Hospital Practice
ISSN: 2154-8331 (Print) 2377-1003 (Online) Journal homepage: http://www.tandfonline.com/loi/ihop20
Congenital Disorders of Neuromuscular Transmission Henry J. Kaminski & Robert L. Ruff To cite this article: Henry J. Kaminski & Robert L. Ruff (1992) Congenital Disorders of Neuromuscular Transmission, Hospital Practice, 27:9, 73-85, DOI: 10.1080/21548331.1992.11705484 To link to this article: https://doi.org/10.1080/21548331.1992.11705484
Published online: 17 May 2016.
Submit your article to this journal
Citing articles: 3 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ihop20
IPIHIYSllOlOGY TIN MIEDllCCllNIE
Congenital Disorders of Neuromuscular Transmission HENRY J. KAMINSKI and ROBERT L. RUFF
Case Western Reserve University
Pathology underlying congenital forms of myasthenia gravis has been delineated through microscopic and electrophysiologic studies over the past 15 years. Differentiation from the immune-mediated disorder is crucial because therapy appropriate for acquired myasthenia may be harmful to patients with congenital disease.
Congenital disorders of neuromuscular transmission are commonly referred to as congenital myasthenias because of their clinical similarity to the immune-mediated disease. Patients often present with fatigable weakness and a propensity for extraocular muscle involvement; electromyography usually reveals a decremental response to repetitive stimulation. However, antibodies against the acetylcholine receptor are not present. By definition, manifestations of the illness may be traced to birth, and symptoms respond poorly to immunosuppressive treatment. The congenital disorders are caused by a variety of defects at the neuromuscular junction and represent experiments of nature that offer unique insights into mechanisms of neurotransmitter release, acetylcholine receptor function, and acetylcholine metabolism. The disorders should be differentiated from neonatal and juvenile myasthenia gravis. Neonatal myasthenia occurs in 10% to 20% of infants consequent to transmission of anti-acetylcholine receptor antibodies across the placenta from mothers with myasthenia gravis. The child shows signs of muscle weakness, a weak cry, poor sucking, and, occasionally, respiratory insufficiency. The disor-
der resolves within days to weeks. Juvenile myasthenia may occur in children under 10 years of age, with manifestations typical of the immune-mediated disease in older patients. The serum contains anti-acetylcholine receptor antibodies, and the disease has the same pathogenesis as the adult form. Fewer than 40 congenital neuromuscular transmission disorder cases have been described in detail, but this number may significantly underrepresent the true frequency of these disorders. As noted, they are easily confused with myasthenia gravis. In addition, extensive evaluation, which may not be performed even at tertiary referral centers, is needed for diagnosis. Nevertheless, every physician should have some familiarity with these disorders,
Dr. Kaminski is Assistant Professor and Dr. Ruff is Associate Professor, Department of Neurology, Case Western Reserve University School of Medicine. Dr. Kaminski is also Staff Physician and Dr. Ruff is Chief, Department of Neurology, Veterans Affairs Medical Center, Cleveland. Their work has been supported by Merit Reviewed Funding from the Department of Veterans Affairs and National Institutes of Health grants NS 26661 and EY09186.This is the third article in a series on neuromuscular transmission disorders.
In cooperation with the American Physiological Society Hospital Practice September 15. 1992
73
PHYSIOLOGY IN MEDICINE which may manifest at any age with severe signs, such as respiratory failure, that may become apparent during acute illnesses or surgery. In the following, we will attempt to categorize the congenital neuromuscular transmission disorders on the basis of the presumed physiologic transmission defects, and to understand the patients' clinical presentations on the basis of observed anatomic and physiologic abnormalities.
Abnormalities of Transmitter Release Normal quantal release. The vesicle hypothesis of synaptic transmission forms the cornerstone for understanding neuromuscular junction mechanisms, and has important implications for evaluation of the sites of the congenital transmission disorders· defects (Figure 1 ). The vesicle hypothesis holds that the neurotransmitter is packaged in
Figure 1. Congenital disorders of neuromuscular transmission may involve defects at any of five possible pre- or postsynaptic sites. As schematized, these are voltage-gated
74
Hospital Practice September 15. 1992
the nerve terminals in synaptic vesicles, which, when fusing with the nerve terminal plasma membrane, cause release of transmitter. The transmitter then diffuses across the synaptic cleft and interacts with postsynaptic receptors, producing depolarization ofthe postsynaptic membrane. The end-plate depolarization triggers action potentials that travel away from the end plate to stimulate muscle contraction. Transmitter release is initiat-
calcium channels (A), acetylcholine-containing vesicles (B), junctional folds (C), acetylcholine receptors (D), and acetylcholinesterase levels in the synaptic cleft (E).
PHYSIOLOGY IN MEDICINE ed when the motor neuron action potential opens voltagegated calcium channels on the nerve terminal. The subsequent influx of calcium initiates fusion of synaptic vesicles with the plasma membrane, resulting in release of acetylcholine. Choline, the hydrolysis product of acetylcholine in the synaptic cleft. is taken up by a high-affinity transport system on the nerve terminal and reused in the synthesis of acetylcholine. The acetylcholine is then repackaged in synaptic vesicles. Most of the synaptic vesicles are formed by endocytosis from nerve terminal membrane. Some are carried to the nerve terminal by axonal transport. The precise mechanisms underlying vesicle membrane recycling and acetylcholine packaging are not clear. Intracellular recording of muscle fibers near the end plate demonstrates spontaneous depolarizations, or miniature endplate potentials (Figure 2). They are thought to represent the depolarization produced by release of acetylcholine from a single vesicle. The depolarization produced by numerous vesicles fusing with the presynaptic membrane is the end-plate potential, the size of which depends on the number of vesicles releasing the transmitter. If the number is above a certain threshold, an action potential is generated, and the muscle contracts. The compound muscle action potential, which represents the summation of individual muscle fiber action potentials, can be determined by electromyography (Figure3). Clinical correlation. The patients who will be described share an abnormality of quantal release that bears clinical similarity to cases of "familial infan-
Figure 2. Miniature end-plate potentials, each thought to represent spontaneous depolarization produced by release of acetylcholine from a single vesicle, can vary in duration or amplitude. To illustrate, a normal miniature end-plate potential is contrasted with the prolonged variant seen in acetylcholinesterase deficiency and slow-channel syndrome, and with the depressed variant seen in disorders characterized by reduced acetylcholine receptor density.
