Immunocytochemical analysis of dystrophin in congenital muscular dystrophy.

Using immunocytochemical methods, we examined the intensity and distribution of dystrophin and spectrin immunostaining of skeletal muscles from 51 congenital muscular dystrophy (CMD) patients including 36 Fukuyama congenital muscular dystrophy (FCMD) and 15 non-FCMD (other CMD). 17 age-matched spinal muscular atrophy (SMA) and 5 Duchenne muscular dystrophy (DMD) patient biopsies were studied as controls. All 15 non-FCMD and SMA patients showed normal localization of dystrophin at the surface membrane of each muscle fiber which was undetectable in DMD. In contrast, 34 of 36 FCMD patients exhibited an unusual immunostaining pattern with occasional (17-43%; mean = 28) negative or abnormally immunoreacted (partially deficient, fluffy or intense) fibers for dystrophin. Dystrophin was absent in 2 of 36 patients having a clinical diagnosis of FCMD, and intragenic deletion of the DMD gene was detected in one. Spectrin, a membrane cytoskeletal protein related to dystrophin, also showed an increased number of abnormally immunostained fibers in FCMD (25%), but not so high in age-matched DMD (9%) or SMA patient muscle (0%). Thus, our results suggested the presence of intrinsic factor(s) that produce abnormality of the plasma membrane of FCMD muscle.     

An electron microscopical study of the T-system in biopsied muscles from fukuyama type congenital muscular dystrophy

The behavior of the tubular system in muscles from six patients with Fukuyama type congenital muscular dystrophy (FCMD) was examined by electron microscopy using a lanthanum nitrate stain for a comparison with that in Duchenne muscular dystrophy (DMD). In FCMD, many fibers showed morphological changes of the T-system as follows: aggregated tubular components forming honeycomb-like structures, focal dilatation of T-tubules with tangle formation, and numerous longitudinally projecting tubules, which were quite similar to those found in cases with DMD. The fibers with abnormal T-systems occasionally showed ultrastructural characteristics of regenerating fibers, including excessive ribosome particles, immaturely organized myofibrils, and an increased number of internal nuclei and satellite cells. The present results suggested that there was no qualitative difference in the behavior of the T-system between FCMD and DMD, and the morphological changes of the T-system in dystrophic muscles were not primary lesions initiating myonecrosis but reflected the behavior of sarcotubular formation in the process of muscle regeneration.     

Congenital muscular dystrophy. A histochemical study with morphometric analysis on biopsied muscles

Muscle biopsies from 10 Japanese patients (9 females and 1 male) with congenital muscular dystrophy (CMD) were studied. Their clinical features varied remarkably in severity; one patient died at 6 years of age. Family history was negative in all but one patient who had an affected sibling. Muscle biopsy findings varied from mild myopathic to advanced dystrophic changes. Hypertrophic fibers associated with occasional fiber splitting were assumed to reflect a chronic dystrophic process. Histochemical examination revealed type 1 fiber predominance in 5 patients, and type 2 fiber predominance in one. Eight patients had a slight to moderate increase in the number of undifferentiated type 2C fibers suggesting a regenerating process after fiber necrosis. Type 2B fibers were fairly well preserved in 8 patients. The overall findings differed from those of the Fukuyama type congenital muscular dystrophy (FCMD) and Duchenne muscular dystrophy (DMD) in which more active fiber necrosis and regeneration are seen. We conclude that the present CMD patients suffered from a chronic dystrophic process similar to that in limb-girdle muscular dystrophy.     

Vascular alterations in Fukuyama type congenital muscular dystrophy

Blood vessels in muscle biopsy specimens from 6 Fukuyama type congenital muscular dystrophy (FCMD) patients were examined by electron microscopy and compared with ones in non-diagnostic biopsy specimens from age-matched controls and patients with childhood neuromuscular disorders. The most striking feature was the blister-like swelling of vascular endothelial cells in the biopsied muscle specimens from 5 of the 6 patients with FCMD. Morphometric analysis of capillaries in biopsied muscles showed the extremely greater capillary, endothelial and pericyte areas in the FCMD patients than in controls. These phenomena are quite similar to those found in Duchenne muscular dystrophy (DMD) at the preclinical stage and suggest an as yet undetermined process in blood vessels in FCMD as well as DMD. An immunohistochemical study involving dystrophin antibodies showed positive staining in FCMD.     

