Comprehensive investigation of RNF213 nonsynonymous variants associated with intracranial artery stenosis.

Intracranial artery stenosis (ICAS) is the most common cause of ischemic stroke worldwide. RNF213 single nucleotide variant c.14429G > A (p.Arg4810Lys, rs112735431) was recently reported to be associated with ICAS in East Asians. However, the disease susceptibility of other RNF213 variants has not been clarified. This study comprehensively investigated ICAS-associated RNF213 variants in a pool of 168 Japanese ICAS patients and 1,194 control subjects. We found 138 nonsynonymous germline variants by target resequencing of all coding exons in RNF213. Association study between ICAS patients and control subjects revealed that only p.Arg4810Lys had significant association with ICAS (P = 1.5 × 10-28, odds ratio = 29.3, 95% confidence interval 15.31-56.2 [dominant model]). Fourteen of 138 variants were rare variants detected in ICAS patients not harboring p.Arg4810Lys variant. Two of these rare variants (p.Cys118Arg and p.Leu2356Phe) consistent with variants previously reported in moyamoya disease patients characterized by stenosis of intracranial artery and association with RNF213, and three rare variants (p.Ser193Gly, p.Val1817Leu, and p.Asp3329Tyr) were found neither in control subjects and Single Nucleotide Polymorphism Database. The present findings may improve our understanding of the genetic background of intracranial artery stenosis.

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.

Expansions of intronic TTTCA and TTTTA repeats in benign adult familial myoclonic epilepsy.

Epilepsy is a common neurological disorder, and mutations in genes encoding ion channels or neurotransmitter receptors are frequent causes of monogenic forms of epilepsy. Here we show that abnormal expansions of TTTCA and TTTTA repeats in intron 4 of SAMD12 cause benign adult familial myoclonic epilepsy (BAFME). Single-molecule, real-time sequencing of BAC clones and nanopore sequencing of genomic DNA identified two repeat configurations in SAMD12. Intriguingly, in two families with a clinical diagnosis of BAFME in which no repeat expansions in SAMD12 were observed, we identified similar expansions of TTTCA and TTTTA repeats in introns of TNRC6A and RAPGEF2, indicating that expansions of the same repeat motifs are involved in the pathogenesis of BAFME regardless of the genes in which the expanded repeats are located. This discovery that expansions of noncoding repeats lead to neuronal dysfunction responsible for myoclonic tremor and epilepsy extends the understanding of diseases with such repeat expansion.

Modeling neurological diseases with induced pluripotent cells reprogrammed from immortalized lymphoblastoid cell lines.

Patient-specific induced pluripotent stem cells (iPSCs) facilitate understanding of the etiology of diseases, discovery of new drugs and development of novel therapeutic interventions. A frequently used starting source of cells for generating iPSCs has been dermal fibroblasts (DFs) isolated from skin biopsies. However, there are also numerous repositories containing lymphoblastoid B-cell lines (LCLs) generated from a variety of patients. To date, this rich bioresource of LCLs has been underused for generating iPSCs, and its use would greatly expand the range of targeted diseases that could be studied by using patient-specific iPSCs. However, it remains unclear whether patient's LCL-derived iPSCs (LiPSCs) can function as a disease model. Therefore, we generated Parkinson's disease patient-specific LiPSCs and evaluated their utility as tools for modeling neurological diseases. We established iPSCs from two LCL clones, which were derived from a healthy donor and a patient carrying PARK2 mutations, by using existing non-integrating episomal protocols. Whole genome sequencing (WGS) and comparative genomic hybridization (CGH) analyses showed that the appearance of somatic variations in the genomes of the iPSCs did not vary substantially according to the original cell types (LCLs, T-cells and fibroblasts). Furthermore, LiPSCs could be differentiated into functional neurons by using the direct neurosphere conversion method (dNS method), and they showed several Parkinson's disease phenotypes that were similar to those of DF-iPSCs. These data indicate that the global LCL repositories can be used as a resource for generating iPSCs and disease models. Thus, LCLs are the powerful tools for generating iPSCs and modeling neurological diseases.

