Mutations in the laminin alpha 2-chain gene (LAMA2) cause merosin-deficient congenital muscular dystrophy.

Congenital muscular dystrophies (CMDs), are heterogeneous autosomal recessive disorders. Their severe manifestations consist of early hypotonia and weakness, markedly delayed motor milestones and contractures, often associated with joint deformities. Histological changes seen in muscle biopsies consist of large variations in muscle fibre size, a few necrotic and regenerating fibres and a marked increase in endomysial collagen tissue. Diagnosis is based on clinical features and on morphological changes. In several CMD cases, we have demonstrated an absence of one of the components of the extracellular matrix around muscle fibres, the merosin M chain, now referred to as the alpha 2 chain of laminin-2 (ref.3). We localized this CMD locus to chromosome 6q2 by homozygosity mapping and linkage analysis. The laminin alpha 2 chain gene (LAMA2) maps to the same region on chromosome 6q22-23 (ref. 5). We therefore investigated LAMA2 for the presence of disease-causing mutations in laminin alpha 2 chain-deficient CMD families and now report splice site and nonsense mutations in two families leading presumably to a truncated laminin alpha 2 protein.

Short GCG expansions in the PABP2 gene cause oculopharyngeal muscular dystrophy.

Autosomal dominant oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease with a world-wide distribution. It usually presents in the sixth decade with progressive swallowing difficulties (dysphagia), eyelid drooping (ptosis) and proximal limb weakness. Unique nuclear filament inclusions in skeletal muscle fibres are its pathological hallmark. We isolated the poly(A) binding protein 2 gene (PABP2) from a 217-kb candidate interval on chromosome 14q11 (B.B. et al., manuscript submitted). A (GCG)6 repeat encoding a polyalanine tract located at the N terminus of the protein was expanded to (GCG)8-13 in the 144 OPMD families screened. More severe phenotypes were observed in compound heterozygotes for the (GCG)9 mutation and a (GCG)7 allele that is found in 2% of the population, whereas homozygosity for the (GCG)7 allele leads to autosomal recessive OPMD. Thus the (GCG)7 allele is an example of a polymorphism which can act either as a modifier of a dominant phenotype or as a recessive mutation. Pathological expansions of the polyalanine tract may cause mutated PABP2 oligomers to accumulate as filament inclusions in nuclei.

Absence of extraocular muscle pathology in Duchenne's muscular dystrophy: role for calcium homeostasis in extraocular muscle sparing.

Duchenne muscular dystrophy (DMD) is characterized by clinical weakness and progressive necrosis of striated muscle as a consequence of dystrophin deficiency. While all skeletal muscle groups are thought to be affected, enigmatically, the extraocular muscles (EOM) appear clinically unaffected. Here we show that dystrophin deficiency does not result in myonecrosis or pathologically elevated levels of intracellular calcium ([Ca2+]i) in EOM. At variance with a previous report, we find no evidence for dystrophin-related protein/utrophin up-regulation in EOM. In vitro experiments demonstrate that extraocular muscles are inherently more resistant to necrosis caused by pharmacologically elevated [Ca2+]i levels when compared with pectoral musculature. We believe that EOM are spared in DMD because of their intrinsic ability to maintain calcium homeostasis better than other striated muscle groups. Our results indicate that modulating levels of [Ca2+]i in muscle may be of potential therapeutic use in DMD.

Deficiency of dystrophin-associated proteins in Duchenne muscular dystrophy patients lacking COOH-terminal domains of dystrophin.

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is a cytoskeletal protein tightly associated with a large oligomeric complex of sarcolemmal glycoproteins including dystroglycan, which provides a linkage to the extracellular matrix component, laminin. In DMD, the absence of dystrophin leads to a drastic reduction in all of the dystrophin-associated proteins, causing the disruption of the linkage between the subsarcolemmal cytoskeleton and the extracellular matrix which, in turn, may render muscle cells susceptible to necrosis. The COOH-terminal domains (cysteine-rich and carboxyl-terminal) of dystrophin have been suggested to interact with the sarcolemmal glycoprotein complex. However, truncated dystrophin lacking these domains was reported to be localized to the sarcolemma in four DMD patients recently. Here we report that all of the dystrophin-associated proteins are drastically reduced in the sarcolemma of three DMD patients in whom dystrophin lacking the COOH-terminal domains was properly localized to the sarcolemma. Our results indicate that the COOH-terminal domains of dystrophin are required for the proper interaction of dystrophin with the dystrophin-associated proteins and also support our hypothesis that the loss of the dystrophin-associated proteins in the sarcolemma leads to severe muscular dystrophy even when truncated dystrophin is present in the subsarcolemmal cytoskeleton.