Immunohistochemical study of calpain and its endogenous inhibitor in the skeletal muscle of muscular dystrophy

A calcium-dependent proteinase (calpain) has been suggested to play an important role in muscle degradation in Duchenne muscular dystrophy (DMD). In immunohistochemical studies, calpain and its endogenous inhibitor (calpastatin) were located exclusively in the cytoplasm in normal human muscles. The intensity of the staining was stronger in type 1 than in type 2 fibers. Quantitative immunohistochemical study showed an increase of calpain in biopsied muscles from the patients with DMD and Becker muscular dystrophy. Abnormal increases in calpain and calpastatin were demonstrated mainly in atrophic fibers, whereas necrotic fibers showed moderate or weak immunoreactions for the enzymes. Opaque fibers and hypertrophic fibers were negative. Not all dystrophin-deficient muscle fibers necessarily showed a strong reaction for calpain. We suggest that calpain may play an important role in muscle fiber degradation, especially in the early stage of muscle degradation in muscular dystrophy.     

Muscle regeneration and satellite cells in Fukuyama type congenital muscular dystrophy

To determine the regenerative capability of muscle fibers in Fukuyama type congenital muscular dystrophy (FCMD), muscle biopsies from nine patients were subjected to morphological examination by histochemical and electron microscopic methods. These results were compared with those from six patients with Duchenne muscular dystrophy (DMD) and nine controls. Degenerating and regenerating fibers were not as frequent in FCMD as in DMD, whereas the number of type 2C fibers in the former was about twice that in the latter. On electron microscopy, numerous immature small fibers with disorganized myofibrils, an increased amount of intermediate filaments, large vesicular nuclei, and prominent nucleoli were observed in FCMD. Satellite cells (SCs), which are known to proliferate and contribute to muscle regeneration after myonecrosis, were not increased in number in FCMD. Their incidence, 7.7% +/- 1.8%, was significantly lower than the 16.2% +/- 4.0% in DMD (P less than 0.005). Moreover, the ratio of area of SC nucleus to the overall area of the SC in FCMD, 64.1% +/- 9.7%, was larger than that in DMD, 58.9% +/- 9.3% (P less than 0.005), suggesting that SCs of FCMD were not in an active state. These findings lead to the conclusion that in FCMD the regenerative process is delayed due to either intrinsic or extrinsic factors, such as dense interstitial fibrosis.     

Coagulation and fibrinolysis disorder in muscular dystrophy

To investigate whether there are any basic abnormalities of coagulation and fibrinolysis in muscular dystrophy, we measured serum levels of the MM isozyme of creatine kinase (CK-MM), fibrin and fibrinogen degradation products (FDP), plasma levels of fibrinogen, antithrombin (AT), and D-dimer in 36 patients with Duchenne muscular dystrophy (DMD), 11 with Becker muscular dystrophy (BMD), 5 with Fukuyama congenital muscular dystrophy (FCMD), 5 with myotonic dystrophy (MyD), and 5 with spinal muscular atrophy (SMA) type 2. FDP levels were elevated in the patients with DMD, BMD, and FCMD (1.0 to 84.9 microg/ml), but not in the patients with MyD and SMA type 2. In DMD, BMD, and FCMD, FDP levels significantly correlated with CK-MM, but not with age, fibrinogen, AT, D-dimer, and type of dystrophy (multiple regression analysis; r(2) = 0.814, P < 0.0001). These findings suggested that enhanced coagulation and fibrinolysis are associated with muscle degeneration in patients with DMD, BMD, and FCMD.     

Selective deficiency of alpha-dystroglycan in Fukuyama-type congenital muscular dystrophy.

BACKGROUND: Fukuyama-type congenital muscular dystrophy (FCMD) is an autosomal recessive disorder characterized by severe dystrophic muscle wasting from birth or early infancy with structural brain abnormalities. The gene for FCMD is located on chromosome 9q31, and encodes a novel protein named fukutin. The function of fukutin is not known yet, but is suggested to be an enzyme that modifies the cell-surface glycoprotein or glycolipids. OBJECTIVE: To elucidate the roles of fukutin gene mutation in skeletal and cardiac muscles and brain. METHODS: Immunohistochemical and immunoblot analyses were performed in skeletal and cardiac muscles and brain tissue samples from patients with FCMD and control subjects. RESULTS: The authors found a selective deficiency of highly glycosylated alpha-dystroglycan, but not beta-dystroglycan, on the surface membrane of skeletal and cardiac muscle fibers in patients with FCMD. Immunoblot analyses also showed no immunoreactive band for alpha-dystroglycan, but were positive for beta-dystroglycan in FCMD in skeletal and cardiac muscles. CONCLUSION: The current findings suggest a critical role for fukutin gene mutation in the loss or modification of glycosylation of the extracellular peripheral membrane protein, alpha-dystroglycan, which may cause a crucial disruption of the transmembranous molecular linkage of muscle fibers in patients with FCMD.     

