ERBB4 mutations that disrupt the neuregulin-ErbB4 pathway cause amyotrophic lateral sclerosis type 19.

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disorder characterized by the degeneration of motor neurons and typically results in death within 3-5 years from onset. Familial ALS (FALS) comprises 5%-10% of ALS cases, and the identification of genes associated with FALS is indispensable to elucidating the molecular pathogenesis. We identified a Japanese family affected by late-onset, autosomal-dominant ALS in which mutations in genes known to be associated with FALS were excluded. A whole- genome sequencing and parametric linkage analysis under the assumption of an autosomal-dominant mode of inheritance with incomplete penetrance revealed the mutation c.2780G>A (p. Arg927Gln) in ERBB4. An extensive mutational analysis revealed the same mutation in a Canadian individual with familial ALS and a de novo mutation, c.3823C>T (p. Arg1275Trp), in a Japanese simplex case. These amino acid substitutions involve amino acids highly conserved among species, are predicted as probably damaging, and are located within a tyrosine kinase domain (p. Arg927Gln) or a C-terminal domain (p. Arg1275Trp), both of which mediate essential functions of ErbB4 as a receptor tyrosine kinase. Functional analysis revealed that these mutations led to a reduced autophosphorylation of ErbB4 upon neuregulin-1 (NRG-1) stimulation. Clinical presentations of the individuals with mutations were characterized by the involvement of both upper and lower motor neurons, a lack of obvious cognitive dysfunction, and relatively slow progression. This study indicates that disruption of the neuregulin-ErbB4 pathway is involved in the pathogenesis of ALS and potentially paves the way for the development of innovative therapeutic strategies such using NRGs or their agonists to upregulate ErbB4 functions.

The TRK-fused gene is mutated in hereditary motor and sensory neuropathy with proximal dominant involvement.

Hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) is an autosomal-dominant neurodegenerative disorder characterized by widespread fasciculations, proximal-predominant muscle weakness, and atrophy followed by distal sensory involvement. To date, large families affected by HMSN-P have been reported from two different regions in Japan. Linkage and haplotype analyses of two previously reported families and two new families with the use of high-density SNP arrays further defined the minimum candidate region of 3.3 Mb in chromosomal region 3q12. Exome sequencing showed an identical c.854C>T (p.Pro285Leu) mutation in the TRK-fused gene (TFG) in the four families. Detailed haplotype analysis suggested two independent origins of the mutation. Pathological studies of an autopsied patient revealed TFG- and ubiquitin-immunopositive cytoplasmic inclusions in the spinal and cortical motor neurons. Fragmentation of the Golgi apparatus, a frequent finding in amyotrophic lateral sclerosis, was also observed in the motor neurons with inclusion bodies. Moreover, TAR DNA-binding protein 43 kDa (TDP-43)-positive cytoplasmic inclusions were also demonstrated. In cultured cells expressing mutant TFG, cytoplasmic aggregation of TDP-43 was demonstrated. These findings indicate that formation of TFG-containing cytoplasmic inclusions and concomitant mislocalization of TDP-43 underlie motor neuron degeneration in HMSN-P. Pathological overlap of proteinopathies involving TFG and TDP-43 highlights a new pathway leading to motor neuron degeneration.

Rapid detection of expanded short tandem repeats in personal genomics using hybrid sequencing.

MOTIVATION: Long expansions of short tandem repeats (STRs), i.e. DNA repeats of 2-6 nt, are associated with some genetic diseases. Cost-efficient high-throughput sequencing can quickly produce billions of short reads that would be useful for uncovering disease-associated STRs. However, enumerating STRs in short reads remains largely unexplored because of the difficulty in elucidating STRs much longer than 100 bp, the typical length of short reads. RESULTS: We propose ab initio procedures for sensing and locating long STRs promptly by using the frequency distribution of all STRs and paired-end read information. We validated the reproducibility of this method using biological replicates and used it to locate an STR associated with a brain disease (SCA31). Subsequently, we sequenced this STR site in 11 SCA31 samples using SMRT(TM) sequencing (Pacific Biosciences), determined 2.3-3.1 kb sequences at nucleotide resolution and revealed that (TGGAA)- and (TAAAATAGAA)-repeat expansions determined the instability of the repeat expansions associated with SCA31. Our method could also identify common STRs, (AAAG)- and (AAAAG)-repeat expansions, which are remarkably expanded at four positions in an SCA31 sample. This is the first proposed method for rapidly finding disease-associated long STRs in personal genomes using hybrid sequencing of short and long reads. AVAILABILITY AND IMPLEMENTATION: Our TRhist software is available at http://trhist.gi.k.u-tokyo.ac.jp/. CONTACT: moris@cb.k.u-tokyo.ac.jp SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Modulation of the age at onset in spinocerebellar ataxia by CAG tracts in various genes.