tile myasthenia," which were described before the advent of sophisticated electrophysiologic techniques and electron microscopy. Impaired acetylcholine packaging. Andrew G. Engel and col-
leagues reported a case that strongly emphasizes the role of synaptic vesicles in producing the end-plate potential. A 23year-old woman had episodic generalized and bulbar fatigable weakness from birth. A deeremental response of the compound muscle action potential was found with repetitive stimulation, which improved with neostigmine treatment. Miniature end-plate potential amplitudes were normal, but the endplate potential at 1-Hz stimulation showed a marked reduction of quantal content. Electron micrographs of the nerve terminal revealed a reduction in synaptic vesicles. The implication was that the small end-plate potentials resulted from a reduction of
readily releasable vesicles containing acetylcholine. Several reports of patients with defects of acetylcholine processing have been described. In 1979, Engel and colleagues reported on an 18-year-old man whose infancy had been marked by ptosis and poor feeding. As he grew older, the patient became easily fatigued and experienced episodic apnea after crying or febrile illness. Anticholinesterase medications palliated his symptoms. The patient's sister had similar difficulties, and three siblings had died of apneic spells. Repetitive stimulation at 2 Hz produced a decrement of the compound muscle action potential. Microelectrode studies of an intercostal muscle biopsy revealed normal resting miniature end-plate potential amplitudes. but after 2-Hz stimulation, miniature end-plate potential amplitude declined significantly. The number of acetylcholine recepHospital Practice September 15. 1992
75
PHYSIOLOGY IN MEDICINE tors was normal as determined by a-bungarotoxin labeling. The only anatomic abnormality was a 60% increase in the number of synaptic vesicles, which may have represented an adaptive change. In a study of three similar patients, other investigators found a reduction in synaptic vesicle size and attempted to correlate it with the reduction of miniature end-plate potential on repetitive stimulation. In controls and patients, the number of vesicles near the nerve terminal synaptic membrane decreased after stim-
ulation. In patients, the vesicle size increased or was unchanged, whereas controls showed a decrease or no change. The normal miniature endplate potential at rest indicates that both quanta! content and postsynaptic function are normal. The decline in miniature end-plate potential amplitude with stimulation suggests an abnormality of "recycling" of functional synaptic vesicles. Such a defect could lie in the choline transporter, acetylcholine resynthesis by choline acetyltransferase, or packaging of acetyl-
choline into vesicles-the net effect being that vesicles contained less releasable acetylcholine. Desensitization of the acetylcholine receptor is unlikely because acetylcholinesterase inhibitors would be expected to worsen such a defect. Impaired vesicle release. An
abnormality of quanta! release has been described in only one patient, who showed an incremental response with repetitive stimulation. Unfortunately, no microelectrode studies could be performed. The patient had been diffusely weak since birth, but without ocular signs. Administration of guanidine hydrochloride, which increases quanta! release, led to improvement in strength. The EMG findings were similar to those in patients with Lambert-Eaton myasthenic syndrome, thought to be a paraneoplastic syndrome or an autoimmune disorder in which antibodies cross-react with nerve terminal calcium channels. The decreased quanta! content in Lambert-Eaton is believed toresult from impaired entry of calcium into the nerve terminal. The defect in this patient was not defined but might have resulted from qualitative or quantitive derangement of presynaptic calcium channels.
Acetylcholinesterase Deficiency
Figure 3. By definition, a compound muscle action potential sum mates individual
muscle fiber action potentials. Its normal shape after a single electrical stimulation is shown at top; the lower curve shows the repetitive shape seen in acetycholinesterase deficiency and slow-channel syndrome.
76
Hospital Practice September 15. 1992
Acetylcholinesterase in neuromuscular transmission. Acetylcholinesterase concentration and kinetics, acetylcholine diffusion, and the electrophysiologic characteristics of the acetylcholine receptor determine the duration and amplitude of the miniature end-plate potential. Several characteristics of the neuromuscular junction lead to
PHYSIOLOGY IN MEDICINE the synchronous opening of many acetylcholine receptors, which is required to produce the rapid rising phase of the miniature end-plate potential. Therelease of acetylcholine into the small area of the synaptic cleft produces local concentrations well in excess of the dissociation constant of the acetylcholine receptor. Acetylcholinesterase is found at concentrations five to eight times lower than acetylcholine receptor on the postsynaptic membrane, and the turnover time of acetylcholinesterase (the time it takes to hydrolyze an acetylcholine molecule) approximates the rising time of the miniature end-plate potential. Therefore, acetylcholine initially binds to receptor before it is hydrolyzed or diffuses from the synaptic cleft. The concentration of acetylcholine must drop quickly so that repeated binding and acetylcholine receptor channel opening do not occur. During the falling phase of the miniature end-plate potential, acetylcholine hydrolysis and diffusion occur more rapidly than acetylcholine receptor channel closure, and the length of the falling phase is therefore determined by the single channel open time of the acetylcholine receptor. Clinical correlation. Using cyto- and immunochemical techniques, Engel and E. H. Lambert demonstrated a severe deficiency of acetylcholinesterase from the neuromuscular junction of a 16-year-old boy. From birth he had ptosis and fatigable weakness and had experienced respiratory insufficiency associated with a viral illness. Anticholinesterases did not improve his symptoms. EMG provided important insights into the physiologic consequences of acetylcholinesterase deficiency.
Figure 4. Congenital acetylcholinesterase deficiency may lead to shrunken nerve terminals (A), shallow junctional folds with reduced numbers of acetylcholine receptors (B), decreased choline uptake from the synaptic cleft (C), and low concentrations of acetylcholine in synaptic vesicles (D).