Diaphragm muscle pathology in Fukuyama type congenital muscular dystrophy

The diaphragm muscle pathology in four patients with Fukuyama type congenital progressive muscular dystrophy (FCMD) was reported. In the diaphragm muscle fibers of three patients aged more than 14 years exhibiting chronic respiratory failure, a lot of electron dense lesions, which varied in size but did not extend over the whole length of the muscle fibers, and a marked increase in mitochondria were observed as well as the dystrophic changes. The dense lesions, consisting of electron dense fibrillary material comparable to Z-band materials in electron density, seemed to be due to Z-band disruption leading to streaming and/or large aggregations of Z-band materials. No mitochondria or other microorganelles were found in these dense lesions. These findings are thought not to be specific to FCMD, but seem to reflect diaphragm muscle fatigue, because, there were no such lesions in the diaphragm muscle of a 2-year-old infant not exhibiting chronic respiratory failure or in muscle at other sites in all patients.     

Clinical significance of serum cardiac myosin light chain I in patients with muscular dystrophy

We examined serum cardiac myosin light chain I (LCI), serum creatine kinase (CK) levels and left ventricular function in patients with muscular dystrophy and secondary cardiac involvement. LCI levels were determined by a two-site immunoradiometric assay method in 25 patients with muscular dystrophy and 10 normal subjects. This study included 15 patients with Duchenne muscular dystrophy (DMD), 8 patients with Fukuyama type congenital muscular dystrophy (FCMD) and 2 sisters with non-Fukuyama type congenital muscular dystrophy (nFCMD). We measured the value of left ventricular fractional shortening (FS) using echocardiography. All patients with DMD and FCMD showed moderate or severe skeletal muscle weakness. The mean values of LCI were significantly higher in patients with DMD (11.0 +/- 8.3 ng/ml, p less than 0.01) and in patients with FCMD (1.6 +/- 1.4 ng/ml, p less than 0.05) than in normal subjects (0.3 +/- 0.2 ng/ml). In patients with DMD, LCI level correlated closely with CK level (r = 0.81, p less than 0.01) but not with FS (r = 0.35, n.s.). In patients with FCMD, LCI level correlated significantly with CK level (r = 0.75, p less than 0.05) but not with FS (r = 0.44, n.s.). Close correlation between LCI and CK levels was thought to result from the cross reaction between cardiac LCI and myosin light chains of skeletal muscle in the assay method we used. Two siblings with nFCMD showed mild skeletal muscle weakness. A 22-year-old sister with mild left ventricular dysfunction (FS = 0.41) showed high level of CK (4794/U/L) and mild elevation of LCI (7.3 ngml).(ABSTRACT TRUNCATED AT 250 WORDS)     

The significance of boiled-egg fibers in biopsied muscles of neuromuscular disorders]

It is believed that one muscle fiber consists of one fiber type determined by its innervating neuron. In biopsied muscles of Duchenne muscular dystrophy (DMD), however, the author has incidentally found a double-typed fiber which is divided into inner and outer parts. The author termed it a "boiled-egg fiber". The author has examined the appearance rate of the boiled-egg fibers on 682 biopsied muscles obtained from patients with various neuromuscular disorders, and classified the types of the inner and outer parts of the boiled-egg fibers by ATPase staining. Boiled-egg fibers were recognized in 17 cases out of 60 with DMD, 5 out of 146 with other types of muscular dystrophy and 6 out of 94 with myositis. No boiled-egg fiber was found in the remaining 382 cases with other disorders which did not represent necrosis with regeneration of muscle fibers. The total number of boiled-egg fibers was 235 with 192 in DMD and 43 in other disorders. 197 of 235 (83.8%) had the same type for both inner and outer parts and remaining 38 (16.2%) had different types for their inner parts. In 133 of 235 (56.6%), the inner parts were type 2C fibers. Boiled-egg fibers were segmentally found with the length of several hundred micrometers. The above findings suggest that boiled-egg fibers reflect an abnormal regenerating process. It remains to be clarified whether or not inner and outer parts of boiled-egg fibers are double-innervated respectively.     

