Biomedical subjects

Yukiko K Hayashi

Publications and source records attributed to Yukiko K Hayashi.

At least 19 recordsLinked to original sources

PubMed2007

Unfolded protein response and aggresome formation in hereditary reducing-body myopathy.

Reducing-body myopathy (RBM) is a rare myopathy characterized by the presence of unique sarcoplasmic inclusions called reducing bodies (RBs). We characterized the aggresomal features of RBs that contained gamma-tubulin, ubiquitin, and endoplasmic reticulum (ER) chaperones, together with a set of membrane proteins, in a family with hereditary RBM. Increased messenger ribonucleic acid and protein levels of a molecular chaperone, glucose-related protein 78, were also observed. These results suggest that the unfolded protein response caused by the accumulation of misfolded proteins in the endoplasmic reticulum plays an important role in the formation of RBs.

Adenosine Triphosphatases↗

PubMed2006

A Gne knockout mouse expressing human V572L mutation develops features similar to distal myopathy with rimmed vacuoles or hereditary inclusion body myopathy.

Distal myopathy with rimmed vacuoles (DMRV) or hereditary inclusion myopathy (h-IBM) is an early adult-onset distal myopathy caused by mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene which encodes for a bifunctional enzyme involved in sialic acid biosynthesis. It is pathologically characterized by the presence of rimmed vacuoles especially in atrophic fibers, which also occasionally contain congophilic materials that are immunoreactive to beta-amyloid, lysosomal proteins, ubiquitin and tau proteins. To elucidate the pathomechanism of this myopathy and to explore the treatment options, we generated a mouse model of DMRV/h-IBM. We knocked out the Gne gene in the mouse, but this resulted in embryonic lethality. We therefore generated a transgenic mouse that expressed the human GNEV572L mutation, which is the most prevalent among Japanese DMRV patients, and crossed this with Gne((+/-)) mouse to obtain Gne((-/-))hGNEV572L-Tg. Interestingly, these mice exhibit marked hyposialylation in serum, muscle and other organs. Reduction in motor performance in these mice can only be seen from 30 weeks of age. A compelling finding is the development of beta-amyloid deposition in myofibers by 32 weeks, which clearly precedes rimmed vacuole formation at 42 weeks. These results show that the Gne((-/-)) hGNEV572L-Tg mouse mimics the clinical, histopathological and biochemical features of DMRV/h-IBM, making it useful for understanding the pathomechanism of this myopathy and for employing different strategies for therapy. Our findings underscore the notion that hyposialylation plays an important role in the pathomechanism of DMRV/h-IBM.

Amyloid beta-Peptides↗

PubMed2006

A novel FKRP gene mutation in a Taiwanese patient with limb-girdle muscular dystrophy 2I.

Limb-girdle muscular dystrophy (LGMD) is a group of hereditary muscle diseases with preferential involvement of the shoulder and pelvic girdle muscles, but with no pathognomonic features as in facioscapulohumeral and congenital muscular dystrophies. We report 18-year-old female with progressive shoulder and pelvic muscle weakness. She had marked restrictive pulmonary dysfunction. Echocardiogram showed mild decrease in ejection fraction of 52% (normal: >55%). She was first seen in our hospital at age 2 years with progressive proximal muscle weakness and elevated creatine kinase (CK) level to 15,290 IU/L, with what clinically and pathologically appeared to be steroid-responsive inflammatory myopathy. She responded dramatically to steroid therapy. Progressive proximal muscle weakness began again at age 8 years. Serum CK was 14,910 IU/L. She was wheelchair-bound by age 12. Muscle biopsy showed dystrophic changes without inflammation with reduced immunoreactivity to an antibody against sugar chain (VIA4-1) of alpha-dystroglycan. On laminin overlay assay, there was a nearly complete loss of laminin-binding activity to alpha-dystroglycan. Genetic analysis of fukutin-related protein (FKRP) gene revealed a novel compound heterozygous mutation of c.823C>T (p.R275C) and c.948delC, confirming the diagnosis of LGMD2I, the first reported case in East Asia.

Adolescent↗

PubMed2006

Central core disease is due to RYR1 mutations in more than 90% of patients.

