Congenital myasthenic syndromes: spotlight on genetic defects of neuromuscular transmission

The neuromuscular junction (NMJ) is a complex structure that efficiently communicates the electrical impulse from the motor neuron to the skeletal muscle to induce muscle contraction. Genetic and autoimmune disorders known to compromise neuromuscular transmission are providing further insights into the complexities of NMJ function. Congenital myasthenic syndromes (CMSs) are a genetically and phenotypically heterogeneous group of rare hereditary disorders affecting neuromuscular transmission. The understanding of the molecular basis of the different types of CMSs has evolved rapidly in recent years. Mutations were first identified in the subunits of the nicotinic acetylcholine receptor (AChR), but now mutations in ten different genes - encoding post-, pre- or synaptic proteins - are known to cause CMSs. Pathogenic mechanisms leading to an impaired neuromuscular transmission modify AChRs or endplate structure or lead to decreased acetylcholine synthesis and release. However, the genetic background of many CMS forms is still unresolved. A precise molecular classification of CMS type is of paramount importance for the diagnosis, counselling and therapy of a patient, as different drugs may be beneficial or deleterious depending on the molecular background of the particular CMS.     

Congenital myasthenic syndrome: a case report

Summary: Congenital myasthenic syndromes (CMS) are diseases of the neuromuscular junction. They usually belong to the disease groups that begin in the infantile or childhood period and carry genetic characteristics. The following is important in establishing the diagnosis of this disease: clinical findings, electromyography, genetic tests, determination of serum acetylcholine receptor antibodies. Acetylcholine esterase inhibitor drugs are used in treatment of CMS. A seven-month old male patient was brought to our department with the complaints of difficult breathing, falling of the eyelids and swallowing difficulty. With clinical and laboratory findings, he was diagnosed with congenital myasthenia and treatment was started. CMS should be suspected in patients with no pathological findings on the physical examination, and normal chest X-rays.     

The spectrum of congenital myasthenic syndromes

The past decade saw remarkable advances in defining the molecular and genetic basis of the congenital myasthenic syndromes. These advances would not have been possible without antecedent clinical observations, electrophysiologic analysis, and careful morphologic studies that pointed to candidate genes or proteins. For example, a kinetic abnormality of the acetylcholine receptor (AChR) detected at the single channel level pointed to a kinetic mutation in an AChR subunit; endplate AChR deficiency suggested mutations residing in an AChR subunit or in rapsyn; absence of acetylcholinesterase (AChE) from the endplate predicted mutations in the catalytic or collagen-tailed subunit of this enzyme; and a history of abrupt episodes of apnea associated with a stimulation dependent decrease of endplate potentials and currents implicated proteins concerned with ACh resynthesis or vesicular filling. Discovery of mutations in endplate-specific proteins also prompted expression studies that afforded proof of pathogenicity, provided clues for rational therapy, lead to precise structure function correlations, and highlighted functionally significant residues or molecular domains that previous systematic mutagenesis studies had failed to detect. An overview of the spectrum of the congenital myasthenic syndromes suggests that most are caused by mutations in AChR subunits, and particularly in the ɛ subunit. Future studies will likely uncover new types of CMS that reside in molecules governing quantal release, organization of the synaptic basal lamina, and expression and aggregation of AChR on the postsynaptic junctional folds.     

An array of diverse microbial genomes

Methods for comparing gene frequencies across large, epidemiologically defined bacterial collections are limited. A novel microarray technology has been developed called 'library on a slide'. In this technology, hundreds of entire microbial genomes are arrayed, rather than sequences of a single genome or sets of genes. These slides can then be probed for the presence of specific genes allowing researchers to draw inferences regarding important differences between related strains that differ in their pathogenic potential.     

Lethal effects of apidaecin on Escherichia coli involve sequential molecular interactions with diverse targets.

Apidaecins, short proline-arginine-rich peptides from insects, are highly bactericidal through a mechanism that includes stereoselective elements but is completely devoid of any pore-forming activity. The spectrum of antibacterial activity, always limited to Gram-negatives, is further dependent on a small number of variable residues and can be manipulated. We show here that mutations in the evolutionary conserved regions result in a more general loss of function, and we have used such analogs to probe molecular interactions in Escherichia coli. First, an assay was developed to measure selectively chiral association with cellular targets. By using this method, we find that apidaecin uptake is energy-driven and irreversible and yet can be partially competed by proline in a stereospecific fashion, results upholding a model of a permease/transporter-mediated mechanism. This putative transporter is not the end point of apidaecin action, for failure of certain peptide analogs to kill cells after entering indicates the existence of another downstream target. Tetracycline-induced loss of bactericidal activity and dose-dependent in vivo inhibition of translation by apidaecin point at components of the protein synthesis machinery as likely candidates. These findings provide new insights into the antibacterial mechanism of a unique group of peptides and perhaps, by extension, for distant mammalian relatives such as PR-39.     

