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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. [ABSTRACT FROM AUTHOR]

Background: The most common form of congenital muscular dystrophy is caused by a deficiency in the alpha2 chain of laminin-211, a protein of the extracellular matrix. A wide variety of mutations, including 20 to 30% of nonsense mutations, have been identified in the corresponding gene, LAMA2. A promising approach for the treatment of genetic disorders due to premature termination codons (PTCs) is the use of drugs to force stop codon readthrough., Methods: Here, we analyzed the effects of two compounds on a PTC in the LAMA2 gene that targets the mRNA to nonsense-mediated RNA decay, in vitro using a dual reporter assay, as well as ex vivo in patient-derived myotubes., Results: We first showed that both gentamicin and negamycin promote significant readthrough of this PTC. We then demonstrated that the mutant mRNAs were strongly stabilized in patient-derived myotubes after administration of negamycin, but not gentamicin. Nevertheless, neither treatment allowed re-expression of the laminin alpha2-chain protein, pointing to problems that may have arisen at the translational or post-translational levels., Conclusions: Taken together, our results emphasize that achievement of a clinical benefit upon treatment with novel readthrough-inducing agents would require several favourable conditions including PTC nucleotide context, intrinsic and induced stability of mRNA and correct synthesis of a full-length active protein.

Tubular aggregates are morphological abnormalities characterized by the accumulation of densely packed tubules in skeletal muscle fibres. To improve knowledge of tubular aggregates, the formation and role of which are still unclear, the present study reports the electron microscopic analysis and protein characterization of tubular aggregates in six patients with 'tubular aggregate myopathy'. Three of the six patients also presented with myasthenic features. A large panel of immunochemical markers located in the sarcoplasmic reticulum, T-tubules, mitochondria, and nucleus was used. Despite differences in clinical phenotype, the composition of tubular aggregates, which contained proteins normally segregated differently along the sarcoplasmic reticulum architecture, was similar in all patients. All of these proteins, calsequestrin, RyR, triadin, SERCAs, and sarcalumenin, are involved in calcium uptake, storage, and release. The dihydropyridine receptor, DHPR, specifically located in the T-tubule, was also present in tubular aggregates in all patients. COX-2 and COX-7 mitochondrial proteins were not found in tubular aggregates, despite being observed close to them in the muscle fibre. The nuclear membrane protein emerin was found in only one case. Electron microscopy revealed vesicular budding from nuclei, and the presence of SAR-1 GTPase protein in tubular aggregates shown by immunochemistry, in all patients, suggests that tubular aggregates could arise from endoplasmic reticulum exit sites. Taken together, these results cast new light on the composition and significance of tubular aggregates., (Copyright (c) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Purpose: To test, by using an electrotransfer protocol for the transfection of skeletal muscle with naked plasmid complementary DNA, whether in vivo magnetic resonance (MR) imaging can help delineate either the spatial extent of the electric field when contrast agent is injected intraperitoneally or the transfection area when contrast agent is injected locally., Materials and Methods: Three groups of five mice each were examined at 4 T. Gadopentetate dimeglumine was injected intraperitoneally before electroporation in group 1 and after electroporation in group 2. In group 3, gadopentetate dimeglumine was coinjected in situ with plasmid pCMV-beta Gal in the gastrocnemius muscle before electroporation. MR imaging and muscle preparation for histologic examination were performed 3 days later. On T1-weighted images, increase of muscle signal intensity was determined in regions of interest (ROIs) of treated legs and compared with contralateral ROIs. Comparison of signal intensity increase between groups 1 and 2 was performed with Kruskal-Wallis test., Results: In groups 1 and 3, T1-weighted images of treated muscle showed zones of strongly increased signal intensity. In corresponding ROIs of groups 1, 2, and 3, the mean T1-weighted signal intensity increase at day 3 was 1.64 +/- 0.20 (SD), 1.16 +/- 0.06, and 1.58 +/- 0.17, respectively. The difference between groups 1 and 2 (ie, gadopentetate dimeglumine injected before and after electrotransfer) was significant (P <.001) both without and with correction for T2 variation (1.47 +/- 0.19 and 1.04 +/- 0.09, respectively). In group 3, after in situ coinjection of gadopentetate dimeglumine and plasmid, the area of increased signal intensity revealed at ex vivo MR imaging of the muscle showed a reasonable concordance with the transfected area revealed with beta-galactosidase on histologic sections., Conclusion: In vivo and ex vivo results indicate that atraumatic visualization of the permeabilized and transfected area is possible.