Ryanodine receptor 1 (RYR1) gene mutations are associated with central core disease (CCD), multiminicore disease (MmD) and malignant hyperthermia (MH), and have been reported to be responsible for 47-67% of patients with CCD and rare cases with MmD. However, to date, the true frequency and distribution of the mutations along the RYR1 gene have not been determined yet, since mutation screening has been limited to three 'hot spots', with particular attention to the C-terminal region. In this study, 27 unrelated Japanese CCD patients were included. Clinical histories and muscle biopsies were carefully reviewed. We sequenced all the 106 exons encoding RYR1 with their flanking exon-intron boundaries, and identified 20 novel and 3 previously reported heterozygous missense mutations in 25 of the 27 CCD patients (93%), which is a much higher mutation detection rate than that perceived previously. Among them, six were located outside the known 'hot spots'. Sixteen of 27 (59%) CCD patients had mutations in the C-terminal 'hot spot'. Three CCD patients had a probable autosomal recessive disease with two heterozygous mutations. Patients with C-terminal mutations had earlier onset and rather consistent muscle pathology characterized by the presence of distinct cores in almost all type 1 fibres, interstitial fibrosis and type 2 fibre deficiency. In contrast, patients with mutations outside the C-terminal region had milder clinical phenotype and harbour more atypical cores in their muscle fibres. We also sequenced two genes encoding RYR1-associated proteins as candidate causative genes for CCD: the 12 kD FK506-binding protein (FKBP12) and the alpha1 subunit of L-type voltage-dependent calcium channel or dihydropyridine receptor (CACNA1S). However, no mutation was found, suggesting that these genes may not, or only rarely, be responsible for CCD. Our results indicate that CCD may be caused by RYR1 mutations in the majority of patients.

Adolescent↗

PubMed2006

Rapid and accurate diagnosis of facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a common muscular disorder, but clinical and genetic complications make its diagnosis difficult. Southern blot analysis detects a smaller sized EcoRI fragment on chromosome 4q35 in most facioscapulohumeral muscular dystrophy patients, that contains integral number of 3.3-kb tandem repeats known as D4Z4. The problems for the genetic diagnosis are that southern blotting for facioscapulohumeral muscular dystrophy is quite laborious and time-consuming, and the D4Z4 number is only estimated from the size of the fragment. We developed a more simplified diagnostic method using a long polymerase chain reaction (PCR) amplification technique. Successful amplification was achieved in all facioscapulohumeral muscular dystrophy patients with an EcoRI fragment size ranging from 10 to 25 kb, and each patient had a specific polymerase chain reaction product which corresponded to the size calculated from the number of D4Z4. Using southern blot analysis, more than 90% of facioscapulohumeral muscular dystrophy patients have a smaller EcoRI fragment than 26kb in our series, and the number of D4Z4 repeats is precisely counted by this polymerase chain reaction method. We conclude that this long polymerase chain reaction method can be used as an accurate genetic screening technique for facioscapulohumeral muscular dystrophy patients.

Blotting, Southern↗

PubMed2006

A case of Fukuyama-type congenital muscular dystrophy with a very mild mental deficit.

A boy had the clinical features of congenital muscular dystrophy with a very mild mental deficit. A muscle biopsy at one year of age showed the typical findings of Fukuyama-type congenital muscular dystrophy, including selective loss of immunoreactions for alpha dystroglycan. Magnetic resonance imaging showed no findings suggestive of migration disorders. The diagnosis of Fukuyama-type congenital muscular dystrophy was confirmed by a molecular assay at 8 years of age, and his haplotype analysis was heterozygous. At 9 years of age, his FIQ on the Wechsler Scale for Children revealed 69, while his IQ on the Tanaka Binnet scale of intelligence was 97. In this report the relationship between mild clinical condition of the studied case and its genotype is discussed.

Biopsy↗

PubMed2006

Fukutin gene mutations cause dilated cardiomyopathy with minimal muscle weakness.

OBJECTIVE: The fukutin gene (FKTN) is the causative gene for Fukuyama-type congenital muscular dystrophy, characterized by rather homogeneous clinical features of severe muscle wasting and hypotonia from early infancy with mental retardation. In contrast with the severe dystrophic involvement of skeletal muscle, cardiac insufficiency is quite rare. Fukuyama-type congenital muscular dystrophy is one of the disorders associated with glycosylation defects of alpha-dystroglycan, an indispensable molecule for intra-extra cell membrane linkage. METHODS: Protein and functional analyses of alpha-dystroglycan and mutation screening of FKTN and other associated genes were performed. RESULTS: Surprisingly, we identified six patients in four families showing dilated cardiomyopathy with no or minimal limb girdle muscle involvement and normal intelligence, associated with a compound heterozygous FKTN mutation. One patient died by rapid progressive dilated cardiomyopathy at 12 years old, and the other patient received cardiac implantation at 18 years old. Skeletal muscles from the patients showed minimal dystrophic features but have altered glycosylation of alpha-dystroglycan and reduced laminin binding ability. One cardiac muscle that underwent biopsy showed altered glycosylation of alpha-dystroglycan similar to that observed in a Fukuyama-type congenital muscular dystrophy patient. INTERPRETATION: FKTN mutations could cause much wider spectrum of clinical features than previously perceived, including familial dilated cardiomyopathy and mildest limb girdle muscular dystrophy.

