Polyglucosan myopathy and functional characterization of a novel GYG1 mutation.

OBJECTIVES: Disorders of glycogen metabolism include rare hereditary muscle glycogen storage diseases with polyglucosan, which are characterized by storage of abnormally structured glycogen in muscle in addition to exercise intolerance or muscle weakness. In this study, we investigated the etiology and pathogenesis of a late-onset myopathy associated with glycogenin-1 deficiency. MATERIALS AND METHODS: A family with two affected siblings, 64- and 66-year-olds, was studied. Clinical examination and whole-body MRI revealed weakness and wasting in the hip girdle and proximal leg muscles affecting ambulation in the brother. The sister had weakness and atrophy of hands and slight foot dorsiflexion difficulties. Muscle biopsy and whole-exome sequencing were performed in both cases to identify and characterize the pathogenesis including the functional effects of identified mutations. RESULTS: Both siblings demonstrated storage of glycogen that was partly resistant to alpha-amylase digestion. Both were heterozygous for two mutations in GYG1, one truncating 1-base deletion (c.484delG; p.Asp163Thrfs*5) and one novel missense mutation (c.403G>A; p.Gly135Arg). The mutations caused reduced expression of glycogenin-1 protein, and the missense mutation abolished the enzymatic function as analyzed by an in vitro autoglucosylation assay. CONCLUSION: We present functional evidence for the pathogenicity of a novel GYG1 missense mutation located in the substrate binding domain. Our results also demonstrate that glycogenin-1 deficiency may present with highly variable distribution of weakness and wasting also in the same family.

[Treatability of sporadic late onset nemaline myopathy]. / Behandelbarkeit der "sporadic late onset nemaline myopathy".

Sporadic late onset nemaline myopathy (SLONM) is an extremely rare disorder which can be associated with monoclonal gammopathy of unclear significance (MGUS). Clinically SLONM appears mostly after the fourth decade of life as rapidly progressing tetraparesis, respiratory insufficiency and features, such as dropped head syndrome, facial and bulbar involvement. Diagnosis is confirmed by muscle biopsy with detection of nemaline bodies and also frequently lobulated fibres. Immunosuppressant and immunomodulating therapies have been shown to be ineffective but clinical improvement accompanied by disappearance of monoclonal gammopathy and even nemaline bodies was reported following autologous stem cell transplantation and chemotherapy with melphalan. This article presents the case of a 53-year-old man with a 4-year history of SLOMN with MGUS in which administration of intravenous immunoglobulin therapy (IVIG) was not successful in reversing gammopathy, histopathological changes or clinical symptoms.

[Muscular dystrophy due to mutations in anoctamin 5: clinical and molecular genetic findings]. / Muskeldystrophie infolge Anoctamin-5-Mutationen : Klinische und molekulargenetische Befunde.

Recessive mutations in the anoctamin 5 (ANO5) gene have been recently identified in families with limb girdle muscular dystrophy (LGMD2L) and distal non-dysferlin Miyoshi myopathy. Anoctamin 5 is supposed to be a putative calcium-activated chloride channel. We report five German patients (four index patients) with muscle dystrophy due to mutations in the ANO5 gene. Sequencing of the ANO5 exons 5, 13 and 20 was performed to screen for a common c.191dupA mutation and two other reported mutations (c.1295C>G and p.R758C). The whole coding region of the ANO5 gene was sequenced to identify new mutations. Phenotypically, three patients showed LGMD and one patient Miyoshi type distal myopathy. One sibling had asymptomatic hyperCKemia. The age at onset was 64, 38 and 40 years in patients with LGMD and 23 years in the patient with distal myopathy. The four symptomatic patients showed remarkable asymmetric muscle involvement. There was marked CK elevation (11 to 30 times). Electron microscopy showed multifocal gaps in the sarcolemmal membrane. All patients harboured the common c.191dupA mutation in at least one allele. Two patients with LGMD were homozygous and the third patient and his asymptomatic sister were compound heterozygous for the c.191dupA mutation and a novel p.T548I mutation. The patient with distal myopathy harboured the p.R758C mutation in the second allele. Mutations in the ANO5 gene seem to be a relatively common cause of muscular dystrophy in Germany. Cases with late onset or asymptomatic hyperCKemia can occur. Clinically, asymmetric manifestation is typical.

