Phenotype genotype analysis in 15 patients presenting a congenital myasthenic syndrome due to mutations in DOK7.

Congenital myasthenic syndromes (CMSs) are a heterogeneous group of diseases caused by genetic defects affecting neuromuscular transmission. Mutations of DOK7 have recently been described in recessive forms of CMS. Dok-7 is a cytoplasmic post-synaptic protein co-activator of the muscle-specific receptor-tyrosine kinase (MuSK) involved in neuromuscular synaptogenesis and maintenance. We report clinical, morphological and molecular data on 15 patients with mutations in DOK7. Eleven different mutations (5 novel) were identified and all patients but one were found to carry at least the common c.1124_1127dupTGCC mutation. Patients with DOK7 mutations have a particular limb-girdle pattern, without tubular aggregates but a frequent lipidosis on the muscle biopsy. Changes in pre- and post-synaptic compartments of the neuromuscular junction were also observed in muscle biopsies: terminal axons showed defective branching which resulted in a unique terminal axon contacting en passant postsynaptic cups. Clinical features, muscle biopsy findings or response to therapy were confusing in several patients. Characterization of this distinct phenotype is essential to provide clues for targeted genetic screening and to predict the therapeutic response to anticholinesterase treatments or ephedrine as has been suggested.

Clinical and histologic findings in autosomal centronuclear myopathy.

Centronuclear myopathy (CNM) is a congenital myopathy characterized by chains of centrally located nuclei in a large number of muscle fibers. Clinically, an early-onset form was reported in several autosomal-recessive (AR) families and many sporadic patients, whereas a late-onset form was found in most autosomal-dominant (AD) families. The boundary between these two forms remains unclear, and the molecular basis of autosomal CNM is still unresolved. To better define the clinical and morphologic characteristics of autosomal CNM, the authors analyzed a series of 29 patients from 12 families. Two subgroups were identified in three AD families: two families had a relatively late onset of disease and a slow progression of diffuse weakness, whereas the third family, who had a similar clinical course, also presented a unique diffuse muscle hypertrophy. Two presumed AR families and seven sporadic patients were analyzed together, and three subgroups were identified: 1) an early-onset form with ophthalmoparesis; 2) an early-onset form without ophthalmoparesis; and 3) a late-onset form without ophthalmoparesis. Overall, 23 muscle biopsies were reviewed; a majority of patients had >20% central nuclei, fiber type 1 predominance, and a radial distribution of sarcoplasmic strands on oxidative stains. A marked endomysial fibrosis was observed in three sporadic patients with a relatively severe clinical course. The classification reported in this study will be useful for the diagnosis and the follow-up evaluation of patients with autosomal CNM and for the research into the molecular defects underlying the condition.

Histopathological differences of myotonic dystrophy type 1 (DM1) and PROMM/DM2.

Muscle biopsy findings in DM2 have been reported to be similar to those in DM1. The authors used myosin heavy chain immunohistochemistry and enzyme histochemistry for fiber type differentiation on muscle biopsies. Their results show that DM2 patients display a subpopulation of type 2 nuclear clump and other very small fibers and, hence, preferential type 2 fiber atrophy in contrast to type 1 fiber atrophy in DM1 patients.

Age-related morphological changes of the deltoid muscle from 50 to 79 years of age.

A quantitative analysis of deltoid muscle biopsy specimens was performed by light microscopy in 26 males and 25 females aged 50-79 years without known neuromuscular disease. Muscle fibre size, fibre type distribution and increase in mitochondrial content in muscle fibres were examined using a semi-automatic image analysis system. This study showed significant age- and gender-related differences. In females, there was marked atrophy of type II fibres with increasing age, specially of type IIb fibres, but no significant change in muscle fibre type distribution. In males, there was diminution in the relative proportion of type IIb fibres with increasing age but no significant muscle fibre atrophy. Mitochondrial aggregates increased with age, and this increase was observed earlier in females than in males. The gender-related morphological changes observed in the present study differ somewhat from those reported in the literature. The differences between males and females may be partially related to gender differences in muscular activity and may reflect an earlier decline in deltoid muscle strength in females. The existence of these age and gender changes should be taken into account in the interpretation of muscle biopsies of aged individuals.