We describe the effects of the neurotoxin 3-nitropropionic acid (3-NPA) on fatty acid oxidation in neonatal rat brain astrocytes in primary culture, using a sensitive assay for beta-oxidation which depends on the release of 3H2O from [9,10(n)-3H]palmitic acid. 3-NPA is a suicide inhibitor of succinate dehydrogenase, a constituent of both Krebs cycle and complex II of the mitochondrial respiratory chain. It is widely distributed in plants and fungi. Neurotoxicity of 3-NPA to humans and animals, leading to selective neuronal cell death, appears mediated by the reduced level of ATP induced by the toxin. We demonstrated that 3-NPA can also impair energy metabolism in astrocytes. Exposure of astroglial cells in culture to 3-NPA leads to inhibition of the release of 3H2O from [9,10(n)-3H]palmitic acid. Addition of 2 mM 3-NPA to the culture medium caused a rapid decrease in beta-oxidation activity, which reached a plateau after 90 min. This inhibition was concentration-dependent. Concentration as low as 0.05 mM for 5 h significantly decreased beta-oxidation activity (25% inhibition). Half-maximal inhibition was obtained after treatment with 0.5 mM 3-NPA, and 3 mM induced a maximal response (63% inhibition) 3-NPA is clearly a potent inhibitor of beta-oxidation activity. We also show that 3-NPA 3 mM inhibits partially complex II (succinate ubiquinone reductase) and aspartate aminotransferase by 60 and 49% after 4 h treatment respectively. It has been shown that fatty acid is the preferred substrate for energy production in cultured astrocytes from developing brain. As astrocytes may also provide substrates alternative for energy metabolism in neurons and oligodendrocytes, it is likely that the inhibition of beta-oxidation by 3-NPA may contribute significantly to the damage induced by this toxin in the central nervous system.

An epidemiological study was conducted in France to estimate the prevalence of adrenoleukodystrophy, a severe neurologic X-linked disorder affecting boys and young men. 129 cases were collected. Analysis of all cases born between 1956 and 1986 with available clinical history allowed the calculation of the lowest estimation of the prevalence: 1 in 100,000 male births.

Adult adrenoleukodystrophy is a X-linked peroxisomal disease associated with the accumulation of very long chain fatty acids (VLCFA) in tissues and body fluids. The diagnosis is established on the demonstration of elevated VLCFA in blood and cultured skin fibroblasts. Women are affected in nearly 15% of cases and neurological symptoms and/or signs develop in 53% of them. Identifying these women is important because of genetic counseling and a possible therapeutic approach. Ten cases of symptomatic heterozygous adult adrenoleukodystrophy are reported. Mean age at the time of diagnosis was 44.6 +/- 9.3 years. All patients presented with spastic paraparesis with inconstant and mild sensory or bladder disturbances. Cognitive impairment was present in 1 case. Cerebrospinal fluid was normal. Adrenal function in response to tetracosactide injection was abnormal in 1/7 cases. Electromyography detected a peripheral neuropathy in 1 case. Somatosensory evoked responses were abnormal in all cases, visual and auditory evoked responses in respectively 3/6 cases and 3/4 cases. Brain MRI detected non specific abnormalities in 3/7 cases; spinal cord MRI was normal in 3/3 cases. The familial history was helpful for the diagnosis in 3/10 cases. Examination of pedigrees detected 5 hemizygous and 1 asymptomatic heterozygous cases. All the patients were enrolled in a dietary study which adret with low VLCFA is currently under evaluation.