While repetitive stimulation produced a decremental response, a single stimulus evoked two or more compound muscle action potentials, and intracellular microelectrode studies demonstrated a prolonged miniature end-plate potential duration and a low normal miniature endplate potential amplitude. Maintenance of the end-plate potential above the level for action potential generation during the refractory period resulted in triggering of multiple action potentials. The acetylcholinesterase deficiency led to an increase in acetylcholine in the synaptic cleft and showed that diffusion was not sufficient to eliminate acetylcholine from the synaptic cleft. In addition to the primary defect of acetylcholinesterase deficiency, investigations revealed several secondary abnormali-
ties, which may have been adaptive. Significant inhibition of acetylcholinesterase in normal subjects may lead to receptor desensitization and the clinical correlate, "cholinergic crisis," which was not observed in this patient. Since the acetylcholine receptor density was normal, the reduced miniature end-plate potential amplitude probably indicated a receptor abnormality or reduced acetylcholine concentration in the vesicles. The reduction of vesicle acetylcholine concentration may offset the lack of acetylcholinesterase and allow diffusion to rapidly terminate the local action of acetylcholine. The decreased acetylcholine content of vesicles may also reflect a decrease in choline reuptake that occurs because of decreased hydrolysis of acetylcholine in the synaptic cleft (Figure 4). Hospital Practice September 15. 1992
77
PHYSIOLOGY IN MEDICINE In some muscles, junctional folds were degenerated. Many nerve terminals were smaller than normal, covering only a portion of the postsynaptic membrane. The quanta! content of the end-plate potential was reduced, in keeping with the decreased size ofthe nerve terminal. The changes in structure of the synapses may also have functioned to increase diffusion of acetylcholine away from the postsynaptic membrane, which would decrease end-plate depolarization and prevent acetylcholine receptor desensitization.
Congenital Paucity of Synaptic Clefts Postsynaptic membrane specialization. The nerve terminal is separated from the postsynaptic region by a narrow space called the primary cleft. The postsynaptic muscle surface is a highly specialized plasma membrane consisting of deep junctional folds, between which is a space continuous with the primary cleft termed the secondary synaptic cleft. While the precise role of the junctional folds in neuromuscular transmission is not fully understood, the folding greatly increases postsynaptic membrane surface area. The acetylcholine receptors are concentrated at the tops of the synaptic folds, and acetylcholinesterase is located within the secondary synaptic clefts. The tops of synaptic folds are located immediately across from the acetylcholine release sites on the presynaptic membrane. Acetylcholine receptors are thus ideally located to receive a high concentration of acetylcholine immediately after it enters the synaptic cleft. The acetylcholinesterase receptor concentration within the secondary synaptic 78
Hospital Practice September 15, 1992
clefts enables rapid hydrolysis of acetylcholine. Voltage-gated sodium channels are located at the bottom of the synaptic folds. The high concentration of sodium channels on the end-plate membrane may increase the safety factor for neuromuscular transmission by reducing the threshold for action potential initiation. The cytoplasm underlying the plasma membrane of the junctional folds consists of a mesh of microtubules and filaments. Actin, a-actinin, and filamin are also concentrated in the postsynaptic membrane. These proteins may maintain the high acetylcholine receptor density or the structural integrity of the folds. Clinical correlation. Several patients have been described with a reduction in the number and depth of secondary synaptic clefts. J.H.J. Wokke and colleagues described two siblings in their sixth and seventh decades who had had ptosis from childhood and gradually generalized weakness and ophthalmoparesis. In contrast, the two brothers reported by L.M.E. Smit and colleagues presented at birth with weakness, ptosis, and contractures. By age three, their strength had improved. All four patients responded to anticholinesterases, and repetitive stimulation produced a decremental response. Miniature end-plate potential amplitudes were reduced, but quanta! content was normal. Acetylcholine receptor densities assessed by a-bungarotoxin labeling were reduced, which explained the reduction in miniature end-plate potential amplitude. The primary defect in these patients appears to be an abnormality of the formation of the
synaptic clefts, which resembled those of fetal muscle (Figure 5). Presumably, the underlying disturbance may be of developmental regulation. In immature muscle, junctional folds develop after acetylcholine receptors have begun to be inserted into the membrane. The invagination of the membrane increases surface area and may serve to increase the capacity of the membrane to accommodate acetylcholine receptor insertion. The cause of the acetylcholine receptor reduction may be due to impaired insertion of receptors into the membrane. Recent investigations have identified regions of the £ subunit of the acetylcholine receptor that play a role in the assembly of the complete receptor on the muscle surface membrane. It is possible that an abnormality in a portion of the acetylcholine receptor associated with insertion underlies the disorder. Increased degradation or decreased synthesis may also account for the reduction in acetylcholine receptor density.
Defects of Acetylcholine Receptor Function Through the use of site-directed mutagenesis, electron microscopy, and electrophysiology, the skeletal muscle nicotinic acetylcholine receptor has become the best understood agonist-gated ion channel. The complete adult receptor contains two a subunits and one copy each of a {3, o, and e subunit, whereas the fetal acetylcholine receptor contains a distinct ysubunit in place of the e subunit. Each subunit is folded to yield four or more membrane-spanning a helices as well as extracellular and intracellular surfaces. Each a subunit possesses an acetylcholine binding site. The subunits are ar-
PHYSIOLOGY IN MEDICINE ranged in a pentamer forming a central pore (Figure 6). Predictions of the acetylcholine receptor structure must explain ion selectiVity, conductance properties, and gating. Acetylcholine receptors pass potassium and sodium ions and other cations, but are impermeable to anions. The acetylcholine receptor is a relatively nonselective channel compared with voltage-gated channels, such as sodium, calcium, and chloride channels. The selectiVity among channels is governed partly by the size of the pore. Based on the size of permeable cations, the size of the acetylcholine receptor pore at its narrowest point is approximately 0.65 nm, which is considerably larger than that of the voltagegated ion channels. Ions do not pass through the channel freely in a bulk-flow fashion, but instead bind to specific sites within the pore and traverse the channel by moving from one site to the next. The charge of the amino acid side chains that line the pore and the inner and outer vestibule contributes to the ion selectivity of the channel and may explain why anions cannot traverse the channel. The binding of acetylcholine to the channel probably leads to a change in pore structure that allows ions to traverse by opening the channel or changing properties of ion binding sites within the pore. Models of acetylcholine receptor quaternary structure predict that the extracellular entrance of the channel possesses a net negative surface charge because of the conformation of amino acid side chains. Site-directed mutagenesis studies have identified a relationship between conductance and net charge of the intracellular or extracellular entrance
Figure 5.1n patients with congenital paucity of synaptic clefts, nerve terminals are normal but postsynaptic junctional folds are immature and ill-formed, with markedly reduced acetylcholine receptor density.