COOH-terminal truncated human cardiac MyBP-C alters myosin filament organization.

Myosin-binding protein C (MyBP-C) is thought to play structural and/or regulatory role in striated muscles. The cardiac isoform of MyBP-C is one of the disease genes associated with familial hypertrophic cardiomyopathy and most of the mutations produce COOH truncated proteins. In order to determine the consequences of these mutations on myosin filament organization, we have characterized the effect of a 52-kDa NH2-terminal peptide of human cardiac MyBP-C on the alpha-myosin heavy chain (alpha-MyHC) filament organization. This peptide lacks the COOH-terminal MyHC-binding site and retains the two MyHC-binding domains located in the N-terminal part of MyBP-C. For this characterization, cDNA constructs (rat alpha-MyHC, full-length and truncated human cardiac MyBP-C) were transiently expressed singly or in pairwise combination in COS cells. In conformity with previous works performed on the skeletal isoform of MyBP-C, we observed that full-length cardiac MyBP-C organizes the MyHC into dense structures of uniform width. While the truncated protein is stable and can interact with MyHC in COS cells, it does not result in the same organization of sarcomeric MyHC that is seen with the full-length MyBP-C. These results suggest that the presence of truncated cardiac MyBP-C could, at least partly, disorganize the sarcomeric structure in patients with familial hypertrophic cardiomyopathy.

[Proximal myotonial myopathy (PROMM): clinical and histology study]. / Myopathie proximale avec myotonie (PROMM): étude clinique et histologique.

We report 13 French patients with proximal myotonic myopathy. PROMM is a recently delineated multisystem disorder with dystrophic myopathy, myotonia and cataracts. This syndrome is genetically distinct from myotonic dystrophy (DM) by the absence of abnormal CTG repeat expansion. The geographical origin varies but 4 families originated from Poland. Of late onset, muscle weakness is diffuse and predominantly affected proximal and axial muscles. Facial involvement and myotonia were moderate or absent, but in all cases myotonic discharges were detected on EMG. 6 patients suffered from myalgia. Cataracts occurred in 11 patients, mainly indistinguishable from those in DM. Cardiac arrythmia occurred in 7 patients. Muscle biopsy revealed rare structural changes of the muscle fibers and selective type I atrophy, common in DM, could not be found on morphometric analysis. PROMM has a distinct clinical spectrum from DM which includes a predominantly proximal muscle weakness, with troubling pain, a more favourable prognosis and a different histopathological pattern.

["MELAS" (A3243G) mutation of mitochondrial DNA: a study of the relationships between the clinical phenotype in 19 patients and morphological and molecular data]. / Mutation <> (A3243G) de l'ADN mitochondrial: étude chez 19 patients des relations entre le phénotype clinique, les données morphologiques et moléculaires.

Nineteen patients were found to harbor the mitochondrial DNA A3243G mutation associated with MELAS syndrome (Mitochondrial myopathy, Encephalopathy, Lactic Acidosis and Stroke-like episodes). Eight of them had presented with stroke-like episodes and therefore had a clinical diagnosis of MELAS syndrome. The other 11 patients had no strokes and presented with generally less severe multisystemic disease. In the two groups, we compared muscle morphology, biochemical activities of muscle respiratory chain, and genetic characteristics: proportion and tissue distribution of the mutation, sequence of the 22 transfer RNA genes of the mitochondrial DNA. The proportion of mutant mtDNA in muscle was always greater than in blood. The number of patients in the two groups was too low to reach significant values. However, the patients with a MELAS syndrome presented with more severe respiratory chain abnormalities and with a proportion of the A3243G mutation that was both higher and more uniformly distributed among tissues. For symptoms others than stroke-like episodes, we did not observe any correlation with the level of mutant mtDNA in muscle. The analysis of the 22 tRNA sequences did not show differences between the two groups, and no co-inherited modifying tRNA genes could explain the variability of severity in our patients.

