A new congenital muscular dystrophy with mitochondrial structural abnormalities

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ABSTRACT: We report a new form of congenital muscular dystrophy (CMD) in 4 patients from three unrelated families with probable autosomalrecessive inheritance. All patients had the clinical characteristics of merosinpositive congenital muscular dystrophy, but had marked mental retardation. The disease was slowly progressive and 1 patient died from dilated cardiomyopathy at the age of 13 years. In addition to dystrophic changes with necrosis and regeneration in muscle, the most striking finding was mitochondrial depletion in the center of the sarcoplasm. Mitochondria at the periphery of fibers were markedly enlarged (‘‘megaconial’’ appearance) with complicated cristae, and contained a normal amount of mitochondrial DNA by in situ hybridization. Mitochondrial enlargement may represent functional compensation for mitochondrial depletion in the central sarcoplasm, where myofibrillar degeneration occurred. © 1998 John Wiley & Sons, Inc. Muscle Nerve, 21: 40–47, 1998.

Key words: congenital muscular dystrophy; mitochondria; enlargement; depletion; selenium


Department of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), 4-1-1 Ogawahigashi-cho, Kodaira, Tokyo 187, Japan 2 Department of Pediatrics, Kanagawa Rehabilitation Center, Atsugi, Kanagawa, Japan 3 Department of Pediatrics, The Second Nihon Medical College Hospital, Kawasaki, Kanagawa, Japan 4 Department of Human Genetics, National Institute of Genetics, Mishima, Japan Accepted 13 August 1997

Congenital muscular dystrophy (CMD) is a group of genetic diseases with muscle weakness and hypotonia from early infancy with dystrophic changes in muscle biopsy. CMD has been classified into two major categories: the Fukuyama type CMD (FCMD) and the classical (Occidental) form. The latter is further subdivided into merosin-deficient and -positive forms. In addition, there are several other distinct clinical entities such as the Walker–Warburg syndrome (WWS) and muscle–eye–brain disease (MEB).15 In the classical form, the ratio of merosindeficient to -positive forms has been reported to be almost equal in Western countries, but the former is *Correspondence to: Dr. I. Nishino CCC 0148-639X/98/010040-08 © 1998 John Wiley & Sons, Inc.



far less frequent in Japan. Only 4 of 134 patients with the classical form (non-FCMD) in our laboratory were merosin-negative. 15 The gene for merosin (LAMA2) has been mapped to 6q22-23 and various mutations have been found in some families.6,13 Although the merosin-positive CMD is a group of heterogeneous diseases, the overall symptoms are mild and more than 90% of patients can walk alone with slow progression.7,15 We studied 4 patients with CMD from three unrelated families among the merosin-positive form; all were mentally retarded and shared the common peculiar morphological changes of mitochondrial depletion in their muscle biopsies in addition to the dystrophic changes. To characterize this new form of CMD, we examined their muscle biopsies by histochemistry, electron microscopy, biochemistry, and molecular genetics.


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A female child was born after 33 weeks gestation, of healthy parents, who were first cousins. The first of two elder sisters had had similar symptoms of muscle weakness and delayed psychomotor development, and died in a traffic accident at the age of 7 years. All other family members including a second sister, were in good health. She was floppy at birth and gained head control at 6 months of age, sat unsupported at 11 months, and walked independently at 2 years 9 months. On examination at the age of 7 years, she had moderate generalized muscle weakness and wasting and excessive lordosis. Gowers’ sign was present and she had a waddling gait. Her intelligence quotient (IQ) was estimated to be 39. Later she developed dilated cardiomyopathy and died of cardiac failure at the age of 13 years. Creatine kinase (CK) was elevated to 370 U/L (normal: 24–195). Serum lactate level was within normal range. Thyroid function was reported to be normal. Complete blood cell count (CBC) was normal. Brain computed tomography (CT) was unremarkable. Patient 1.