Immunohistochemical analysis of dystrophin-associated proteins in Becker/Duchenne muscular dystrophy with huge in-frame deletions in the NH2-terminal and rod domains of dystrophin.

The absence of dystrophin causes the drastic reduction of the dystrophin-associated proteins (DAPs) in the sarcolemma and the loss of the linkage between the subsarcolemmal cytoskeleton and the extracellular matrix in Duchenne muscular dystrophy (DMD) skeletal muscle. Here, we report a mild reduction of the DAPs in the unique Becker muscular dystrophy patients with huge deletions in the rod domain of dystrophin and a moderate reduction of the DAPs in patients with huge deletions that involve both the NH2-terminal and rod domains of dystrophin. The phenotype of the latter patients was more severe than that of the former. In both cases, however, the reduction in the DAPs was milder than in typical DMD patients or DMD patients lacking the COOH-terminal domains of dystrophin. Our results suggest that (a) the NH2-terminal and rod domains of dystrophin may not be essential for the interaction with the sarcolemmal glycoprotein complex; and (b) defects in the actin binding activity of dystrophin may cause disruption of the anchorage of the dystrophin-glycoprotein complex to the subsarcolemmal cytoskeleton, which may render muscle fibers susceptible to degeneration.

A homologue of dystrophin is expressed at the neuromuscular junctions of normal individuals and DMD patients, and of normal and mdx mice. Immunological evidence.

Polyclonal and monoclonal antibodies, which recognize different regions and epitopes of the dystrophin molecule, bind to a protein of Mr 400,000 which is present in extracts of mdx muscle from regions which contain neuromuscular junctions (NMJ) and is absent from those which do not. This NMJ-associated homologue of dystrophin has at least 2 epitopes which are different to usual Xp21 form of dystrophin expressed along the sarcolemma of muscle fibres in normal muscles. This protein is also expressed at the NMJ of a DMD patient who lacks the first 52 exons of the Xp21 dystrophin gene and it must therefore be translated from a different gene transcript.

Expression of utrophin and its mRNA in denervated mdx mouse muscle.

Utrophin is a large cytoskeletal protein which shows high homology to dystrophin. In contrast to the sarcolemmal distribution of dystrophin, utrophin accumulates at the postsynaptic membrane of the neuromuscular junction. Because of its localization within this compartment of muscle fibers, expression of utrophin may be significantly influenced by the presence of the motor nerve. We tested this hypothesis by denervating muscles of mdx mouse and monitoring levels of utrophin and its mRNA by immunofluorescence, immunoblotting and RT-PCR. A significant increase in the number of utrophin positive fibers was observed by immunofluorescence 3 to 21 days after sectioning of the sciatic nerve. Quantitative analyses of utrophin and its transcripts in hindlimb muscles denervated for two weeks showed only a moderate increase in the levels of both utrophin (approximately 2-fold) and its transcript (approximately 60 to 90%). The present data suggest that although utrophin is a component of the postsynaptic membrane, its neural regulation is distinct from that of the acetylcholine receptor.

Absence of gamma-sarcoglycan (35 DAG) in autosomal recessive muscular dystrophy linked to chromosome 13q12.

We have partially sequenced rabbit skeletal muscle gamma-sarcoglycan, an integral component of the dystrophin-glycoprotein complex. Specific antibodies were produced against a gamma-sarcoglycan peptide and used to examine the expression of gamma-sarcoglycan in skeletal muscle of patients with severe childhood autosomal muscular dystrophy linked to chromosome 13q12 (SCARMD). We show by immunofluorescence and Western blotting that in skeletal muscle from these patients gamma-sarcoglycan is completely absent and alpha- and beta-sarcoglycan are greatly reduced in abundance, whereas other components of the DGC are preserved. In addition, we show that in normal muscle alpha-, beta-, and gamma-sarcoglycan constitute a tightly associated sarcolemma complex which cannot be disrupted by SDS treatment.

