The myopathic form of coenzyme Q10 deficiency is caused by mutations in the electron-transferring-flavoprotein dehydrogenase (ETFDH) gene.

Coenzyme Q10 (CoQ10) deficiency is an autosomal recessive disorder with heterogenous phenotypic manifestations and genetic background. We describe seven patients from five independent families with an isolated myopathic phenotype of CoQ10 deficiency. The clinical, histological and biochemical presentation of our patients was very homogenous. All patients presented with exercise intolerance, fatigue, proximal myopathy and high serum CK. Muscle histology showed lipid accumulation and subtle signs of mitochondrial myopathy. Biochemical measurement of muscle homogenates showed severely decreased activities of respiratory chain complexes I and II + III, while complex IV (COX) was moderately decreased. CoQ10 was significantly decreased in the skeletal muscle of all patients. Tandem mass spectrometry detected multiple acyl-CoA deficiency, leading to the analysis of the electron-transferring-flavoprotein dehydrogenase (ETFDH) gene, previously shown to result in another metabolic disorder, glutaric aciduria type II (GAII). All of our patients carried autosomal recessive mutations in ETFDH, suggesting that ETFDH deficiency leads to a secondary CoQ10 deficiency. Our results indicate that the late-onset form of GAII and the myopathic form of CoQ10 deficiency are allelic diseases. Since this condition is treatable, correct diagnosis is of the utmost importance and should be considered both in children and in adults. We suggest to give patients both CoQ10 and riboflavin supplementation, especially for long-term treatment.

Pathological consequences of VCP mutations on human striated muscle.

Mutations in the valosin-containing protein (VCP, p97) gene on chromosome 9p13-p12 cause a late-onset form of autosomal dominant inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia (IBMPFD). We report on the pathological consequences of three heterozygous VCP (R93C, R155H, R155C) mutations on human striated muscle. IBMPFD skeletal muscle pathology is characterized by degenerative changes and filamentous VCP- and ubiquitin-positive cytoplasmic and nuclear protein aggregates. Furthermore, this is the first report demonstrating that mutant VCP leads to a novel form of dilatative cardiomyopathy with inclusion bodies. In contrast to post-mitotic striated muscle cells and neurons of IBMPFD patients, evidence of protein aggregate pathology was not detected in primary IBMPFD myoblasts or in transient and stable transfected cells using wild-type-VCP and R93C-, R155H-, R155C-VCP mutants. Glutathione S-transferase pull-down experiments showed that all three VCP mutations do not affect the binding to Ufd1, Npl4 and ataxin-3. Structural analysis demonstrated that R93 and R155 are both surface-accessible residues located in the centre of cavities that may enable ligand-binding. Mutations at R93 and R155 are predicted to induce changes in the tertiary structure of the VCP protein. The search for putative ligands to the R93 and R155 cavities resulted in the identification of cyclic sugar compounds with high binding scores. The latter findings provide a novel link to VCP carbohydrate interactions in the complex pathology of IBMPFD.

Novel splice site mutation in the caveolin-3 gene leading to autosomal recessive limb girdle muscular dystrophy.

Mutations in CAV3 gene encoding the protein caveolin-3 are associated with autosomal dominant limb girdle muscular dystrophy 1C, rippling muscle disease, hyperCKemia, distal myopathy, hypertrophic cardiomyopathy and rare autosomal recessive limb girdle muscular dystrophy phenotypes. In a 57-year-old patient with asymmetric limb girdle weakness, we detected a novel homozygous intronic mutation (IVS1 + 2T > C) of the CAV3 gene. This is the first splicing mutation reported for CAV3. These findings add to the clinical and genetic variability of CAV3 mutations.

Myotonic dystrophies type 1 and 2: a summary on current aspects.

Myotonic dystrophies (DMs) encompass at least 2 forms: myotonic dystrophy type 1 and 2. In general, DMs are late-onset autosomal dominant disorders characterized by a variety of multisystemic features including myotonia, muscular dystrophy, cardiac conduction defects, dilated cardiomyopathy, posterior iridescent cataracts, frontal balding, insulin-resistance and disease-specific serological abnormalities such as gamma-glutamyltransferase and creatine kinase elevations, hyperglycemia, hypotestosteronism, and reduced immunoglobulin (Ig) G and IgM levels. Beyond the adult forms, in the classic DM1, a congenital form and an early-onset form is recognized. Here we summarize current aspects of the myotonic dystrophy pathogenesis and review the core features of both types of myotonic dystrophies, including the congenital DM1.

