voltage-dependent calcium channel (T-type) and is expressed abundantly in the central nervous system, including the cerebellum. The mutation p.Arg1715His, identified in this study, was found to be located at S4 of repeat IV, the voltage sensor of the CaV3.1. Electrophysiological analyses revealed that the membrane potential dependency of the mutant CaV3.1 transfected into HEK293T cells shifted toward a positive potential. We established induced pluripotent stem cells (iPSCs) from fibroblasts of the patient, and to our knowledge, this is the first report of successful differentiation from the patient-derived iPSCs into Purkinje cells. There was no significant difference in the differentiation status between control- and patient-derived iPSCs.ConclusionsTo date, several channel genes have been reported as causative genes for SCA. Our findings provide important insights into the pathogenesis of SCA as a channelopathy.

Exome sequencing reveals a novel TTC19 mutation in an autosomal recessive spinocerebellar ataxia patient

BackgroundSpinocerebellar ataxias (SCAs) are heterogeneous diseases characterized by progressive cerebellar ataxia associated with dysarthria, oculomotor abnormalities, and mental impairment. To identify the causative gene, we performed exome sequencing on a Japanese patient clinically diagnosed with recessive SCA.MethodThe patient is a 37-year-old Japanese woman with consanguineous parents. The head magnetic resonance imaging (MRI) showed cerebellar atrophy and T1 low/T2 high intensity at the bilateral inferior olives. Single-nucleotide polymorphism (SNP) genotyping and next-generation sequencing were performed, and the variants obtained were filtered and prioritized.ResultsAfter these manipulations, we identified a homozygous nonsense mutation of the TTC19 gene (p.Q277*). TTC19 has been reported to be a causative gene of a neurodegenerative disease in Italian and Portuguese families and to be involved in the pathogenesis of mitochondrial respiratory chain complex III (cIII) deficiency. This report is the first description of a TTC19 mutation in an Asian population. Clinical symptoms and neuroimaging are consistent with previous reports. The head MRI already showed abnormal features four years before her blood lactate and pyruvate levels were elevated.ConclusionsWe should consider the genetic analysis of TTC19 when we observe such characteristic MRI abnormalities. Genes associated with mitochondrial function cause many types of SCAs; the mutation we identified should help to elucidate the pathology of these disorders.

Exome sequencing reveals a novel ANO10 mutation in a Japanese patient with autosomal recessive spinocerebellar ataxia

To the Editor : Spinocerebellar ataxia autosomal recessive type 10 (SCAR10, OMIM 613728) is caused by the mutation of ANO10 (1). The clinical phenotype was characterized by ataxia, hyper-reflexia, normal plantar reflex, downbeat nystagmus and lower motor neuron involvement. Here, we report a novel ANO10 mutation in a patient with autosomal recessive spinocerebellar ataxia (ARSCA), using exome sequencing. The research procedure was approved by the Ethics Committee of Hiroshima University. All of the examinations were performed after obtaining informed consent from the patient. Control exomes were obtained from patients undergoing exome analysis for diseases other than SCA. A Japanese patient with cerebellar ataxia born to consanguineous parents had a homozygous nonsense ANO10 (c.609C>G, p.Y203X) mutation. The identified mutation was validated with conventional Sanger sequencing. This mutation was not detected in our control exomes. A 58-year-old man had presented with loss of consciousness at age 42 and was treated with 800 mg of sodium valproate (further information could not be obtained). He had noticed a tendency to fall at age 46 and dysarthria at age 54. However, he continued to satisfactorily function at work. At age of 58, he presented with saccadic eye movement, hyper-reflexia, decreased vibration sense and constipation. Muscle atrophy, nystagmus and tortuosity of the conjunctival vessels were not observed. Electromyography was normal. His MiniMental State Examination score was 29. Brain magnetic resonance imaging (MRI) showed mild cerebellar atrophy, and brain stem was slightly atrophic at age 57 (Fig. 1). Single photon emission computed tomography showed a decrease in cerebellar flow. Previously reported SCAR10 cases have originated from the Netherlands, Serbia and France (1, 2), and this is the first case reported outside Europe. In contrast to our case, previous reported cases also presented muscle atrophy, nystagmus, tortuosity of the conjunctival vessels and intellectual deficit. In addition, in our case, decreased vibration sense and constipation were detected and the age of onset was relatively late. These characteristics should be considered when diagnosing SCAR10. Previously, homozygous missense mutation, homozygous frame-shift mutation and compound Fig. 1. Brain magnetic resonance imaging at age of 57: T1 sagittal image. Mild cerebellar atrophy was observed.