Protracted course of juvenile ceroid lipofuscinosis associated with a novel CLN3 mutation (p.Y199X).

The juvenile neuronal ceroid lipofuscinosis (JNCL, Batten disease, MIM 204200), is an autosomal recessive lysosomal storage disease, which is characterized by ubiquitous accumulation of the lipopigment material ceroid-lipofuscin. It manifests with loss of vision in childhood due to retinal degeneration, followed by seizures and parkinsonism leading to premature death at around 30 years. Eighty-five percent of JNCL patients carry a disease-causing 1.02 kb deletion in the CLN3 gene on chromosome 16. Here we report on a large consanguineous Lebanese family with five affected siblings. Electron microscopy of lymphocytes revealed the presence of fingerprint profiles suggesting JNCL. However, disease progression, especially of mental and motor function was slower as expected for 'classic' JNCL. We thus confirmed the diagnosis by genetic testing and found a new c.597C>A transversion in exon 8, homozygous in all affected family members and not present in 200 alleles of normal controls. The mutation generates a premature termination codon (p.Y199X) truncating the CLN3 protein by 55%. In heterozygous state mutant mRNA transcripts are expressed at the same levels as the wild-type ones, suggesting the absence of nonsense mediated messenger decay. We discuss a potential residual catalytic function of the truncated protein as a cause for the mild phenotype.

Electron microscopy in myofibrillar myopathies reveals clues to the mutated gene.

We studied the ultrastructural characteristics in patients with myofibrillar myopathy (MFM) and differentiated between MFM-subtypes using electron microscopic (EM) findings. The ultrastructural findings in 19 patients with different genetically proven MFMs (9 desmin, 5 alphaB-crystallin, 3 ZASP, 2 myotilin) were analyzed. In one ZASPopathy, we additionally performed an immunoEM study, using antibodies against desmin, alphaB-crystallin, ZASP and myotilin. The ultrastructural findings in desminopathies and alphaB-crystallinopathies were very similar and consisted of electrondense granulofilamentous accumulations and sandwich formations. They differed in the obvious presence of early apoptotic nuclear changes in alphaB-crystallinopathies. ZASPopathies were characterized by filamentous bundles (labeled with the myotilin antibody on immunoEM), and floccular accumulations of thin filamentous material. Tubulofilamentous inclusions in sarcoplasm and myonuclei in combination with filamentous bundles were characteristic for myotilinopathies. We conclude that MFMs ultrastructural findings can direct diagnostic efforts towards the causal gene mutated, and that EM should be included in the diagnostic workup of MFMs.

Phenolic and tyrosyl ring iodothyronine deiodination and thyroid hormone concentrations in the human central nervous system.