Patient 2. This 13-year-old boy was born at full term of unrelated healthy parents. He had no siblings, and no one else in the family had neuromuscular problems. He attained head control at the age of 5 months, sat unsupported at 9 months, and walked alone at 1 year 9 months. A first muscle biopsy was performed at the age of 1 year and 2 months because of ‘‘floppiness.’’ He had a generalized convulsion at age 10 years and was placed on valproic acid. He did not speak any meaningful words. On examination at age 13 years, he showed generalized muscle weakness with reduced muscle tone. To stand up, he had to ‘‘climb up’’ his legs. He was mentally retarded with an IQ of 44. There were no physical signs indicating central nervous system involvement other than mental retardation. CK was elevated ranging from 465 to 2676 U/L; serum lactate was 13.4 mg/dL (normal: 4–16). Thyroid function was reported to be normal. CBC was normal. Serum selenium level was within normal limits. Electroencephalography (EEG) showed multifocal spikes. Brain CT showed mild brain atrophy. Electrocardiography and chest x-ray were normal. A second muscle biopsy was performed at the age of 13 years.

ter of patient 4. A male sibling born between her and patient 4 was stillborn after 9-month gestation. No other family members were symptomatic. She was floppy in infancy. She gained head control at age 7 months, sat alone at 1 year, and walked alone at 2 years. She spoke a few meaningful words at age 5 years, but became mute at 6 years. On examination at the age of 8 years, she had mild hypotonia and predominantly proximal muscle weakness. Gowers’ sign was positive. Facial muscles were mildly affected. She showed precocious sexual development. Her developmental quotient at the age of 9 years was equivalent to that of a 15-monthold infant. CK was elevated to 502 U/L. Serum lactate was normal. Urinary excretion of 17-OHCS and 17-KS was increased to 6.4 mg/day (normal: 1.7–3.3) and 2.1 mg/day (normal: 0.7–1.6), respectively. Secretion of pituitary hormones including ACTH, LH, and FSH was reported to be normal. Thyroid function was normal. Serum selenium level was decreased to less than 25 µg/L (normal: 151–289). EEG showed multifocal spikes although she never experienced seizures. Brain CT was normal except for the presence of cavum septi pellucidi. Chest x-ray showed an increased cardiothoracic ratio of 60%. A muscle biopsy was performed at age 8 years. This 8-year-old boy, a younger brother of patient 3, was born after a 31-week gestation. He was floppy from birth. He gained head control at age 7 months, sat alone at 1 year 1 month, and walked alone at 2 years 6 months. He never spoke any meaningful words. On examination at the age of 5 years he showed generalized muscle weakness and hypotonia. Facial muscles were mildly affected. He always used the Gowers’ maneuver to stand up. He could not run. At the age of 5 years, his developmental quotient was equivalent to that of a 12-month-old boy. He developed generalized seizures at age 6 years and was placed on valproic acid. CK was mildly elevated to 230 U/L. CBC was normal. Serum lactate was normal on several occasions. Hormonal tests including thyroid function were within normal range. Serum selenium was decreased to 110 µg/mL. Brain CT was reported to be normal although EEG showed a focal abnormality. A muscle biopsy was performed at the age of 5 years.

Patient 4.


A 13-year-old girl was born at full term of nonconsanguinous healthy parents. She was the sis-

Patient 3.


All the biopsied specimens were obtained from the biceps brachii muscle. They were

Muscle Biopsies.


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immersed in 2-methylbutane cooled in liquid nitrogen for fixation. Transverse serial sections at 10 µm were stained with hematoxylin and eosin (H&E), modified Gomori trichrome, and various histochemical methods. We also screened muscle biopsies with monoclonal antibodies to dystrophin, a-sarcoglycan (adhalin), b-dystroglycan, and merosin using a direct immunohistochemical method,9,21 and with monoclonal antibodies to desmin (Dako) and mitochondria (Chemicon) using an indirect streptoavidin– biotin immunohistochemical method, as previously described.11,17 For electron microscopic examination, small portions of biopsied muscle specimens were fixed in 2% glutaraldehyde solution and embedded in epoxy resin. In Situ Hybridization. To clarify the localization of mtDNA on muscle fibers, we used the mtDNA probe corresponding to nt531–718. Transverse sections (6 µm thick) from the patients’ and from control muscles were placed side-by-side on the same glass and fixed in freshly prepared 4% paraformaldehyde for 30 min. After treatment with 0.5 µg/mL proteinase K for 10 min, the sections were postfixed in 4% paraformaldehyde for 5 min. The samples were then treated with 20 µg/mL RNase A at 37°C for 30 min and overlaid with hybridization solution (2 × SSC/ 5% dextran sulfate/0.2% skim milk) combined with the digoxigenin-labeled probe. The specimens were heated to 90°C for 10 min and then incubated at 42°C overnight. After washing in 2 × SSC at 60°C for 20 min, 0.2 × SSC at 60°C for 20 min, and 0.1 × SSC at room temperature for 5 min, signals were detected according to the manufacturer’s instruction (Boehringer Mannheim). Mitochondrial DNA Analysis. Total DNA was prepared from muscle biopsies according to a previously described method.5 We quantitated mtDNA by the method of Moraes et al.10 Briefly, approximately 2-µg DNA aliquots from the patients and from 6 age-matched controls were digested with PvuII (Takara), electrophoresed through 0.8% agarose gel, and transferred to a nylon membrane (Hybond-N+, Amersham). The membrane was hybridized with mtDNA probe and 18S rDNA probe, and the densities of each band were compared. We sequenced the entire mtDNA in patient 2, and all mitochondrial tRNA genes in patients 1 and 3, by a previously described method.12 The resulting data were compared with the reference sequence,1