Adult↗

PubMed2006

Mutation analysis of the GNE gene in distal myopathy with rimmed vacuoles (DMRV) patients in Thailand.

Distal myopathy with rimmed vacuoles (DMRV) is an early-adult-onset, distal myopathy caused by a mutation of the UDP-N-acetylglucosamine 2 epimerase/N-acetylmannosamine kinase (GNE) gene. We herein report four Thai patients with DMRV who carried compound heterozygous mutations of the GNE gene including three novel (p.G89R, p.P511T, and p.I656N) and two known mutations (p.A524V and p.V696M). All patients shared p.V696M in one allele. Our study demonstrates the mutation spectrum of the GNE gene in Thai patients with DMRV.

Adult↗

PubMed2006

Malignant hyperthermia in Japan: mutation screening of the entire ryanodine receptor type 1 gene coding region by direct sequencing.

BACKGROUND: Malignant hyperthermia (MH) is a disorder of calcium homeostasis in skeletal muscle triggered by volatile anesthetics or succinylcholine in susceptible persons. More than 100 mutations in the ryanodine receptor type 1 gene (RYR1) have been associated with MH susceptibility, central core disease, or both. RYR1 mutations may account for up to 70% of MH-susceptible cases. The authors aimed to determine the frequency and distribution of RYR1 mutations in the Japanese MH-susceptible population. METHODS: The authors selected 58 unrelated Japanese diagnosed as MH-susceptible for having an enhanced Ca-induced Ca release rate from the sarcoplasmic reticulum on chemically skinned muscle fibers. They sequenced the entire RYR1 coding region from genomic DNA. Muscle pathology was also characterized. RESULTS: Seven previously reported and 26 unknown RYR1 potentially pathogenic sequence variations were identified in 33 patients (56.9%). Of these patients, 48% had cores on muscle biopsy. The mutation detection rate was higher in patients with clear enhancement of Ca-induced Ca release rate (72.4%), whereas all patients with central core disease had RYR1 mutations. Six patients harbored potentially causative compound heterozygous sequence variations. CONCLUSIONS: Distribution and frequency of RYR1 mutations differed markedly from those of the North American and European MH-susceptible population. Comprehensive screening of the RYR1 gene is recommended for molecular investigations in MH-susceptible individuals, because many mutations are located outside the "hot spots." Based on the observed occurrence of compound heterozygous state, the prevalence of a possibly predisposing phenotype in the Japanese population might be as high as 1 in 2,000 people.

Adolescent↗

PubMed2006

Emerin-lacking mice show minimal motor and cardiac dysfunctions with nuclear-associated vacuoles.

Emery-Dreifuss muscular dystrophy is an inherited muscular disorder clinically characterized by slowly progressive weakness affecting humero-peroneal muscles, early joint contractures, and cardiomyopathy with conduction block. The X-linked recessive form is caused by mutation in the EMD gene encoding an integral protein of the inner nuclear membrane, emerin. In this study, mutant mice lacking emerin were produced by insertion of a neomycin resistance gene into exon 6 of the coding gene. Tissues taken from mutant mice lacked emerin. The mutant mice displayed a normal growth rate indistinguishable from their littermates and were fertile. No marked muscle weakness or joint abnormalities were observed; however, rotarod test revealed altered motor coordination. Electrocardiography showed mild prolongation of atrioventricular conduction time in emerin-lacking male mice older than 40 weeks of age. Electron microscopic analysis of skeletal and cardiac muscles from emerin-lacking mice revealed small vacuoles, which mostly bordered the myonuclei. Our results suggest that emerin deficiency causes minimal motor and cardiac dysfunctions in mice with a structural fragility of myonuclei.

Animals↗

PubMed2005

Characterization of MTM1 mutations in 31 Japanese families with myotubular myopathy, including a patient carrying 240 kb deletion in Xq28 without male hypogenitalism.