In the present study we investigated the biochemical properties of in vitro phenolic (5'D) and tyrosyl (5D) ring deiodination and the tissue concentrations of T4, T3, and rT3 in adult human central nervous system (CNS) tissue. All samples were obtained from nontumoral tissue at autopsy (n = 6) or neurosurgical operation (n = 5). Both phenolic and tyrosyl ring deiodinase activities were demonstrable in all samples obtained intraoperatively, whereas only tyrosyl ring deiodination was evident in the tissues obtained postmortem. The phenolic ring deiodination pathway corresponded to the type II 5'-deiodinase isoenzyme with regard to its high affinity for T4 and rT3 (Km = 2.2 and 2.4 nmol/L, respectively), its insensitivity to 6-propyl-n-2-thiouracil (PTU), and the sequential reaction mechanism. No PTU-sensitive 5'-deiodination of rT3 was demonstrable. Tyrosyl ring deiodination of both T4 and T3 showed typical type III 5D kinetics (Ka, 6.5 nmol/L for T4 and 3.4 nmol/L for T3) and was PTU insensitive. Nanomolar concentrations of tissue T4, T3, and rT3 were detected in samples obtained both intraoperatively and postmortem. They were very similar to the absolute values of the apparent Km for T4, T3, and rT3 in the phenolic and tyrosyl ring deiodination pathways. In conclusion, we have demonstrated the coexistence of both phenolic and tyrosyl ring deiodinase activities in the human CNS. Their kinetic characteristics, substrate specificity, and reaction mechanisms are very similar to the corresponding type II 5'- and type III 5-iodothyronine deiodinase activities in rat brain. In contrast to the findings in the rat CNS, no PTU-sensitive phenolic ring deiodinase (i.e. type I 5'D) activity was found in the human CNS. This may explain the relatively high tissue concentrations of rT3.

Brain-IL-1beta induces local inflammation but systemic anti-inflammatory response through stimulation of both hypothalamic-pituitary-adrenal axis and sympathetic nervous system.

It is well established that systemic inflammation induces a counter-regulatory anti-inflammatory response particularly resulting in deactivation of monocytes/macrophages. However, recently we demonstrated a systemic anti-inflammatory response without preceding signs of systemic inflammation in patients with brain injury/surgery and release of cytokines into the cerebrospinal fluid (CSF). In order to analyze the mechanisms and pathways of systemic immunodepression resulting from sterile cerebral inflammation we established an animal model using continuous intra-cerebroventricular (i.c.v.) or intra-hypothalamic (i.h.) infusion of rat recombinant (rr) tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta for 48 h. Controls received intra-venous (i.v.) cytokine administration. Interestingly, i.c.v. and i.h. infusion of IL-1beta but not TNF-alpha produced distinct signs of central nervous system (CNS) inflammation. Correspondingly, i.c.v. infusion of IL-1beta particularly diminished the TNF-alpha but increased the IL-10 concentration in whole blood cultures after endotoxin stimulation. All parameters normalized within 48 h after termination of the infusion. Blocking the hypothalamic-pituitary-adrenal (HPA) axis by hypophysectomy (HPX) led to complete recovery of the diminished TNF-alpha concentration and temporarily inhibited the IL-10 increase. Blocking the sympathetic nervous system (SNS) transmission by application of the beta2-adrenoreceptor antagonist propranolol not only inhibited the increase but further downregulated the endotoxin induced IL-10 concentration in the media of whole blood cell cultures, whereas the TNF-alpha decrease was only partially prevented. Interestingly, HPX and propranolol also diminished the cell invasion into the CSF. In summary, activation of both the HPA axis and the SNS plays an important role in systemic anti-inflammatory response resulting from cytokines in brain and cerebral inflammation.

High levels of human chymase expression in the pineal and pituitary glands.

The brain renin-angiotensin system plays a role in both cardiovascular homeostasis and neurosecretory functions. Since the mechanisms of angiotensin (Ang) II formation in the human brain have not been clarified, the aims of the present study were to determine the presence of human chymase and angiotensin I-converting enzyme (ACE) in human and non-human brains. In the human brain, the total Ang II-forming activity was significantly higher in the pineal and pituitary glands than those in other regions. In other species (rat, bovine and porcine), the level of chymase as well as total Ang II-forming activities in pineal glands were significantly lower than those in human glands. High levels of chymase-like immunoreactivity (ir) were found in the arteriolar endothelial cells, adventitial mesenchymal cells and in parenchymal cells of the human pineal and pituitary glands while ACE-ir was mostly observed in the endothelial cells and occasionally found in parenchymal cells. Our study provides the first evidence that human chymase exists in the pineal and pituitary glands. The remarkable regional and species differences in mechanisms of Ang II formation suggest a specific role of chymase or ACE in the human brain.