and with previous reports of polymorphism (Mitochondrial Human Genome Database, Emory University, Atlanta, GA [http://www.gen.emory.edu/ mitomap.html]). This was not done in patient 4 because the sequence of mtDNA was presumably identical to patient 3. Biochemical Analysis. Only the second muscle biopsy of patient 2 was analyzed because the amount of tissue in other cases was limited. Mitochondrial respiratory chain enzyme activities were determined by spectrophotometric assays as previously described.8 RESULTS

All muscle biopsies showed similar pathologic findings. There was moderate variation in fiber size, with values ranging from 10 to 90 µm in diameter, and minimal-to-mild endomysial fibrosis. The most striking findings on modified Gomori trichrome stain were the presence of red-colored large granules at the periphery of and the absence of normal granules in the center of sarcoplasm (Fig. 1A). These granules had high enzyme activities for NADH-tetrazolium reductase (NADH-TR), SDH and cytochrome c oxidase (COX), suggesting that they were mitochondria (Fig. 1B, C). These mitochondrial abnormalities were seen in most type 2 fibers and in some small-calibered type 1 fibers (Fig. 1D). In longitudinal sections, this anomalous mitochondrial distribution was maintained along the longitudinal axis of the fiber, resulting in a tubelike appearance (Fig. 1E). In addition to the mitochondrial abnormalities, there was evidence of both necrosis and regeneration (Fig. 1F). There were neither ragged-red fibers (RRF), nemaline bodies, rimmed vacuoles, nor tubular aggregates. Strongly succinate dehydrogenase (SDH)-reactive blood vessels (SSV) were not present. Intermyofibrillar networks were well-organized except in necrotic and regenerating fibers. There was type 1 fiber predominance ranging from 56% to 61%. In patient 2 who had two muscle biopsies with a 12-year interval, the mitochondrial abnormalities had not changed with time. What had changed was the fiber size which had increased in proportion with age, and the number of necrotic and regenerating fibers, which were increased in the second biopsy. Dystrophin, a-sarcoglycan, b-dystroglycan, and merosin were normally present along the surface membrane by immunohistochemistry. Desmin also stained normally. The distribution of mitochondria identified by antimitochondria antibody was essen-



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FIGURE 1. Abnormal granules were seen at the periphery of the sarcoplasm by modified Gomori trichrome stain (A). Those granules have high SDH (B) and COX (C) activities, suggesting that they are mitochondria. Note markedly decreased to absent mitochondria in the center of sarcoplasm. Routine ATPase (preincubation at pH 10.5) shows abnormal granules in most of the type 2 fibers (asterisks) and some small type 1 fibers (arrow) (D). (A–D: patient 3; original magnification: ×230.) In longitudinal sections, the abnormal mitochondrial distribution was maintained along the long axis of the fiber (asterisks), resulting in a tubelike appearance (E) (the second biopsy of patient 2; COX; original magnification: ×500). In addition to variation in fiber size, necrotic fibers (arrows) are seen in the muscle biopsy, showing dystrophic muscle changes (F) (the first biopsy of patient 2; H&E; original magnification: ×480).

tially identical to that seen on histochemical staining (Fig. 2A). Electron Microscopy. Many fibers had no mitochondria in the deep sarcoplasm (Fig. 3A). The residual mitochondria located under the sarcolemma were markedly enlarged, measuring approximately 1 µm in diameter and 4–5 µm in length, and had a ‘‘megaconial’’ appearance (Fig. 3B). Cristae were redundant and disoriented. There were no inclusions or dense granules in the matrix. The giant mito-


chondria were present between myofibrils, but not necessarily at the A–I junction, where normal mitochondria are located. There was no increase in lipid droplets or glycogen granules. They myofibrils were well-organized except in the necrotic fibers. There were scattered necrotic fibers with macrophage invasion. In the vicinity of the necrotic segments, the myofibrils were markedly distorted, predominantly in the deep sarcoplasm (Fig. 4), suggesting that mitochondrial depletion may play a role in the degenerative process.