Myoclonic epilepsy with ragged-red fibers (MERRF) syndrome is a neuromuscular disorder characterized by mitochondrial myopathy and progressive myoclonus epilepsy. A heteroplasmic A to G transition mutation in the mitochondrial encoded tRNA(Lys) gene at nucleotide pair 8344 has been suggested to be linked to the MERRF-syndrome. We have investigated biochemically and histochemically muscle biopsies and studied the mitochondrial genomes of hair, blood and muscle tissue of a family including three cases of MERRF-syndrome as well as unaffected relatives within the maternal lineage. Sequence analysis of the mtDNAs, performed after amplification by the polymerase chain reaction (PCR), confirmed the A to G transition mutation in the tRNA(Lys) gene at position 8344. The additional point mutation at nucleotide pair 750 in the 12 S rRNA gene, which was also found by Shoffner et al. (1990), however, was absent in all investigated tissues. Quantitative analysis of the percentage of mutated mtDNA by mispairing PCR (Seibel et al., 1990) revealed variable contents in different tissues and individuals, including unaffected family members. Mitochondrial protein synthesis in cultured fibroblasts from MERRF patients revealed diminished incorporation of 35S-methionine into lysine-containing peptides.

Evidence that inherited disorders of mitochondrial fatty acid beta-oxidation can cause sudden infant death syndrome (SIDS) comes from case reports, systematic autopsy studies, and family studies. Family studies are important when no pediatric autopsy has been done, which is still frequent. After reviewing the fatty acid beta-oxidation, and its pathophysiology, we present the results of our metabolic study on 189 siblings of SIDS victims, and on 84 'near-miss' infants. We have found evidence for a disorder of fat oxidation in 28 (15%) infants in the first group, and in 14 (17%) infants in the second group. Diagnosing and treating such disorders early in infancy may prevent some cases of SIDS to occur.

Incorporation of malonyl-CoA or acetyl-CoA is studied in mouse brain mitochondrial fatty acids. Rupture of mitochondria is necessary ; Triton X-100 gives the best result. Other detergents or sonication are of lesser efficiency. Cofactor requirements have been studied : NADH and NADPH have been tested ; ATP increases biosynthesis and CoA causes an inhibition. Two systems of biosynthesis are involved : -- One is a de novo system using malonyl-CoA. Malonyl-CoA alone is incorporated and synthesizes mainly C16, indicating the existence of a malonly-CoA decarboxylase although elongation of short chain fatty acids cannot be excluded. Addition of acetyl-CoA increases the biosynthesis and palmityl-CoA when added causes an inhibition. -- The other system, using acetyl-CoA, elongates exogenous palmityl-CoA ; endogenous acyl-CoAs are not elongated by acetyl-CoA. All these results are confirmed by radiogas chromatographic studies of the reactions products.

In order to determine precisely the respective roles of linolenic acid and linoleic acid in the maternal diet on rat brain subcellular fractions during development, we used two diets with different percentages of linolenic acid (18:3 n-3). The animals were fed peanut oil (group A) or soybean oil (group B) during pregnancy and throughout lactation. Nature and amount of essential fatty acids had no incidence on saturated and monounsaturated fatty acid distributions in myelin, synaptosomal, mitochondrial and microsomal fractions. In adult rats, all subcellular fractions are marked by an increase of n-3 fatty acid and a decrease of n-6 fatty acid levels in group B compared to group A. In 15-day-old animals, on the contrary, only the synaptosomal fractions are significantly affected by the diet. Independent of diet, brain development is marked by a decrease of n-6 fatty acids in all subcellular fractions; on the other hand, the n-3 fatty acid level is increased in the synaptosomal and mitochondrial fractions, and decrease in the myelin and microsomal fractions. The sum of (n-3 + n-6) fatty acids remains constant in group B and in group A in all subcellular fractions. Finally, under our experimental conditions, we found no marked effect of diet composition upon linoleic acid conversion to arachidonic acid; only the delta 4-7-10-13-16-docosapentaenoic acid (22:5 n-6) level decreased in group B. delta 7-10-13-16-19-Docosapentaenoic acid (22:5 n-3) seemed to be a better substrate for delta 4 desaturase than delta 7-10-13-16-docosatetraenoic acid (22:4 n-6).