Congenital syndromes and leukemia: clues to pathogenesis

The association of specific congenital syndromes with leukemia provides an opportunity to study the process of leukemogenesis on the background of known genetic alterations. The role of the intracellular DNA damage response system in suppressing leukemia is demonstrated by the congenital disorders of genomic instability. Specific collaborations between survival and differentiation pathways characterize the leukemias observed in Down, Noonan and neurofibromatosis syndromes. As these syndromes clearly reveal, childhood leukemia arises when the delicate balance between growth, development and differentiation of the fetal and early post-natal hematopoietic system is disrupted.     

Pathophysiological characterization of congenital myasthenic syndromes: the example of mutations in the MUSK gene

Congenital myasthenic syndromes (CMS) are rare genetic diseases affecting the neuromuscular junction (NMJ) and are characterized by a dysfunction of the neurotransmission. They are heterogeneous at their pathophysiological level and can be classified in three categories according to their presynaptic, synaptic and postsynaptic origins. We report here the first case of a human neuromuscular transmission dysfunction due to mutations in the gene encoding a postsynaptic molecule, the muscle-specific receptor tyrosine kinase (MuSK). Gene analysis identified two heteroallelic mutations, a frameshift mutation (c.220insC) and a missense mutation (V790M). The muscle biopsy showed dramatic pre- and postsynaptic structural abnormalities of the neuromuscular junction and severe decrease in acetylcholine receptor (AChR) epsilon-subunit and MuSK expression. In vitro and in vivo expression experiments were performed using mutant MuSK reproducing the human mutations. The frameshift mutation led to the absence of MuSK expression. The missense mutation did not affect MuSK catalytic kinase activity but diminished expression and stability of MuSK leading to decreased agrin-dependent AChR aggregation, a critical step in the formation of the neuromuscular junction. In electroporated mouse muscle, overexpression of the missense mutation induced, within a week, a phenotype similar to the patient muscle biopsy: a severe decrease in synaptic AChR and an aberrant axonal outgrowth. These results strongly suggest that the missense mutation, in the presence of a null mutation on the other allele, is responsible for the dramatic synaptic changes observed in the patient.     

A nomenclature and classification for the congenital myasthenic syndromes: preparing for FAIR data in the genomic era

Background

Congenital myasthenic syndromes (CMS) are a heterogeneous group of inherited neuromuscular disorders sharing the common feature of fatigable weakness due to defective neuromuscular transmission. Despite rapidly increasing knowledge about the genetic origins, specific features and potential treatments for the known CMS entities, the lack of standardized classification at the most granular level has hindered the implementation of computer-based systems for knowledge capture and reuse. Where individual clinical or genetic entities do not exist in disease coding systems, they are often invisible in clinical records and inadequately annotated in information systems, and features that apply to one disease but not another cannot be adequately differentiated.

Results

We created a detailed classification of all CMS disease entities suitable for use in clinical and genetic databases and decision support systems. To avoid conflict with existing coding systems as well as with expert-defined group-level classifications, we developed a collaboration with the Orphanet nomenclature for rare diseases, creating a clinically understandable name for each entity and placing it within a logical hierarchy that paves the way towards computer-aided clinical systems and improved knowledge bases for CMS that can adequately differentiate between types and ascribe relevant expert knowledge to each.

Conclusions

We suggest that data science approaches can be used effectively in the clinical domain in a way that does not disrupt preexisting expert classification and that enhances the utility of existing coding systems. Our classification provides a comprehensive view of the individual CMS entities in a manner that supports differential diagnosis and understanding of the range and heterogeneity of the disease but that also enables robust computational coding and hierarchy for machine-readability. It can be extended as required in the light of future scientific advances, but already provides the starting point for the creation of FAIR (Findable, Accessible, Interoperable and Reusable) knowledge bases of data on the congenital myasthenic syndromes.

     

A unique and universal molecular barcode array

Molecular barcode arrays allow the analysis of thousands of biological samples in parallel through the use of unique 20-base-pair (bp) DNA tags. Here we present a new barcode array, which is unique among microarrays in that it includes at least five replicates of every tag feature. The use of smaller dispersed replicate features dramatically improves performance versus a single larger feature and allows the correction of previously undetectable hybridization defects.     

Cancer-relevant biochemical targets of cytotoxic Lonchocarpus flavonoids: A molecular docking analysis

A molecular docking investigation has been carried out on cytotoxic prenylated flavonoids from Lonchocarpus haberi with cancer-relevant chemotherapeutic targets known to be inhibited by flavonoids. Two molecular docking programs, Molegro and ArgusDock, were used to compare the binding energies of Lonchocarpus flavonoids with other flavonoids, inhibitors, or known ligands, to aromatase (CYP 19), fatty acid synthase (FAS), xanthine oxidase (XO), cyclooxygenases (COX-1 and COX-2), lipoxygenase (LOX-3), ornithine decarboxylase (ODC), protein tyrosine kinase (PTK), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), topoisomerase II (ATP binding site), ATP binding cassette (ABC) transporter, and phospholipase A₂ (PLA). The Lonchocarpus flavonoids examined in this study exhibited docking energies comparable to or stronger than other flavonoids that had been previously shown to be effective inhibitors of these enzymes. Furthermore, prenylated flavonoids, such as the Lonchocarpus flavonoids and xanthohumol, generally showed greater binding energies than the non-prenylated flavonoids. We conclude, therefore, that the Lonchocarpus flavonoids possibly owe their cytotoxic activity by inhibition of one or more of these enzymes.      