Age related profiles of free amino acids in human skeletal muscle.

Sarcopenia describes the involuntary decline in muscle mass with aging, coupled with fatigue, and loss of force and function. We investigated 113 human muscle biopsy specimens obtained from patients with neuromuscular diseases and controls. We measured 21 amino acids in these muscle biopsies. Age emerged as a significant negative predictor of cytosolic concentration ratio of glutamine to total branched chain amino acids and of glutamine to total aromatic amino acids using stepwise multiple linear regression analysis. This pattern of alteration corresponds well to documented alterations in skeletal muscle of critically ill patients and after immobilization. Additionally, in myositis, citrulline was significantly elevated, while glutamate, lysine and taurine were significantly reduced. Furthermore, in sporadic amyotrophic lateral sclerosis (sALS) the total aromatic amino acids, arginine, glutamate, threonine, and tyrosine were significantly elevated. This study provides evidence, that alteration of glutamine is correlated to aging and might reflect increased proteolysis in aged and diseased human skeletal muscle.

Extraocular mitochondrial myopathies and their differential diagnoses.

The diagnosis of mitochondrial myopathy depends upon a constellation of findings, family history, type of muscle involvement, specific laboratory abnormalities, and the results of histological, pathobiochemical and genetic analysis. In the present paper, the authors describe the diagnostic approach to mitochondrial myopathies manifesting as extraocular muscle disease. The most common ocular manifestation of mitochondrial myopathy is progressive external ophthalmoplegia (PEO). To exclude myasthenia gravis, ocular myositis, thyroid associated orbitopathy, oculopharyngeal muscular dystrophy, and congenital fibrosis of the extraocular muscles in patients with an early onset or long-lasting very slowly progressive ptosis and external ophthalmoplegia, almost without any diplopia, and normal to mildly elevated serum creatine kinase and lactate, electromyography, nerve conduction studies and MRI of the orbits should be performed. A PEO phenotype forces one to look comprehensively for other multisystemic mitochondrial features (e.g., exercise induced weakness, encephalopathy, polyneuropathy, diabetes, heart disease). Thereafter, and presently even in familiar PEO, a diagnostic muscle biopsy should be taken. Histological and ultrastructural hallmarks are mitochondrial proliferations and structural abnormalities, lipid storage, ragged-red fibers, or cytochrome-C negative myofibers. In addition, Southern blotting may reveal the common deletion, or molecular analysis may verify specific mutations of distinct mitochondrial or nuclear genes.

Differential expression of nitric oxide synthases (NOS 1-3) in human skeletal muscle following exercise countermeasure during 12 weeks of bed rest.

Adaptive changes of major body systems in astronauts during spaceflight can be simulated by strict anti-orthostatic head-down tilt (HDT) bed rest (BR), a ground-based microgravity (microG) model that provides a meaningful opportunity to study atrophy mechanisms and possible countermeasures under controlled experimental conditions. As nitric oxide (NO) signaling is linked to muscle activity, we investigated altered expression of the three major isoforms of nitric oxide synthase (NOS 1-3) at cellular compartments during prolonged HDT BR without (control group) and with resistance exercise interventions (exercise group) using a flywheel ergometer (FWE). Atrophy detected in mixed (fast-slow) m. vastus lateralis (VL) and slow-type m. soleus (SOL) myofiber Types I and II (minus 35-40% of myofiber cross-sectional area) was prevented by FWE training. Concomitant to muscle atrophy, reduced NOS 1 protein and immunostaining was found in VL not in SOL biopsies. In trained VL, NOS 1 protein and immunostaining at myofibers II were significantly increased at the end of BR. Exercise altered NOS 2/caveolin 3 co-immunostaining patterns of subsarcolemmal focal accumulations in VL or SOL myofibers, which suggests reorganization of sarcolemmal microdomains. In trained VL, increased capillary-to-fiber (C/F) ratio and NOS 3 protein content were documented. Activity-linked NO signaling may be widespread in skeletal muscle cellular compartments that may be directly or indirectly impacted by adequate exercise countermeasure protocols to offset the negative effects induced by disuse, immobilization, or extended exposure to microgravity.

Evoked potentials during active horizontal head rotations in patients with vertigo.

