Andrew G. Engel

Mutation of the acetylcholine receptor α subunit causes a slow-channel myasthenic syndrome by enhancing agonist binding affinity

In five members of a family and another unrelated person affected by a slow-channel congenital myasthenic syndrome (SCCMS), molecular genetic analysis of acetylcholine receptor (AChR) subunit genes revealed a heterozygous G to A mutation at nucleotide 457 of the alpha subunit, converting codon 153 from glycine to serine (alpha G153S). Electrophysiologic analysis of SCCMS end plates revealed prolonged decay of miniature end plate currents and prolonged activation episodes of single AChR channels. Engineered mutant AChR expressed in HEK fibroblasts exhibited prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Single-channel kinetic analysis of engineered alpha G153S AChR revealed a markedly decreased rate of ACh dissociation, which causes the mutant AChR to open repeatedly during ACh occupancy. In addition, ACh binding measurements combined with the kinetic analysis indicated increased desensitization of the mutant AChR. Thus, ACh binding affinity can dictate the time course of the synaptic response, and alpha G153 contributes to the low binding affinity for ACh needed to speed the decay of the synaptic response.

Mode Switching Kinetics Produced by a Naturally Occurring Mutation in the Cytoplasmic Loop of the Human Acetylcholine Receptor ε Subunit

We describe the genetic and kinetic defects in a congenital myasthenic syndrome caused by heteroallelic mutations of the acetylcholine receptor (AChR) epsilon subunit gene. The mutations are an in-frame duplication of six residues in the long cytoplasmic loop (epsilon1254ins18) and a cysteine-loop null mutation (epsilonC128S). The epsilon1254 ins18 mutation causes mode switching in the kinetics of receptor activation in which three modes activate slowly and inactivate rapidly. The epsilon1245ins18-AChR at the endplate shows abnormally brief activation episodes during steady state agonist application and appears electrically silent during the synaptic response to acetylcholine. The phenotypic consequences are endplate AChR deficiency, simplification of the postsynaptic region, and compensatory expression of fetal AChR that restores electrical activity at the endplate and rescues the phenotype.

Thyroid function and periodic paralysis.

Abstract The effects of 1-triiodothyronine and thyrotropin were investigated in a case of familial periodic paralysis. Hypermetabolism, first induced by the administration of 1-triiodothyronine then by thyrotropin, had no immediate adverse effects on the periodic paralysis. Exacerbations of the disease occurred twice, each time with return of the basal metabolic rate to normal. The increased weakness following withdrawal of l-triiodothyronine was promptly reversed by the administration of thyrotropin. The increased weakness following thyrotropin withdrawal disappeared gradually over twentyfive days. Thyroid hormones rather than pituitary thyrotropin secretion determined the effects on the periodic paralysis. Exacerbations of the disease were not associated with depression of the serum potassium levels. Decreased urinary excretion of potassium was noted during the first exacerbation, and decreased excretion of potassium and sodium were noted preceding the second one. No support could be found for the assumption that the two exacerbations were caused by temporary hyperaldosteronism. The present study points to a difference in the basic abnormality between the thyrotoxic and the familial forms of periodic paralysis.

Experimental autoimmune myasthenia gravis: a sequential and quantitative study of the neuromuscular junction ultrastructure and electrophysiologic correlations.

Neuromuscular junction ultrastructure in rat forelimb digit extensor muscle was sequentially and quantitatively investigated in experimental autoimmune myasthenia gravis (EAMG). Experimental animals were immunized with highly purified eel electroplax acetylcholine receptor protein plus complete Freunds adjuvant and B. pertussis vaccine; control animals received only adjuvant and vaccine. During the first 7 days (latent period) after immunization end-plate structure and neuromuscular transmission remained normal in the experimental group. Between day 7 and 11 (acute phase) mononuclear cells infiltrated those regions of muscle where the end-plates were located and there was intense degeneration of the postsynaptic regions with splitting away of abnormal junctional folds from the underlying muscle fibers. Macrophages entered the gaps thus formed and removed the degenerating folds by phagocytosis. The nerve terminals were displaced from their usual location but maintained their structural integrity. Neuromuscular transmission was blocked in many muscle fibers. Miniature end-plate potentials (MEPPs), detectable in only a few fibers, were of abnormally low amplitude. After day 11 (chronic phase) the nerve terminals returned to the highly simplified postsynaptic regions and the inflammatory reaction subsided. Subsequently the postsynaptic folds became reconstituted and again degenerated. Immature junctions with poorly differentiated postsynaptic regions and nerve sprouts near end-plates were also observed. In two animals relapsing

Acid maltase deficiency in adults presenting as respiratory failure.

During the past nine years 10 patients with the adult form of acid maltase deficiency have been observed at the Mayo Clinic. Three of the adults presented with respiratory failure. In all three the respiratory manifestations dominated the clinical picture and the cause of the respiratory failure (muscle weakness) and the underlying myopathy (glycogen storage disease) were initially unsuspected. Careful evaluation of the respiratory function tests, including the maximal static respiratory pressures, electromyographic examination and histochemical and biochemical studies of muscle biopsy specimens eventually led to the correct diagnosis.

