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Regular health checkups for mothers of patients with Duchenne muscular dystrophy have been performed at National Hospital Organization Tokushima Hospital since 1994. Among 43 mothers participated in this study, 28 dystrophinopathy carriers were identified. Skeletal and cardiac muscle functions of these subjects were examined. High serum creatine kinase was found in 23 subjects (82.1%). Obvious muscle weakness was present in 5 (17.8%) and had progressed from 1994 to 2015. Cardiomyopathy was observed in 15 subjects (60.0%), including dilated cardiomyopathy-like damage that was more common in the left ventricular (LV) posterior wall. Late gadolinium enhancement on cardiac MRI was found in 5 of 6 subjects, suggesting fibrotic cardiac muscle. In speckle tracking echocardiography performed seven years later, global longitudinal strain was decreased in these subjects, indicating LV myocardial contractile abnormality. These results suggest that female dystrophinopathy carriers should receive regular checkups for detection and treatment of cardiomyopathy, even if they have no cardiac symptoms.

In this study, we compared the clinicopathological findings of two autopsy cases of patients with calpainopathy (LGMD2A) from different families. The patient in case 1 was a 72-year-old man with a history of type 2 diabetes mellitus. He exhibited recent memory impairments from the age of 70. ECG revealed an incomplete right bundle branch block. A homozygous frameshift mutation c.1796dupA was found in the CAPN3 gene. Cause of death was respiratory insufficiency and heart failure. The patient in case 2 was a 70-year-old man with a history of hypertension. ECG revealed an incomplete right bundle branch block. A homozygous missense mutation c.1080GC (p.Trp360Cys) in CAPN3 gene was identified. Cause of death was ischemic cardiomyopathy and systemic circulatory failure. In both cases, muscle pathology revealed severe dystrophic changes. In case 2, cardiac hypertrophy and old myocardial infarcts with stenosis of coronary arteries were observed. Histological examination of the sinoatrial node showed fatty infiltration with ischemic changes in case 2. In both cases, the patients' brains showed cerebral atrophy and well preserved neurons. Calpain 3 abnormality was correlated with skeletal muscle involvement. It should be considered that LGMD2A might be complicated by dysfunction of the cardiac conduction system.

We report on the clinical, pathological, and genetic features of 7 patients with limb-girdle muscular dystrophy type 2A (LGMD2A) from three Japanese families. The mean age of onset was 9.7+/-3.1 years (mean+/-SD), and loss of ambulance occurred at 38.5+/-2.1 years. Muscle atrophy was predominant in the pelvic and shoulder girdles, and proximal limb muscles. Muscle pathology revealed dystrophic changes. In two families, an identical G to C mutation at position 1080 the in calpain 3 gene was identified, and a frameshift mutation (1796insA) was found in the third family. The former mutation results in a W360R substitution in the proteolytic site of calpain 3, and the latter in a deletion of the Ca2+-binding domain.

alpha-Mannosidosis is an autosomal recessive lysosomal-storage disorder caused by a deficiency of lysosomal alpha-mannosidase activity. This disease shows a wide range of clinical phenotypes, from a severe, infantile form (type I), which is fatal at

A new homozygous alpha-sarcoglycan (adhalin) gene mutation was found in a Japanese patient with severe childhood autosomal recessive muscular dystrophy (SCARMD). Muscle biopsy specimens from the patient showed marked reduction but not complete deficiency of alpha-sarcoglycan. The sequence of part of exon 3 of the alpha-sarcoglycan gene exhibited a cytosine to thymidine substitution at nucleotide position 220. Since the same mutation was not found in 100 normal control samples, this new alpha-sarcoglycan gene mutation is not a polymorphism but is presumed to be responsible for the marked reduction of alpha-sarcoglycan in skeletal muscle. Most patients with homozygous alpha-sarcoglycan gene mutation were reported to show complete alpha-sarcoglycan deficiency. Present case showed the homozygous missense mutation of alpha-sarcoglycan and associated with incomplete alpha-sarcoglycan deficiency and severe clinical phenotype.

The authors present a sixteen-year-old girl with blue rubber bleb nevus syndrome (BRBNS) associated with disseminated hemangiomas involving the skin, oral cavity, skeletal muscle, and cerebrum. Although she denied neurologic symptoms, magnetic resonance imaging of the brain demonstrated dilatated cerebral veins and the Chiari I malformation. Examination of hemostasis revealed disseminated intravascular coagula tion (DIC) manifesting as Kasabach-Merritt syndrome, with the potential for life-threat ening bleeding or thrombosis in the central nervous system. Since successful management of life-threatening hemangiomas with interferon alpha-2a (IFN α-2a) has been reported, the authors administered IFN α-2a with an improvement in hemostasis. These findings suggest that IFN α-2a therapy is beneficial for relieving the life-threatening consumptive coagulopathy associated with BRBNS.

The level of plasma brain natriuretic peptide (BNP) was elevated in 8 of 15 female gene carriers of Duchenne muscular dystrophy (DMD), and the level correlated with indices of cardiac function. In one of these carriers, whose clinical course was followed for one year, the plasma BNP level was elevated before the development of cardiac symptoms, further increased with the evolution of cardiac symptoms, and then decreased after treatment for cardiac failure. These results suggest that the plasma BNP level may be useful for the early detection of cardiac dysfunction and for evaluating the efficacy of cardiac treatment in female DMD carriers.