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FIGURE 2. Immunohistochemistry using antimitochondria antibody (A) and in situ hybridization using mtDNA probe (B) shows that the distribution of mitochondria and mtDNA is identical to that of the abnormal mitochondria, indicating that the abnormal mitochondria possess normal amounts of mtDNA (original magnification: ×400). In Situ Hybridization. The signal for mtDNA was distributed in the same pattern as mitochondria; that is, there was no signal in the center of affected fibers, whereas it was present only at the peripheral sarcoplasm (Fig. 2B). mtDNA Analysis. None of the patients had deletion or duplication of mtDNA. Southern blot analysis using double probes showed two bands in all samples: 16.6-kb bands for mtDNA and 12.0-kb bands for 18S rDNA. The ratio of signal intensities for mtDNA to 18S rDNA was 3.70 ± 1.19 in the patients (mean ± SD; n = 4) and 5.34 ± 2.24 in controls (mean ± SD; n = 6). The amount of mtDNA seemed mildly decreased in the patients but the difference was not statistically significant. On sequencing, patient 1 had two nucleotide substitutions: T to C at nt4386 and G to A at nt5821, both of which have been reported as neutral polymorphisms. Patients 2 and 3 had no mutations in mitochondrial tRNA genes. In patient 2, all other regions of mtDNA were also sequenced. Conserved sequence blocks I, II, III, promoter sites, and mtTF1 binding sites in the D-loop2 were all spared. In rRNA regions, there were five nucleotide substitutions, T to C at nt681, A to G at nt750, C to T at nt1048, T to C at nt1107, and A to G at nt1438, and deletion of C at nt3106. Of these, all but the 681 mutation had been previously reported as polymorphisms. Several nucleotide substitutions were found in the protein coding region, most of them known polymorphisms. The rest were synonymous substitutions except for one, a T to C at nt6253. To evaluate the 681 and 6253 mutations, we designed mismatch primers of 58-TGGTCCTAGCCTTTCTATTAGAGCT-38 (mismatch sites underlined), which corresponded to nt656–680 in mtDNA, and of 58-CTCCTACTCCTGCTCGCATCT-



TGTA-38, which corresponded to nt6228–6252 in mtDNA. PCR was performed together with the reverse primers corresponding to nt805–787 and nt6403–6384, respectively. The amplified fragments were digested with SacI overnight for the 681 mutation and with BsrGI for the 6253 mutation. We found that one healthy individual among 121 Japanese people had both nucleotide substitutions. These two mutations were homoplasmic both in patient 2 and in the normal individual. Other patients did not have either mutation. Therefore, these nucleotide substitutions were presumably polymorphisms rather than disease-related mutations. There was no decrease in respiratory chain enzyme activities in patient 2: NADH cytochrome c reductase was 110.9 (27.3 ± 11.6, n = 5); succinate cytochrome c reductase 71.5 (76.6 ± 17.7, n = 7); and cytochrome c oxidase 28.9 (33.0 ± 16.1, n = 7) (nanomoles per minute per milligram of mitochondrial protein).

Biochemical Analysis.


The disease reported here is quite different from other CMDs in both clinical and pathologic aspects. Although the muscle symptoms of our patients are similar to those seen in the merosin-positive CMD, all had notable mental retardation. All patients walked by the age of 3 years, although the disease was slowly progressive thereafter. One patient died of dilated cardiomyopathy and another had cardiac dilatation by chest x-ray, suggesting early cardiac involvement in this disorder. In addition to necrosis and regeneration, which are commonly seen in progressive muscular dystrophies, the most striking finding in these patients’ muscles was the absence of mitochondria in the


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FIGURE 3. Electron micrographs of the fibers with mitochondrial abnormalities. Mitochondria are exclusively present at the periphery of sarcoplasm (A, right half of the figure). In contrast, there are no mitochondria in the deep sarcoplasm (A, left half of the figure). Mitochondria in the vicinity of the sarcolemma are enlarged up to 5 µm and extend over several sarcomeres (B). The enlarged mitochondria have complicated cristae but no inclusions (bar = 1 µm).

deep sarcoplasm and the presence of giant mitochondria under the sarcolemma. Because myofibrillar degeneration was predominantly seen in the areas of sarcoplasm lacking mitochondria (Fig. 4), it is conceivable that mitochondrial depletion may trigger muscle fiber necrosis.


