Mimicking human riboflavin responsive neuromuscular disorders by silencing flad-1 gene in C. elegans: Alteration of vitamin transport and cholinergic transmission.

Riboflavin (Rf), or vitamin B2, is the precursor of FMN and FAD, redox cofactors of several dehydrogenases involved in energy metabolism, redox balance and other cell regulatory processes. FAD synthase, coded by FLAD1 gene in humans, is the last enzyme in the pathway converting Rf into FAD. Mutations in FLAD1 gene are responsible for neuromuscular disorders, in some cases treatable with Rf. In order to mimic these disorders, the Caenorhabditis elegans (C. elegans) gene orthologue of FLAD1 (flad-1) was silenced in a model strain hypersensitive to RNA interference in nervous system. Silencing flad-1 resulted in a significant decrease in total flavin content, paralleled by a decrease in the level of the FAD-dependent ETFDH protein and by a secondary transcriptional down-regulation of the Rf transporter 1 (rft-1) possibly responsible for the total flavin content decrease. Conversely an increased ETFDH mRNA content was found. These biochemical changes were accompanied by significant phenotypical changes, including impairments of fertility and locomotion due to altered cholinergic transmission, as indicated by the increased sensitivity to aldicarb. A proposal is made that neuronal acetylcholine production/release is affected by alteration of Rf homeostasis. Rf supplementation restored flavin content, increased rft-1 transcript levels and eliminated locomotion defects. In this aspect, C. elegans could provide a low-cost animal model to elucidate the molecular rationale for Rf therapy in human Rf responsive neuromuscular disorders and to screen other molecules with therapeutic potential.

Novel CAPN3 variant associated with an autosomal dominant calpainopathy.

AIMS: The most common autosomal recessive limb girdle muscular dystrophy is associated with the CAPN3 gene. The exclusively recessive inheritance of this disorder has been recently challenged by the description of the recurrent variants, c.643_663del21 [p.(Ser215_Gly221del)] and c.598_612del15 [p.(Phe200_Leu204del)], associated with autosomal dominant inheritance. Our objective was to confirm the existence of autosomal dominant calpainopathies. METHODS: Through our activity as one of the reference centres for genetic diagnosis of calpainopathies in France and the resulting collaborations through the French National Network for Rare Neuromuscular Diseases (FILNEMUS), we identified four families harbouring the same CAPN3 heterozygous variant with supposedly autosomal dominant inheritance. RESULTS: We identified a novel dominantly inherited CAPN3 variant, c.1333G>A [p.(Gly445Arg)] in 14 affected patients from four unrelated families. The complementary phenotypic, functional and genetic findings correlate with an autosomal dominant inheritance in these families, emphasizing the existence of this novel transmission mode for calpainopathies. The mild phenotype associated with these autosomal dominant cases widens the phenotypic spectrum of calpainopathies and should therefore be considered in clinical practice. CONCLUSIONS: We confirm the existence of autosomal dominant calpainopathies as an entity beyond the cases related to the in-frame deletions c.643_663del21 and c.598_612del15, with the identification of a novel dominantly inherited and well-documented CAPN3 missense variant, c.1333G>A [p.(Gly445Arg)]. In addition to the consequences for genetic counselling, the confirmation of an autosomal dominant transmission mode for calpainopathies underlines the importance of re-assessing other myopathies for which the inheritance is considered as strictly autosomal recessive.

Antisense Oligonucleotide-Based Therapy for Neuromuscular Disease.

Neuromuscular disorders such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy are neurodegenerative genetic diseases characterized primarily by muscle weakness and wasting. Until recently there were no effective therapies for these conditions, but antisense oligonucleotides, a new class of synthetic single stranded molecules of nucleic acids, have demonstrated promising experimental results and are at different stages of regulatory approval. The antisense oligonucleotides can modulate the protein expression via targeting hnRNAs or mRNAs and inducing interference with splicing, mRNA degradation, or arrest of translation, finally, resulting in rescue or reduction of the target protein expression. Different classes of antisense oligonucleotides are being tested in several clinical trials, and limitations of their clinical efficacy and toxicity have been reported for some of these compounds, while more encouraging results have supported the development of others. New generation antisense oligonucleotides are also being tested in preclinical models together with specific delivery systems that could allow some of the limitations of current antisense oligonucleotides to be overcome, to improve the cell penetration, to achieve more robust target engagement, and hopefully also be associated with acceptable toxicity. This review article describes the chemical properties and molecular mechanisms of action of the antisense oligonucleotides and the therapeutic implications these compounds have in neuromuscular diseases. Current strategies and carrier systems available for the oligonucleotides delivery will be also described to provide an overview on the past, present and future of these appealing molecules.

Diagnosis and management of adult hereditary cardio-neuromuscular disorders: A model for the multidisciplinary care of complex genetic disorders.