The intracellular localization of dystrophin and beta-dystroglycan mRNA in skeletal muscles of patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) and normal subjects was examined by in situ hybridization using biotinylated oligonucleotide probes. These mRNAs were found preferentially in sarcolemma in the skeletal muscles of both normal subjects and affected patients. Quantitative analysis of mRNA signals demonstrated no prominent reduction of dystrophin or beta-dystroglycan mRNA in DMD/BMD muscles. These results suggest that even mRNAs with deletions contain specific information that affects their localization, and the characteristic defect of dystrophin in DMD/BMD muscles seems to be caused mainly by the instability of dystrophin protein, as a post-transcriptional event.

Cardiac function was examined in 21 patients with Becker muscular dystrophy (BMD) and compared with 43 patients with Duchenne muscular dystrophy (DMD) and 37 healthy control subjects. Electrocardiography showed myocardial damage was most frequently found in the lateral wall, compatible with autopsy findings. The ratio of the preejection period to the ejection time was higher in patients with BMD (0.37 +/- 0.07, mean +/- SD) than in patients with DMD (0.28 +/- 0.05) and healthy controls (0.23 +/- 0.04). Left ventricular dimension and mitral annular size at end diastole in patients with BMD increased to 52.3 +/- 7.7 mm and 28.8 +/- 5.3 mm with age, respectively. In patients with cardiac failure and BMD, mitral regurgitation was observed at a rate of 66.7%. No definite relation between the deleted locus of the dystrophin gene and cardiac failure was found. Because motor dysfunction progresses more slowly in BMD than in DMD, a prolonged work load on the morbid myocardium may lead to dilated cardiomyopathy with mitral regurgitation.

To investigate the relationship between human T-lymphotropic virus (HTLV) types I and II and the pathogenesis of autoimmune thyroid diseases, we examined serum anti-thyroid antibodies in 1019 blood donors with or without serum anti-HTLV-I antibody as well as proviral DNA for HTLV-II in leukocyte DNA by the polymerase chain reaction in 395 blood donors with or without anti-thyroid antibodies. The frequency of donors with anti-HTLV-I antibody who also showed anti-thyroid antibodies (7.9%) tended to be higher than that (6.3%) among donors who did not have the anti-HTLV-I antibody. The frequency of anti-thyroid antibodies in 125 young male donors aged 16–39 years with anti-HTLV-I antibody (4.8%) was significantly higher (P

We purified the nicotinic acetylcholine receptor from digitonin-solubilized rabbit skeletal muscle by affinity chromatography and detected many proteins linked to AChR, including dystrophin, adhalin, beta-dystroglycan, utrophin, rapsyn, and actin. To determine whether or not AChR links to dystrophin-associated proteins (DAPs) without dystrophin, we studied the effects of denervation on AChR and DAPs in the skeletal muscle of a mdx mouse. Following surgical denervation, the levels of adhalin and beta-dystroglycan dramatically increased at the extrajunctional sarcolemma with AChR, suggesting that their association is independent of dystrophin. Furthermore, the diffuse extrajunctional appearance of adhalin, beta-dystroglycan, and AChR was observed after pharmacological denervation through the subcutaneous administration of succinylcholine. Since the depletion of DAPs and the subsequent disruption of sarcolemmal linkage are believed to be a primary cause of muscle cell necrosis in dystrophinopathies, pharmacological denervation may have some beneficial effect on these diseases.

Human T-lymphotropic virus type l (HTLV-I) protein and messenger RNA (mRNA) for HTLV-I were examined in thyroid tissues from two patients with Hashimoto's thyroiditis and serum anti-thyroid antibody. The virus envelope protein and signals for the mRNA were detected in many of the follicular epithelial cells of the thyroid tissue from one of the patients, respectively, by immunohistochemistry and in situ hybridization. PCR-Southern blotting revealed the presence of HTLV-I DNA in the thyroid tissue, in which the viral protein and mRNA were detected, although no virus particles were found in the epithelial cells by electron microscopy. HTLV-I virus was not present in the thyroid tissue from the second patient. The present findings suggest that infection of thyroid tissue with HTLV-I is associated with the pathogenesis of Hashimoto's thyroiditis in some patients.

No etiologic role of human T-lymphotropic virus type II (HTLV-II) in any disease is yet known. The present study showed a high incidence of HTLV-II proviral DNA fragments in DNA of peripheral blood leukocytes from patients with autoimmune thyroid diseases. Using primers for the pol and tax regions of HTLV-II proviral DNA, amplified DNA fragments were demonstrated in 51.5% of the patients with Hashimoto's thyroiditis and in 11.8% of those with Graves' disease examined, but in only 1.9% of the disease controls and 1.0% of healthy individuals. These amplified DNA fragments hybridized with each of the inner probes. The nucleotide sequences of the DNA fragments of the pol and tax regions showed high homology with those of the prototype of HTLV-II. No antibodies for HTLV-II could, however, be detected in the patients examined. Because of the absence of its antibody, HTLV-II infection was not confirmed in these patients, but the presence of HTLV-II proviral DNA or its related DNA at high frequency suggests a relationship of HTLV-II with the development of autoimmune thyroid diseases.