Polyglutamine-coding (CAG)n repeat expansions in seven different genes cause spinocerebellar ataxias. Although the size of the expansion is negatively correlated with age at onset, it accounts for only 50-70% of its variability. To find other factors involved in this variability, we performed a regression analysis in 1255 affected individuals with identified expansions (spinocerebellar ataxia types 1, 2, 3, 6 and 7), recruited through the European Consortium on Spinocerebellar Ataxias, to determine whether age at onset is influenced by the size of the normal allele in eight causal (CAG)n-containing genes (ATXN1-3, 6-7, 17, ATN1 and HTT). We confirmed the negative effect of the expanded allele and detected threshold effects reflected by a quadratic association between age at onset and CAG size in spinocerebellar ataxia types 1, 3 and 6. We also evidenced an interaction between the expanded and normal alleles in trans in individuals with spinocerebellar ataxia types 1, 6 and 7. Except for individuals with spinocerebellar ataxia type 1, age at onset was also influenced by other (CAG)n-containing genes: ATXN7 in spinocerebellar ataxia type 2; ATXN2, ATN1 and HTT in spinocerebellar ataxia type 3; ATXN1 and ATXN3 in spinocerebellar ataxia type 6; and ATXN3 and TBP in spinocerebellar ataxia type 7. This suggests that there are biological relationships among these genes. The results were partially replicated in four independent populations representing 460 Caucasians and 216 Asian samples; the differences are possibly explained by ethnic or geographical differences. As the variability in age at onset is not completely explained by the effects of the causative and modifier sister genes, other genetic or environmental factors must also play a role in these diseases.

Molecular epidemiology and clinical spectrum of hereditary spastic paraplegia in the Japanese population based on comprehensive mutational analyses.

Hereditary spastic paraplegia (HSP) is one of the most genetically heterogeneous neurodegenerative disorders characterized by progressive spasticity and pyramidal weakness of lower limbs. Because >30 causative genes have been identified, screening of multiple genes is required for establishing molecular diagnosis of individual patients with HSP. To elucidate molecular epidemiology of HSP in the Japanese population, we have conducted mutational analyses of 16 causative genes of HSP (L1CAM, PLP1, ATL1, SPAST, CYP7B1, NIPA1, SPG7, KIAA0196, KIF5A, HSPD1, BSCL2, SPG11, SPG20, SPG21, REEP1 and ZFYVE27) using resequencing microarrays, array-based comparative genomic hybridization and Sanger sequencing. The mutational analysis of 129 Japanese patients revealed 49 mutations in 46 patients, 32 of which were novel. Molecular diagnosis was accomplished for 67.3% (33/49) of autosomal dominant HSP patients. Even among sporadic HSP patients, mutations were identified in 11.1% (7/63) of them. The present study elucidated the molecular epidemiology of HSP in the Japanese population and further broadened the mutational and clinical spectra of HSP.

Roles of the cerebellum and basal ganglia in temporal integration: Insights from a synchronized tapping task.

