Hirosato Tanaka

Unstable expansion of CAG repeat in hereditary dentatorubral–pallidoluysian atrophy (DRPLA)

Hereditary dentatorubral–pallidoluysian atrophy (DRPLA) is an autosomal dominant neurologic disorder characterized by variable combinations of myoclonus, epilepsy, cerebellar ataxia, choreoathetosis and dementia. By specifically searching published brain cDNA sequences for the presence of CAG repeats we identified unstable expansion of a CAG in a gene on chromosome 12 in all the 22 DRPLA patients examined. A good correlation between the size of the CAG repeat expansion and the ages of disease onset is found in this group. Patients with earlier onset tended to have a phenotype of progressive myoclonus epilepsy and larger expansions. We propose that the wide variety of clinical manifestations of DRPLA can now be explained by the variable unstable expansion of the CAG repeat.

Identification of the spinocerebellar ataxia type 2 gene using a direct identification of repeat expansion and cloning technique, DIRECT.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant, neurodegenerative disorder that affects the cerebellum and other areas of the central nervous system. We have devised a novel strategy, the direct identification of repeat expansion and cloning technique (DIRECT), which allows selective detection of expanded GAG repeats and cloning of the genes involved. By applying DIRECT, we identified an expanded CAG repeat of the gene for SCA2. CAG repeats of normal alleles range in size from 15 to 24 repeat units, while those of SCA2 chromosomes are expanded to 35 to 59 repeat units. The SCA2 cDNA is predicted to code for 1,313 amino acids — with the CAG repeats coding for a polyglutamine tract. DIRECT is a robust strategy for identification of pathologically expanded trinucleotide repeats and will dramatically accelerate the search for causative genes of neuropsychiatric diseases caused by trinucleotide repeat expansions.

Close Associations between Prevalences of Dominantly Inherited Spinocerebellar Ataxias with CAG-Repeat Expansions and Frequencies of Large Normal CAG Alleles in Japanese and Caucasian Populations

To test the hypothesis that the frequencies of normal alleles (ANs) with a relatively large number of CAG repeats (large ANs) are related to the prevalences of the dominant spinocerebellar ataxias (SCAs)-SCA types 1, 2, 3 (Machado-Joseph disease), 6, and dentatorubral-pallidoluysian atrophy (DRPLA)-we investigated the relative prevalences of these diseases in 202 Japanese and 177 Caucasian families and distributions of the number of CAG repeats of ANs at these disease loci in normal individuals in each population. The relative prevalences of SCA1 and SCA2 were significantly higher in Caucasian pedigrees (15% and 14%, respectively) than in Japanese pedigrees (3% and 5%, respectively), corresponding to the observation that the frequencies of large ANs of SCA1 (alleles >30 repeats) and of SCA2 (alleles >22 repeats) were significantly higher in Caucasians than in Japanese. The relative prevalences of MJD/SCA3, SCA6, and DRPLA were significantly higher in Japanese pedigrees (43%, 11%, and 20%, respectively) than in Caucasian pedigrees (30%, 5%, and 0%, respectively), corresponding to the observation that the frequencies of large ANs of MJD/SCA3 (>27 repeats), SCA6 (>13 repeats), and DRPLA (>17 repeats) were significantly higher in Japanese than in Caucasians. The close correlations of the relative prevalences of the dominant SCAs with the distributions of large ANs strongly support the assumption that large ANs contribute to generation of expanded alleles (AEs) and the relative prevalences of the dominant SCAs.

Japanese families with autosomal dominant pure cerebellar ataxia map to chromosome 19p13.1-p13.2 and are strongly associated with mild CAG expansions in the spinocerebellar ataxia type 6 gene in chromosome 19p13.1.

Autosomal dominant cerebellar ataxia is a group of clinically and genetically heterogeneous disorders. We carried out genomewide linkage analysis in 15 families with autosomal dominant pure cerebellar ataxia (ADPCA). Evidence for linkage to chromosome 19p markers was found in nine families, and combined multipoint analysis refined the candidate region to a 13.3-cM interval in 19p13.1-p13.2. The remaining six families were excluded for this region. Analysis of CAG-repeat expansion in the alpha1A-voltage-dependent calcium channel (CACNL1A4) gene lying in 19p13.1, recently identified among 8 small American kindreds with ADPCA (spinocerebellar ataxia type 6 [SCA6]), revealed that 8 of the 15 families studied had similar, very small expansion in this gene: all affected individuals had larger alleles (range of CAG repeats 21-25), compared with alleles observed in neurologically normal Japanese (range 5-20 repeats). Inverse correlation between the CAG-repeat number and the age at onset was found in affected individuals with expansion. The number of CAG repeats in expanded chromosomes was completely stable within each family, which was consistent with the fact that anticipation was not statistically proved in the SCA6 families that we studied. We conclude that more than half of Japanese cases of ADPCA map to 19p13.1-p13.2 and are strongly associated with the mild CAG expansion in the SCA6/CACNL1A4 gene.

Atrophy of the cerebellum and brainstem in dentatorubral pallidoluysian atrophy. Influence of CAG repeat size on MRI findings.