Magnetic resonance imaging scans of the brain were obtained in 13 patients with myotonic dystrophy, seven with congenital myotonic dystrophy and six with adult-type myotonic dystrophy. All seven patients with congenital myotonic dystrophy had ventriculomegaly and a low IQ (DQ). Cerebral white matter lesions were observed in six cases, a small corpus callosum in four cases, a small brainstem in two cases, and a cerebellar white matter lesion in one case. Cerebral white matter lesions were observed in five of the six cases with adult-type myotonic dystrophy of which one had ventriculomegaly. The IQ (DQ) was significantly lower in patients with congenital myotonic dystrophy than in those with adult-type myotonic dystrophy. The incidence of a small corpus callosum or ventricular enlargement was higher in congenital myotonic dystrophy than in adult-type myotonic dystrophy. These findings may be related to the presence of neurologic impairment in congenital myotonic dystrophy.

The brain of patients with adult type myotonic dystrophy was measured using MRI, and compared with age- and sex-matched controls. The width of the cerebrum, and the areas of the corpus callosum, pons and pituitary gland were significantly smaller in adult-type myotonic dystrophy than in the controls. The IQ of the patients with adult-type myotonic dystrophy was lower than that of controls. These results demonstrate a decreased volume of the brain and pituitary glands in myotonic dystrophy. These findings suggest a causal relationship for the mental retardation and endocrine dysfunction that may occur.

Two siblings who exhibited hereditary parkinsonism with pyramidal signs and cerebellar ataxia are reported. Anticholinergics had a dramatic beneficial effect in both cases, but levodopa did not. This responsiveness, which is similar to that reported in patients with Joseph's disease, suggests dysfunction of an “indirect pathway” involving the globus pallidus and the subthalamic nucleus, in addition to that of the nigrostriatal system. We propose a new hereditary variant of early onset Parkinson's disease, distinct from the levodopa sensitive forms of juvenile Parkinson's disease.

The intracellular localization of myoglobin mRNA in the skeletal muscles of normal subjects was examined by in situ hybridization using a biotin-labeled cDNA probe. By phase-contrast microscopy, myoglobin mRNA signals were demonstrated to be located preferentially on the A-band. Two different methods of tissue preparation, i.e., pre-embedding method and post-embedding method, were used for the electron microscopic study. With the pre-embedding method, only a few gold particles were found to be associated with cytoskeletal filaments in the intermyofibrillar space. With the post-embedding method, superior preservation of sections and higher signal intensities were obtained. Although most of the gold particles were localized on the A-band, some were seen in other regions; i.e., in the intermyofibrillar space, perinuclear space, or the I-band, where myoglobin is localized. These findings suggest that myoglobin is primarily synthesized on the A-band, where ribosomes predominantly exist, although myoglobin is also localized on the I-band. The predominant localization of myoglobin mRNA on the A-band may aid in the mRNA transcription and may be related to the regulation of myoglobin synthesis in skeletal muscle cells.

We present an overview of autosomal recessive distal muscular dystrophy (ARDMD), including recent molecular genetic findings. ARDMD is often referred to as Miyoshi-type distal muscular dystrophy (MDMD) or Miyoshi myopathy (MM). The onset of MDMD occurs in early adulthood. Muscle atrophy is most dominant in distal leg muscles, especially the flexor muscles, i.e., gastrocnemius and soleus. As MDMD advances, muscle atrophy progresses to the thigh and hip muscles. Toe standing is impaired but heel standing can still be accomplished early in the disease course. This is followed by difficulty in standing and walking. The patients rarely become confined to bed. Serum creatine kinase level is markedly elevated, e.g., 100 times the upper limit of the normal range early in the disease course. Pre-symptomatic patients may also have high creatine kinase levels. Heterozygous individuals may have only slightly elevated creatine kinase levels. Recent development revealed that MDMD and LGMD2B are both caused by mutations in the dysferlin gene (DYSF). C1939G, G3370T, 3746delG, and 4870delT are reported to be common mutations among patients with MDMD. The dysferlin protein is presumably involved in the repair of muscle cell membranes. Among the patients reported originally by Miyoshi et al., 3 affected individuals from 3 different families were confirmed carriers of dysferlin mutations. Additionally, 1 heterozygous individual was identified. Although MDMD and LGMD2B are caused by the mutation of the same gene, ARDMD is characterized by initial involvement of leg flexors while LGMD2B is characterized by involvement of the proximal leg muscles. The difference in the distribution becomes obscure as the 2 diseases progress. The temporal profiles of functional impairment in the 2 diseases are reportedly very similar. When MDMD is suspected, it is important to carefully observe the relevant leg, more specially the flexor muscle group.

The intracellular localization of myoglobin(Mb) mRNA in the skeletal muscles of normal subjects and patients with Duchenne muscular dystrophy(DMD) or amyotrophic lateral sclerosis(ALS) was examined by in situ hybridization using a biotin-labeled cDNA probe. In cross sections of normal muscles, Mb mRNA signals were demonstrated to be diffusely distributed as granular reaction products throughout the sarcoplasm, and in longitudinal sections the products were observed preferentially on the A-band. In DMD or ALS muscles, the distribution of granular mRNA signals showed some similarities with that in normal muscles, although degenerated fibers revealed a heterogenous distribution of the signals. In DMD muscles, the optical density(OD) of stained signal was higher in non-atrophic fibers and lower in atrophic fibers than in normal muscles. In ALS muscles, the OD was lower than in normal muscles. These results suggest that Mb mRNA is distributed preferentially on the A-band of the muscle fibers, and that in diseased muscle fibers Mb synthesis is affected by pathological changes.