Toxic polyneuropathies after sniffing a glue thinner.

In West Berlin in the autumn of 1975 through the following 5 months we observed 18 juvenile patients who had a toxic polyneuropathy and had sniffed a glue thinner. The neurological picture consisted of a symmetrical, progressive, ascending, mainly motor, polyneuropathy with pronounced muscle atrophy and characteristic vegetative alterations. The height of the disease was reached after 1 1/2-2 1/2 months and was characterized by tetraplegia in 7 patients. After 8 months all patients still had a motor deficit. Nerve biopsy showed paranodal axon swelling, dense masses of neurofilaments and secondary myelin retraction. The neurological and morphological data correspond to the "glue sniffer's neuropathy" and the n-hexane and MBK polyneuropathy after industrial exposure, as described in 10 cases to date. However, there was no MBK in the glue thinner. The polyneuropathies occurred in close time relation with the denaturation of the thinner with MEK (2-butanone). It is concluded from the data n-hexane and MBK have a common toxic mechanism with primary axonal changes and that there is an additional synergistic effect of MEK.

Experimental studies on hydrocarbon neuropathies induced by methyl-ethyl-ketone (MEK).

An outbreak of neuropathies among Berlin solvent sniffers was closely related to the denaturation by methyl-ethyl-ketone (MEK) of the mixture used. The solvent was composed of n-hexane, toluene and ethyl-acetate. Nervous system responses to chronic repeated exposure to 10,000 ppm pure n-hexane, 10,000 ppm MEK/n-hexane (ratio 1:9) and 6000 ppm pure MEK were investigated in rats. Motor neuropathy of the dying back type with giant swelling of axons in the peripheral and central nervous system developed in animals exposed to MEK/n-hexane and n-hexane. Severe potentiation of n-hexane neurotoxicity and shortened onset of morphological and clinical signs were demonstrated in animals exposed to MEK/n-hexane. MEK alone did not produce neuropathy under these conditions. The findings suggest that commercial solvent mixtures containing MEK/n-hexane should be avoided.

Nervous system responses of rats to subchronic inhalation of N-hexane and N-hexane + methyl-ethyl-ketone mixtures.

The effects of long-term continuous and intermittent inhalation exposure to selected concentrations of n-hexane and mixtures of n-hexane and methyl-ethyl-ketone (MEK) on the nervous system of rats were investigated. Animals exposed continuously (24 h/d, 7 d/week) to 500 ppm n-hexane displayed complete hindlimb paralysis after 9 weeks. Histological examination showed hexacarbon-specific axonal lesions in peripheral nerves, particularly tibial branches to calf muscles, and in the gracile tract at cervical levels of the spinal cord. Similar clinical and pathological signs of neuropathy appeared one week earlier in animals treated with a mixture of 500 ppm n-hexane/MEK (4:1 or 3:2) and 5 weeks earlier with 700 n-hexane/MEK mixture (5:2) or 700 ppm of n-hexane alone. Rats exposed to the latter concentrations intermittently, 8 hours daily for 40 weeks, did not develop clinical or morphological signs of a hexacarbon neuropathy.

PCBs have a predominantly neurotoxic effect on dissociated cultures of the nervous system.

Although their manufacture and use have been restricted or banned in Europe and the United States since the 1970s, polychlorinated biphenyls (PCBs) are still an ubiquitous environmental contaminant whose low-term effects are as yet not completely clear. Clinical case studies of patients with occupational exposure report cognitive impairment and peripheral neuropathy. In our defined nerve cell culture models in which we use pure neurons, pure glia and mixed cultures prepared from dorsal root ganglia of chick embryos we observed a neurotoxic effect after the application of a PCB compound (Clophen). It was only at higher concentrations that an additional gliatoxic effect could be observed.

Developmental delay of astrocytes in hippocampus of rhesus monkeys reflects the effect of pre- and postnatal chronic low level lead exposure.