OBJECTIVES: The purpose of this study was to investigate whether evoked potentials by active head rotation help to verify and topographically differentiate patients with the major symptom vertigo. METHODS: Twenty-four healthy human subjects and 43 patients with either infratentorial or supratentorial brain lesions were analysed. RESULTS: The evoked response in normal subjects was composed of six peaks, indicated by polarization and time difference from the trigger points P100, N30, P0, N50, P155 and N320. The EEG pattern was independent of the direction, type of target and whether the eyes were open or closed. In contrast, the evoked response, especially P155, was dependent on the chosen trigger point and acceleration. P155 was the most stable and significant component of the evoked potentials. Thus, we chose P155 as the reference for studying patients with vertigo. DISCUSSION: In peripheral vestibular disorders, cerebellar and diffuse supratentorial cerebral lesions and P155 latencies remain non-significantly altered. However, P155 latencies significantly increase in pontine lesions homolaterally, and space occupying tumors contralaterally. CONCLUSION: Active horizontal head rotations differentially stimulate the vestibulocortical pathways and may contribute to the analysis of vertigo.

Homozygosity for CCTG mutation in myotonic dystrophy type 2.

Myotonic dystrophy type 2 (DM2) is caused by a dominantly transmitted CCTG repeat expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene on chromosome 3q. DM2 patients with two mutant alleles have not been reported so far. In one large consanguineous family from Afghanistan, we found three homozygotes for the DM2 mutation. The oldest patient was clinically more severely affected, compared with the two younger homozygotes, but for the clinical course of symptoms all three homozygotes were within the range expected for heterozygotes. Further investigations, such as mutation repeat length, muscle histology, anti-muscleblind-like 1 stainings or brain imaging studies, at least at short-term observation, showed no differences between heterozygotes and homozygotes. Twenty of 24 children, aged 2-21 years, were available for clinical examination. None of these children have signs or symptoms of disease until the age of 18 years. Homozygosity for the DM2 expansion does not seem to alter the disease phenotype as compared with the heterozygous state.

Motor excitability in myopathy.

OBJECTIVE: To explore whether patients with myopathy present changes in motoneuronal excitability. METHODS: Patients with well-defined myopathies were studied with single and paired pulse transcranial magnetic stimulations and electrical nerve stimulations to explore neuronal motor excitability. Motor-evoked potentials were recorded from the clinically unaffected first dorsal interosseous muscle (n=10) and the paretic deltoid muscle (n=8). RESULTS: Compared to an age-matched healthy control group, myopathic patients showed a reduction of intracortical inhibition, enhancements of alpha-motoneuron excitability and increased amplitudes of motor-evoked potentials during target muscle contraction. These alterations were present in clinically affected and clinically unaffected muscles. CONCLUSION: In myopathy, nervous system excitability may be altered, presenting as a motor disinhibition on cortical and subcortical levels.

Rippling muscle disease in childhood.

Rippling muscle disease is a rare autosomal dominant disorder first described in 1975. Recently, it could be classified as a caveolinopathy; in European families, mutations in the caveolin-3 gene were revealed as causing this disease. Although clinical symptoms were almost all described in adulthood, we are now reporting clinical data of seven children with rippling muscle disease owing to mutations in the caveolin-3 gene. Initial symptoms were frequent falls, inability to walk on heels, tiptoe walking with pain and a warm-up phenomenon, calf hypertrophy, and an elevated serum creatine kinase level. Percussion-/pressure-induced rapid contractions, painful muscle mounding, and rippling could be observed even in early childhood. The diagnosis can be confirmed by molecular genetic analysis. Muscle biopsy must be considered in patients without muscle weakness or mechanical hyperirritability to differentiate between rippling muscle disease and limb-girdle muscular dystrophy 1C.

Ocular myositis: diagnostic assessment, differential diagnoses, and therapy of a rare muscle disease - five new cases and review.

Ocular myositis represents a subgroup within the idiopathic orbital inflammatory syndrome, formerly termed orbital pseudotumor. Ocular myositis describes a rare inflammatory disorder of single or multiple extraocular eye muscles. Unilateral or sequential bilateral subacute painful diplopia is the leading symptom of eye muscle myositis. There are at least two major forms, a limited oligosymptomatic ocular myositis (LOOM) with additional conjunctival injections only, and a severe exophthalmic ocular myositis (SEOM) with additional ptosis, chemosis, and proptosis. Eye muscle myositis is an idiopathic inflammation of the extraocular muscles in the absence of thyroid disease, ocular myasthenia gravis, and other systemic, particularly autoimmune mediated diseases, resembling CD4(+) T cell-mediated dermatomyositis. Contrast-enhanced orbital magnetic resonance imaging most sensitively discloses swelling, signal hyperintensity, and enhancement of isolated eye muscles. Typically, corticosteroid treatment results in prompt improvement and remission within days to weeks in most patients. Compiled data of five patients and a review of the clinical pattern, diagnostic procedures, differential diagnoses, and current treatment options are given.