Assessment of the upper limb muscles in patients with Fukuyama muscular dystrophy: Noninvasive assessment using visual ultrasound muscle analysis and shear wave elastography

Fukuyama-type congenital muscular dystrophy (FCMD) is severe, childhood-onset muscular dystrophy. Recently, our group has discovered a potential treatment using antisense oligonucleotides. Therefore, an effective, reliable, and objective method of assessing muscle is needed. Ultrasound is a minimally invasive tool that can be applied without radiation exposure or pain. Evaluating tissue stiffness by shear wave elastography (SWE) has especially recently attracted attention. Here, we aimed to evaluate SWE value of the upper limb muscles: biceps brachii, triceps brachii, brachioradialis, abductor pollicis brevis, and abductor finger muscle in patients with FCMD. Upper extremity function was evaluated by visual muscle ultrasound analysis (VMUA) and SWE in 13 patients with FCMD and 20 healthy controls. The motor function evaluation tool was used to evaluate motor function, and the correlation with the dynamics of the SWE was determined. VMUA scaled using the Heckmatt scale was higher in patients with FCMD. SWE was also significantly higher and stiffer in the biceps brachii and brachioradialis in patients with FCMD. Furthermore, the severity of FCMD symptoms was correlated with muscle stiffness. We conclude that VMUA and SWE can be useful tools for monitoring muscle atrophy and upper limb function in patients with FCMD.     

Major alteration of the pathological phenotype in gamma irradiated mdx soleus muscles

Two thousand rads of gamma irradiation delivered to the lower legs of ten day old normal and x-chromosome linked muscular dystrophy (mdx) mice caused significant inhibition of tibial bone and soleus muscle fiber growth. In the irradiated mdx solei, there was a major loss of muscle fibers, lack of central nucleation, and some endomysial fibrosis. These features were caused by a failure of regeneration of muscle fibers due to impaired proliferative capacity of satellite cells. Gamma irradiation transforms the late pathological phenotype of mdx muscles, so that in one major aspect (muscle fiber loss) they resemble muscles in Duchenne muscular dystrophy. However, extensive endomysial fibrosis which is another characteristic feature of Duchenne muscular dystrophy did not develop. This experimental model could be useful for the functional investigation of possible beneficial effects of therapeutic interventions in mdx dystrophy.     

Becker 型肌营养不良症患者肌纤维组织化学特征的研究

目的 观察Becker型肌营养不良症（BMD）患者病变肌纤维组织化学特征及其临床意义。方法 3例BMD患者肌肉活检标本分别进行HE染色和还原型辅酶Ⅰ-四唑氮还原酶（NADH—TR酶）染色,光镜下观察,并与正常对照组比较。结果 BMD病例的肌肉切片镜下均可见肌纤维大小不等及显著肥大纤维、劈裂纤维、类靶子纤维、分叶状纤维、轮状纤维和环形纤维;NADH—TR染色可见类靶子纤维。结论 BMD病变肌纤维坏死程度轻,以变性、增生为主,其临床症状轻、病程长,是预后较好的病理学基础。     

Accumulation of collagen and altered fiber-type ratios as indicators of abnormal muscle gene expression in the mdx dystrophic mouse

The growth and development of the X-linked muscular dystrophy mutant mouse (mdx) was compared with a control group from 3 weeks to 1 year old. Quantitative cytological analysis of the soleus muscle revealed cycles of degeneration, regeneration, and hypertrophy, and at any one time it was difficult to assess the extent of the disease based on muscle fiber size. One noticeable difference even in the youngest muscles studied was the reduced numbers of slow oxidative fibers and the increased number of fast glycolytic fibers in the mdx soleus muscles. The collagen of the connective tissue components of selectively stained sections was determined by computerized image analysis. Marked accumulation of collagen was found in both the endomysium and perimysium of the dystrophic muscles as compared with age-matched controls. Since the mdx mouse is a result of the same type of genetic defect as in human Duchenne muscular dystrophy, this model could thus be used to assess the effectiveness of various therapeutic approaches, including gene therapy using muscle fibrosis and fiber type proportions as the indicators.     

Experimental intrauterine infection of akabane virus. Pathological studies of skeletal muscles and central nervous system of newborn hamsters with relevances to the Fukuyama type congenital muscular dystrophy

A vertical infection system in hamsters produced by inoculating with Akabane virus was established as an experimental model of congenital muscular dystrophy (Fukuyama type) (FCMD) and arthrogryposis multiplex congenita (AMC) in humans. Swollen fetuses, mummified fetuses, arthrogryposis and cranial deformities were produced in 13 of 415 newborn hamsters inoculated transplacentally (3.1%). The incidence was significantly higher than that in the control group (p less than 0.05). Eight cases presenting apparent abnormalities were examined histologically and virologically. Pictures of skeletal muscles showing such immature features as chains of internal nuclei and myotubular muscle fibers were demonstrated in all cases. In addition, perivascular infiltration of small round cells and thickening of vascular walls were seen in 5 cases, while myogenic changes such as broken myofibrils, small muscle fibers and changes in fiber size were observed in 6 cases. In the anterior horn of the spinal cord, swelling and loss of nuclei and cell matrices were noticed in 4 cases. In the cerebral cortex, disarrangement of cell layers, edematous changes and loss of nerve cells were revealed in 5 cases. In 4 cases virus particles were found on electron microscopy in the cerebral cortex. The authors considered that this experimental system of intrauterine viral infection would be useful for the etiological study of FCMD and AMC in humans in which not only skeletal muscles but also the central nervous system is affected congenitally.     