Genetic disorders that disrupt the structure and function of the cardiovascular system and the peripheral nervous system are common enough to be encountered in routine cardiovascular practice. Although often these patients are diagnosed in childhood and come to the cardiologist fully characterized, some patients with hereditary neuromuscular disease may not manifest until adulthood and will present initially to the adult cardiologist for an evaluation of an abnormal ECG, unexplained syncope, LV hypertrophy, and or a dilated cardiomyopathy of unknown cause. Cardiologists are often ill-equipped to manage these patients due to lack of training and exposure as well as the complete absence of practice guidelines to aid in the diagnosis and management of these disorders. Here, we review three key neuromuscular diseases that affect the cardiovascular system in adults (myotonic dystrophy type 1, Friedreich ataxia, and Emery-Dreifuss muscular dystrophy), with an emphasis on their clinical presentation, genetic and molecular pathogenesis, and recent important research on medical and interventional treatments. We also advocate the development of interdisciplinary cardio-neuromuscular clinics to optimize the care for these patients.

Neuromuscular disorders in zebrafish: state of the art and future perspectives.

Neuromuscular disorders are a broad group of inherited conditions affecting the structure and function of the motor system with polymorphic clinical presentation and disease severity. Although individually rare, collectively neuromuscular diseases have an incidence of 1 in 3,000 and represent a significant cause of disability of the motor system. The past decade has witnessed the identification of a large number of human genes causing muscular disorders, yet the underlying pathogenetic mechanisms remain largely unclear, limiting the developing of targeted therapeutic strategies. To overcome this barrier, model systems that replicate the different steps of human disorders are increasingly being developed. Among these, the zebrafish (Danio rerio) has emerged as an excellent organism for studying genetic disorders of the central and peripheral motor systems. In this review, we will encounter most of the available zebrafish models for childhood neuromuscular disorders, providing a brief overview of results and the techniques, mainly transgenesis and chemical biology, used for genetic manipulation. The amount of data collected in the past few years will lead zebrafish to became a common functional tool for assessing rapidly drug efficacy and off-target effects in neuromuscular diseases and, furthermore, to shed light on new etiologies emerging from large-scale massive sequencing studies.

Renal, ocular, and neuromuscular involvements in patients with CLDN19 mutations.

BACKGROUND AND OBJECTIVES: The objective of this study was to describe the renal and extrarenal findings in patients with recessively inherited familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) associated with CLDN19 mutations. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Medical records of three patients from two French unrelated families with CLDN19 mutations were retrospectively examined. RESULTS: Direct sequencing of CLDN19 identified a known variant (p.Gly20Asp) in all patients and a new missense mutation (p.Val44Met) in one (compound heterozygous). The patients' renal phenotype closely mimicked CLDN16-related nephropathy: low serum Mg2+ (<0.65 mmol/L) despite oral supplementation, hypercalciuria partly thiazide-sensitive, and progressive renal decline with ESRD reached at age 16 and 22 years in two individuals. Primary characteristics (failure to thrive, recurrent urinary tract infections, or abdominal pain), age at onset (0.8 to 16 years), and rate of renal decline were highly heterogeneous. Ocular involvement was identified in all patients, although two patients did not have visual loss. Additionally, exercise intolerance with pain, weakness, and electromyographical alterations mimicking a Ca2+/K+ channelopathy (pattern V) were observed in two of three individuals. These features persisted despite the normalization of serum K+ and Mg2+ after renal transplantation. CONCLUSIONS: Ocular manifestations, even subtle, and exercise intolerance mimicking mild to moderate periodic paralysis are two symptoms that need to be searched for in patients with FHHNC and may indicate CLDN19 mutations.

Knockout of the trcp3 gene causes a recessive neuromotor disease in mice.

Delta202 mice carry a transgene encoding the SV40 T antigen. Mice homozygous for the transgene develop paralysis and atrophy starting at week 4 and die around week 12. To determine the molecular basis of the neurological syndrome, we identified the transgene insertion site by sequencing two successive nested PCR products amplified with reverse primers from circularized Delta202 mouse DNA fragments generated through XbaI digestion. From the cloned products a consensus 542 bp sequence was obtained, with 409 bp corresponding to the transgene ends surrounding a 133 bp sequence formed by a left 128 bp segment and a right 8 bp segment. The 128 bp sequence matched the chr3:36811347-364811421 sequence corresponding to the promoter region of the trpc3 gene between nucleotides -54 and -53 from the transcriptional start point (+1). Complementary DNA amplification from total brain RNA demonstrated a lack of TRPC3 transcripts in Delta202 mouse brain. The neurologic syndrome of Delta202 mice thus appears to be a monogenic recessive neuromotor disease caused by interruption of the trpc3 gene promoter due to the transgene insertion which in turn blocks the transcription and knocks out TRPC3 calcium channels leading to a failure in the postnatal development of the central nervous system.

Deficiencies in C20 polyunsaturated fatty acids cause behavioral and developmental defects in Caenorhabditis elegans fat-3 mutants.