OBJECTIVE: The aim of this study was to clarify the roles of the cerebellum and basal ganglia for temporal integration. METHODS: We studied 39 patients with spinocerebellar degeneration (SCD), comprising spinocerebellar atrophy 6 (SCA6), SCA31, Machado-Joseph disease (MJD, also called SCA3), and multiple system atrophy (MSA). Thirteen normal subjects participated as controls. Participants were instructed to tap on a button in synchrony with isochronous tones. We analyzed the inter-tap interval (ITI), synchronizing tapping error (STE), negative asynchrony, and proportion of delayed tapping as indicators of tapping performance. RESULTS: The ITI coefficient of variation was increased only in MSA patients. The standard variation of STE was larger in SCD patients than in normal subjects, especially for MSA. Negative asynchrony, which is a tendency to tap the button before the tones, was prominent in SCA6 and MSA patients, with possible basal ganglia involvement. SCA31 patients exhibited normal to supranormal performance in terms of the variability of STE, which was surprising. CONCLUSIONS: Cerebellar patients generally showed greater STE variability, except for SCA31. The pace of tapping was affected in patients with possible basal ganglia pathology. SIGNIFICANCE: Our results suggest that interaction between the cerebellum and the basal ganglia is essential for temporal processing. The cerebellum and basal ganglia and their interaction regulate synchronized tapping, resulting in distinct tapping pattern abnormalities among different SCD subtypes.

Increased gene dosage of myelin protein zero causes Charcot-Marie-Tooth disease.

OBJECTIVE: On the basis of the hypothesis that copy number mutations of the genes encoding myelin compact proteins are responsible for myelin disorders in humans, we have explored the possibility of copy number mutations in patients with Charcot-Marie-Tooth disease (CMT) whose responsible genes remain undefined. METHODS: A family with 6 affected members in 3 consecutive generations, presenting with motor and sensory demyelinating polyneuropathy, was investigated. Characteristic clinical features in this pedigree include Adie pupils and substantial intrafamilial variability in the age at onset, electrophysiological findings, and clinical severity. Nucleotide sequence analyses of PMP22, MPZ, or GJB1 and gene dosage study of PMP22 did not reveal causative mutations. Hence, we applied a custom-designed array for comparative genomic hybridization (CGH) analysis to conduct a comprehensive screening of copy number mutations involving any of the known causative genes for CMT other than PMP22. RESULTS: The array CGH analyses revealed increased gene dosage involving the whole MPZ, and the flanking genes of SDHC and C1orf192. The gene dosage is estimated to be 5 copies. This mutation showed complete cosegregation with the disease phenotype in this pedigree. INTERPRETATION: The increased gene dosage of MPZ and increased expression level of MPZ mRNA emphasize the important role of the dosage of the MPZ protein in the functional integrity of peripheral nerve myelin in humans, and provide a new insight into the pathogenic mechanisms underlying CMT.

Mutations in KCND3 cause spinocerebellar ataxia type 22.

OBJECTIVE: To identify the causative gene in spinocerebellar ataxia (SCA) 22, an autosomal dominant cerebellar ataxia mapped to chromosome 1p21-q23. METHODS: We previously characterized a large Chinese family with progressive ataxia designated SCA22, which overlaps with the locus of SCA19. The disease locus in a French family and an Ashkenazi Jewish American family was also mapped to this region. Members from all 3 families were enrolled. Whole exome sequencing was performed to identify candidate mutations, which were narrowed by linkage analysis and confirmed by Sanger sequencing and cosegregation analyses. Mutational analyses were also performed in 105 Chinese and 55 Japanese families with cerebellar ataxia. Mutant gene products were examined in a heterologous expression system to address the changes in protein localization and electrophysiological functions. RESULTS: We identified heterozygous mutations in the voltage-gated potassium channel Kv4.3-encoding gene KCND3: an in-frame 3-nucleotide deletion c.679_681delTTC p.F227del in both the Chinese and French pedigrees, and a missense mutation c.1034G>T p.G345V in the Ashkenazi Jewish family. Direct sequencing of KCND3 further identified 3 mutations, c.1034G>T p.G345V, c.1013T>C p.V338E, and c.1130C>T p.T377M, in 3 Japanese kindreds. Immunofluorescence analyses revealed that the mutant p.F227del Kv4.3 subunits were retained in the cytoplasm, consistent with the lack of A-type K(+) channel conductance in whole cell patch-clamp recordings. INTERPRETATION: Our data identify the cause of SCA19/22 in patients of diverse ethnic origins as mutations in KCND3. These findings further emphasize the important role of ion channels as key regulators of neuronal excitability in the pathogenesis of cerebellar degeneration.

An open trial of long-term testosterone suppression in spinal and bulbar muscular atrophy.