Gene delivery to the myocardium by intrapericardial injection.

Several studies have demonstrated the feasibility of gene transfer into the heart muscle. However, all the available data also indicate that the extent of transfection remains limited. As an alternative method to intravascular administration, we have developed a novel strategy which uses the pericardial sac. When a replication-deficient adenovirus containing the cDNA encoding a bacterial beta-galactosidase is injected into the pericardial sac of adult Wistar rats the staining is exclusively restricted to the pericardial cell layers. However, injecting a mixture of collagenase and hyaluronidase together with the virus, leads to a large diffusion of the transgene activity, reaching up to 40% of the myocardium. Transgene expression is predominant in the left ventricle and the interventricular septum but limited in the right ventricle. In vivo echocardiographic measurements of the left ventricular diameters at end diastolic and end systolic times show no difference between virus- and sham-injected animals, thus indicating a good clinical tolerance to this strategy of virus delivery. The same protocol has been used with the same efficiency in mice, which leads us to propose injection into the pericardial sac as an effective and harmless method for gene transfer into the heart muscle.

[Progressive external ophthalmoplegia of mitochondrial origin: contribution of morphological and molecular studies]. / Ophtalmoplégies externes progressives d'origine mitochondriale: apport des investigations morphologiques et moléculaires.

We performed a comparative analysis of the clinical, morphological and molecular characteristics of 62 patients affected with progressive external ophthalmoplegia with ragged-red fibres in muscle. Twenty-seven patients had only muscular disease, and 35 had a multisystemic disease with neurological, cardiac, sensory, or endocrine symptoms. Quantitation of mitochondrial accumulation and numbering of cytochrome c oxidase deficient muscle fibres were done in 43 patients. Muscle mitochondrial DNA deletions were detected, quantitated and localised by Southern Blot analysis. Point mutations were screened in five mitochondrial DNA transfer RNA genes by denaturing gradient gel electrophoresis technique. This study further emphasized the relationships between progressive external ophthalmoplegia and mitochondrial DNA mutations that were present in 46/62 patients (40 deletions, 4 h point mutations in the tRNA leucine gene and 2 further families with maternal inheritance but no mutation identified to-date). Family history was positive in 12 patients: 4 with a maternally inherited disease (2 of whom had an identified mitochondrial DNA mutation), and 4 with an autosomal dominant inherited disease, none of which was associated with multiple mitochondrial DNA deletions. Interestingly, 2 of our patients with an identified mitochondrial DNA mutation appeared as sporadic cases. No morphological or molecular parameters was correlated with the tissular extension of the disease. However, mitochondrial DNA deletions were significantly associated with ocular symptoms which had an earlier onset and were more severe. Clinical features of the patients with a multisystemic disease and a mitochondrial DNA deletion were essentially related to Kearns-Sayre syndrome. In particular, a cardiac conduction defect was present in 12 patients out of 18 with a multisystemic disease associated with a mitochondrial DNA deletion; it was never encountered in 17 patients with a multisystemic disease but no mitochondrial DNA deletion.

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.

Chronic progressive external ophthalmoplegia with ragged-red fibers: clinical, morphological and genetic investigations in 43 patients.

The evaluation of the severity of progressive external ophthalmoplegia (PEO) with ragged-red fibers in muscle, at the onset of the disease, when PEO is most often the only presenting symptom, is a difficult problem in neurological practice. In order to address that issue, we have performed a comparative analysis of the clinical, morphological and molecular characteristics of 43 patients affected with that form of ocular myopathy. Quantification of mitochondrial accumulation was performed with an image analysis application on muscle sections stained with succinate dehydrogenase histochemical reaction. The proportion of muscle fibres appearing as cytochrome c oxidase deficient was used as an index of the muscle-energy defect. Muscle mitochondrial DNA deletions were detected, localized and quantitated by Southern blot analysis. Point mutations were screened in five transfer RNA genes in the mtDNA (tRNA(Leucine (UUR)), tRNA(Lysine), tRNA(Glutamine), tRNA(Isoleucine) and tRNA(Formylmethionine)) by a denaturing gradient gel electrophoresis technique. This investigation confirmed the high frequency of mtDNA deletions or point mutations in PEO. At the onset of the disease, no clinical, morphological or molecular features could predict whether PEO would remain isolated or become part of a more severe multisystem disease. However, patients with mtDNA deletions were characterized by more severe ophthalmoplegia of earlier onset. Their muscle alterations were roughly parallel in severity to the proportion of deleted mtDNA molecules in muscle. Patients with a multitissular disease and mtDNA deletions were always sporadic cases and their clinical presentation was, most often, closely related to Kearns Sayre syndrome.