The clinical characteristics of spinocerebellar ataxia 36: A study of 2121 Japanese ataxia patients†‡

Spinocerebellar ataxia 36 is caused by the expansion of the intronic GGCCTG hexanucleotide repeat in NOP56. The original article describing this condition demonstrated that patients with spinocerebellar ataxia 36 present with tongue atrophy, a finding that had not been seen in previous types of spinocerebellar ataxias. A total of 2121 patients with clinically diagnosed spinocerebellar ataxia participated in the study. We screened our patient samples for spinocerebellar ataxia 36 using the repeat‐primed polymerase chain reaction method and also determined the clinical features of spinocerebellar ataxia 36. Of the ataxia cases examined, 12 were identified as spinocerebellar ataxia 36. Of these, 7 cases (6 families) were autosomal dominant, 4 cases (three families) had a positive family history but were not autosomal dominant, and 1 case was sporadic. The average age of onset was 51.7 years, and disease progression was slow. The main symptoms and signs of disease included ataxia, dysarthria, and hyperreflexia. Approximately half the affected patients demonstrated nystagmus, bulging eyes, and a positive pathological reflex, although dysphagia, tongue atrophy, and hearing loss were rare. Moreover, the observed atrophy of the cerebellum and brain stem was not severe. The patients identified in this study were concentrated in western Japan. The frequency of spinocerebellar ataxia 36 was approximately 1.2% in the autosomal dominant group, and the age of onset for this condition was later in comparison with other spinocerebellar ataxia subtypes.

Exome sequencing reveals a novel MRE11 mutation in a patient with progressive myoclonic ataxia

Progressive myoclonic ataxia (PMA) is a clinical syndrome defined as progressive ataxia and myoclonus and infrequent seizures in the absence of progressive dementia. Due to the extremely heterogeneous nature of PMA, a large proportion of PMA cases remain molecularly undiagnosed. The aim of this study was to clarify the molecular etiology of PMA. The patient was a 52-year-old female from consanguineous parents. She developed a jerking neck movement at age 9, which gradually expanded to her entire body. On physical examination at age 47, she exhibited generalized, spontaneous myoclonus that occurred continuously. She also presented with mild limb and truncal ataxia. An electroencephalogram revealed no abnormalities. A brain MRI displayed no atrophy of the cerebellum. Electrophysiological studies suggested myoclonus of a subcortical origin. For further evaluation, we performed exome sequencing, and we identified a novel homozygous missense mutation in the MRE11 gene (NM_005590:c.140C>T:p.A47V). Subsequently, we analyzed the expression of MRE11 and related proteins (RAD50 and NBS1) via Western blot, and they were markedly decreased compared to a healthy control. Mutations in the MRE11 gene have been known to cause an ataxia-telangiectasia-like (ATLD) disorder. Accumulating evidence has indicated that its wide phenotypic variations in ATLD correspond to genotypic differences. Interestingly, our case exhibited a relatively mild decrease in NBS1 compared to previously reported cases of a homozygous missense mutation, which may account for the milder phenotype in this patient. Moreover, together with a recently reported case of an MRE11 mutation, it is suggested that MRE11 mutations can present as PMA.

Detecting gene mutations in Japanese Alzheimer's patients by semiconductor sequencing

Alzheimers disease (AD) is the most common form of dementia. To date, several genes have been identified as the cause of AD, including PSEN1, PSEN2, and APP. The association between APOE and late-onset AD has also been reported. We here used a bench top next-generation sequencer, which uses an integrated semiconductor device, detects hydrogen ions, and operates at a high-speed using nonoptical technology. We examined 45 Japanese AD patients with positive family histories, and 29 sporadic patients with early onset (<60-year-old). Causative mutations were detected in 5 patients in the familial group (11%). Three patients had a known heterozygous missense mutation in the PSEN1 gene (p.H163R). Two patients from 1 family had a novel heterozygous missense mutation in the PSEN1 gene (p.F386L). In the early onset group, 1 patient carrying homozygous APOEε4 had a novel heterozygous missense mutation in the PSEN2 gene (p.T421M). Approximately 43% patients were APOEε4 positive in our study. This new sequencing technology is useful for detecting genetic variations in familial AD.