Rhesus monkeys were pre- and postnatally exposed to lead-acetate at 0, 350, or 600 ppm in diet for nine years, followed by a period of lead-free diet for 32 months. During this time blood lead levels declined to normal, but still showed dose-related differences. In behavioral and neurophysiological studies the rhesus monkeys exhibited dose-related cognitive and functional deficits. After sacrifice hippocampal sections were processed for immunohistological staining. GFAP, introduced as a marker of neurotoxicity and Vimentin, which is expressed by immature or reactive astrocytes were investigated. A dose-dependent increase of GFAP due to prenatal and chronic low level lead exposure was not observed. We found a dose-related increase of GFAP-positive radial glia and star-shaped Vimentin-positive astrocytes in the high lead group. We consider these findings as indication of immature astrocytes, which are not able to react with gliosis in respond to pre- and postnatal low level lead exposure. The lack of pronounced glial response due to low level lead exposure may result in a delay of astrocytic differentiation, shown by persistence of radial glia.

Clinical data on three cases of occupationally induced PCB-intoxication.

Since the ban of PCBs at the end of the 1970s, extensive measures have been undertaken in Germany to dispose of PCB contaminated transformers. We report on three patients with considerable skin exposure to PCBs, in particular to Clophen A 30, while repairing or dismounting transformers. The periods of exposure range from 4 and 5 to 20 years. All patients presented with distal-symmetrical sensorimotor polyneuropathy as well as encephalopathy. In one of the cases, the neuropathy and encephalopathy progressed for a period of over 8 years after termination of exposure. In the two other cases the neurological deficits persisted over an observed period of 2-3 years. The reported clinical results may suggest that the long half-life of Clophen and its accumulation in fatty tissue can lead to persistence of PNS and CNS impairment long after the period of exposure.

Effects of chronic low level lead exposure on the expression of GFAP and vimentin mRNA in the rat brain hippocampus analysed by in situ hybridization.

In this study we used in situ hybridization to examine the effects of chronic low level lead toxicity during different periods of brain development. Low level lead is known to affect astroglia. GFAP and Vimentin were chosen as glialtypic markers for neurotoxicity. The effects of lead were investigated on male Wistar rats. Animals were divided into four groups: a control group, a permanent group exposed during gestation, lactation and post-weaning (E0-P100), a perinatal group exposed during gestation and postnatally until weaning (E0-P16), and a post-weaning exposed group (P16-P100). All experimental animals were fed a diet containing 750 ppm lead acetate. With respect to Vimentin mRNA no major differences could be detected among the treatment groups. Significant differences in GFAP mRNA levels were detected in the post-weaning group relative to controls. In this group we observed a strong increase of GFAP mRNA in the polymorphic zone of the dentate gyrus and in the CA1 region of the hippocampus. Permanent and perinatal groups showed no overt changes compared to controls. Our findings suggest that an irritation of the mature astrocyte results in a change from the quiescent to the reactive state. The majority of astrocytes that have been exposed during their development and differentiation fail to react even if the exposure is continued to adulthood. This suggests an irreversible insult by low level lead exposure during this period of time.

Tuberous sclerosis complex with disseminated telencephalic distribution of atypical cells and their relation to corticogenesis.

In 2 cases of tuberous sclerosis complex (TSC), a disseminated distribution of atypical cells throughout the white matter and cortex of the telencephalon has been found. No cortical tubera were observed. In 1 of the cases, ventricular wall tumors (giant astrocytomas) were present. Stripes and candle guttering excrescences of groups of atypical cells perpendicular to the ventricular wall and to the cortical surface indicate erroneous genetic information in sets of neuroepithelial germ cells. This is compatible with the somatic second hit hypothesis effective in addition to the basic defect of TSC1 and TSC2 genes. The normal age-correspondend corticogenesis with regular layering and regular differentiation of neurons and glia without any cortical malformation or dysplasia and the sparing of allocortical parts of the telencephalon (hippocampus) as well as of basal ganglia, cerebellum, brain stem and spinal cord point to the rather late appearance of atypical cells which manifest in loco and do not interfere with corticogenesis. The bidirectional potential of atypical cells is obvious by their strong GFAP and APP surface staining. This coexpression indicates glial as well as neuronal features and emphasizes the relatively low level of differentiation of these cells. In their disseminated localization in our cases, these cells do not form tumors in the telencephalic white matter or cortex thus escaping sonographic detection before birth.

