Nozomu Sato

Pentanucleotide repeats at the spinocerebellar ataxia type 31 (SCA31) locus in Caucasians

Spinocerebellar ataxia type 31 (SCA31) was recently discovered to be caused by 2.5- to 3.8-kb-long complex pentanucleotide repeats containing (TGGAA)n, (TAGAA)n, and (TAAAA)n in an intronic region shared by 2 different genes, BEAN (brain expressed, associated with Nedd4) and TK2 (thymidine kinase 2), in chromosome 16q22.1.1,2 Among the 3 pentanucleotide repeats, (TGGAA)n was the only one in which large repeats segregated with the phenotype, suggesting its importance in pathogenesis.2 SCA31 is considered one of a growing number of neuromuscular diseases with RNA-mediated gain-of-function mechanism such as myotonic dystrophies type 1 and 2, SCA8, SCA10, and fragile X–tremor ataxia syndrome.3 While SCA31 is one of the most common SCAs in Japan, it is not known whether it is prevalent in Caucasians. In this study, we directly tested the SCA31 mutation in large European cohorts of SCA families. ### Methods. French and German cohorts of SCA index patients without known mutations were analyzed. The French cohort (n = 271 patients) was collected through a national ataxia network, and the German cohort (n = 49 patients) was collected at the Ludwig-Maximilians-University Munich, Germany. Healthy controls comprising 285 French and 303 German individuals were also tested. The SCA31 locus and its nearby markers were analyzed as previously described.2 #### Standard protocol approvals, registrations, and patient consents. This study was approved by the respective institutional review boards. ### Results. One out of 49 German and 31 out of 271 French SCA index patients carried abnormal expansions (figure,A). Two of these patients carried expansions homozygously. DNA sequencing revealed that the expansions were located at the same position as in Japanese …

Regulatory Role of RNA Chaperone TDP-43 for RNA Misfolding and Repeat-Associated Translation in SCA31

Microsatellite expansion disorders are pathologically characterized by RNA foci formation and repeat-associated non-AUG (RAN) translation. However, their underlying pathomechanisms and regulation of RAN translation remain unknown. We report that expression of expanded UGGAA (UGGAAexp) repeats, responsible for spinocerebellar ataxia type 31 (SCA31) in Drosophila, causes neurodegeneration accompanied by accumulation of UGGAAexp RNA foci and translation of repeat-associated pentapeptide repeat (PPR) proteins, consistent with observations in SCA31 patient brains. We revealed that motor-neuron disease (MND)-linked RNA-binding proteins (RBPs), TDP-43, FUS, and hnRNPA2B1, bind to and induce structural alteration of UGGAAexp. These RBPs suppress UGGAAexp-mediated toxicity in Drosophila by functioning as RNA chaperones for proper UGGAAexp folding and regulation of PPR translation. Furthermore, nontoxic short UGGAA repeat RNA suppressed mutated RBP aggregation and toxicity in MND Drosophila models. Thus, functional crosstalk of the RNA/RBP network regulates their own quality and balance, suggesting convergence of pathomechanisms in microsatellite expansion disorders and RBP proteinopathies.

Spinocerebellar ataxia type 36 exists in diverse populations and can be caused by a short hexanucleotide GGCCTG repeat expansion

Objective Spinocerebellar ataxia 36 (SCA36) is an autosomal-dominant neurodegenerative disorder caused by a large (>650) hexanucleotide GGCCTG repeat expansion in the first intron of the NOP56 gene. The aim of this study is to clarify the prevalence, clinical and genetic features of SCA36. Methods The expansion was tested in 676 unrelated SCA index cases and 727 controls from France, Germany and Japan. Clinical and neuropathological features were investigated in available family members. Results Normal alleles ranged between 5 and 14 hexanucleotide repeats. Expansions were detected in 12 families in France (prevalence: 1.9% of all French SCAs) including one family each with Spanish, Portuguese or Chinese ancestry, in five families in Japan (1.5% of all Japanese SCAs), but were absent in German patients. All the 17 SCA36 families shared one common haplotype for a 7.5 kb pairs region flanking the expansion. While 27 individuals had typically long expansions, three affected individuals harboured small hexanucleotide expansions of 25, 30 and 31 hexanucleotide repeat-units, demonstrating that such a small expansion could cause the disease. All patients showed slowly progressive cerebellar ataxia frequently accompanied by hearing and cognitive impairments, tremor, ptosis and reduced vibration sense, with the age at onset ranging between 39 and 65 years, and clinical features were indistinguishable between individuals with short and typically long expansions. Neuropathology in a presymptomatic case disclosed that Purkinje cells and hypoglossal neurons are affected. Conclusions SCA36 is rare with a worldwide distribution. It can be caused by a short GGCCTG expansion and associates various extracerebellar symptoms.