X-linked myotubular myopathy is a congenital muscle disorder due to MTM1 mutation, and is characterized clinically by generalized muscle weakness and hypotonia at birth usually resulting in early death. We newly identified 26 unrelated Japanese patients with MTM1 mutations by genomic DNA and transcript analysis, including 12 novel mutations. Among 31 patients, including our previously reported five patients, the c.1261-10A>G splice site mutation was the most frequent mutation. Three mutations, one missense and two splice site, were associated with milder phenotype. Of particular interest, one boy had a 240 kb deletion in Xq28 encompassing CXorf6 (formerly F18), MTM1 and MTMR1 but was not accompanied by hypogenitalism. CXorf6, which have been implicated in male sexual development, was not entirely deleted in this boy, resulting in the fusion with the MTMR1 gene. A chimeric fusion transcript was detected in patient's muscle by RT-PCR, suggesting this fusion gene product avoids the phenotype. This deletion led us to refine the critical region of CXorf6 for the development of male genitalia.

Adolescent↗

PubMed2005

Congenital muscular dystrophy with glycosylation defects of alpha-dystroglycan in Japan.

Glycosylation defects of alpha-dystroglycan (alpha-DG) cause various muscular dystrophies. We performed clinical, pathological and genetic analyses of 62 Japanese patients with congenital muscular dystrophy, whose skeletal muscle showed deficiency of glycosylated form of alpha-DG. We found, the first Japanese patient with congenital muscular dystrophy 1C with a novel compound heterozygous mutation in the fukutin-related protein gene. Fukuyama-type congenital muscular dystrophy was genetically confirmed in 54 of 62 patients. Two patients with muscle-eye-brain disease and one Walker-Warburg syndrome were also genetically confirmed. Four patients had no mutation in any known genes associated with glycosylation of alpha-DG. Interestingly, the molecular mass of alpha-DG in the skeletal muscle was similar and was reduced to approximately 90 kDa among these patients, even though the causative gene and the clinico-pathological severity were different. This result suggests that other factors can modify clinical features of the patients with glycosylation defects of alpha-DG.

Blotting, Western↗

PubMed2005

Dysferlin interacts with affixin (beta-parvin) at the sarcolemma.

The dysferlin gene is defective in Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B). Dysferlin is a sarcolemmal protein that is implicated in calcium-dependent membrane repair. Affixin (beta-parvin) is a novel, integrin-linked kinase-binding protein that is involved in the linkage between integrin and the cytoskeleton. Here we show that affixin is a dysferlin binding protein that colocalizes with dysferlin at the sarcolemma of normal human skeletal muscle. The immunoreactivity of affixin was reduced in sarcolemma of MM and LGMD2B muscles, although the total amount of the affixin protein was normal. Altered immunoreactivity of affixin was also observed in other muscle diseases including LGMD1C, where both affixin and dysferlin showed quite similar changes with a reduction of sarcolemmal staining with or without cytoplasmic accumulations. Colocalization of dysferlin and affixin was confirmed by immunofluorescence analysis using dysferlin-expressing C2 myoblasts. Wild-type and mutant dysferlin colocalized with endogenous affixin. The interaction of dysferlin and affixin was confirmed by immunoprecipitation study using normal human and mouse skeletal muscles. Using immunoprecipitation with deletion mutants of dysferlin, we have identified that C-terminal region of dysferlin is an apparent binding site for affixin. We also found N-terminal calponin homology domain of affixin as a binding site for dysferlin. Our results suggest that affixin may participate in membrane repair with dysferlin.

Actinin↗

PubMed2005

[Molecular pathomechanism of distal myopathy with rimmed vacuoles].

Distal myopathy with rimmed vacuoles (DMRV) and hereditary inclusion body myopathy (HIBM) are genetically identical autosomal recessive muscle disorders caused by mutations in the GNE gene. This gene encodes a bifunctional protein with UDP-GlcNAc 2-epimerase and ManNAc kinase activities that catalyze the rate limiting step and the succeeding step, respectively, in the sialic acid biosynthetic pathway. V572L mutation is the most prevalent among Japanese DMRV patients and accounts for about 60% of mutant alleles. Clinical spectrum of DMRV/HIBM seems to be wider than previously thought in terms of both the severity of the disease and the range of affected organs. There are rare asymptomatic homozygotes with missense GNE mutations, indicating the presence of mitigating factors. Surprisingly, more than 10% of the patients had a variety of cardiac abnormalities, suggesting that skeletal muscle may not be the only organ involved. Studies on recombinant GNE demonstrate a loss-of-function nature of the missense mutations identified. Patients' cells show decreased sialylation status which can be recovered by adding GNE metabolites, such as ManNAc and NeuAc. This indicates the possibility of developing a therapy for DMRV/HIBM by giving these metabolites to patients although we have to await the model mice that are currently being produced at several laboratories.