Unusual manifestations in two cases of necrotizing myopathy associated with SRP-antibodies.

Anti-SRP (signal recognition particle) positive necrotizing myopathy is commonly not associated with neoplasms. We demonstrate two histologically confirmed cases with unusual manifestations of anti-SRP positive necrotizing myopathy. A 65-year-old man presented with rapidly progressing weakness and mild difficulties in swallowing and speaking. Screening for underlying disorders revealed a moderately differentiated renal adenocarcinoma. The muscular symptoms partially improved after tumor nephrectomy and prednisone treatment. However, the patient developed pulmonary metastases and died of the sequelae of pneumonia 11 months after the diagnosis of renal cancer. The second patient developed rapidly complete external ophthalmoplegia, severe bulbar dysarthrophonia and dysphagia, bilateral facial palsy, loss of patellar and ankle jerk reflexes, and severe symmetrical tetraparesis of both proximal and distal muscles. CSF showed mildly increased protein levels, neurography axonal impairment of motor nerves. Screening revealed no evidence for infections, ganglioside antibodies, and carcinoma. MRI was normal. The disease course suggested an overlap syndrome of Miller-Fisher-syndrome, axonal Guillain-Barré-syndrome and Bickerstaff brainstem encephalitis. In conclusion SRP antibodies might be found in necrotizing myopathies associated with autoimmune mediated overlap syndromes and neoplasms. The pathomechanism is not clear. Any otherwise unexplained evidence of necrotizing myopathy should prompt the screening for SRP antibodies.

Phenotype variability and histopathological findings in centronuclear myopathy due to DNM2 mutations.

Autosomal-dominant centronuclear myopathy (CNM) due to mutations in the dynamin 2 gene (DNM2) is a rare congenital myopathy histopathologically characterized by centrally located nuclei and a radial arrangement of sarcoplasmic strands around the central nuclei. A total of 1,582 consecutive muscle biopsies of adult patients (age ≥ 18 years) were screened for morphologically characteristic signs of CNM. Patients with CNM were screened for mutations in DNM2. Clinical data and complementary neurophysiologic, respiratory, cardiac, and muscle MRI data in these patients were analyzed. Six index patients had histopathological signs of CNM (0.38%). Three had the heterozygous p.R465W and 2 siblings the heterozygous p.E368K DNM2 mutation. In 2 patients mutational screening for DNM2, BIN1, MTM1, and RYR1 was negative. Apart from the siblings, there was no positive history, parental mutation screening in 2 cases was negative. Both the percentage of muscle fibers with centralized nuclei and the ratio of muscle fibers with centralized to internalized nuclei were higher in DNM2-CNM compared to non-DNM2-CNM (50% vs. 18% and 94% vs. 63%). The onset was already neonatal or in infancy in 3/5 patients with DNM2 mutation. Symptoms in DNM2-CNM included bilateral ptosis (n = 3), paresis of the external ocular muscles (n = 2), axonal neuropathy (n = 4), restrictive ventilatory involvement (n = 5), and contractures (n = 5), including muscular torticollis (n = 1) and masticatory muscles (n = 2). DNM2-CNM patients and non-DNM2-CNM patients could not be distinguished by clinical features. DNM2-CNM often shows de novo mutations. In addition to the feature of radial sarcoplasmic strands, the ratio of centrally to internalized nuclei might help to differentiate DNM2-CNM from other forms of CNM. Other genes than currently known seem to cause the clinical and histopathological phenotype of CNM.

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.