Prevalence of inositol 1, 4, 5-triphosphate receptor type 1 gene deletion, the mutation for spinocerebellar ataxia type 15, in Japan screened by gene dosage

Spinocerebellar ataxia type 15 (SCA15) is an autosomal dominant neurodegenerative disorder clinically characterized by late-onset, slowly progressive pure cerebellar ataxia. This disease is caused by a heterozygous deletion of the inositol 1, 4, 5-triphosphate receptor type 1 (ITPR1) gene, suggesting that haploinsufficiency of the receptor function is the plausible disease mechanism. To clarify the prevalence of SCA15 in Japan, we designed four sets of probes and primers in different regions of ITPR1 and performed TaqMan PCR assay to search for gene deletions in 226 index SCA patients excluded for repeat expansion disorders. Deletion was found in only one patient, in whom gait ataxia started at 51 years of age and progressed to show cerebellar ataxia. This study demonstrates a simple but efficient method for screening ITPR1 deletion. We also conclude that ITPR1 gene deletions are much rare in Japan than in Europe, comprising only 0.3% in all SCAs in Japan.

Redefining the disease locus of 16q22.1-linked autosomal dominant cerebellar ataxia

AbstractThe 16q22.1-linked autosomal dominant cerebellar ataxia (16q-ADCA; Online Mendelian Inheritance in Man [OMIN] #117210) is one of the most common ADCAs in Japan. Previously, we had reported that the patients share a common haplotype by founder effect and that a C-to-T substitution (−16C>T) in the puratrophin-1 gene was strongly associated with the disease. However, recently, an exceptional patient without the substitution was reported, indicating that a true pathogenic mutation might be present elsewhere. In this study, we clarified the disease locus more definitely by the haplotype analysis of families showing pure cerebellar ataxia. In addition to microsatellite markers, the single nucleotide polymorphisms (SNPs) that we identified on the disease chromosome were examined to confirm the borders of the disease locus. The analysis of 64 families with the −16C>T substitution in the puratrophin-1 gene revealed one family showing an ancestral recombination event between SNP04 and SNP05 on the disease chromosome. The analysis of 22 families without identifiable genetic mutations revealed another family carrying the common haplotype centromeric to the puratrophin-1 gene, but lacking the −16C>T substitution in this gene. We concluded that the disease locus of 16q-ADCA was definitely confined to a 900-kb genomic region between the SNP04 and the −16C>T substitution in the puratrophin-1 gene in 16q22.1.

A Novel Mutation in ELOVL4 Leading to Spinocerebellar Ataxia (SCA) With the Hot Cross Bun Sign but Lacking Erythrokeratodermia: A Broadened Spectrum of SCA34

IMPORTANCE Although mutations in 26 causative genes have been identified in the spinocerebellar ataxias (SCAs), the causative genes in a substantial number of families with SCA remain unidentified. OBJECTIVE To identify the causative gene of SCA in 2 Japanese families with distinct neurological symptoms and radiological presentations. DESIGN, SETTING, AND PARTICIPANTS Clinical genetic study at a referral center of 11 members from 2 Japanese families, which started in 1997. MAIN OUTCOMES AND MEASURES Results of neurological examinations and radiological evaluations. The causative mutation was identified using genome-wide linkage analysis and next-generation sequencing. RESULTS Affected members (9 of 11 members [81.8%]) showed slowly progressive cerebellar ataxia (all 9 members [100%]), ocular movement disturbance (all 9 members [100%]), and pyramidal tract signs (8 of 9 members [88.9%]) with an age at onset between the second and sixth decades of life. Besides cerebellar and pontine atrophy, magnetic resonance imaging of the brain revealed the hot cross bun sign (4 of 6 members [66.7%]), pontine midline linear hyperintensity (2 of 6 members [33.3%]), or high intensity in the middle cerebellar peduncle (1 of 6 members [16.7%]), which are all reminiscent of multiple system atrophy in tested patients. Using linkage analysis combined with exome and whole-genome sequencing, we identified a novel heterozygous mutation in the ELOVL fatty acid elongase 4 (ELOVL4) gene (c.736T>G, p.W246G) in both families. Haplotype analysis indicated that it was unlikely that these 2 Japanese families shared a common ancestor. Although a missense mutation in ELOVL4 (c.504G>C, p.L168F) was recently reported to be associated with SCA with erythrokeratodermia variabilis (SCA34) in a French-Canadian family, signs of erythrokeratodermia variabilis were absent in our families. CONCLUSIONS AND RELEVANCE Combined with the results of the family with SCA34 reported previously, this report confirms that mutations in ELOVL4 can cause dominantly inherited neurodegeneration severely affecting the cerebellum and brainstem. We should be aware that the presence of multiple system atrophy-like features on magnetic resonance imaging scans, together with cerebellar and brainstem atrophy, suggests SCA34, even when erythrokeratodermia variabilis is absent. The present study further broadened the spectrum of the clinical presentations of SCA34 associated with mutations in ELOVL4, which is involved in the biosynthesis of very long-chain fatty acids.