Seven patients, aged 10–17 years, with Duchenne muscular dystrophy were treated orally with prednisolone (PSL) at a dose of 0.8–1.0 mg/kg per day for 8 weeks. During the treatment their muscle strength, serum creatine kinase (CK) activity, serum levels of myoglobin (Mb), and urinary excretion of 3-methyl-histidine (3-MeH) and glycine (Gly) were measured serially. In all the patients, the motor function or muscle strength improved, and the serum CK activity and Mb level decreased during PSL treatment. Urinary excretion of 3-MeH, a unique constituent of muscle contractile proteins, decreased to 51–63% of the baseline value in weeks 6–9 after the start of PSL administration, and returned to the baseline level in week 12. The ratios of 3-MeH to creatinine and to Gly also decreased during the treatment. Urinary excretion of Gly, which is ubiquitous in all tissues including muscle, did not decrease during the treatment. These findings suggest that PSL inhibits proteolysis of muscle contractile protein.

症例は58歳,女性.主訴は吐血.上部消化管内視鏡検査で食道静脈瘤と胃前庭部にびまん性に小斑状の発赤を認め,胃前庭部毛細血管拡張症(GAVE)と診断した.口腔乾燥症状があり,顔面,背部に血管拡張像,手指に皮膚硬化を認め抗セントロメア抗体,抗ミトコンドリア抗体,抗SS-A, B抗体陽性で,腹腔鏡検査,肝生検,唾液腺造影等から強皮症,原発性胆汁性肝硬変(PBC), Sjogren症候群(SjS)に合併したGAVEと診断した.

An autopsied case of Takayasu's arteritis associated with complete atrioventricular (AV) block is described for the first time. The findings of scar formation and diffuse infiltration of lymphocytes into the cardiac conduction system, particularly the AV node, were similar to those in patients with connective tissue diseases or congenital complete heart block. The degree of AV block progressed with aggravation of the disease. These findings suggest that complete AV block may have been induced by acquired autoimmunity involving the cardiac conduction system.

Background: Chronic progressive external ophthalmoplegia (CPEO), which includes Kearns-Sayre syndrome, is a mitochondrial disorder with large deletions of mitochondrial DNA. Recently, mtDNA deletions in cardiac muscle cells were thought to be a cause of dilated cardiomyopathy. However, the cardiac involvement in patients with CPEO is generally considered to be limited to the cardiac conduction system. Hypothesis: The purpose of this study was to evaluate left ventricular function in patients with CPEO. Methods: We evaluated the cardiac function of five patients with CPEO by means of carotid pulse recording and Doppler echocardiography. Results: The ratio of the pre-ejection period to ejection time was increased to 0.67 in one patient and to 0.50 in another. Echocardiography showed left ventricular dilatation and diffuse hypokinetic wall motion in both cases. Left ventricular fractional shortening was decreased to 5 and 19%, respectively, and the mean rate of circumferential shortening was decreased to 0.12 and 0.63 circ/s, respectively. One of the two patients died of congestive heart failure 2 months after the study. The Doppler pattern of left ventricular filling in the three remaining patients showed a decrease in the ratio of peak flow velocity in early diastole to that in late diastole, with an increase in deceleration time. Conclusion: Although cardiac involvement in patients with CPEO is generally considered to be limited to the cardiac conduction system, left ventricular dysfunction may be present and should receive more attention in the management of patients with CPEO.

The plasma concentration of atrial natriuretic peptide was measured in patients with muscular dystrophies to study its relationship with congestive heart failure. In patients with Duchenne muscular dystrophy, the plasma atrial natriuretic peptide concentration was 35.5 +/- 3.3 pg/mL (mean +/- SE), which was higher than that in age-matched normal subjects (9.8 +/- 0.6 pg/mL). It increased with progression of disability and showed significant correlations with the cardiothoracic ratio and the ratio of the preejection period to the left ventricular ejection time. In patients with other types of muscular dystrophy, the plasma atrial natriuretic peptide concentration showed no significant change. Immunohistochemical examination demonstrated many atrial natriuretic peptide-positive cells in atrial muscle of an autopsied patient, indicating preservation of the peptide until the end stage. These findings suggest that measurement of the plasma atrial natriuretic peptide concentration is useful for evaluating heart failure in patients with Duchenne muscular dystrophy.

Alpha-dystroglycan is a cell surface peripheral membrane protein which binds to the extracellular matrix (ECM), while beta-dystroglycan is a type I integral membrane protein which anchors alpha-dystroglycan to the cell membrane via the N-terminal extracellular domain. The complex composed of alpha-and beta-dystroglycan is called the dystroglycan complex. We reported previously a matrix metalloproteinase (MMP) activity that disrupts the dystroglycan complex by cleaving the extracellular domain of beta-dystroglycan. This MMP creates a characteristic 30 kDa fragment of beta-dystroglycan that is detected by the monoclonal antibody 43DAG/8D5 directed against the C-terminus of beta-dystroglycan. We also reported that the 30 kDa fragment of beta-dystroglycan was increased in the skeletal and cardiac muscles of cardiomyopathic hamsters, the model animals of sarcoglycanopathy, and that this resulted in the disruption of the link between the ECM and cell membrane via the dystroglycan complex. In this study, we investigated the proteolysis of beta-dystroglycan in the biopsied skeletal muscles of various human muscular diseases, including sarcoglycanopathy, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, Fukuyama congenital muscular dystrophy, Miyoshi myopathy, LGMD2A, facioscapulohumeral muscular dystrophy, myotonic dystrophy and dermatomyositis/polymyositis. We show that the 30 kDa fragment of beta-dystroglycan is increased significantly in sarcoglycanopathy and DMD, but not in the other diseases. We propose that the proteolysis of beta-dystroglycan may contribute to skeletal muscle degeneration by disrupting the link between the ECM and cell membrane in sarcoglycanopathy and DMD.