to the acetylcholine receptor channel. When amino acids with negatively charged side chains are modified to have a nonpolar or a positively charged side chain, the conductance of the channel decreases. The internal surface of the pore is lined by uncharged, mostly nonpolar, amino acids. Accordingly, cations would be attracted to the entrance of the pore, but passage of ions would not be impeded by strong electrostatic interactions between the ion and the internal portion of the pore. Substitution of the y by the £ subunit produces a channel with a greater conductance and a shorter open time. The £subunit has fewer net positive charges in the region of the channel entrance, which may explain the difference in conductance. The specific differences between the £ andy subunits that account for
the different channel open time of adult- and fetal-type acetylcholine receptor have not yet been delineated. Clinical correlation. Several patients have been reported who appear to have had primary derangements of acetylcholine receptor function. High-conductance fast channel syndrome. A nine-year-old girl had delayed motor development and fatigable weakness since infancy. Her EMG revealed a decremental response, but her symptoms did not respond to anticholinesterases. Miniature end-plate potential amplitude was large, and the rate of decay was abnormally short. Singlechannel studies revealed increased conductance and decreased channel open time. The increase in conductance may be explained by a mutation Hospital Practice September 15. 1992
79
PHYSIOLOGY IN MEDICINE
Figure 6. The acetylcholine receptor is a pentamer consisting of two a subunits and one copy of each of a f3, 8, and £(or y) subunit. Acetylcholine binding to the a subunits is believed to open an ion channel through the pentamer's center. The narrowest region of the channel is wider than that of voltage-gated ion channels. Amino acids with negatively charged side chains that are clustered on the channel's extracellular and cytosolic surfaces contribute to cationic conductivity.
that results in an increase in the net negative charge of the external surface of the acetylcholine receptor channel. The shorter channel open time could result if the mutation destabilized the open-state conformation of the acetylcholine receptor. Anatomic studies of the postsynaptic region demonstrated some degeneration and immaturity of the junctional folds, which may explain the weakness expressed clinically. The degenerative changes could have resulted in part from the increased influx of calcium through the larger conductance channel. Short channel open time and acetylcholine receptor deficiency. A single patient has been described. At birth, she required mechanical ventilation and was hypotonic and ophthalmoparetic. Her fatigable weakness improved with anticholinesterases. EMG showed a decremental re80
Hospital Practice September 15. 1992
sponse with repetitive stimulation. Miniature end-plate potential amplitude was low, but increased with neostigmine treatment. The acetylcholine receptor open time was decreased, but the conductance was normal. Bungarotoxin binding revealed a decrease in acetylcholine receptor density, although there were no abnormalities of the postsynaptic membrane. The reduced miniature endplate potential amplitude was probably secondary to fewer acetylcholine receptors. The shorter channel open time and miniature end-plate potential duration suggest a mutation that destabilized the open state. Low conductance, increased open time, and acetylcholine receptor deficiency. In another patient with acetylcholine receptor deficiency, a very different clinical presentation was described. A 48-year-old woman
had had generalized and extraocular muscle weakness since childhood, and a deeremental response was identified by EMG. Miniature end-plate potential amplitudes were decreased, and miniature endplate potential time course was prolonged. Single-channel conductance was decreased by 20%, and channel open time was nearly twice that of controls. Acetylcholine receptor density was decreased, but the pattern of the junctional folds was normal. The findings are similar to what one would expect if the ysubunit replaced the e subunit. However, the channel open time was much longer than would be expected by a simple subunit substitution. Slow channel syndrome. Patients with this syndrome have presented from infancy to adulthood with intermittent weakness and a history of a slowly progressive myopathy. Involvement appears to be greater in the arms and trunk than it is in the legs. Weakness does not improve with anticholinesterase treatment, and in some cases anticholinesterase medications actually exacerbate the condition. EMG shows decremental responses to repetitive stimulation, and single electrical stimulation produces repetitive compound muscle action potentials, as seen in acetylcholinesterasedeficient patients. Microelectrode studies have demonstrated prolonged miniature end-plate potential duration and a decreased amplitude. The electrophysiologic studies resemble those of acetylcholinesterase deficiency, but stains reveal the presence of functional acetylcholinesterase. Presumably, prolongation of the miniature end-plate potential results from prolongation of the acetylcholine receptor's open time.
PHYSIOLOGY IN MEDICINE The increased open time would therefore prolong depolarization of the membrane beyond the refractory period for action potential generation and produce the observed repetitive compound muscle action potentials. The only anatomic abnormality identified is degeneration of junctional folds; acetylcholine receptor density is normal. The cause of the membrane damage has been hypothesized to result from prolonged depolarization of the end plate. Prolonged depolarization could lead to increased influx of calcium and activation of calcium-sensitive proteases. The hypothesis remains speculative. Abnormality of acetylcholine and acetylcholine receptor interaction. From birth, a 21-year-
old woman had experienced severe fatigable generalized weakness, which responded partially to anticholinesterases. EMG revealed a decremental response to 2-Hz stimulation. Miniature endplate potential amplitudes were decreased. No reduction in acetylcholine receptor density or synaptic vesicle abnormality was identified to explain the reduction in miniature end-plate potential. Acetylcholine-induced end-plate current fluctuations were used to study acetylcholine receptor kinetic properties. These studies found normal single-channel conductance, but more detailed analysis indicated complex channel closure. Three possibilities may be considered to explain the current fluctuation data: 1) 1\vo populations of acetylcholine receptors with different open times may be present; 2) the open ion channel may be transiently blocked by acetylcholine or in some other way; or 3) a decrease in the rate of acetylcholine dissociation from the receptor, or a receptor
abnormality, may exist so that closure of the channel is no longer the rate-limiting step. Since the channel recordings revealed normal single-channel conductance, the first possibility would not explain the low miniature end-plate potential amplitude. Blockage of the channel by an agonist would not explain the clinical improvement with anticholinesterase treatment, and an increase in miniature endplate potential amplitude with neostigmine would not be expected. Reduced affinity of the receptor for acetylcholine could explain the decreased miniature end-plate potential in the setting of normal acetylcholine receptor conductance and a normal number of acetylcholine receptors, as well as the improvement with anticholinesterases. Acetylcholine receptor with altered d-tubocurartne binding.