Clinical and electromyographic studies in a patient with primary hypokalemic periodic paralysis

Abstract A patient with severe primary hypokalemic periodic paralysis improved significantly on a regimen low in carbohydrate, restricted in sodium and high in potassium. Moderate weakness persisted in the proximal muscles and was associated with persistent vacuolation of muscle fibers. Enlargement of the motor end-plate zone is described in this case of primary hypokalemic periodic paralysis. Considerable weakness occurred even when serum potassium values were within the normal range. On an isocaloric nonreducing diet, the patient lost weight when the carbohydrate intake was reduced. Most of the weight loss could be accounted for by a loss of body water that was not associated with a negative sodium balance. Data obtained in the course of an acute attack are compatible with an increase in the total body water content and a shift of fluid from the extracellular into the intracellular compartment. Renal retention of potassium, sodium and chloride preceded changes in the patients strength and the decrease in serum potassium. Not more than 10 mEq. of potassium could have been lost from the patients erythrocytes during the attack despite the decreasing serum potassium levels. A fully paralyzed muscle and a muscle only somewhat weak were similar in their electrolyte and water content, except for the higher calcium and magnesium contents of the weaker muscle. Both muscles had chloride space and water levels which were higher than most normal values; sodium, chloride and magnesium levels higher than normal; and a potassium level which was lower than most normal values. The chloride space values were too high to be a measure of the extracellular space and could not be used for calculating intracellular electrolyte concentrations. Small doses of epinephrine, administered intra-arterially, had a direct adverse effect on the muscles of the perfused forearm. These effects were not mediated by systemic hypokalemia or hyperglycemia but may have been related to an acceleration of glycogenolysis and glycolysis in muscles. An increased uptake of sodium by the forearm tissues followed rather than preceded the decrease in action potential and twitch tension. Maximal voluntary exercise followed by rest caused a transient potentiation and then a depression of the action potential and of the twitch tension in the patient and in healthy subjects, but the patients response was at times quantitatively greater. The deviation from normal tended to be greatest when the patient was weakest. Excessive accumulation of lactic and pyruvic acids in the blood occurred after a standard work load. This may indicate a greater than normal release of these metabolites from muscle or an impaired metabolism by muscle, or both. There appears to be no impairment in glycogenolysis or glycolysis in muscle during exercise. A close relationship was again observed between carbohydrate metabolism and primary hypokalemic periodic paralysis. The present study points to a possible metabolic lesion, or a contributing mechanism, that can be activated by rapidly induced glycolysis.

Novel congenital myasthenic syndromes associated with defects in quantal release

Background: Most congenital myasthenic syndromes are caused by defects in postsynaptic or synaptic basal lamina–associated proteins; congenital myasthenic syndromes (CMSs) associated with presynaptic defects are uncommon. Here, the authors describe clinical, electrophysiologic, and morphologic features of two novel and highly disabling CMSs, one determined by presynaptic and the other determined by combined presynaptic and postsynaptic defects. Methods: Microelectrode, single channel patch clamp, immunocytochemical, [125I]α-bungarotoxin binding, and quantitative electron microscopy studies of endplates were performed. Candidate genes were directly sequenced. Results: Patient 1, a 7-year-old boy, had severe myasthenic symptoms since infancy. Patient 2, a 48-year-old man, had delayed motor milestones and became progressively weaker after age 2 years. Both used wheelchairs and had a 30-50% EMG decrement on 2-Hz stimulation. Evoked quantal release was reduced to approximately 25% of normal in both. In Patient 2, the synaptic response to acetylcholine was further compromised by degeneration of the junctional folds with concomitant loss of the acetylcholine receptor (AChR). A search for mutations in components of the synaptic vesicle release complex and in other candidate proteins failed to identify the molecular basis of the two syndromes. Conclusions: Combined clinical, morphologic, and in vitro electrophysiologic findings define two novel congenital myasthenic syndromes. The molecular basis of these syndromes awaits discovery.

Congenital myopathy associated with the triadin knockout syndrome

Objective: Triadin is a component of the calcium release complex of cardiac and skeletal muscle. Our objective was to analyze the skeletal muscle phenotype of the triadin knockout syndrome. Methods: We performed clinical evaluation, analyzed morphologic features by light and electron microscopy, and immunolocalized triadin in skeletal muscle. Results: A 6-year-old boy with lifelong muscle weakness had a triadin knockout syndrome caused by compound heterozygous null mutations in triadin. Light microscopy of a deltoid muscle specimen shows multiple small abnormal spaces in all muscle fibers. Triadin immunoreactivity is absent from type 1 fibers and barely detectable in type 2 fibers. Electron microscopy reveals focally distributed dilation and degeneration of the lateral cisterns of the sarcoplasmic reticulum and loss of the triadin anchors from the preserved lateral cisterns. Conclusions: Absence of triadin in humans can result in a congenital myopathy associated with profound pathologic alterations in components of the sarcoplasmic reticulum. Why only some triadin-deficient patients develop a skeletal muscle phenotype remains an unsolved question.

Molecular insights into acetylcholine receptor structure and function revealed by mutations causing congenital myasthenic syndromes

Publisher Summary This chapter provides molecular insights into acetylcholine receptor structure and function revealed by mutations causing congenital myasthenic syndromes (CMS). The investigation of CMS proceeds from a generic clinical diagnosis to defining the morphologic and electrophysiologic phenotype. If these studies point to a candidate gene whose sequence is known, then mutation analysis becomes feasible. In addition, if a mutation in a relevant protein is identified, then appropriate expression studies are designed. A generic clinical diagnosis of a CMS is based on the history of myasthenic symptoms from birth or early childhood, similarly affected relatives, a decremental electromyographic (EMG) response of the compound muscle fiber action potential on low-frequency (2–3 Hz) stimulation, and a negative test for anti-AChR antibodies. Some CMS cases, however, are sporadic or present in later life and the decremental EMG response may not be present in all muscles or at all times. Conventional microelectrode studies on muscle specimens excised from origin to insertion readily reveal whether the defect of neuromuscular transmission is presynaptic, synaptic, or postsynaptic and define the factors that impair the safety margin of neuromuscular transmission.