To determine the relationship between myoglobin (Mb) and the defense system against reactive oxygen species in various myopathies, we performed immunohistochemical analyses of Mb and various antioxidant enzymes, including manganese superoxide dismutase (Mn-SOD), copper zinc SOD (CuZn-SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). Biopsied muscle specimens were obtained from patients with chronic progressive external ophthalmoplegia (CPEO), Kearns-Sayre syndrome (KSS), Duchenne muscular dystrophy (DMD), and polymyositis (PM). In patients with CPEO/KSS, stainings of Mb, SOD, CAT, and GSH-Px in nonatrophic ragged-red fibers (RRFs) were more intense than those in non-RRFs. These pronounced stainings corresponded to ragged-red lesions. The staining intensities of these antioxidant enzymes were significantly correlated with that of Mb (P < 0.001). Atrophic RRFs in specimens from patients with CPEO/KSS showed intense stainings of these antioxidant enzymes but not intense staining of Mb. In specimens from patients with DMD/PM, the antioxidant enzymes but not Mb were overexpressed in degenerative fibers. These results suggest that oxidative stress is associated with Mb expression specifically in mitochondrial diseases. The antioxidant enzymes seem to be upregulated to protect against muscle damage in nonatrophic RRFs. However, the Mb-mediated oxidative damage may become more extensive and result in further mitochondrial dysfunction and progressive atrophy of RRF with impaired upregulation of Mb.

OBJECTIVES Mutational analysis of the sterol 27-hydroxylase ( CYP27 ) gene was performed on three patients from two Japanese families who had cerebrotendinous xanthomatosis (CTX) associated with parkinsonism. METHODS Clinical evaluations, brain MRI studies, and laboratory analyses were completed on the three patients. The CYP27 gene was analysed for mutations by PCR amplification of gene segments followed by direct sequencing. RESULTS Two different, homozygous mutations were identified in these families. One is a novel transition, substituting T for G at Glu162 (GAG) resulting in a stop codon (TAG). The other is also a transition, substituting T for C at Arg441 (CGG) resulting in Trp (TGG). The second is located in two amino acids ahead of the heme ligand binding site (Cys443) of the protein likely rendering it non-functional. It is the most common CTX mutation in Japanese patients. CONCLUSIONS CTX with parkinsonism is caused by mutations with a severe impact on enzyme function. The two mutations described here are likely to cause loss of function because they are chain terminating or affect an essential site in the protein.

Object: The aim of this study was to reveal variations in the patterns of expression of the cell surface proteins in regenerating fibers and those in the number of satellite cells to gain an understanding of the pathological processes involved in sarcoglycanopathy. Methods: We have reported that there is a reduction of the beta-1 subunit of laminin, heparan sulfate proteoglycan (HSPG), and HCAM (CD44) in Japanese patients with sarcoglycanopathy. Here, we investigated immunohistocheniically the expression of the neural cell adhesion molecule (NCAM), which is a marker for human regenerating muscle and satellite cell, and CD24, which appears to be expressed in the early stages of the regeneration process. Patients: We investigated six Japanese patients with sarcoglycanopathy, and compared to age-matched Becker muscular dystrophy. Results: We found that the incidences of muscle fibers with increased NCAM were not statistically different between the two groups. However, the incidences of muscle fibers with increased CD24 and those of NCAM positive satellite cells were very low in sarcoglycanopathy and were statistically different between sarcoglycanopathy and age-matched Becker muscular dystrophies. Conclusion: The poor expression of CD24 and the fewer satellite cells in sarcoglycanopathy without significant difference in the number of total regenerating fibers suggest that a different regeneration process is involved in sarcoglycanopathy compared to that in other types of muscular dystrophy.(Interal Medicine 38: 412-415, 1999)

Quantitative analysis of signal intensities in immunostained sections has been performed in only a few studies owing to difficulties with quantifying amounts of antigen present. We determined correlations between fluorescent signal intensities and amounts of antigen in muscle cryosections by altering section thickness from 4 to 10 microm. Fluorescent signals of dystrophin. beta-dystroglycan and myosin were detected with monoclonal and/or polyclonal primary antibodies using routine procedures. Confocal laser microscopy demonstrated that these signals were distributed uniformly along the z-axis suggesting that the antibodies permeated well through the sections. Epifluorescence microscopy with microfluorometry demonstrated a positive correlation between the optical density of signals and section thickness. These findings suggest that immunofluorescent signals can be quantitated by epifluorescence microscopy.