J. A. Morgan-Hughes and coworkers described a 32-year-old man with a 14-month history of fluctuating generalized and bulbar weakness. The patient's parents were first cousins, and two siblings had died during infancy. An EMG showed a slight decrement in compound muscle action potential after 5-Hz stimulation. Light microscopic analysis of a triceps biopsy revealed tubular aggregates. Electron microscopy of some end plates revealed reduced synaptic vesicles and short, irregular junctional folds. Microelectrode recordings were not performed. The acetylcholine receptor density was reduced, as measured by a-bungarotoxin binding. The acetylcholine receptor affinity for binding of d-tubocurarine was increased 10-fold, compared with normal and myasthenic controls. Presumably, a change in acetylcholine receptor structure led to altered sensitivity to
d-tubocurarine, a reduction of acetylcholine receptor density, and alterations of the end plate.
Identification ofPatients Distinction between patients with autoimmune myasthenia gravis and forms of congenital disorders of neuromuscular transmission is of utmost importance for appropriate management. The treatment of myasthenia gravis often involves long-term administration of immunosuppressive agents, plasmapheresis, or thymectomy, which would not benefit patients with the congenital disorders and could be harmful. Several clinical and diagnostic features should alert the physician to the presence of a congenital disorder of neuromuscular transmission. Since myasthenia gravis and Lambert-Eaton myasthenic syndrome are rare in childhood, the physician should consider a congenital disorder of neuromuscular transmission in an infant or child with weakness and an EMG that shows a deeremental or incremental response to repetitive stimulation. A long history of muscle weakness, often from early childhood, is also suggestive. Distinct fluctuations of strength are common. Patients with myasthenia gravis usually have been symptomatic for a year or more before diagnosis and may have frequently been misdiagnosed, but their complaints tend to be of relatively recent onset. A diagnosis of myasthenia gravis in a family member may also serve as a diagnostic clue. A poor or oversensitive response to anticholinesterase treatment is seen in some of the congenital disorders, but improvement is consistent with the diagnosis. A posi(continues)
Hospital Practice September 15, 1992
81
PHYSIOLOGY IN MEDICINE (continued)
tive edrophonium chloride test is consistent with acquired or congenital diseases of neuromuscular transmission. An EMG is imperative; it will confirm a disorder of neuromuscular transmission and repetitive stimulations. An incremental response usually indicates the presence of Lambert-Eaton, so that a search for a neoplasm or autoimmune disorder should be initiated. Only one patient with a congenital form has been reported with an EMG response similar to that associated with Lambert-Eaton. The single most helpful finding may be the repetitive compound muscle action potential observed after a single nerve stimulation. This finding suggests maintenance of the endplate depolarization beyond the refractory period for action potential generation. The only acquired condition that could produce a prolonged end-plate potential is poisoning or overdose with anticholinesterase agents. All congenital disorders of neuromuscular transmission involve a lack of serum antibodies to the acetylcholine receptor; however, roughly 10% of patients with generalized myasthenia and 50% of those with ocular myasthenia also lack acetylcholine receptor antibodies. The majority of seronegative myasthenic patients probably have an autoimmune disorder and respond to standard therapy for myasthenia gravis. If the features we have described exist in a seronegative patient, however, a congenital neuromuscular transmission disorder should be strongly considered. When the history, physical examination, EMG, and serologic
tests (including acetylcholine receptor antibody titers) suggest a diagnosis of congenital disorder of neuromuscular transmission, the patient should be referred to a center that can perform specialized testing. Muscle biopsy is required in such cases, as is electron microscopic analysis of the neuromuscular junction, which allows for characterization of postsynaptic anatomy, quantitative estimates of acetylcholine receptor density, and synaptic vesicle morphology and concentration. Microelectrode studies of end-plate currents are helpful to further delineate the defect. Also, sufficient quantities of muscle should be removed and frozen at -70 oc to allow for future studies, including genetic analysis.