Abnormal mitochondrial distribution in muscle is seen in many disorders and is associated with RRF, central core, and target/targetoid fibers. The mitochondrial anomalies in the present patients resemble the ‘‘cores’’ seen in central core disease but differ completely in their morphologic details, as fol-


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FIGURE 4. An electron micrograph showing a necrotic process. Myofibrils are disorganized and mostly lost in the center of the sarcoplasm, where mitochondria are sparse (left half of the figure). Giant mitochondria are present at the periphery of the sarcoplasm (right half of the figure) (bar = 1 µm).

lows: (1) central cores, either structured or unstructured, are exclusively present in type 1 fibers,3 whereas the abnormalities in our patients are predominantly seen in type 2 fibers; (2) mitochondria in central core fibers have a normal size and morphology, whereas residual mitochondria in our patients are enlarged and contain abnormal cristae; (3) the sarcomere in the core region is disorganized with Z-streaming, whereas nonnecrotic muscle fibers in our patients has normal striation; and (4) sarcoplasmic reticulum is sparse in the cores, whereas it is well preserved in muscles from our patients. The muscle fibers in our patients are also distinctly different from the target/targetoid fibers, which have morphologic characteristics similar to those of central core fibers. There is a subgroup of mitochondrial encephalomyopathies associated with mtDNA mutations.14,19 In our patients, however, all the mutations found in sequence analyses of mtDNA were either polymorphisms or synonymous mutations, and deletions were not found by Southern blot analysis. Therefore, the mitochondrial disorder described here is not due to alterations in mtDNA sequence. There is another group of mitochondrial diseases in which the amount of mtDNA is decreased to less than 34% of the normal value10 and RRF are usually seen in muscle biopsies (mtDNA depletion). How-



ever, in our patients, there was no significant reduction in the number of mtDNA nor mitochondrial dysfunction because serum lactate levels were not increased and respiratory chain enzyme activities were normal. Therefore, it appears that the residual mitochondria at the periphery of the sarcoplasm were hypertrophied to functionally compensate for the mitochondrial depletion at the periphery of fibers. The dystrophic changes appear to be responsible for the weakness rather than the mitochondrial abnormalities. The cause of the abnormal mitochondrial distribution observed in these patients is probably a defect in mitochondrial fixation or in mitochondrial division in the sarcoplasm. A muscle-specific intermediate filament, desmin, is known to attach to mitochondria for fixation,20 but desmin was normally expressed by immunohistochemistry. Similar mitochondrial abnormalities were described in familial patients with adult-onset postexercise muscle pain and weakness.4 Although the clinical presentation of those patients differed significantly from ours, the pathological findings are almost identical, suggesting that mitochondrial dysgenesis can produce different clinical phenotypes. Dystrophic changes, the most distinct feature in our patients, was absent in the patients of Genge et al.4 Servidei et al. reported a 66-year-old patient, with


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similar mitochondrial depletion labeled as ‘‘oligomitochondrial myopathy,’’18 who had recurrent encephalopathy, lactic acidosis, and decreased respiratory enzyme activities and mtDNA. Different from our patients, mitochondria in Servidei’s patient were depleted in both type 1 and 2 fibers, and residual mitochondria were not hypertrophied. Recently, we encountered a 37-year-old patient with selenium-deficient myopathy due to long-term parenteral nutrition secondary to duodenal pseudoobstruction.16 He developed muscle pain and proximal weakness unrelated to exercise. In his muscle biopsy, approximately 10% of the fibers, most of them type 2 fibers, showed mitochondrial depletion in the center and mitochondrial enlargement at the periphery of the sarcoplasm. There were a few necrotic and regenerating fibers. These findings were quite similar to those presented here, raising the possibility that the new CMD reported here may share a common pathogenetic mechanism with selenium-deficient myopathy. We are presently studying selenium metabolism in our patients, because 2 of the 3 patients we examined had decreased serum selenium levels. We are grateful to Drs. Salvatore DiMauro and Eduardo Bonilla, Department of Neurology, H. Houston Merritt Clinical Research Center for Muscular Dystrophy and Related Diseases, Columbia– Presbyterian Medical Center, for their advice and critical reading of the manuscript. We thank Dr. James E. Sylvester, Department of Genetics, Hahnemann University, for providing human ribosomal DNA clones, and Ms. Kumiko Murayama, Department of Ultrastructural Research, National Center of Neurology and Psychiatry, for expert assistance.






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14. 15. 16.

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