There are no detailed reports about the clinical and pathological findings in the central nervous system (CNS) of patients with Becker’s muscular dystrophy (BMD), although dysfunctions of the skeletal and cardiac muscles are well known in BMD. Since dystrophin is present in the CNS, and usual medical treatment is often influenced by CNS manifestations in patients, we studied the clinical and pathological features of BMD, with particular focus on the CNS manifestations, and also examined the relationships of these features with skeletal and cardiac muscle dysfunctions. We examined eight BMD patients aged 24-77 years who were admitted to our hospital, and six autopsied BMD patients aged 39-58 years. In addition to family histories of the patients, clinical findings, and disease progression, BMD was diagnosed based on the presence of dystrophin revealed by immunohistochemical examination and examination of the dystrophin gene. Brain computed tomography (CT), magnetic resonance imaging (MRI), and CNS pathohistology were also performed. Gene analyses by multiplex PCR revealed that exons 3-4, 3-6, 12-19, 45-47,45-49, and 45-52 were deleted; all the deletions occurred in hot spots in the dystrophin gene. With regard to the clinical CNS manifestations of BMD, 2 of the 14 patients experienced hallucinations, but there was no patient with severe mental retardation. Brain CT and MRI scans revealed various grades of mild atrophy of the front-parietal lobe for all the patients. Low-magnification microscopy images of the brain of three autopsied patients with mild BMD showed that the gyri of the brain were separated, suggesting brain atrophy. The nerve cells of the frontal lobe, however, seemed to be unaffected histologically. These results show that there are mild abnormalities in the CNS of BMD patients. It is well known that BMD patients demonstrate different levels of skeletal and cardiac muscle dysfunctions. In the current study as well, the BMD patients demonstrated CNS abnormalities, although these abnormalities were mild compared to the skeletal and cardiac muscle dysfunctions.

We investigated the expression of alpha-sarcoglycan, beta-sarcoglycan, gamma-sarcoglycan, and delta-sarcoglycan immunohistochemically in three patients with mutations of the alpha-sarcoglycan gene and a patient with a mutation of the gamma-sarcoglycan gene. Although each of the four sarcoglycans were decreased on the muscle membranes of all the patients, different expression patterns for each were seen among the patients. In patients with mutations of the alpha-sarcoglycan gene, beta-, gamma- and delta-sarcoglycans were relatively preserved as compared to greatly reduced alpha-sarcoglycan. However, the patient with a mutation of the gamma-sarcoglycan gene showed marked reduction of gamma-sarcoglycan as compared to partially preserved alpha- and beta-sarcoglycans, and well-preserved delta-sarcoglycan. These results suggest that each sarcoglycan component in sarcoglycanopathy does not decrease in the same manner, and that mutations of the sarcoglycan gene can be predicted, at least in part, by means of sensitive immunohistochemistry for each sarcoglycan.

Genomic clones of human MANB gene encoding the lysosomal enzyme, alpha-mannosidase, have been isolated, sequenced and analyzed. The human MANB gene spans approximately 22 kb and consists of 24 exons. The 5' flanking region of the gene shows a high G+C content and has two Sp1 and three AP-2 sites. Promoter analysis using deletion constructs of the 5' flanking region fused to the bacterial CAT gene showed that 150 bp of 5' sequence could drive the expression of MANB in COS 7 cells. Determination of the sequence of the 5' end of the alpha-mannosidase mRNA by 5' RACE protocol showed that transcription is initiated from a cluster of sites centered -28 and -20 bp from the first in-frame ATG. These data demonstrate that, like other lysosomal enzyme genes such as those for beta-glucuronidase or beta-hexosaminidase, the human MANB gene is controlled by a short 5' flanking sequence located near the initiation codon.

We examined the effect of the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) on the neurotoxicity of L-glutamate in organotypic cultures of rat spinal cord. Eighteen-day-old cultures were incubated with 500 microM L-glutamate, 1 mM PDC, or both. After 72 hours, the tissues were stained for acetylcholinesterase (AChE), and the ventral horn AChE-positive neurons (VHANs) analyzed using morphometry. Neither L-glutamate nor PDC affected AChE staining, but in combination they produced markedly reduced AChE staining in the dorsal horn and a significant decrease in the number of VHANs (especially the smaller VHANs) as compared with the control. Moreover, treatment with 200 microM PDC for 2 weeks preferentially affected the smaller VHANs. The neurotoxicity of L-glutamate plus PDC was blocked by the N-methyl-D-aspartate (NMDA) antagonist 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP). Results suggest that glutamate uptake system has an important protective function in the aggravation of acute neuronal damage.

The precise localization and semiquantitative correlation of dystrophin, utrophin and {beta}-dystroglycan expression on the sarcolemma of skeletal muscle cells obtained from patients with Becker muscular dystrophy (BMD) was studied using three types of double immunofluorescence. Staining intensity was measured using a confocal laser microscope. Each of these proteins was identified at the same locus on the sarcolemma. The staining intensities of dystrophin and utrophin were approximately reciprocal at sarcolemmal sites where dystrophin expression was obviously observed. The staining intensity of {beta}-dystroglycan was strong in areas where dystrophin staining was also strong and utrophin expression was weak. Quantitative analysis revealed that the staining intensity of {beta}-dystroglycan minus that of dystrophin approximated the staining intensity of utrophin, indicating that the sum of dystrophin and utrophin expression corresponds to that of {beta}-dystroglycan. These results suggest that utrophin may compensate for dystrophin deficiency found in BMD by binding to {beta}-dystroglycan. 35 refs., 3 figs., 1 tab.

To estimate the oxidative damage to skeletal muscle DNA in mitochondrial encephalomyopathies, we studied the amount of 8-hydroxy-deoxyguanosine (8-OH-dG) and the localization of superoxide dismutase (SOD) in the skeletal muscles of patients with progressive external ophthalmoplegia (PEO) or Kearns-Sayre syndrome (KSS). The molar ratio of 8-OH-dG/deoxyguanosine in skeletal muscle from PEO or KSS patients was significantly higher than the control value. The ratio from patients with polymyositis or Duchenne's muscular dystrophy was not significantly elevated. Immunohistochemical staining for both Mn-SOD and Cu,Zn-SOD showed pronounced staining in the subsarcolemmal and intermyofibrillar regions of cytochrome-oxidase-negative ragged red fibers of KSS or PEO muscles. Our findings suggest that overproduction of 8-OH-dG and mitochondrial dysfunction with gene deletions are associated with each other in muscle cells of patients with PEO or KSS, and that free radicals may play an important role in the pathophysiology of mitochondrial encephalomyopathies.