Future Prospects Congenital forms of myasthenia gravis were characterized clinically in the 1950s, but delineation of the underlying pathology has followed advances in microscopy and electrophysiology in the past 15 years. Application of molecular biology techniques to these diseases will further improve understanding of the neuromuscular junction and may be particularly important in the analysis of acetylcholine receptor function, as these disorders appear to offer natural site-directed mutagenesis studies. With the advent of gene therapy for inherited disorders, specific therapy for congenital disorders of neuromuscular transmission may be close at hand. D
Selected Reading Bady B, Chauplannaz G. Carrier H: Congenital Lambert-Eaton myasthenic syndrome. J Neurol NeurosurgPsychiatry 50:476, 1987 Dani JA: Site-directed mutagenesis and single-channel currents define the ionic channel of the nicotinic acetylcholine receptor. Trends Neurosci 12: 125, 1989 Engel AG eta!: Newly recognized congenital myasthenic syndromes: I. Congenital paucity of synaptic vesicles and reduced quanta! release; II. High conductance fast-channel syndrome; III. Abnormal acetylcholine receptor (AChR) interaction with acetylcholine; IV. AChR deficiency and short channel open time. ProgBrain Res 84: 125. 1990 Engel AG et al: Recently recognized congenital myasthenic syndromes: (A) End-plate acetylcholinesterase deficiency, (B) Putative abnormality of the ACh induced ion channel. (C) Putative defect of ACh resynthesis or mobilization: Clinical features, ultrastructure and cytochemistry. NY AcadSci377:614, 1981 Guy HR. Hucho F: The ion channel of the nicotinic acetylcholine receptor. TrendsNeurosci 10:318, 1987 Kaminski HJ et al: Why are eye muscles frequently involved in myasthenia graVis? Neurology 40: 1663, 1990 Morgan-Hughes JA et al: Alterations in the number and affinity of junctional acetylcholine receptors in myopathy with tubular aggregates: A newly recognized receptor defect. Brain 104 : 2 79. 1981 Ruff RL: Ionic channels: I. The biophysical basis for ion passage and channel gating; II. Voltage and agonist-modified channel properties and structure. Muscle Nerve 9: 675, 767. 1986 Salpeter MM (Ed): The Vertebrate Neuromuscular Junction. Alan R Liss. NewYork, 1987 Toyoshima C. Unwin N: Ion channel of acetylcholine receptor reconstructed from images of postsynaptic membranes. Nature 336: 24 7, 1988
Hospital Practice September 15, 1992
85
ISSN: 2154-8331 (Print) 2377-1003 (Online) Journal homepage: http://www.tandfonline.com/loi/ihop20
Congenital Disorders of Neuromuscular Transmission Henry J. Kaminski & Robert L. Ruff To cite this article: Henry J. Kaminski & Robert L. Ruff (1992) Congenital Disorders of Neuromuscular Transmission, Hospital Practice, 27:9, 73-85, DOI: 10.1080/21548331.1992.11705484 To link to this article: https://doi.org/10.1080/21548331.1992.11705484
Published online: 17 May 2016.
Submit your article to this journal
Citing articles: 3 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ihop20
IPIHIYSllOlOGY TIN MIEDllCCllNIE
Congenital Disorders of Neuromuscular Transmission HENRY J. KAMINSKI and ROBERT L. RUFF
Case Western Reserve University
Pathology underlying congenital forms of myasthenia gravis has been delineated through microscopic and electrophysiologic studies over the past 15 years. Differentiation from the immune-mediated disorder is crucial because therapy appropriate for acquired myasthenia may be harmful to patients with congenital disease.
Congenital disorders of neuromuscular transmission are commonly referred to as congenital myasthenias because of their clinical similarity to the immune-mediated disease. Patients often present with fatigable weakness and a propensity for extraocular muscle involvement; electromyography usually reveals a decremental response to repetitive stimulation. However, antibodies against the acetylcholine receptor are not present. By definition, manifestations of the illness may be traced to birth, and symptoms respond poorly to immunosuppressive treatment. The congenital disorders are caused by a variety of defects at the neuromuscular junction and represent experiments of nature that offer unique insights into mechanisms of neurotransmitter release, acetylcholine receptor function, and acetylcholine metabolism. The disorders should be differentiated from neonatal and juvenile myasthenia gravis. Neonatal myasthenia occurs in 10% to 20% of infants consequent to transmission of anti-acetylcholine receptor antibodies across the placenta from mothers with myasthenia gravis. The child shows signs of muscle weakness, a weak cry, poor sucking, and, occasionally, respiratory insufficiency. The disor-
der resolves within days to weeks. Juvenile myasthenia may occur in children under 10 years of age, with manifestations typical of the immune-mediated disease in older patients. The serum contains anti-acetylcholine receptor antibodies, and the disease has the same pathogenesis as the adult form. Fewer than 40 congenital neuromuscular transmission disorder cases have been described in detail, but this number may significantly underrepresent the true frequency of these disorders. As noted, they are easily confused with myasthenia gravis. In addition, extensive evaluation, which may not be performed even at tertiary referral centers, is needed for diagnosis. Nevertheless, every physician should have some familiarity with these disorders,
Dr. Kaminski is Assistant Professor and Dr. Ruff is Associate Professor, Department of Neurology, Case Western Reserve University School of Medicine. Dr. Kaminski is also Staff Physician and Dr. Ruff is Chief, Department of Neurology, Veterans Affairs Medical Center, Cleveland. Their work has been supported by Merit Reviewed Funding from the Department of Veterans Affairs and National Institutes of Health grants NS 26661 and EY09186.This is the third article in a series on neuromuscular transmission disorders.
In cooperation with the American Physiological Society Hospital Practice September 15. 1992
73
PHYSIOLOGY IN MEDICINE which may manifest at any age with severe signs, such as respiratory failure, that may become apparent during acute illnesses or surgery. In the following, we will attempt to categorize the congenital neuromuscular transmission disorders on the basis of the presumed physiologic transmission defects, and to understand the patients' clinical presentations on the basis of observed anatomic and physiologic abnormalities.
Abnormalities of Transmitter Release Normal quantal release. The vesicle hypothesis of synaptic transmission forms the cornerstone for understanding neuromuscular junction mechanisms, and has important implications for evaluation of the sites of the congenital transmission disorders· defects (Figure 1 ). The vesicle hypothesis holds that the neurotransmitter is packaged in
Figure 1. Congenital disorders of neuromuscular transmission may involve defects at any of five possible pre- or postsynaptic sites. As schematized, these are voltage-gated
74
Hospital Practice September 15. 1992
the nerve terminals in synaptic vesicles, which, when fusing with the nerve terminal plasma membrane, cause release of transmitter. The transmitter then diffuses across the synaptic cleft and interacts with postsynaptic receptors, producing depolarization ofthe postsynaptic membrane. The end-plate depolarization triggers action potentials that travel away from the end plate to stimulate muscle contraction. Transmitter release is initiat-
calcium channels (A), acetylcholine-containing vesicles (B), junctional folds (C), acetylcholine receptors (D), and acetylcholinesterase levels in the synaptic cleft (E).