Lack of C9orf72 expansion in 406 sporadic and familial cases of idiopathic dystonia in Japan

C9orf72 hexanucleotide repeat expansions have been identified as the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Growing evidence suggests that C9orf72 expansions are associated with other neurodegenerative diseases, such as Alzheimer’s disease, sporadic Creutzfeldt-Jakob disease, and Huntington’s disease (HD) phenocopy syndromes, which denote a group of patients with clinically suspected HD, but without a cytosine-adenine-guanine repeat in the HTT gene. A recent report found C9orf72 expansions in approximately 2% (10 of 514) of HD phenocopies, including 1 patient with a pure movement disorder. Given the evidence suggesting that C9orf72 expansions have importance in movement disorders, we analyzed a Japanese cohort of idiopathic dystonia patients for C9orf72 expansions. This genetic study was approved by the ethics committee of The University of Tokushima, and all participants provided written informed consent. Four hundred six Japanese patients with idiopathic dystonia (320 sporadic cases and 86 familial cases from 57 families), mainly from Western Japan, were analyzed. No patients had documented cognitive or behavioral abnormalities or reported a family history of motor neuron diseases. Dystonia subtypes were classified as follows: 254 patients with focal; 68 with segmental; 75 with generalized; and 9 with multifocal. Mean age of onset was 41.7 years (range, 1-89). To test for the presence of a GGGGCC expansion in C9orf72, amplicon-length analysis and repeat-primed polymerase chain reaction (RP-PCR) were performed as previously described. Fragment length analysis was performed on an ABI 3130 genetic analyzer and analyzed using ABI GeneMapper (v4.1; Applied Biosystems, Foster City, CA). We found no C9orf72 expansions in these 406 idiopathic dystonia patients. After identification of C9orf72 expansions as the cause of chromosome 9p-related ALS in 2011, accumulating evidence has been widening the clinical spectrum associated with C9orf72 expansions to corticobasal syndrome, olivopontocerebellar degeneration, and HD phenocopies, which demonstrates that a variety of movement disorders may exhibit C9orf72 expansions. Hensman Moss et al. reported that 4 of 10 C9orf72 expansion-positive HD phenocopies showed dystonia, 4 showed myoclonus, 3 showed tremor, and 3 showed chorea. Importantly, 1 patient presenting with dystonia lacked cognitive/psychiatric symptoms, and this finding was quite unique, given that the hallmark of the C9orf72related disease is upper/lower motor neuron signs and/or cognitive/psychiatric impairments. This knowledge prompted us to perform genetic screening on our panel of idiopathic dystonia patients; however, no pathological expansion was observed. This negative result suggests that idiopathic dystonia is not associated with C9orf72 expansions. Our results may be influenced by the disproportionate distribution of C9orf72 ALS throughout the world; the general frequency of C9orf72 ALS in Japan is lower than Europe, which suggests that the screening results in other populations could be different from our own. Additionally, even a combination of amplicon-length analysis and RP-PCR has a risk of yielding false-negative results; however, none of our samples showed a questionable pattern in RP-PCR. Further genetic screenings for C9orf72 expansions in movement disorders with or without cognitive/psychiatric symptoms may help in the understanding of C9orf72-related movement disorders.

DYT6 in Japan-genetic screening and clinical characteristics of the patients

dissynergic, propulsive pattern emerged, resulting in a festinating gait with lateral displacement of the body (see Video Segment 1). Skilled movements of the upper limbs were preserved. The boy’s score on the Leiter-R IQ test was 78, and his performance on the Tower of London test was within normal range (z 5 20.50). A trial with levodopa (L-dopa)/ carbidopa was ineffective. Starting from age 9 years, brain magnetic resonance imaging revealed progressive cerebral and cerebellar atrophy. Proton magnetic resonance spectroscopy detected a decline in N-acetylaspartate levels and increased lipid signals in both the cerebral white matter and the basal ganglia. Generalized myoclonic and tonic seizures, which were triggered by photic stimulation, were observed first at age 11 years. Somatosensory stimulation of the limbs evoked giant cortical potentials, suggesting cortical myoclonus. At age 12 years, the boy lost the ability to read and write, although social interaction and verbal comprehension were relatively preserved (Vineland Adaptive Behavior Scale: socialization domain, 6 years and 2 months; receptive subdomain, 7 years and 9 months). Between ages 12 and 15 years, he experienced a further clinical decline, with severe hypokinesia and bradykinesia, impairment of postural reactions, ataxia, continuous rest and action myoclonic jerks, hypomimia, anarthric mutism, and upper limb-kinetic apraxia (see Video Segment 2). Visual functions remained unaffected. CLN6 gene sequencing revealed a homozygous c.700T!C transition (p.Phe234Leu), which involved a transmembrane domain (TM6) of the protein. The alteration was confirmed in the parents’ DNA, thus suggesting possible consanguinity.