Carbohydrate Epimerases↗

PubMed2004

FSHD-like patients without 4q35 deletion.

Facioscapulohumeral muscular dystrophy (FSHD) is characterized by progressive weakness and wasting of facial, shoulder-girdle and upper arm muscles. Despite of the characteristic clinical features, the diagnosis of FSHD is sometimes difficult because clinical symptoms are extremely variable including facial sparing type, limb-girdle type, and distal myopathy type. Most of the FSHD patients have a deletion in the subtelomeric region of chromosome 4q35 (FSHMD1A), however the linkage analysis in some families suggested genetic heterogeneity. In the present study, we identified 40 patients without a deletion in the 4q35 region (non-4q35del) among 200 Japanese patients who were clinically suspected to have FHSD. All non-4q35del patients had shoulder-girdle weakness and 75% also had facial weakness. Eight patients showed clinical features that were indistinguishable from FSHD, but two of them had Becker muscular dystrophy. FSHD is clinically, and most likely genetically, as well, variable. Other forms of muscular dystrophy can also mimic FSHD.

Adolescent↗

PubMed2004

Reduction of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase activity and sialylation in distal myopathy with rimmed vacuoles.

Distal myopathy with rimmed vacuoles is an autosomal recessive muscle disease with preferential involvement of the tibialis anterior that spares the quadriceps muscles in young adulthood. In a Japanese patient with distal myopathy with rimmed vacuoles, we identified pathogenic mutations in the gene encoding the bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase, which catalyzes the initial two steps in the biosynthesis of sialic acid. In this study, we demonstrated the relationship between the genetic mutations and enzymatic activities using an in vitro expression assay system. Furthermore, we also showed that the levels of sialic acid in muscle and primary cultured cells from DMRV patients were reduced to 60-75% of control. The reactivities to lectins were also variable in some myofibers, suggesting that hyposialylation and abnormal glycosylation in muscles may contribute to the focal accumulations of autophagic vacuoles, amyloid deposits, or both in patient muscle tissue. The addition of ManNAc and NeuAc to primary cultured cells normalized sialylation levels, thus demonstrating the therapeutic potential of these compounds for this disease.

Animals↗

PubMed2004

Two novel CAV3 gene mutations in Japanese families.

Caveolin-3 deficiency is a rare, autosomal dominant, muscle disorder caused by caveolin-3 gene (CAV3) mutations and consists of four clinical phenotypes: limb-girdle muscular dystrophy type 1C (LGMD-1C), rippling muscle disease, distal myopathy, and familial hyperCKemia. So far, only 13 mutations have been reported. We here report two novel heterozygous mutations, 96C>G (N32K) and 128T>A (V43E), in the CAV3 gene in two unrelated Japanese families with LGMD-1C. Both probands presented with elevated serum CK level with calf muscle hypertrophy in their childhood but without apparent muscle weakness. However, their mothers showed mild limb-girdle weakness in addition to high CK level. Caveolin-3 was deficient and caveolae were lacking in muscles from both patients. Our data confirm that caveolin-3 deficiency causes LGMD-1C and expand the variability in CAV3 gene mutations.

Adult↗

PubMed2004

Subcellular localization of fukutin and fukutin-related protein in muscle cells.

Fukuyama-type congenital muscular dystrophy and congenital muscular dystrophy 1C are congenital muscular dystrophies that commonly display reduced levels of glycosylation of alpha-dystroglycan in skeletal muscle. The genes responsible for these disorders are fukutin and fukutin-related protein (FKRP), respectively. Both gene products are thought to be glycosyltransferases, but their functions have not been established. In this study, we determined their subcellular localizations in cultured skeletal myocytes. FKRP localizes in rough endoplasmic reticulum, while fukutin localizes in the cis-Golgi compartment. FKRP was also localized in rough endoplasmic reticulum in skeletal muscle biopsy sample. Our data suggest that fukutin and FKRP may be involved at different steps in O-mannosylglycan synthesis of alpha-dystroglycan, and FKRP is most likely involved in the initial step in this synthesis.

Adult↗