Cytoplasmic location of α1A voltage-gated calcium channel C-terminal fragment (Cav2.1-CTF) aggregate is sufficient to cause cell death.

The human α1A voltage-dependent calcium channel (Cav2.1) is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C)-tail contains a small poly-glutamine (Q) tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6). A recent study has shown that a 75-kDa C-terminal fragment (CTF) containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (r)CTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12) cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range) than with Q13 (normal-length). Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB) and phosphorylated-CREB (p-CREB) in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei.

Reduced brain-derived neurotrophic factor (BDNF) mRNA expression and presence of BDNF-immunoreactive granules in the spinocerebellar ataxia type 6 (SCA6) cerebellum

Spinocerebellar ataxia type 6 (SCA6) is an autosomal‐dominant neurodegenerative disorder caused by a small expansion of tri‐nucleotide (CAG) repeat encoding polyglutamine (polyQ) in the gene for α1A voltage‐dependent calcium channel (Cav2.1). Thus, this disease is one of the nine neurodegenerative disorders called polyQ diseases. The Purkinje cell predominant neuronal loss is the characteristic neuropathology of SCA6, and a 75‐kDa carboxy‐terminal fragment (CTF) of Cav2.1 containing polyQ, which remains soluble in normal brains, becomes insoluble in the cytoplasm of SCA6 Purkinje cells. Because the suppression of the brain‐derived neurotrophic factor (BDNF) expression is a potentially momentous phenomenon in many other polyQ diseases, we implemented BDNF expression analysis in SCA6 human cerebellum using quantitative RT‐PCR for the BDNF mRNA, and by immunohistochemistry for the BDNF protein. We observed significantly reduced BDNF mRNA levels in SCA6 cerebellum (n = 3) compared to controls (n = 6) (Mann–Whitney U‐test, P = 0.0201). On immunohistochemistry, BDNF protein was only weakly stained in control cerebellum. On the other hand, we found numerous BDNF‐immunoreactive granules in dendrites of SCA6 Purkinje cells. We did not observe similar BDNF‐immunoreactive granules in other polyQ diseases, such as Huntingtons disease or SCA2. As we often observed that the 1C2‐positive Cav2.1 aggregates existed more proximally than the BDNF‐positive granules in the dendrites, we speculated that the BDNF protein trafficking in dendrites may be disturbed by Cav2.1 aggregates in SCA6 Purkinje cells. We conclude that the SCA6 pathogenic mechanism associates with the BDNF mRNA expression reduction and abnormal localization of BDNF protein.

Sequence configuration of spinocerebellar ataxia type 8 repeat expansions in a Japanese cohort of 797 ataxia subjects

Spinocerebellar ataxia type 8 (SCA8), an autosomal dominant neurodegenerative disorder showing slowly progressive cerebellar ataxia, is caused by a tri-nucleotide CTG repeat expansion (CTGexp) in the SCA8 gene. As the CTGexp is not fully penetrant, the significance of screening CTGexp in ataxia subjects remains obscure. We tested SCA8 CTGexp in a cohort of 797 ataxia subjects, and if present, its sequence configuration was analyzed. CTGexp was found in 16 alleles from 14 individuals, 2 of which was homozygous for CTGexp. Nucleotide sequencing disclosed 3 types of CTGexp sequence configurations: uninterrupted CTGexp, tri-nucleotide CTA interruption and CCG interruption. The 2 individuals with homozygous expansions were both sporadic cases with clinical features compatible with SCA8, supporting gene dosage effect. Seven out of 14 CTGexp-positive subjects were also carriers of other SCA expansions [Machado-Joseph disease (n=1), SCA6 (n=3) and SCA31 (n=3)], whereas 7 others were not complicated with such major SCAs. Ages of onset in subjects with pure CTGexp tended to be earlier than those with interrupted CTGexp among the 7 subjects not complicated by major SCAs, suggesting that pure CTGexp have stronger pathogenic effect than interrupted CTGexps. The present study underscores importance of disclosing sequence configuration when testing SCA8.

Spinocerebellar Ataxia Type 31 with Blepharospasm

A 58-year-old man consulted our hospital due to a 2-year history of dysarthria and a 1-month history of blepharospasm. In addition to the ataxic dysarthria and blepharospasm, a neurological examination demonstrated slight ataxia of the trunk and lower limbs. Brain MRI demonstrated atrophy of the upper portion of the cerebellar vermis. Gene analysis established a diagnosis of spinocerebellar ataxia type 31 (SCA31). Single photon emission computed tomography (SPECT) with the three-dimensional stereotaxic ROI template (3DSRT) software program demonstrated hyperperfusion in the lenticular nucleus and thalamus. Although the association between SCA31 and blepharospasm in our patient remains unclear, we considered that this combination might be more than coincidental.