PHYSIOLOGY IN MEDICINE ed when the motor neuron action potential opens voltagegated calcium channels on the nerve terminal. The subsequent influx of calcium initiates fusion of synaptic vesicles with the plasma membrane, resulting in release of acetylcholine. Choline, the hydrolysis product of acetylcholine in the synaptic cleft. is taken up by a high-affinity transport system on the nerve terminal and reused in the synthesis of acetylcholine. The acetylcholine is then repackaged in synaptic vesicles. Most of the synaptic vesicles are formed by endocytosis from nerve terminal membrane. Some are carried to the nerve terminal by axonal transport. The precise mechanisms underlying vesicle membrane recycling and acetylcholine packaging are not clear. Intracellular recording of muscle fibers near the end plate demonstrates spontaneous depolarizations, or miniature endplate potentials (Figure 2). They are thought to represent the depolarization produced by release of acetylcholine from a single vesicle. The depolarization produced by numerous vesicles fusing with the presynaptic membrane is the end-plate potential, the size of which depends on the number of vesicles releasing the transmitter. If the number is above a certain threshold, an action potential is generated, and the muscle contracts. The compound muscle action potential, which represents the summation of individual muscle fiber action potentials, can be determined by electromyography (Figure3). Clinical correlation. The patients who will be described share an abnormality of quantal release that bears clinical similarity to cases of "familial infan-
Figure 2. Miniature end-plate potentials, each thought to represent spontaneous depolarization produced by release of acetylcholine from a single vesicle, can vary in duration or amplitude. To illustrate, a normal miniature end-plate potential is contrasted with the prolonged variant seen in acetylcholinesterase deficiency and slow-channel syndrome, and with the depressed variant seen in disorders characterized by reduced acetylcholine receptor density.
tile myasthenia," which were described before the advent of sophisticated electrophysiologic techniques and electron microscopy. Impaired acetylcholine packaging. Andrew G. Engel and col-
leagues reported a case that strongly emphasizes the role of synaptic vesicles in producing the end-plate potential. A 23year-old woman had episodic generalized and bulbar fatigable weakness from birth. A deeremental response of the compound muscle action potential was found with repetitive stimulation, which improved with neostigmine treatment. Miniature end-plate potential amplitudes were normal, but the endplate potential at 1-Hz stimulation showed a marked reduction of quantal content. Electron micrographs of the nerve terminal revealed a reduction in synaptic vesicles. The implication was that the small end-plate potentials resulted from a reduction of
readily releasable vesicles containing acetylcholine. Several reports of patients with defects of acetylcholine processing have been described. In 1979, Engel and colleagues reported on an 18-year-old man whose infancy had been marked by ptosis and poor feeding. As he grew older, the patient became easily fatigued and experienced episodic apnea after crying or febrile illness. Anticholinesterase medications palliated his symptoms. The patient's sister had similar difficulties, and three siblings had died of apneic spells. Repetitive stimulation at 2 Hz produced a decrement of the compound muscle action potential. Microelectrode studies of an intercostal muscle biopsy revealed normal resting miniature end-plate potential amplitudes. but after 2-Hz stimulation, miniature end-plate potential amplitude declined significantly. The number of acetylcholine recepHospital Practice September 15. 1992
75
PHYSIOLOGY IN MEDICINE tors was normal as determined by a-bungarotoxin labeling. The only anatomic abnormality was a 60% increase in the number of synaptic vesicles, which may have represented an adaptive change. In a study of three similar patients, other investigators found a reduction in synaptic vesicle size and attempted to correlate it with the reduction of miniature end-plate potential on repetitive stimulation. In controls and patients, the number of vesicles near the nerve terminal synaptic membrane decreased after stim-
ulation. In patients, the vesicle size increased or was unchanged, whereas controls showed a decrease or no change. The normal miniature endplate potential at rest indicates that both quanta! content and postsynaptic function are normal. The decline in miniature end-plate potential amplitude with stimulation suggests an abnormality of "recycling" of functional synaptic vesicles. Such a defect could lie in the choline transporter, acetylcholine resynthesis by choline acetyltransferase, or packaging of acetyl-
choline into vesicles-the net effect being that vesicles contained less releasable acetylcholine. Desensitization of the acetylcholine receptor is unlikely because acetylcholinesterase inhibitors would be expected to worsen such a defect. Impaired vesicle release. An
abnormality of quanta! release has been described in only one patient, who showed an incremental response with repetitive stimulation. Unfortunately, no microelectrode studies could be performed. The patient had been diffusely weak since birth, but without ocular signs. Administration of guanidine hydrochloride, which increases quanta! release, led to improvement in strength. The EMG findings were similar to those in patients with Lambert-Eaton myasthenic syndrome, thought to be a paraneoplastic syndrome or an autoimmune disorder in which antibodies cross-react with nerve terminal calcium channels. The decreased quanta! content in Lambert-Eaton is believed toresult from impaired entry of calcium into the nerve terminal. The defect in this patient was not defined but might have resulted from qualitative or quantitive derangement of presynaptic calcium channels.
Acetylcholinesterase Deficiency
Figure 3. By definition, a compound muscle action potential sum mates individual
muscle fiber action potentials. Its normal shape after a single electrical stimulation is shown at top; the lower curve shows the repetitive shape seen in acetycholinesterase deficiency and slow-channel syndrome.
