Yaeko Ichikawa

Mutations in COQ2 in familial and sporadic multiple-system atrophy the multiple-system atrophy research collaboration

BACKGROUND Multiple-system atrophy is an intractable neurodegenerative disease characterized by autonomic failure in addition to various combinations of parkinsonism, cerebellar ataxia, and pyramidal dysfunction. Although multiple-system atrophy is widely considered to be a nongenetic disorder, we previously identified multiplex families with this disease, which indicates the involvement of genetic components. METHODS In combination with linkage analysis, we performed whole-genome sequencing of a sample obtained from a member of a multiplex family in whom multiple-system atrophy had been diagnosed on autopsy. We also performed mutational analysis of samples from members of five other multiplex families and from a Japanese series (363 patients and two sets of controls, one of 520 persons and one of 2383 persons), a European series (223 patients and 315 controls), and a North American series (172 patients and 294 controls). On the basis of these analyses, we used a yeast complementation assay and measured enzyme activity of parahydroxybenzoate-polyprenyl transferase. This enzyme is encoded by the gene COQ2 and is essential for the biosynthesis of coenzyme Q10. Levels of coenzyme Q10 in lymphoblastoid cells and brain tissue were measured on high-performance liquid chromatography. RESULTS We identified a homozygous mutation (M78V-V343A/M78V-V343A) and compound heterozygous mutations (R337X/V343A) in COQ2 in two multiplex families. Furthermore, we found that a common variant (V343A) and multiple rare variants in COQ2, all of which are functionally impaired, are associated with sporadic multiple-system atrophy. The V343A variant was exclusively observed in the Japanese population. CONCLUSIONS Functionally impaired variants of COQ2 were associated with an increased risk of multiple-system atrophy in multiplex families and patients with sporadic disease, providing evidence of a role of impaired COQ2 activities in the pathogenesis of this disease. (Funded by the Japan Society for the Promotion of Science and others.).

Mutations in KCND3 Cause Spinocerebellar Ataxia Type 22

To identify the causative gene in spinocerebellar ataxia (SCA) 22, an autosomal dominant cerebellar ataxia mapped to chromosome 1p21‐q23.

C9ORF72 Repeat Expansion in Amyotrophic Lateral Sclerosis in the Kii Peninsula of Japan

BACKGROUND In the Kii peninsula of Japan, high prevalences of amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia complex have been reported. There are 2 major foci with a high prevalence, which include the southernmost region neighboring the Koza River (Kozagawa and Kushimoto towns in Wakayama prefecture) and the Hohara district (Mie prefecture). OBJECTIVE To delineate the molecular basis of ALS in the Kii peninsula of Japan, we analyzed hexanucleotide repeat expansion in the chromosome 9 open reading frame 72 (C9ORF72) gene, which has recently been identified as a frequent cause of ALS and frontotemporal dementia in the white population. DESIGN Case series. SETTING University hospitals. PATIENTS Twenty-one patients (1 familial patient and 20 sporadic patients) with ALS from Wakayama prefecture, and 16 patients with ALS and 16 patients with parkinsonism-dementia complex originating from Mie prefecture surveyed in 1994 through 2011 were enrolled in the study. In addition, 40 probands with familial ALS and 217 sporadic patients with ALS recruited from other areas of Japan were also enrolled in this study. MAIN OUTCOME MEASURES After screening by repeat-primed polymerase chain reaction, Southern blot hybridization analysis was performed to confirm the expanded alleles. RESULTS We identified 3 patients with ALS (20%) with the repeat expansion in 1 of the 2 disease foci. The proportion is significantly higher than those in other regions in Japan. Detailed haplotype analyses revealed an extended shared haplotype in the 3 patients with ALS, suggesting a founder effect. CONCLUSIONS Our findings indicate that the repeat expansion partly accounts for the high prevalence of ALS in the Kii peninsula.

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.

The genomic structure and expression of MJD, the Machado-Joseph disease gene

AbstractMachado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder that is clinically characterized by cerebellar ataxia and various associated symptoms. The disease is caused by an unstable expansion of the CAG repeat in the MJD gene. This gene is mapped to chromosome 14q32.1. To determine its genomic structure, we constructed a contig composed of six cosmid clones and eight bacterial artificial chromosome (BAC) clones. It spans approximately 300kb and includes MJD. We also determined the complete sequence (175,330bp) of B445M7, a human BAC clone that contains MJD. The MJD gene was found to span 48,240bp and to contain 11 exons. Northern blot analysis showed that MJD mRNA is ubiquitously expressed in human tissues, and in at least four different sizes; namely, 1.4, 1.8, 4.5, and 7.5kb. These different mRNA species probably result from differential splicing and polyadenylation, as shown by sequences of the 21 independent cDNA clones isolated after the screening of four human cDNA libraries prepared from whole brain, caudate, retina, and testis. The sequences of these latter clones relative to the MJD gene in B445M7 indicate that there are three alternative splicing sites and eight polyadenylation signals in MJD that are used to generate the differently sized transcripts.

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.

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.

Variants associated with Gaucher disease in multiple system atrophy

Glucocerebrosidase gene (GBA) variants that cause Gaucher disease are associated with Parkinson disease (PD) and dementia with Lewy bodies (DLB). To investigate the role of GBA variants in multiple system atrophy (MSA), we analyzed GBA variants in a large case–control series.

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 SMRTTM 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.

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 proteins 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.