Effects of calcitonin gene-related peptide (CGRP) and interleukin 6 (IL-6) on muscle cell differentiation were studied using cultured rat myoblasts (L6 cells). Cell morphology and the amounts of the messenger RNAs (mRNAs) of myogenin and Myf-5, DNA content, creatine kinase (CK) activity, and myoglobin (Mb) content in the cultured cells were examined serially over 10 days of culture. In the presence of CGRP or IL-6, the mRNAs of myogenin and Myf-5 were expressed earlier and at a higher concentration in the treated cells than in the control cells. The ratios of CK activity to DNA content (CK/DNA) and of Mb content to DNA content (Mb/DNA) on day 10 of culture also were greater than in the control cells. Furthermore, the mRNAs of myogenin and Myf-5 in cultured cells incubated with both CGRP and IL-6 increased more rapidly than in cells cultured with CGRP or IL-6 alone, and the ratios of CK/DNA and Mb/DNA on day 10 were more than twice those in the presence of CGRP or IL-6. These findings indicate that both CGRP and IL-6 facilitate the differentiation of myoblasts and may have an additive effect.

The localization and amounts of myoglobin (Mb) and Mb mRNA in ragged-red fibers (RRF) in skeletal muscle of 6 patients with mitochondrial encephalomyopathy were examined immunohistochemically and by in situ hybridization. The amounts of Mb and Mb mRNA were expressed in terms of optical densities (ODs) of Mb immunostaining and Mb mRNA signals. In nonatrophic RRF, Mb was predominantly seen in the ragged-red region and Mb mRNA signals were increased throughout the sarcoplasm. The amounts of Mb and Mb mRNA in nonatrophic RRF were greater than those in nonatrophic non-RRF. In contrast, the localization and amount in atrophic RRF were similar to those in atrophic non-RRF. Thus, Mb synthesis in nonatrophic RRF may increase to compensate for mitochondrial dysfunction and to supply sufficient oxygen to mitochondria, but this compensatory function may be impaired in atrophic RRF.

Homozygous adhalin gene mutations were found in three patients from two consanguineous families with autosomal recessive childhood onset muscular dystrophy. Muscle biopsies from patients in each family showed complete absence of adhalin. Sequencing of adhalin cDNA prepared from skeletal muscle by reverse transcription PCR demonstrated a cytosine to thymidine substitution at nt 229 in the patient in family 1 and an adenine to guanine substitution at nt 410 and a 15-base insertion between nt 408 and 409 in the two patients in family 2. Sequencing of genomic DNA prepared from peripheral blood leukocytes by PCR confirmed these mutations. The parents in each family were found to be heterozygous for the respective mutations. These adhalin gene mutations are presumed to be responsible for the absence of adhalin in the skeletal muscle. Adhalin deficiency likely causes disruption of the muscle cell membrane, resulting in dystrophic changes in the skeletal muscle similar to dystrophin deficiency in Duchenne muscular dystrophy.

Activities of nine lysosomal enzymes and pH-dependent isozyme patterns of α-mannosidase were examined in the skeletal muscle of patients with neuromuscular diseases, and the ratios of these enzyme activities in leukocytes to those in myocytes (UM ratio) were determined. The activities of enzymes with a high UM ratio were markedly increased in the muscles of patients with Duchenne muscular dystrophy (DMD), myotonic dystrophy (MyD), or polymyositis (PM). In contrast, those which showed a low UM ratio were increased in the muscles of amyotrophic lateral sclerosis (ALS) and disuse muscle atrophy (DUA). The isozyme pattern of α-mannosidase in DMD muscle resembled that in leukocytes, while those in ALS and DUA muscle resembled that in normal muscle. These results may suggest that the increased activity of lysosomal enzymes in the muscles of patients with DMD, MyD, or PM is due primarily to infiltrating leukocytes, while that in patients with ALS or DUA is due to intramyofiber lysosomes. 1995 John Wiley & Sons, Inc. . neuromuscular disease MUSCLE & NERVE 18 :1009-10151995

We report a Japanese family with chronic progressive external ophthal-moplegia (CPEO) with autosomal dominant inheritance, and review 54 reported CPEO patients in seven families (including the present family) with autosomal dominant inheritance and mtDNA deletions in the skeletal muscle. Mean age at onset in the CPEO was 26 years, which is older than that in published solitary cases. In addition to blepharoptosis and external ophthalmoplegia, proximal muscle atrophy and weakness were found in 62%, hearing loss in 25%, and ataxia in 17% of the patients. Retinal degeneration was not found, and cardiac involvement was very rare. mtDNA deletions in the muscle were multiple and large scale, and all such deletions were located in the non–D-loop region. Autosomal dominant CPEO has unique clinical features which differ from those of solitary CPEO, and is associated with multiple large-scale mtDNA deletions. Thus, autosomal dominant CPEO can be considered a clinical and genetic entity of mitochondrial diseases. © 1995 John Wiley & Sons, Inc.