Arachidonic acid and other long-chain polyunsaturated fatty acids (PUFAs) are important structural components of membranes and are implicated in diverse signaling pathways. The Delta6 desaturation of linoleic and linolenic acids is the rate-limiting step in the synthesis of these molecules. C. elegans fat-3 mutants lack Delta6 desaturase activity and fail to produce C20 PUFAs. We examined these mutants and found that development and behavior were affected as a consequence of C20 PUFA deficiency. While fat-3 mutants are viable, they grow slowly, display considerably less spontaneous movement, have an altered body shape, and produce fewer progeny than do wild type. In addition, the timing of an ultradian rhythm, the defecation cycle, is lengthened compared to wild type. Since all these defects can be ameliorated by supplementing the nematode diet with gamma-linolenic acid or C20 PUFAs of either the n6 or the n3 series, we can establish a causal link between fatty acid deficiency and phenotype. Similar epidermal tissue defects and slow growth are hallmarks of human fatty acid deficiency.

A transgenic mouse model of the slow-channel syndrome.

To investigate the effect of acetylcholine receptor (AChR) mutations on neuromuscular transmission and to develop a model for the human neuromuscular disease, the slow-channel syndrome, we generated transgenic mice with abnormal AChRs using a delta subunit with a mutation in the ion channel domain. In three transgenic lines, nerve-evoked end-plate currents and spontaneous miniature end-plate currents (MEPCs) had prolonged decay phases and MEPC amplitudes were reduced by 33%. Single nerve stimuli elicited repetitive compound muscle action potentials in vivo. Transgenic mice were abnormally sensitive to the neuromuscular blocker, curare. These observations demonstrate that we can predictably alter AChR function, synaptic responses, and muscle fiber excitation in vivo by overexpressing subunits containing well-defined mutations. Furthermore these data support the hypothesis that the electrophysiological findings in the neuromuscular disorder, the slow-channel syndrome, are due to mutant AChRs.

Therapeutic stereotactic procedures on the thalamus for motor movement disorders.

The value of functional neurosurgery in the treatment of motor movement disorders is emphasized. The two methods of stereotactic procedures, namely a destructive one with small lesions centered on specific targets, and a non-destructive one with chronically inserted electrodes connected with an also implanted programmable neuropacemaker are described in detail. The results in Parkinsonian tremor, essential tremor, tremor of multiple sclerosis, post-traumatic tremor and in other involuntary movement disorders are reported and demonstrate that stereotactic neurosurgical treatment of these conditions is a safe and efficacious method.

[Sanatorium-health resort treatment of children with hereditary diseases of the neuromuscular system]. / O sanatorno-kurortnom lechenii detei s nasledstvennymi zabolevaniiami nervno-myshechnoi sistemy.

The study was aimed at determining the efficacy of sanatorium treatment of patients with hereditary neuromuscular abnormalities. Sanatorium methods of treatment combined with drug therapy were found to be more effective as compared with outpatient or hospital treatment. The sanatorium treatment proved most effective in children with congenital myopathy, Charcot-Marie neural amyotrophy, and Roussy-Levy polyneuropathy. Improvement in the general status and motor functions was correlated with certain improvement in dynamometric findings, locomotor tests, stabilization of a number of parameters of electroneuromyographic examination, and biochemical findings. The results point to the necessity of setting up specialized departments in childhood neurologic sanatoria or sanatoria specializing in the treatment of children with individual forms of neuromuscular diseases at health resorts which can provide treatment with sulphide waters or therapeutic mud.

Emic accounts of a mystery illness: the Groote Eylandt syndrome.

The Aboriginal people of Groote Eylandt, in the Northern Territory of Australia, are suffering from an unusual disease complex having neurological, psychiatric and teratological features, which admits no ready explanation. The island people at various times blame it on the spirits, or accuse enemies, or take some responsibility upon themselves. In this paper, 'emic' accounts of the illness (those current among the members of the society) are described in order to compare them with 'etic' accounts of those who study the society from the outside. Since emic views regulate people's behaviours toward illness, it is suggested these views should complement and inform etic views of researchers and therapists. This principle might apply to all mysterious or poorly understood illness.

Familial intrahepatic cholestasis associated with progressive neuromuscular disease and vitamin E deficiency.

Three Japanese patients with familial progressive intrahepatic cholestasis developed complications involving neurologic abnormalities characterized by ataxia and pigmentary retinopathy. Serum vitamin E concentrations were extremely low in all patients, suggesting a long-term vitamin E deficiency. High dose oral supplementation of alpha-tocopherol produced normal serum vitamin E levels in two patients. Parenteral administration of vitamin E resulted in no clinical improvement in one patient who first received the treatment at 14 years of age. In the other two patients, the progression of neurological abnormalities was slowed by vitamin E supplementation. Cholestyramine treatment resulted in an apparent decrease in serum vitamin E levels despite oral alpha-tocopherol supplementation.