A large German kindred with cold-aggravated myotonia and a heterozygous A1481D mutation in the SCN4A gene.

Muscle sodium-channel disorders cover a spectrum of rare myotonic diseases. In a German family with 17 affected individuals in four generations, we identified a heterozygous missense mutation in exon 24 A1481D (c.4442 C>A) of the voltage-gated sodium channel gene (SCN4A) alpha subunit. Phenotypes of 12 family members were characterized by a mild myotonia with cold sensitivity but without paramyotonia. The index patient presented with fluctuating cold- and exercise-induced stiffness of ocular, facial, and distal muscles. The myotonia became more severe at the age of 22 years. His father had had cold- and exercise-induced periodic weakness with fluctuating myotonia since age 10. Later he developed a more severe, purely exercise- and cold-aggravated myotonia of arms, hands, and facial muscles. The father's mother presented with cold-induced myotonia until age 65, when progressive weakness of proximal limb muscles developed. Her muscle biopsies revealed considerable myopathic changes with a variety of fine structural alterations. This study presents a family with cold-aggravated myotonia and progression of myopathic changes in the muscle biopsy with increasing age. In older patients, sodium channelopathies may mimic the phenotypic features of myotonic dystrophy type 2.

Mutations of the LMNA gene can mimic autosomal dominant proximal spinal muscular atrophy.

The molecular basis of autosomal dominant spinal muscular atrophy (AD-SMA) is largely unknown. Because the phenotypic spectrum of diseases caused by LMNA mutations is extremely broad and includes myopathies, neuropathies, and cardiomyopathies designated as class 1 laminopathies, we sequenced the LMNA gene in index patients with the clinical picture of proximal SMA, who had a family history suggestive of autosomal dominant inheritance. Among the 19 families investigated, two showed pathogenic mutations of the LMNA gene, resulting in the diagnosis of a class 1 laminopathy in about 10% of our series. We found one novel truncating mutation (c.1477C > T, Q493X) and one previously described missense mutation (c.1130G > T, R377H) in the LMNA gene of two unrelated patients with adult-onset proximal SMA followed by cardiac involvement 14 and 22 years after the onset of weakness. The pedigrees of both families revealed a high frequency of cardiac abnormalities or sudden deaths. Our findings extend the spectrum of laminopathies and are of relevance for genetic counseling and clinical care of families presenting with adult-onset proximal SMA. Particularly, if neurogenic atrophy is combined with a cardiac disease in a family, this should prompt LMNA mutation analysis.

Deconstructing Pompe disease by analyzing single muscle fibers: to see a world in a grain of sand...

Autophagy is a major pathway for delivery of proteins and organelles to lysosomes where they are degraded and recycled. We have previously shown excessive autophagy in a mouse model of Pompe disease (glycogen storage disease type II), a devastating myopathy caused by a deficiency of the glycogen-degrading lysosomal enzyme acid alpha-glucosidase. The autophagic buildup constituted a major pathological component in skeletal muscle and interfered with delivery of the therapeutic enzyme. To assess the role of autophagy in the pathogenesis of the human disease, we have analyzed vesicles of the lysosomal-degradative pathway in isolated single muscle fibers from Pompe patients. Human myofibers showed abundant autophagosome formation and areas of autophagic buildup of a wide range of sizes. In patients, as in the mouse model, the enormous autophagic buildup causes greater skeletal muscle damage than the enlarged, glycogenfilled lysosomes outside the autophagic regions. Clearing or preventing autophagic buildup seems, therefore, a necessary target of Pompe disease therapy.

Commonality of TRIM32 mutation in causing sarcotubular myopathy and LGMD2H.