Mutations in MME cause an autosomal-recessive Charcot-Marie-Tooth disease type 2.

OBJECTIVE: The objective of this study was to identify new causes of Charcot-Marie-Tooth (CMT) disease in patients with autosomal-recessive (AR) CMT. METHODS: To efficiently identify novel causative genes for AR-CMT, we analyzed 303 unrelated Japanese patients with CMT using whole-exome sequencing and extracted recessive variants/genes shared among multiple patients. We performed mutation screening of the newly identified membrane metalloendopeptidase (MME) gene in 354 additional patients with CMT. We clinically, genetically, pathologically, and radiologically examined 10 patients with the MME mutation. RESULTS: We identified recessive mutations in MME in 10 patients. The MME gene encodes neprilysin (NEP), which is well known to be one of the most prominent beta-amyloid (Aß)-degrading enzymes. All patients had a similar phenotype consistent with late-onset axonal neuropathy. They showed muscle weakness, atrophy, and sensory disturbance in the lower extremities. All the MME mutations could be loss-of-function mutations, and we confirmed a lack/decrease of NEP protein expression in a peripheral nerve. No patients showed symptoms of dementia, and 1 patient showed no excess Aß in Pittsburgh compound-B positron emission tomography imaging. INTERPRETATION: Our results indicate that loss-of-function MME mutations are the most frequent cause of adult-onset AR-CMT2 in Japan, and we propose that this new disease should be termed AR-CMT2T. A loss-of-function MME mutation did not cause early-onset Alzheimer's disease. Identifying the MME mutation responsible for AR-CMT could improve the rate of molecular diagnosis and the understanding of the molecular mechanisms of CMT.

Recurrent DUX4 fusions in B cell acute lymphoblastic leukemia of adolescents and young adults.

The oncogenic mechanisms underlying acute lymphoblastic leukemia (ALL) in adolescents and young adults (AYA; 15-39 years old) remain largely elusive. Here we have searched for new oncogenes in AYA-ALL by performing RNA-seq analysis of Philadelphia chromosome (Ph)-negative AYA-ALL specimens (n = 73) with the use of a next-generation sequencer. Interestingly, insertion of D4Z4 repeats containing the DUX4 gene into the IGH locus was frequently identified in B cell AYA-ALL, leading to a high level of expression of DUX4 protein with an aberrant C terminus. A transplantation assay in mice demonstrated that expression of DUX4-IGH in pro-B cells was capable of generating B cell leukemia in vivo. DUX4 fusions were preferentially detected in the AYA generation. Our data thus show that DUX4 can become an oncogenic driver as a result of somatic chromosomal rearrangements and that AYA-ALL may be a clinical entity distinct from ALL at other ages.

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.

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 medaka draft genome and insights into vertebrate genome evolution.

Teleosts comprise more than half of all vertebrate species and have adapted to a variety of marine and freshwater habitats. Their genome evolution and diversification are important subjects for the understanding of vertebrate evolution. Although draft genome sequences of two pufferfishes have been published, analysis of more fish genomes is desirable. Here we report a high-quality draft genome sequence of a small egg-laying freshwater teleost, medaka (Oryzias latipes). Medaka is native to East Asia and an excellent model system for a wide range of biology, including ecotoxicology, carcinogenesis, sex determination and developmental genetics. In the assembled medaka genome (700 megabases), which is less than half of the zebrafish genome, we predicted 20,141 genes, including approximately 2,900 new genes, using 5'-end serial analysis of gene expression tag information. We found single nucleotide polymorphisms (SNPs) at an average rate of 3.42% between the two inbred strains derived from two regional populations; this is the highest SNP rate seen in any vertebrate species. Analyses based on the dense SNP information show a strict genetic separation of 4 million years (Myr) between the two populations, and suggest that differential selective pressures acted on specific gene categories. Four-way comparisons with the human, pufferfish (Tetraodon), zebrafish and medaka genomes revealed that eight major interchromosomal rearrangements took place in a remarkably short period of approximately 50 Myr after the whole-genome duplication event in the teleost ancestor and afterwards, intriguingly, the medaka genome preserved its ancestral karyotype for more than 300 Myr.