Familial fingerprint body myopathy.

Muscle biopsy specimens from two half brothers with a congenital benign muscle disorder and from their mother, clinically unaffected, were studied by histochemistry and electron microscopy. In the children's biopsy specimens, the ultrastructural examination showed numerous fingerprint bodies located at the periphery of the muscle fibers. The histochemical pattern was different in these two specimens. In the mother's biopsy specimen, while fingerprint bodies were not found, the muscle fibers showed slight but definite changes. Even if the fingerprint bodies by themselves are not specific for a particular muscle disease, their occurrence in two half brothers is a valid argument in favor of the individalization of the fingerprint body myopathy, which has been previously described.

Nuclear inclusions in innervated cultured muscle fibers from patients with oculopharyngeal muscular dystrophy.

We established monolayer muscle fiber cultures from muscle biopsies of 3 patients with oculopharyngeal muscular dystrophy (OPMD) who had characteristic intranuclear inclusions (INI-A) in their muscle fibers. Aneural cultures had normal morphology, except for a few muscle fibers that contained small vacuoles. Innervated cultures had large cytoplasmic vacuoles in a number of muscle fibers. Those muscle fibers were breaking easily, and could not be maintained longer than 2 months. Electron microscopy showed unusual intranuclear inclusions (INI-B) not previously reported in aneurally cultured muscle fibers of OPMD or in any normal or disease-control aneural or innervated cultured human muscle fibers. They resembled, but were not identical to, the INI-A, and they occurred in both the cultured fibers and the original muscle biopsies of all 3 patients. Our study demonstrate that (1) nuclear inclusions in OPMD reflect an intrinsic genetic defect; and (2) neuronal influence, advanced maturation, or both, seem to be essential for their induction in muscle fibers.

Intranuclear inclusions in oculopharyngeal muscular dystrophy among Bukhara Jews.

We studied, by electron microscopy, muscle biopsies from seven patients with autosomal dominant oculopharyngeal muscular dystrophy (OPMD) belonging to the recently described Bukhara-Jewish cluster. Typical tubulofilamentous intranuclear inclusions (INI) of 8.5 nm outer diameter were present in all cases. The INI were observed in 4.5 +/- 1.8% of the nuclei in five patients. In the other two, they occurred in 9.5 +/- 0.5% of the nuclei and often occupied a larger nuclear area. These two patients, offspring of intermarriage between affected cousins, had an unusually severe form of OPMD beginning in their early 30s, suggesting homozygote state. Our results confirm that INI are pathognomonic for OPMD and suggest that their frequency may be quantitatively related to the number of abnormal DNA copies.

Oculopharyngeal muscular dystrophy in two unrelated Japanese families.

The occurrence of oculopharyngeal muscular dystrophy (OPMD) in Orientals is uncertain. We identified two unrelated Japanese families, including 30 affected individuals (14 men, 16 women, mean age 58 years) of OPMD through four generations, with complete penetrance. Their major clinical manifestations were late-onset bilateral ptosis and dysphagia. Histologic studies of slightly affected muscles reveal mild myogenic changes, occasional rimmed vacuoles, and small angulated fibers. By contrast, the severely involved cricopharyngeal muscle showed marked loss of fibers and massive proliferation of connective tissue. Ultrastructural studies of four different biopsied muscles disclosed subsarcolemmal intranuclear tubulofilamentous inclusions, identical to those of non-Japanese OPMD patients.

Congenital muscular dystrophy with primary partial laminin alpha2 chain deficiency: molecular study.

The authors report a case of congenital muscular dystrophy with mild nonprogressive muscle weakness, white matter hypodensity, and absence of the laminin alpha2 chain in muscle fibers with two antibodies, but not with four others. They identified mutations in LAMA2, which explain the partial laminin alpha2 deficiency. Analysis of this case and two others allows us to refine the epitopes of two of the commercial antibodies, and illustrate the importance of using antibodies directed against different domains of the protein.