INTRODUCTION: We investigated the long-term effects of leuprorelin on leg-muscle strength in spinal and bulbar muscular atrophy (SBMA). We hypothesized that testosterone suppression by leuprorelin would prevent the progression of muscle weakness. METHODS: In a prospective, long duration, open trial, 16 SBMA patients underwent medical castration with leuprorelin for 3.5 years. Chlormadinone was coadministered initially to prevent a testosterone surge. The strength of knee extension and flexion were quantitated using a torque machine. RESULTS: Our hypothesis was rejected. The leg strength measures decreased significantly with the mean reduction of 22.3-27.8%. In a post hoc analysis, the leg strength of 4 patients with higher pretreatment baseline total testosterone levels and short disease duration of 1-6 years were stronger at baseline and decreased by only 12.3-15.7% after treatment. CONCLUSIONS: Leuprorelin was not effective in this small long-term treatment trial in SBMA. The possibility that earlier treatment might be beneficial may deserve further study.

Genome-wide association study identifies a new susceptibility locus in PLA2G4C for Multiple System Atrophy.

To elucidate the molecular basis of multiple system atrophy (MSA), a neurodegenerative disease, we conducted a genome-wide association study (GWAS) in a Japanese MSA case/control series followed by replication studies in Japanese, Korean, Chinese, European and North American samples. In the GWAS stage rs2303744 on chromosome 19 showed a suggestive association ( P = 6.5 × 10 -7 ) that was replicated in additional Japanese samples ( P = 2.9 × 10 -6 . OR = 1.58; 95% confidence interval, 1.30 to 1.91), and then confirmed as highly significant in a meta-analysis of East Asian population data ( P = 5.0 × 10 -15 . Odds ratio= 1.49; 95% CI 1.35 to 1.72). The association of rs2303744 with MSA remained significant in combined European/North American samples ( P =0.023. Odds ratio=1.14; 95% CI 1.02 to 1.28) despite allele frequencies being quite different between these populations. rs2303744 leads to an amino acid substitution in PLA2G4C that encodes the cPLA2γ lysophospholipase/transacylase. The cPLA2γ-Ile143 isoform encoded by the MSA risk allele has significantly decreased transacylase activity compared with the alternate cPLA2γ-Val143 isoform that may perturb membrane phospholipids and α-synuclein biology.

Posterior column ataxia with retinitis pigmentosa in a Japanese family with a novel mutation in FLVCR1.

Posterior column ataxia with retinitis pigmentosa (PCARP) is an autosomal recessive neurodegenerative disorder characterized by retinitis pigmentosa and sensory ataxia. Previous studies of PCARP in two families showed a linkage to 1q31-q32. However, detailed investigations on the clinical presentations as well as molecular genetics of PCARP have been limited. Here, we describe a Japanese consanguineous family with PCARP. Two affected siblings suffered from childhood-onset retinitis pigmentosa and slowly progressive sensory ataxia. They also showed mild mental retardation, which has not been described in patients with PCARP. Parametric linkage analysis using high-density single nucleotide polymorphism arrays supported a linkage to the same locus. Target capture and high-throughput sequencing technologies revealed a novel homozygous c.1477G>C (G493R) mutation in FLVCR1, which cosegregated with the disease. A recent study has identified three independent mutations in FLVCR1 in the original and other families. Our results further confirmed that PCARP is caused by mutations in FLVCR1.

Cerebellar Ataxia as a Common Clinical Presentation Associated with DNMT1 p.Y511H and a Review of the Literature.

The phenotypes of patients with disease-associated variants in DNMT1 have been classified into two syndromes: hereditary sensory and autonomic neuropathy type 1E (HSAN1E, MIM614116, https://www.omim.org/ ) and autosomal dominant cerebellar ataxia, deafness, and narcolepsy (ADCA-DN, MIM604121). The amino acid codon 511 is a hotspot, and p.Y511C is the most frequently observed disease-associated variant among those in HSAN1E patients, whereas there have been only a few reports on patients with p.Y511H. In this study, we report on the cases of a kindred carrying the DNMT1 variant NM_001130823.2:c.1531 T > C (p.Y511H) presenting with the ADCA-DN phenotype. The review of the literature further revealed that later ages at onset and the presence of cerebellar ataxia are the main characteristics of patients carrying the DNMT1 p.Y511H as compared with those carrying DNMT1 p.Y511C. Although HSAN1E and ADCA-DN are proposed to be called DNMT1-complex disorders owing to their overlapping symptoms, this finding suggests a distinct genotype-phenotype correlation regarding the DNMT1 p.Y511H and p.Y511C variants.