Hint and luck for identification of a gene for Fukuyama muscular dystrophy, fukutin]

Fukuyama type congenital muscular dystrophy (FCMD), the second most common form of childhood muscular dystrophy in Japan, is an autosomal recessive severe muscular dystrophy, associated with brain anomalies due to neuronal overmigration. By taking advantages of the presence of a consanguineous patient with both FCMD and xeroderma pigmentosum group A, we performed homozygosity mapping using consanguineous FCMD families mainly, and localized the FCMD locus to chromosome 9q31-33. Subsequently, we have identified the gene responsible for FCMD on 9q31, which encodes a novel 461-amino-acid protein termed fukutin. Most FCMD-bearing chromosomes are derived from a single ancestral founder (87%), and a 3kb-retrotransposal insertion was found to be a founder mutation. Two independent point mutations in this gene have also been detected on chromosomes carrying the non-founder haplotype. FCMD is the first human disease to be caused by an ancient retrotransposal integration. We further identified the gene for muscle-eye-brain (MEB) disease, which encodes POMGnT1. Recent studies have revealed that posttranslational modification of alpha-dystroglycan is associated with congenital muscular dystrophy with brain malformations. Since hypoglycosylation of alpha-dystroglycan is common amongst several other disorders, a new clinical entity called alpha-dystroglycanopathy is proposed. However, only POMGnT1 (MEB) and POMT1 (WWS) are shown to have a definite enzymatic activity, and no enzymatic activity has been detected in fukutin. We show positive interactions between fukutin and POMGnT1. Fukutin may form a protein complex with POMGnT1 and modulate POMGnT1's enzymatic activity. Through cDNA microarray, we also show aberrant neuromuscular junction formation and delayed muscle fiber maturation in alpha-dystroglycanopathies, suggesting a new pathomechanism.     

Degradation of connectin (titin) in Fukuyama type congenital muscular dystrophy: immunochemical study with monoclonal antibodies

Connectin (also called titin) is a myofibrillar elastic filament which links a thick filament to a neighbouring Z line in a sarcomere and thus contributes significantly to the elasticity of myofibrils. In a previous study, we demonstrated by Western blot analysis of the biopsied skeletal muscles using an anti-connectin monoclonal antibody that connectin was degraded extensively after 5 years of age in Duchenne muscular dystrophy (DMD), while it was degraded mildly in Becker muscular dystrophy and only minimally in myotonic dystrophy, limb girdle dystrophy, amyotrophic lateral sclerosis and Charcot-Marie-Tooth disease. In the present study, we investigated the degradation state of connectin in Fukuyama type congenital muscular dystrophy (FCMD) by a similar method using 2 distinct anti-connectin monoclonal antibodies. In FCMD, connectin degradation began much earlier than in DMD: Definite degradation was already observed in 5-8-month-old patients. It was presumed that connectin degradation would play an important role in the myofibrillar degeneration in the early stage of FCMD.     

Muscular dystrophy in a litter of golden retriever dogs

Clinicopathologic findings from two golden retriever dogs with an inherited, progressive, degenerative muscle disease that were studied until 27 and 40 months of age are described. Initial clinical signs included stilted gait and simultaneous advancement of their pelvic limbs. Further gait restriction and muscle hypertrophy eventually occurred. Serum creatine kinase was dramatically elevated (greater than 10,000 U/L). There were persistent "spontaneous" high-frequency discharges (pseudomyotonia) on electromyographic evaluation. Features of both muscle fiber degeneration (hyaline fibers, myophagocytosis) and regeneration (small basophilic fibers) were seen on light microscopy. Similar ultrastructural changes (fiber hypercontraction, increased myoblasts) were present. On morphometric histochemical evaluation, mean fiber diameter of both type 1 and 2 fibers was increased compared with controls in two of three muscles examined. There was no apparent fiber type predominance. Scattered ragged red fibers were seen, but this appeared to be a nonspecific finding of either muscle fiber regeneration or degeneration. These findings and potential contributing pathophysiologic mechanisms are discussed in relation to Duchenne muscular dystrophy.