Myoblast transplantation between monozygotic twin girl carriers of Duchenne muscular dystrophy.

Monozygotic twin girls, both carriers of Duchenne muscular dystrophy, only one a severe symptomatic carrier and the other asymptomatic due to opposite lyonization, were studied. Myoblast clones were obtained from a muscle biopsy of the asymptomatic carrier. PCR amplification showed that most (94%) of these clones produced normal dystrophin mRNA. Roughly 704 million myoblasts were produced from 119 clones. These myoblasts were transplanted into the extensor carpi radialis (ECR) and in the biceps of one arm of the manifesting carrier while the other arm acted as the control. The strength of the patient was evaluated in a series of pre- and post-tests and a biopsy was obtained about 1 yr after the transplantation. The myoblast injections produced a significant force gain (12%-31%) in wrist extension but no force gain for elbow flexion. Muscle biopsies on the injected and control muscles obtained 1 yr after the injections showed only a small increase in the number of dystrophin positive fibers and the presence of numerous small type II fibers. The small beneficial effect of this transplantation cannot be attributed to immune problems, the donor and the recipient being identical twins, but may be due to a low level of spontaneous muscle regeneration.

Changes in surface morphology and basal lamina of cultured muscle cells from Duchenne muscular dystrophy patients.

Cultured muscle cells from Duchenne muscular dystrophy (DMD) patients show altered growth from the mononucleated stage: abnormal morphology, decreased adhesiveness, reduced number of population doublings and delayed fusion. On the basis of these findings, a study was undertaken to observe cell shape and surface morphology by scanning electron microscopy and to define the immunocytochemical localization of 4 basal lamina components (type IV collagen, laminin, fibronectin, heparan sulfate proteoglycan (HSPG]. Eight DMD muscle cultures with fusion indices higher than 65% were compared to muscle cultures from 10 age-matched controls. The following results were noted for the dystrophic muscle cells: (1) the cell surface was smooth with a few slender cell processes and anchorage extensions; (2) distribution of type IV collagen and laminin was heterogenous, with large patches (type IV collagen) or a reticulum (laminin); (3) in contrast, fibronectin and HSPG levels were clearly decreased. These molecules did not form a network but rather were arranged in thick filaments and patches. Cell surface morphology may be related to the decreases in fibronectin and HSPG, which could reflect a more general decrease in basal lamina. Such findings could explain the low adhesiveness of the cells from dystrophic cultures and the delayed fusion of myoblasts. Although these abnormalities were maximally expressed after myoblast fusion, they were already present in mononucleated cells and their connection with the primary defect in DMD, i.e., lack of dystrophin, must now be clarified.

Scanning electron microscopic study of nerve-muscle junctions in embryonic rat cell cultures.

The morphology of neuromuscular junctions between embryonic rat spinal cord and muscle cells grown in vitro has been studied by scanning electron microscopy (SEM). Histochemical detection of acetylcholinesterase (AChE) spots and autoradiographic detection of acetylcholine receptor protein (AChR) were performed in parallel. At the scanning EM level the contacts exhibit a marked polymorphism; the nervous endings may present as a bulbous swelling, or often as a plexiform network on the surface of the myotubes. Many neuromuscular contacts seem to occur 'passing by'; in other places, neurites processes slide along myotubes without any differentiated contacts. The muscle cell surface does not look substantially modified in the contact area. The distribution of the contacts between nerve and muscle cells is at the same time convergent and divergent.