Time Distortion in Parkinsonism.

Although animal studies and studies on Parkinson's disease (PD) suggest that dopamine deficiency slows the pace of the internal clock, which is corrected by dopaminergic medication, timing deficits in parkinsonism remain to be characterized with diverse findings. Here we studied patients with PD and progressive supranuclear palsy (PSP), 3-4 h after drug intake, and normal age-matched subjects. We contrasted perceptual (temporal bisection, duration comparison) and motor timing tasks (time production/reproduction) in supra- and sub-second time domains, and automatic versus cognitive/short-term memory-related tasks. Subjects were allowed to count during supra-second production and reproduction tasks. In the time production task, linearly correlating the produced time with the instructed time showed that the "subjective sense" of 1 s is slightly longer in PD and shorter in PSP than in normals. This was superposed on a prominent trend of underestimation of longer (supra-second) durations, common to all groups, suggesting that the pace of the internal clock changed from fast to slow as time went by. In the time reproduction task, PD and, more prominently, PSP patients over-reproduced shorter durations and under-reproduced longer durations at extremes of the time range studied, with intermediate durations reproduced veridically, with a shallower slope of linear correlation between the presented and produced time. In the duration comparison task, PD patients overestimated the second presented duration relative to the first with shorter but not longer standard durations. In the bisection task, PD and PSP patients estimated the bisection point (BP50) between the two supra-second but not sub-second standards to be longer than normal subjects. Thus, perceptual timing tasks showed changes in opposite directions to motor timing tasks: underestimating shorter durations and overestimating longer durations. In PD, correlation of the mini-mental state examination score with supra-second BP50 and the slope of linear correlation in the reproduction task suggested involvement of short-term memory in these tasks. Dopamine deficiency didn't correlate significantly with timing performances, suggesting that the slowed clock hypothesis cannot explain the entire results. Timing performance in PD may be determined by complex interactions among time scales on the motor and sensory sides, and by their distortion in memory.

A novel ferritin light chain gene mutation in a Japanese family with neuroferritinopathy: description of clinical features and implications for genotype-phenotype correlations.

Neuroferritinopathy is a hereditary neurodegenerative disorder caused by mutations in the ferritin light chain gene (FTL1). The cardinal features are progressive movement disturbance, hypoferritinemia, and iron deposition in the brain. To date, five mutations have been described in Caucasian and Japanese families, but the genotype-phenotype correlations remain to be established. We identified a novel FTL1 mutation (exon 4, c.641/642, 4-nucletotide duplication) in a Japanese family and compared the clinical traits with those previously reported. All mutations but one are insertions in exon 4, resulting in frameshifts. Clinical features are similar among patients with the same mutations. Middle-age onset chorea is common in patients with insertions in the 5' portion of exon 4 including our cases, whereas patients with insertions in the 3' portion of exon 4 develop early-onset tremor, suggesting genotype-phenotype correlations. In this family, male predominance and normal serum ferritin levels are characteristic.

Novel SLC20A2 variant in a Japanese patient with idiopathic basal ganglia calcification-1 (IBGC1) associated with dopa-responsive parkinsonism.

Idiopathic basal ganglia calcification-1 (IBGC1) is an autosomal dominant disorder characterized by calcification in the basal ganglia, which can manifest a range of neuropsychiatric symptoms, including parkinsonism. We herein describe a 64-year-old Japanese IBGC1 patient with bilateral basal ganglia calcification carrying a novel SLC20A2 variant (p.Val322Glufs*92). The patient also presented with dopa-responsive parkinsonism with decreased dopamine transporter (DAT) density in the bilateral striatum and decreased cardiac 123I-meta-iodobenzylguanidine uptake.

Prominent Spasticity and Hyperreflexia of the Legs in a Nepalese Patient with Friedreich Ataxia.