Two groups of rats of different lines were divided into two lots. The four lots, whose dams received a fat-controlled diet, were fed with diets containing different levels of essential unsaturated fatty acids, namely linoleic (18:2 (n-6)) and linolenic (18:2 (n-3)) acid, in the oil. Nerve and muscle fatty acids were analyzed at adulthood. When the linolenic acid level was less than 0.6 p. 100 of the total fatty acids in the diets (peanut oil and sunflower oil), the level of long-chain polyunsaturated fatty acids (PUFA) of the (n-3) series decreased. This decrease was associated with an increase in the PUFA of the (n-6) series. Total PUFA ((n-3) + (n-6)) remained similar in the same group of rats, whatever the diet. On the contrary, when dietary linolenic acid reached 7.6 p. 100 (soya oil) or 8.5 p. 100 (rapeseed oil) of the total fatty acids, in the presence of a very high linoleic acid level, the PUFA of the (n-6) series decreased and that of the (n-3) series increased in nerve and especially in muscle. It appears that preferential PUFA biosynthesis from linolenic acid occurred when the ratio (n-6)/(n-3) was low enough in the dietary oil.

Thirty-three children presenting with "primitive" cardiomyopathy observed from January 1984 to December 1985 underwent a protocol of investigations consisting of histo-enzymatic study of the deltoid muscle, metabolic studies (glucose, free fatty acids, lactate, pyruvate, 3-hydroxybutyrate, aceto-acetate, carnitine, amino-acids blood levels after a 15 hour-fast; urinary organic acids chromatography) and a study of the fatty acids oxidation in cultured fibroblasts. In all children cardiac involvement was predominant and had been the cause for hospitalization. Cardiomyopathies of the hypertrophic type have an early onset, most often are part of a complex picture of extra-cardiac involvement and frequently have a lethal evolution. On the contrary, hypokinetic dilated cardiomyopathies are most often isolated, have a later onset and a less severe course. In 2 cases, an early hypokinetic dilated cardiomyopathy evolved toward hypertrophy. Peripheral muscular involvement is very frequent (lipidosis, mitochondrial aggregates or specific aspects) (60% of cases) in dilated as well as hypertrophic types. A precise etiological diagnosis or a strong presumption was possible in 12 of 33 cases: 2 with hereditary deficiency of the fatty acids beta-oxidation, 1 carnitine systemic deficiency, 1 Friedreich ataxia, 1 central core disease, 1 coxsackie B1 myocarditis, 6 strong suspicions of respiratory chain deficiency.

We report a case of adult adrenoleukodystrophy. The patient, originating from North Africa, had no clinical history until age 23. The first disorder, a spastic paraparesis, occurred after a 5 days coma following a cranial traumatism. The course of the disease was then progressive remitting and the patient died at age 30. Total duration was 7 years. The diagnosis of A.L.D. was ascertained by the determination of excessive very long chain fatty acid level in plasma. Endocrinological study revealed adrenal insufficiency but darkening of the skin was masked by racial pigmentation. The patient was the oldest of 10 children. 3 of the brothers died of childhood A.L.D. around the age of 12 after a progressive 2 years course. The mother and 2 asymptomatic brothers have increased plasma levels of hexacosanoic acid and A.C.T.H. The particularity of this adult case is the lack of spinal demyelination. Pathological studies revealed a widespread cerebral demyelination with an inflammatory process. The features of this case are compared to those of the 13 other reported cases of adult A.L.D.