Oculopharyngeal MD among Bukhara Jews is due to a founder (GCG)9 mutation in the PABP2 gene.

OBJECTIVE: To determine whether all cases of oculopharyngeal muscular dystrophy (OPMD) among Bukhara Jews share the same founder mutation. BACKGROUND: Autosomal dominant OPMD is caused by a (GCG)8-13 repeat expansion in the polyadenylation binding protein 2 (PABP2) gene. The disease has a worldwide distribution but is particularly prevalent in Bukhara Jews and in French Canadians, in whom it was introduced by three sisters in 1648. METHODS: We established the size of the PABP2 mutation in 23 Bukhara Jewish patients belonging to eight unrelated families. In all families, we constructed haplotypes for the carrying chromosomes composed of the alleles for eight chromosome 14q polymorphic markers. RESULTS: All patients share a (GCG)9 PABP2 mutation and a four-marker haplotype. Furthermore, a shared intron single nucleotide polymorphism (SNP) in the PABP2 gene 2.6Kb from the mutation was not observed in 22 families with (GCG)9 mutations from nine different countries. The smaller size of the chromosomal region in linkage disequilibrium around the mutation in Bukhara Jews, as compared with French Canadians, suggests a founder effect that occurred more than 350 years ago. Based on the Luria-Delbrück corrected "genetic clock," we estimate that the mutation appeared or was introduced once in the Bukhara Jewish population between AD 872 and 1512 (mean, AD 1243). CONCLUSION: OPMD among Bukhara Jews is the result of a shared, historically distinct, PABP2 (GCG)9 mutation that likely arose or was introduced in this population at the time they first settled in Bukhara and Samarkand during the 13th or 14th centuries.

Primary adhalinopathy (alpha-sarcoglycanopathy): clinical, pathologic, and genetic correlation in 20 patients with autosomal recessive muscular dystrophy.

Primary adhalin (or alpha-sarcoglycan) deficiency due to a defect of the adhalin gene localized on chromosome 17q21 causes an autosomal recessive myopathy. We evaluated 20 patients from 15 families (12 from Europe and three from North Africa) with a primary adhalin deficiency with two objectives: characterization of the clinical phenotype and analysis of the correlation with the level of adhalin expression and the type of gene mutation. Age at onset and severity of the myopathy were heterogeneous: six patients were wheel-chair bound before 15 years of age, whereas five other patients had mild disease with preserved ambulation in adulthood. The clinical pattern was similar in all the patients with symmetric characteristic involvement of trunk and limb muscles, calf hypertrophy, and absence of cardiac dysfunction. Immunofluorescence and immunoblot studies of muscle biopsy specimens showed a large variation in the expression of adhalin. The degree of adhalin deficiency was fairly correlated with the clinical severity. There were 15 different mutations (10 missense, five null). Double null mutations (three patients) were associated with severe myopathy, but in the other cases (null/missense and double missense) there was a large variation in the severity of the disease.

Oculopharyngeal muscular dystrophy in France.

The clinical, histopathological, ultrastructural and geographical data on 29 cases of oculopharyngeal muscular dystrophy (OPMD) identified by the authors in France is briefly presented. The mean age of the patients was 53.8 +/- 8.1 years. Onset symptoms were ptosis (14/29), dysphagia (12/29) and limb girdle weakness (3/29). The evolution of the disease was always progressive and followed different clinical patterns. The main histological changes in muscle biopsies were atrophic angulated fibers (29/29) and rimmed vacuoles (25/29); muscle fiber necrosis was very rare (1/29). The characteristic nuclear inclusions made of 8.5-nm filaments were observed in all cases, and found in 2-5% of the nuclei in a given ultrathin section. They are the morphological marker of the disease.

Ubiquitin and beta-amyloid-protein in inclusion body myositis (IBM), familial IBM-like disorder and oculopharyngeal muscular dystrophy: an immunocytochemical study.