Neonicotinoids show selective and diverse actions on their nicotinic receptor targets: electrophysiology, molecular biology, and receptor modeling studies

Neonicotinoid insecticides, which act selectively on insect nicotinic acetylcholine receptors (nAChRs), are used worldwide for insect pest management. Studies that span chemistry, biochemistry, molecular biology, and electrophysiology have contributed to our current understanding of the important physicochemical and structural properties essential for neonicotinoid actions as well as key receptor residues contributing to the high affinity of neonicotinoids for insect nAChRs. Research to date suggests that electrostatic interactions and possibly hydrogen bond formation between neonicotinoids and nAChRs contribute to the selectivity of these chemicals. A rich diversity of neonicotinoid-nAChR interactions has been demonstrated using voltage-clamp electrophysiology. Computational modeling of nAChR-imidacloprid interaction has assisted in the interpretation of these results.     

Ion channels: molecular targets of neuroactive insecticides

Many of the insecticides in current use act on molecular targets in the insect nervous system. Recently, our understanding of these targets has improved as a result of the complete sequencing of an insect genome, i.e., Drosophila melanogaster. Here we examine the recent work, drawing on genetics, genomics and physiology, which has provided evidence that specific receptors and ion channels are targeted by distinct chemical classes of insect control agents. The examples discussed include, sodium channels (pyrethroids, p,p′-dichlorodiphenyl-trichloroethane (DDT), dihydropyrazoles and oxadiazines); nicotinic acetylcholine receptors (cartap, spinosad, imidacloprid and related nitromethylenes/nitroguanidines); γ-aminobutyric acid (GABA) receptors (cyclodienes, γ-BHC and fipronil) and L-glutamate receptors (avermectins). Finally, we have examined the molecular basis of resistance to these molecules, which in some cases involves mutations in the molecular target, and we also consider the future impact of molecular genetic technologies in our understanding of the actions of neuroactive insecticides.     

Anticancer properties of tocotrienols: A review of cellular mechanisms and molecular targets

Vitamin E is composed of two groups of compounds: α-, β-, γ-, and δ-tocopherols (TPs), and the corresponding unsaturated tocotrienols (TTs). TTs are found in natural sources such as red palm oil, annatto seeds, and rice bran. In the last decades, TTs (specifically, γ-TT and δ-TT) have gained interest due to their health benefits in chronic diseases, based on their antioxidant, neuroprotective, cholesterol-lowering, anti-inflammatory activities. Several in vitro and in vivo studies pointed out that TTs also exert a significant antitumor activity in a wide range of cancer cells. Specifically, TTs were shown to exert antiproliferative/proapoptotic effects and to reduce the metastatic or angiogenic properties of different cancer cells; moreover, these compounds were reported to specifically target the subpopulation of cancer stem cells, known to be deeply involved in the development of resistance to standard therapies. Interestingly, recent studies pointed out that TTs exert a synergistic antitumor effect on cancer cells when given in combination with either standard antitumor agents (i.e., chemotherapeutics, statins, “targeted” therapies) or natural compounds with anticancer activity (i.e., sesamin, epigallocatechin gallate (EGCG), resveratrol, ferulic acid). Based on these observations, different TT synthetic derivatives and formulations were recently developed and demonstrated to improve TT water solubility and to reduce TT metabolism in cancer cells, thus increasing their biological activity. These promising results, together with the safety of TT administration in healthy subjects, suggest that these compounds might represent a new chemopreventive or anticancer treatment (i.e., in combination with standard therapies) strategy. Clinical trials aimed at confirming this antitumor activity of TTs are needed.     

A diverse array of genetic factors contribute to the pathogenesis of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease with variable clinical presentation frequently affecting the skin, joints, haemopoietic system, kidneys, lungs and central nervous system. It can be life threatening when major organs are involved. The full pathological and genetic mechanisms of this complex disease are yet to be elucidated; although roles have been described for environmental triggers such as sunlight, drugs and chemicals, and infectious agents. Cellular processes such as inefficient clearing of apoptotic DNA fragments and generation of autoantibodies have been implicated in disease progression. A diverse array of disease-associated genes and microRNA regulatory molecules that are dysregulated through polymorphism and copy number variation have also been identified; and an effect of ethnicity on susceptibility has been described.