We report histochemical, immunocytochemical, biochemical and molecular studies of skeletal muscle from a 23-year-old man with Kearns-Sayre syndrome. Southern blot analysis revealed a 4.7 kb heteroplasmic deletion of the mitochondrial DNA mapping within genes coding for subunits of complexes I, IV and V of the respiratory chain and for tRNA. Cytochrome c oxidase activity was decreased by 30% in isolated muscle mitochondria, without alteration of the Km. Histochemical and immunocytochemical correlation studies for cytochrome c oxidase revealed a lack of activity in 34% of individual muscle fibers including all the typical ragged-red fibers and a low percentage of immunodeficient fibers.

Age-related changes in the activities of microsomal and mitochondrial elongating systems have been determined in mouse brain from birth to maturity. In microsomes, the components necessary for behenyl-CoA (docosanoly-CoA) elongation have been found to be NADPH and malonyl-CoA. In mitochondria, both NADH and NADPH are used and acetyl-CoA is the only donor of two-carbon-atoms unit. The synthesised fatty acids were identified by thin-layer and gas chromatography. The specific activity is higher in microsomes than in mitochondria. In microsomes, the specific activity for malonyl-Co-A incorporation reached a maximum at 15 - 20 days of age; this peak was not obtained in the Quaking and Jimpy mutants. The increase in enzyme activity (specific activity and total activity per brain) paralleled the myelin deposition. The activity of the mitochondrial system increases regularly during development: it is not correlated to myelination and it is not affected in the Quaking mutant. The interplay between microsomal and mitochondrial elongation systems is studied.

In microsomes, biosynthesis of lignoceric acid from its direct precursor (behenyl-CoA) is largely increased during myelination. The peak is hardly detectable in Quaking; in Jimpy, the synthesis is nearly absent (3% of normal value); in the adult Quaking, the synthesis is normal. Mitochondria are capable of synthesizing lignoceric acid. This synthesis increases regularly during brain development and is normal in both mutants. Saturated fatty acid analysis is brain mitochondria shows that these organelles contain mainly palmitic and stearic acids. However, very long chains are also detectable. Thus it appears that mitochondria synthesize their own acids. Microsomes synthesize their own acids also and myelin fatty acids. There is no interplay between microsomal and mitochondrial metabolisms.

This is a report of a case of the adult cerebral form of X-linked ALD. The 27-year-old patient presented with psychiatric disturbances. NMR was performed and compared to CT scan to define cerebral demyelination. The level of hexacosanoate was found to be increased in the patient's serum. Biochemical analysis of the patient's mother's serum and cultured fibroblasts and of serum samples from 10 other members of the family who could have been carriers of this X-linked disease, produced negative results. Hence, it is most likely that this case has occurred sporadically. HLA determination revealed the DR2 antigen which is often associated with multiple sclerosis.

Peroxisomes are ubiquitous subcellular organelles varying in number, size and enzymatic content according to species, tissues or physiological states. Microperoxisomes are present in the central nervous system and in muscle. Peroxisomes participate in anabolic and catabolic processes, including ether-lipid synthesis, bêta-oxidation, bile acid synthesis, prostaglandin catabolism. Very long chain fatty acids are specific substrates of peroxisomal acyl-CoA oxidase. Peroxisomal disorders occur as two main groups: 1/ disorders with multiple deficiencies of peroxisomal functions: Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease, rhizomelic chondrodysplasia punctata; 2/ disorders with a single peroxisomal enzyme defect: X-linked adrenoleukodystrophy, acatalasemia, type 1 hyperoxaluria, pseudo-Zellweger syndrome. Present therapy is tentative with some limited success. It includes peroxisomal inductors and lipid-controlled diet. Prenatal diagnosis and heterozygote detection allow genetic counselling in some peroxisomal disorders.