Friedreich ataxia (FRDA) is an autosomal recessive spinocerebellar ataxia caused by mutations of FXN. Hypotonus and hyporeflexia of the lower extremities are observed in most FRDA patients. Patients with hyperreflexia, called Friedreich ataxia with retained reflexes (FARR), have also been identified. We herein report the case of a 16-year-old Nepalese boy presenting with early-onset ataxia with prominent spasticity and hyperreflexia of the legs. Mutational analyses established the diagnosis of FRDA presenting as FARR. A haplotype analysis revealed that expanded alleles of the patient shared a common haplotype with Indian and European FRDA patients, suggesting that the mutation descended from a common founder.

Noncoding CGG repeat expansions in neuronal intranuclear inclusion disease, oculopharyngodistal myopathy and an overlapping disease.

Noncoding repeat expansions cause various neuromuscular diseases, including myotonic dystrophies, fragile X tremor/ataxia syndrome, some spinocerebellar ataxias, amyotrophic lateral sclerosis and benign adult familial myoclonic epilepsies. Inspired by the striking similarities in the clinical and neuroimaging findings between neuronal intranuclear inclusion disease (NIID) and fragile X tremor/ataxia syndrome caused by noncoding CGG repeat expansions in FMR1, we directly searched for repeat expansion mutations and identified noncoding CGG repeat expansions in NBPF19 (NOTCH2NLC) as the causative mutations for NIID. Further prompted by the similarities in the clinical and neuroimaging findings with NIID, we identified similar noncoding CGG repeat expansions in two other diseases: oculopharyngeal myopathy with leukoencephalopathy and oculopharyngodistal myopathy, in LOC642361/NUTM2B-AS1 and LRP12, respectively. These findings expand our knowledge of the clinical spectra of diseases caused by expansions of the same repeat motif, and further highlight how directly searching for expanded repeats can help identify mutations underlying diseases.

Novel SIL1 mutations and exclusion of functional candidate genes in Marinesco-Sjögren syndrome.

Marinesco-Sjögren syndrome (MSS) is a rare autosomal recessively inherited neurodegenerative disorder characterized by cerebellar ataxia, cataracts, mental retardation, and progressive myopathy. Recently, mutations in the SIL1 gene, which encodes an endoplasmic reticulum (ER) resident cochaperone, were identified as a major cause of MSS. We here report four novel mutations in SIL1, including the first missense substitution p.Leu457Pro described in MSS. In addition, we excluded three functional candidate genes, HSPA5, HYOU1, and AARS, as causative genes in SIL1 mutation-negative patients. To understand the mechanisms of disturbed SIL1 function, we studied the subcellular localization of the missense mutant Leu457Pro protein in COS-1 cells. Moreover, we studied a mutant protein lacking the putative C-terminal ER retrieval signal. In contrast to the wild-type protein's localization to ER and Golgi apparatus, both mutant proteins formed aggregates within the ER depending on the expression level. These data imply that aggregation of mutant proteins may contribute to MSS pathogenesis. The genetic background of a subgroup of patients with MSS remains uncovered.

Pure alexia for kana. Characterization of alexia with lesions of the inferior occipital cortex.

OBJECTIVE: To characterize reading impairments caused by lesions in the posterior occipital cortices. METHODS: We gave six patients with these lesions reading and writing tests and located a critical site for alexia using MRI and SPECT. RESULTS: The patients read three-character kana (Japanese syllabograms) nonwords, and five-character kana nonwords significantly or at a near significant level more poorly and slowly than normal subjects, whereas they read kanji (Japanese morphograms) almost correctly but more slowly. Letter-by-letter reading with a single-kana character identification impairment (in five patients), a word-length effect, kinesthetic facilitation, a lexicality effect, and minor to mild agraphia for kanji (in three patients) were observed. These deficits were characteristic of pure alexia. Alexia disappeared within a few months except in one patient who had extensive hypoperfusion in the left occipital lobe. A shared lesion was located in the left posterior fusiform/inferior occipital gyri (Area 18/19) on MRI, and there was blood flow reduction around this area on SPECT. This area coincided with the activation site for kana word covert reading in our previous study. CONCLUSIONS: These results suggest that pure alexia particularly for kana, or more generally pure alexia for letters, is caused by a lesion in the posterior inferior occipital cortex, characterized primarily by impaired kana character or letter identification, with relatively preserved kanji or word recognition.