We used immunocytochemistry to identify ubiquitin and beta-amyloid-protein in muscle biopsies from patients with three neuromuscular disorders characterized by the presence of rimmed vacuoles in muscle fibres: inclusion body myositis (IBM), familial IBM-like disorder and oculopharyngeal muscular dystrophy (OPMD). Labelling with anti-ubiquitin antibodies was observed in all three diseases, but it was frequent in IBM, less common in familial IBM-like disorder and rare in OPMD. This labelling is thought to correspond to the presence of IBM-type filaments (16-18 nm in external diameter) which are characteristic but not specific for IBM or familial IBM-like disorder, as they may also occur in other diseases including OPMD. Labelling with anti-beta-amyloid-protein antibody was seen in a few fibres in IBM but not in the other two conditions. The structures labelled with this antibody have yet to be determined. Labelling with anti-ubiquitin or anti-beta-amyloid-protein antibodies was not correlated with the presence of acid phosphatase activity.

Autosomal recessive oculopharyngodistal myopathy in light of distal myopathy with rimmed vacuoles and oculopharyngeal muscular dystrophy.

We investigated two Japanese siblings presenting with oculopharyngodistal myopathy, whose healthy parents were consanguineous. To clarify their disease characteristics, we compared them with four patients with distal myopathy with rimmed vacuoles linked to chromosome 9p1-q1, and 36 patients with oculopharyngeal muscular dystrophy linked to 14q11.2-q13. The first symptom in the patients with autosomal recessive oculopharyngodistal myopathy was weakness of the tibialis anterior muscle. Their biceps muscles showed initial and advanced myogenic changes, with rimmed vacuoles in 3% and 6% of the muscle fibers, respectively. In contrast, patients with distal myopathy with rimmed vacuoles revealed many rimmed vacuoles, on average in 20% of the fibers, and their oculopharyngeal muscles were spared. None of the patients with oculopharyngeal muscular dystrophy showed distal dominant weakness and the occurrence of rimmed vacuoles was rare. Ultrastructural studies in groups of autosomal recessive oculopharyngodistal myopathy and distal myopathy with rimmed vacuoles disclosed a collection of cytoplasmic filaments of 16-18 nm, but oculopharyngeal muscular dystrophy-specific intranuclear inclusions of 8.5 nm were not found. Thus, the phenotype of autosomal recessive oculopharyngodistal myopathy is distinct from distal myopathy with rimmed vacuoles and oculopharyngeal muscular dystrophy, but shares some ultrastructural characteristics with distal myopathy with rimmed vacuoles and hereditary inclusion body myopathy.

Morphological changes in muscle fibers in oculopharyngeal muscular dystrophy.

The study of muscle biopsies of 29 cases of oculopharyngeal muscular dystrophy (OPMD) showed the two main morphological features of this disease: rimmed vacuoles (in 26 cases) and intranuclear inclusions (in all cases). These inclusions are made of 8.5 nm tubular filaments and the areas occupied by them are lighter than the surrounded nucleoplasm. This can be seen by light microscopy, facilitating the detection of the tubulo-filamentous inclusions which can only be identified with certitude by electron microscopy. In a given ultrathin section the area occupied by these inclusions varied from 2% to 5% of the nuclei. The intranuclear inclusions are the morphological marker of OPMD and their finding in a muscle biopsy allows the exact diagnosis of this disease. The origin and biochemical nature of the intranuclear inclusions is unknown.

Expression of dystrophin-associated glycoproteins during human fetal muscle development: a preliminary immunocytochemical study.

An immunocytochemical study was performed on quadriceps muscle from eight fetuses ranging from 12 weeks of gestation to term, using antibodies against the dystrophin-associated proteins, in order to evaluate the developmental expression of these proteins. For comparison, antibodies against dystrophin and utrophin were also used. The expression of the 59 kDa dystrophin-associated protein was simultaneous with that of dystrophin, which is also a subsarcolemmal protein. The extracellular glycoprotein of 156 kDa (alpha-dystroglycan) and the transmembrane glycoprotein of 43 kDa (beta-dystroglycan) appeared to be expressed later. The transmembrane glycoproteins of 50 kDa (adhalin) and 35 kDa were fully expressed at an even later stage of fetal muscle development. This study suggests that the subsarcolemmal proteins may have an essential role in the assembly of the transmembrane and extracellular components of the dystrophin-glycoprotein complex during fetal muscle development. The knowledge obtained from observing the developmental expression of these proteins may contribute to the understanding of the molecular mechanism of their different involvement in muscle disorders.