Sarcotubular myopathy (OMIM 268950) is a rare autosomal recessive myopathy first described in two Hutterite brothers from South Dakota and in two non-Hutterite brothers from Germany. We report that sarcotubular myopathy (STM) is caused by mutation in TRIM32, the gene encoding the tripartite motif-containing protein 32. TRIM32 was found to be the gene mutated in limb girdle muscular dystrophy type 2H (LGMD2H [OMIM 254110]), a disorder that has been confined to the Hutterite population. The TRIM32 mutation found in the STM patients is identical to the causative mutation for LGMD2H (D487N), Haplotype analysis shows that the disease chromosomes share common ancestry.

Myotonic dystrophy type 2: human founder haplotype and evolutionary conservation of the repeat tract.

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, can be caused by a mutation on either chromosome 19 (DM1) or 3 (DM2). In 2001, we demonstrated that DM2 is caused by a CCTG expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene. To investigate the ancestral origins of the DM2 expansion, we compared haplotypes for 71 families with genetically confirmed DM2, using 19 short tandem repeat markers that we developed that flank the repeat tract. All of the families are white, with the majority of Northern European/German descent and a single family from Afghanistan. Several conserved haplotypes spanning >700 kb appear to converge into a single haplotype near the repeat tract. The common interval that is shared by all families with DM2 immediately flanks the repeat, extending up to 216 kb telomeric and 119 kb centromeric of the CCTG expansion. The DM2 repeat tract contains the complex repeat motif (TG)(n)(TCTG)(n)(CCTG)(n). The CCTG portion of the repeat tract is interrupted on normal alleles, but, as in other expansion disorders, these interruptions are lost on affected alleles. We examined haplotypes of 228 control chromosomes and identified a potential premutation allele with an uninterrupted (CCTG)(20) on a haplotype that was identical to the most common affected haplotype. Our data suggest that the predominant Northern European ancestry of families with DM2 resulted from a common founder and that the loss of interruptions within the CCTG portion of the repeat tract may predispose alleles to further expansion. To gain insight into possible function of the repeat tract, we looked for evolutionary conservation. The complex repeat motif and flanking sequences within intron 1 are conserved among human, chimpanzee, gorilla, mouse, and rat, suggesting a conserved biological function.

Muscle pathology in 57 patients with myotonic dystrophy type 2.

We evaluated muscle biopsies from 57 patients with genetically confirmed myotonic dystrophy type 2/proximal myotonic myopathy (DM2/PROMM). Light microscopy showed myopathic together with "denervation-like" changes in almost all biopsies obtained from four different muscles: increased fiber size variation, internal nuclei, small angulated fibers, pyknotic nuclear clumps, and predominant type 2 fiber atrophy. Quantitative morphometry in 18 biopsies that were immunostained for myosin heavy chain confirmed a predominance of nonselective type 2 fiber atrophy. These histological changes were similar in all patients regardless of the site of biopsy, the predominant clinical symptoms and signs, and the clinical course. It is likely that, in a number of undiagnosed patients, DM2 is the underlying disorder. With a better understanding of the histopathological pattern in DM2, biopsies from patients with undiagnosed neuromuscular disorders can now be reevaluated.

Homozygous mutations in caveolin-3 cause a severe form of rippling muscle disease.

Heterozygous missense mutations in the caveolin-3 gene (CAV3) cause different muscle disorders. Most patients with CAV3 alterations present with rippling muscle disease (RMD) characterized by signs of increased muscle irritability without muscle weakness. In some patients, CAV3 mutations underlie the progressive limb-girdle muscular dystrophy type 1C (LGMD1C). Here, we report two unrelated patients with novel homozygous mutations (L86P and A92T) in CAV3. Both presented with a more severe clinical phenotype than usually seen in RMD. Immunohistochemical and immunoblot analyses of muscle biopsies showed a strong reduction of caveolin-3 in both homozygous RMD patients similar to the findings in heterozygous RMD. Electron microscopy studies showed a nearly complete absence of caveolae in the sarcolemma in all RMD patients analyzed. Additional plasma membrane irregularities (small plasmalemmal discontinuities, subsarcolemmal vacuoles, abnormal papillary projections) were more pronounced in homozygous than in heterozygous RMD patients. A stronger activation of nitric oxide synthase was observed in both homozygous patients compared with heterozygous RMD. Like in LGMD1C, dysferlin immunoreactivity is reduced in RMD but more pronounced in homozygous as compared with heterozygous RMD. Thus, we further extend the phenotypic variability of muscle caveolinopathies by identification of a severe form of RMD associated with homozygous CAV3 mutations.