Three carriers of human T-lymphotropic virus type I (HTLV-I) with Graves' disease are reported. All three cases were complicated with uveitis, and one also showed chronic arthropathy. Anti-HLTV-I antibody was found in the serum by the particle agglutination method and western blotting, and HTLV-I proviral DNA was detected in peripheral lymphocytes by the polymerase chain reaction and Southern blotting. HTLV-I is a causal agent of adult T-cell leukemia and HTLV-I associated myelopathy/tropical spastic paraparesis, and is believed to be related to the pathogenesis of diseases such as chronic arthropathy, uveitis, chronic bronchoalveolitis, and Sjogren's syndrome. On the other hand, retrovirus infection is considered to cause autoimmune diseases. Thus, the pathogenesis of Graves' disease in the present patients might be associated with HTLV-I infection.

Expression of the myoglobin (Mb) gene in skeletal muscle was studied in patients with Duchenne muscular dystrophy (DMD), polymyositis (PM), or amyotrophic lateral sclerosis (ALS) by measuring Mb concentration by radioimmunoassay and Mb messenger ribonucleic acid (RNA) (MbmRNA) levels by Northern blot analysis. Mb concentrations in the muscle cells (Mb/noncollagenous protein) were decreased in patients with DMD, PM, or ALS. However, while Mb concentrations per MbmRNA content (Mb/MbmRNA) were decreased in DMD and PM patients, these values were normal in ALS patients. These results suggest that Mb synthesis is increased in muscles of DMD and PM patients, but is not sufficient to compensate for the excessive loss of Mb from the affected muscles, and that the synthesis is decreased in the muscles of ALS patients. © 1994 John Wiley & Sons, Inc.

We describe two HTLV-I virus carriers who have biopsy-proven Hashimoto's thyroiditis. The first patient, a 64-year-old female, has had goiter and hypothyroidism since the age of 56. The second patient, a 66-year-old male, developed hyperthyroidism and goiter at the age of 44, but at present he is hypothyroid. Both patients are positive for anti-thyroid antibodies and anti-HTLV-I virus antibody. Findings of the thyroid biopsy specimens were consistent with Hashimoto's thyroiditis. These data suggest that Hashimoto's thyroiditis develops in HTLV-I carriers who have no clinical evidence of HAM/TSP.

To investigate the possible relationship of HTLV-I virus infection to autoimmune thyroid disease, we examined, firstly, the frequency of HTLV-I seropositivity among patients with Hashimoto's thyroiditis and, secondly, the frequency of Hashimoto's thyroiditis in patients with HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP). Of 144 patients with Hashimoto's thyroiditis in the Tokushima and Kochi Prefectures, Japan, 9 (6.3%) were positive for serum HTLV-I virus antibody 2 of whom were confirmed histologically to have Hashimoto's thyroiditis. This percentage is significantly higher (P < 0.01) than the estimated prevalence (2.2%) of HTLV-I carriers among the general population in this region. Of 9 patients with HAM/TSP, 3 (33.3%), including 2 biopsy-proven cases, had evidence of Hashimoto's thyroiditis. This proportion is apparently much higher than the prevalence (1.7%) of Hashimoto's thyroiditis in the general population. These findings suggest that HTLV-I virus may be related to the development of Hashimoto's thyroiditis. © 1992 Wiley-Liss, Inc.

X-linked spinal and bulbar muscular atrophy (SBMA), an adult-onset form of motor neuron disease, was recently reported to be caused by amplification of the CAG repeats in the androgen receptor gene. We report here a simple and rapid strategy to detect the precise number of the CAGs. After the DNA fragment containing the CAG repeats is amplified by the polymerase chain reaction, a primer extension is carried out; the extension of the end-labelled reverse primer adjacent to 3' end of CAG repeats stops at the first T after CAG repeats with the incorporation of dideoxy ATP in the reaction mixture. The resultant primer products are analysed by denaturing polyacrylamide gel electrophoresis and autoradiography. This method could be quite useful to detect not only CAG repeats in SBMA but also other polymorphic dinucleotide and trinucleotide repeats.

The localizations of myoglobin in skeletal muscle cells of patients with Duchenne muscular dystrophy (DMD), myotonic dystrophy (MyD), and amyotrophic lateral sclerosis (ALS) were studied by immunohistochemistry and immunoelectron microscopy. In normal skeletal muscle cells, myoglobin was localized mainly in the I-band region. In degenerating muscle cells of patients with DMD and MyD, myoglobin was also demonstrated in the distended lumen of the internal membrane system and in the intermyofibrillar space, through which it seemed to pass into the extracellular space. No myoglobin was detected in opaque fibers or in some of small-sized fibers in DMD muscle. In patients with ALS the staining intensities of myoglobin varied in different muscle cells, but myoglobin was restricted to the I-band region in many muscle cells. These findings suggest that changes in the localization of myoglobin in skeletal muscle cell sensitively reflect the pathologic status of muscle cells.

A 38-year-old man with asthma developed eosinophilic polymyositis following the administration of Tranilast, an antiasthmatic agent. Low grade fever, erythematous rashes on the entire body, dysphagia, blood eosinophilia, elevations of serum creatine phosphokinase and myoglobin levels, and inverted T waves in the electrocardiogram were noted. A muscle biopsy showed focal degeneration of muscle fibers with an infiltrate of eosinophils and lymphocytes. A rechallenge with Tranilast resulted in erythema formation, blood eosinophilia, and elevations of some serum muscle enzymes and myoglobin levels. Tranilast was considered to be the causative agent. This is the first reported case of Tranilast-induced eosinophilic polymyositis.