To elucidate how the size of the expanded CAG repeat of the gene for dentatorubral pallidoluysian atrophy (DRPLA) and other factors affect the atrophy of the brainstem and cerebellum, and the appearance of high-intensity signals on T2-weighted MRI of the cerebral white matter of patients with DRPLA, we quantitatively analyzed the MRI findings of 26 patients with DRPLA, the diagnosis of which was confirmed by molecular analysis of the DRPLA gene. When we classified the patients into two groups based on the size of the expanded CAG repeat of the DRPLA gene (group 1, number of CAG repeat units≥66; group 2, number of CAG repeat units ≤65), we found strong inverse correlations between the age at MRI and the areas of midsagittal structures of the cerebellum and brainstem in group 1 but not in group 2. Multiple regression analysis, however, revealed that both the patients age at MRI and the size of the expanded CAG repeat correlated with the areas of midsagittal structures. Involvement of the cerebral white matter as detected on T2-weighted images was observed more frequently in patients belonging to group 2 than in group 1 patients. Furthermore it was demonstrated that high-intensity signals can be detected on T2-weighted images of the cerebral white matter of patients with a largely expanded CAG repeat (group 1) in their thirties. These results suggest that patient age as well as the size of the expanded CAG repeat are related to the degree of atrophy of the brainstem and cerebellum, and the white matter changes in patients with DRPLA.

Structure of the data encoding β-1,3-glucanase A1 of Bacillus circulans WL-12

Abstract The nucleotide sequence of the glcA gene encoding the precursor of extracellular β-1,3-glucanase (βGl), Al, a polysaccharidase produced by Bacillus circulans WL-12, was determined. The putative glcA gene was 2046 bp long, encoding a polypeptide of 682 amino acids (aa). The N-terminal aa sequence of βGl produced in Escherichia coli harboring the glcA plasmid was identical to that of βGl A1 prepared from the culture fluid of BitB. circulans WL-12. In both proteins, cleavage of the signal sequence of pre-βGl occurred between Ala-38 and Ala-39 of the predicted sequences.

Linkage mapping of the gene for Charcot-Marie-Tooth disease type 2 to chromosome 1p (CMT2A) and the clinical features of CMT2A

Charcot-Marie-Tooth disease type 2 (CMT2) is characterized by a motor conduction velocity of the median nerve of >38 m/sec and is a genetically heterogeneous disorder with at least three loci identified: CMT2A (1p35-36), CMT2B (3q13-22), CMT2C (not linked to any known loci), and CMT2D (7p14). In this study, we performed linkage analysis of two Japanese CMT2 families using markers flanking the CMT2A, CMT2B, and CMT2D loci. The highest cumulative multipoint lod score of 3.69 was obtained at D1S244. The CMT2B and CMT2D loci were excluded by the results of linkage analysis performed using markers D3S1551, D3S1290, and D7S484. The clinical features of the CMT2A affecting the two families include similar levels of muscle weakness of the posterior and anterior tibial muscles, tendon reflexes preserved in upper extremities but reduced or absent in lower extremities, no enlargement of the peripheral nerves, and mild sensory disturbance in only 20% of affected individuals.

Cloning and Sequencing of chiC Gene of Bacillus circulans WL-12 and Relationship of Its Product to Some Other Chitinases and Chitinase-Like Proteins

Abstract To clarify the roles of individual chitinases comprising in the chitinase system of Bacillus circulans WL-12, the gene ( chiC ) encoding chitinase C, a minor constituent of the chitinase system, was cloned and expressed in Escherichia coli . The predicted product of the chiC gene is 491 amino acids long with a calculated molecular mass of 53,447 Da. The N-terminal portion of the deduced polypeptide exhibited 46.8% amino acid homology with the catalytic domain of chitinase A1 of this bacterium, suggesting that the chitinase encoded by the chiC gene is comprised of an N-terminal catalytic domain and a C-terminal domain with unknown function. Two bands of chitinases with estimated molecular masses of 55 kDa and 40 kDa were detected on SDS-PAGE of the periplasmic fraction of E. coli carrying the cloned chiC gene. Chitinase C of B. circulans WL-12 is practically identical to the 40 kDa chitinase detected in E. coli based on their N-terminal amino acid sequences, isoelectric points and estimated sizes, and corresponds to the catalytic domain of the initial product of the chiC gene (55 kDa chitinase, designated as chitinase C1). Comparison of domain organizations of chitinases, of this bacterium, sequenced so far suggested that they represent two types of catalytic domains and that domain shuffling occurred relatively recently giving rise to three chitinases. Sequence comparison and the evolutionary relationship of chitinase C1 with other chitinases and chitinase-like proteins are also discussed.

Machado-Joseph disease in four Chinese pedigrees Molecular analysis of 15 patients including two juvenile cases and clinical correlations

Article abstract-Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder associated with the expansion of a (CAG)n array in the MJD1 gene. We analyzed the sizes of the (CAG)n array using DNA samples from 61 members of four Chinese MJD families and 18 Chinese normal control subjects and confirmed that the (CAG)n array in 15 MJD chromosomes was expanded to 72-86 repeat units. There were no subjects with (CAG)n array sizes intermediate between those of normal and MJD affected groups. Meanwhile, we found a significant negative correlation between the age of onset of symptoms and (CAG)n array size. The largest (CAG)n array of 86 repeat units was in the youngest patient, whose age of onset was 5 years. The intergenerational increase in number of CAG repeat units was associated with the clinical phenomenon of anticipation. NEUROLOGY 1997;48: 482-485

C-Terminal domain of β-1,3-glucanase H in Bacillus circulans IAM1165 has a role in binding to insoluble β-1,3-glucan

The deduced amino acid sequences of 72‐kDa β‐1,3‐glucanase from Bacillus circulans WL‐12 (GlcA) and 91‐kDa enzyme from B. circulans IAM1165 (BglH) are highly homologous, except that the latter has an additional long C‐terminal region composed of 192 amino acid residues. Two mutant enzymes (BglH deprived of the C‐terminal region and GlcA with the C‐terminal region added) were constructed. The enzymes possessing the C‐terminal region bound more abundantly to pachyman (insoluble β‐1,3‐glucan) and Aspergillus oryzae cell wall than those not possessing the region. This indicates that the C‐terminal region participated in binding of the enzymes to insoluble β‐1,3‐glucan.