Compound heterozygote mutations in the SIGMAR1 gene in an oldest-old patient with amyotrophic lateral sclerosis: Letters to the Editor

Dear Editor, Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by progressive loss of upper and lower motor neurons with or without frontotemporal dementia (FTD). Just 10% of ALS cases are familial or inherent. The survival of patients with the oldest-old onset (aged ≥80 years) is particularly short. The present patient was an 89-year-old man who presented with a slowly progressive gait disturbance, which began at age 80 years. He had no significant family history, and his parents were not consanguineous. At age 83 years, he showed almost normal muscle strength, hyperactive deep tendon reflexes and bilateral positive Babinski’s signs. Mini-Mental State Examination and Frontal Assessment Battery scores were 28 out of 30 and 14 out of 18. Two years later, he developed weakness in the left hand and legs. Needle electromyography showed both active and chronic denervations in the lumbosacral region, and chronic denervation in the cervical and truncal regions. He was diagnosed with probable ALS based on the revised El Escorial criteria. At age 88 years, he showed mild bulbar signs and a reduced percentage vital capacity of 64.8%. At age 89 years, he could still walk with a cane and eat by himself. To investigate the genetic etiology, we used an ion conduction sequencer. This investigation was approved by the institutional ethics committees of Tokushima University Hospital and Hiroshima University. Written informed consent was obtained from the patient. Genomic DNA was extracted from patient-derived peripheral lymphocytes. We chose 10 ALS genes to investigate: FUS, SQSTM1, SOD1, VCP, SIGMAR1, TARDBP, OPTN, ANG, PFN1 and UBQLN2. All coding exons were simultaneously polymerase chain reaction amplified and sequenced. We determined the novelty of variants using data from the National Center for Biotechnology Information database for short genetic variations: dbSNP137, the 1000 Genomes Website and the Human Genetic Variation Database from Top Global University Japan. The large repeat expansion in the C9orf72 gene was assessed using repeatprimed polymerase chain reaction, as previously described. Using ion conduction sequencing, a novel heterozygous missense variation (c.505T>A, p.W169R) located in exon 4 of the SIGMAR1 gene was identified. Furthermore, another variant (c.622C>T, p.R208W, rs11559048) in the same exon of SIGMAR1 was also identified. We confirmed c.505T>A and c.622C>T substitutions by Sanger sequencing (Fig. 1). We also confirmed that these substitutions are located on the different alleles by subcloning. Variant prediction algorithms (Sorting Intolerant From Tolerant, Polymorphism Phenotyping v2 and Mutation Taster) predicted both variants to be damaging. The large repeat expansion in the C9orf72 gene was not detected. Luty et al. reported heterozygous mutations in the 30-UTR of SIGMAR1 in three families presenting ALS/FTD or FTD. However, one of these families was subsequently identified as positive for the C9orf72 mutation, suggesting that the SIGMAR1 mutation does not exclusively explain the etiology at least in this family. Homozygous SIGMAR1 mutations cause juvenile or young-onset ALS with no cognitive deficit. Here, we identified a novel SIGMAR1 mutation together with a common, but damaging, singlenucleotide polymorphism in an ALS patient with the oldest-old onset. ALS symptoms mainly begin during mid or late life. Just 15.5% of patients start to show symptoms at age ≥80 years. The median survival of the oldest-old patients appeared to be 10 months less compared with patients aged <80 years (7.7 vs 17.4 months). This case was unique in terms of very slow progression, despite the oldest-old onset. The present patient harbored double deleterious heterozygous variants: p.W169R and p.R208W. These mutations potentially represent two causes for the development of ALS. For example, p.W169R dominantly contributes to the disease. Luty et al. suggested that a heterozygous SIGMAR1 variant might cause ALS/FTD. More recently, a different heterozygous variant in the 30-UTR region was identified in an ALS patient showing slow progression. These findings suggest that monoallelic SIGMAR1 mutations might cause ALS/FTD. Similarly, the p.W169R variant in the present case might be an exclusive genetic factor involved in the development of ALS. The second possibility, which we consider more likely, is that both p.R208W and p.W169R variants contribute to the pathogenesis of ALS. The p.R208W variant is a common single-nucleotide polymorphism that accounts for ~5% of genetic variants observed in Asian populations. However, multiple prediction programs predict this variant to be damaging. Furthermore, a common single-nucleotide polymorphism can contribute to biallelic neurological diseases with a concurrent pathological mutation, and the present case might fit this type of compound heterozygote model. In summary, we identified double heterozygous SIGMAR1 variants in a clinically