76
Hospital Practice September 15. 1992
Acetylcholinesterase in neuromuscular transmission. Acetylcholinesterase concentration and kinetics, acetylcholine diffusion, and the electrophysiologic characteristics of the acetylcholine receptor determine the duration and amplitude of the miniature end-plate potential. Several characteristics of the neuromuscular junction lead to
PHYSIOLOGY IN MEDICINE the synchronous opening of many acetylcholine receptors, which is required to produce the rapid rising phase of the miniature end-plate potential. Therelease of acetylcholine into the small area of the synaptic cleft produces local concentrations well in excess of the dissociation constant of the acetylcholine receptor. Acetylcholinesterase is found at concentrations five to eight times lower than acetylcholine receptor on the postsynaptic membrane, and the turnover time of acetylcholinesterase (the time it takes to hydrolyze an acetylcholine molecule) approximates the rising time of the miniature end-plate potential. Therefore, acetylcholine initially binds to receptor before it is hydrolyzed or diffuses from the synaptic cleft. The concentration of acetylcholine must drop quickly so that repeated binding and acetylcholine receptor channel opening do not occur. During the falling phase of the miniature end-plate potential, acetylcholine hydrolysis and diffusion occur more rapidly than acetylcholine receptor channel closure, and the length of the falling phase is therefore determined by the single channel open time of the acetylcholine receptor. Clinical correlation. Using cyto- and immunochemical techniques, Engel and E. H. Lambert demonstrated a severe deficiency of acetylcholinesterase from the neuromuscular junction of a 16-year-old boy. From birth he had ptosis and fatigable weakness and had experienced respiratory insufficiency associated with a viral illness. Anticholinesterases did not improve his symptoms. EMG provided important insights into the physiologic consequences of acetylcholinesterase deficiency.
Figure 4. Congenital acetylcholinesterase deficiency may lead to shrunken nerve terminals (A), shallow junctional folds with reduced numbers of acetylcholine receptors (B), decreased choline uptake from the synaptic cleft (C), and low concentrations of acetylcholine in synaptic vesicles (D).
While repetitive stimulation produced a decremental response, a single stimulus evoked two or more compound muscle action potentials, and intracellular microelectrode studies demonstrated a prolonged miniature end-plate potential duration and a low normal miniature endplate potential amplitude. Maintenance of the end-plate potential above the level for action potential generation during the refractory period resulted in triggering of multiple action potentials. The acetylcholinesterase deficiency led to an increase in acetylcholine in the synaptic cleft and showed that diffusion was not sufficient to eliminate acetylcholine from the synaptic cleft. In addition to the primary defect of acetylcholinesterase deficiency, investigations revealed several secondary abnormali-
ties, which may have been adaptive. Significant inhibition of acetylcholinesterase in normal subjects may lead to receptor desensitization and the clinical correlate, "cholinergic crisis," which was not observed in this patient. Since the acetylcholine receptor density was normal, the reduced miniature end-plate potential amplitude probably indicated a receptor abnormality or reduced acetylcholine concentration in the vesicles. The reduction of vesicle acetylcholine concentration may offset the lack of acetylcholinesterase and allow diffusion to rapidly terminate the local action of acetylcholine. The decreased acetylcholine content of vesicles may also reflect a decrease in choline reuptake that occurs because of decreased hydrolysis of acetylcholine in the synaptic cleft (Figure 4). Hospital Practice September 15. 1992
77
PHYSIOLOGY IN MEDICINE In some muscles, junctional folds were degenerated. Many nerve terminals were smaller than normal, covering only a portion of the postsynaptic membrane. The quanta! content of the end-plate potential was reduced, in keeping with the decreased size ofthe nerve terminal. The changes in structure of the synapses may also have functioned to increase diffusion of acetylcholine away from the postsynaptic membrane, which would decrease end-plate depolarization and prevent acetylcholine receptor desensitization.
Congenital Paucity of Synaptic Clefts Postsynaptic membrane specialization. The nerve terminal is separated from the postsynaptic region by a narrow space called the primary cleft. The postsynaptic muscle surface is a highly specialized plasma membrane consisting of deep junctional folds, between which is a space continuous with the primary cleft termed the secondary synaptic cleft. While the precise role of the junctional folds in neuromuscular transmission is not fully understood, the folding greatly increases postsynaptic membrane surface area. The acetylcholine receptors are concentrated at the tops of the synaptic folds, and acetylcholinesterase is located within the secondary synaptic clefts. The tops of synaptic folds are located immediately across from the acetylcholine release sites on the presynaptic membrane. Acetylcholine receptors are thus ideally located to receive a high concentration of acetylcholine immediately after it enters the synaptic cleft. The acetylcholinesterase receptor concentration within the secondary synaptic 78
Hospital Practice September 15, 1992
clefts enables rapid hydrolysis of acetylcholine. Voltage-gated sodium channels are located at the bottom of the synaptic folds. The high concentration of sodium channels on the end-plate membrane may increase the safety factor for neuromuscular transmission by reducing the threshold for action potential initiation. The cytoplasm underlying the plasma membrane of the junctional folds consists of a mesh of microtubules and filaments. Actin, a-actinin, and filamin are also concentrated in the postsynaptic membrane. These proteins may maintain the high acetylcholine receptor density or the structural integrity of the folds. Clinical correlation. Several patients have been described with a reduction in the number and depth of secondary synaptic clefts. J.H.J. Wokke and colleagues described two siblings in their sixth and seventh decades who had had ptosis from childhood and gradually generalized weakness and ophthalmoparesis. In contrast, the two brothers reported by L.M.E. Smit and colleagues presented at birth with weakness, ptosis, and contractures. By age three, their strength had improved. All four patients responded to anticholinesterases, and repetitive stimulation produced a decremental response. Miniature end-plate potential amplitudes were reduced, but quanta! content was normal. Acetylcholine receptor densities assessed by a-bungarotoxin labeling were reduced, which explained the reduction in miniature end-plate potential amplitude. The primary defect in these patients appears to be an abnormality of the formation of the
synaptic clefts, which resembled those of fetal muscle (Figure 5). Presumably, the underlying disturbance may be of developmental regulation. In immature muscle, junctional folds develop after acetylcholine receptors have begun to be inserted into the membrane. The invagination of the membrane increases surface area and may serve to increase the capacity of the membrane to accommodate acetylcholine receptor insertion. The cause of the acetylcholine receptor reduction may be due to impaired insertion of receptors into the membrane. Recent investigations have identified regions of the £ subunit of the acetylcholine receptor that play a role in the assembly of the complete receptor on the muscle surface membrane. It is possible that an abnormality in a portion of the acetylcholine receptor associated with insertion underlies the disorder. Increased degradation or decreased synthesis may also account for the reduction in acetylcholine receptor density.
Defects of Acetylcholine Receptor Function Through the use of site-directed mutagenesis, electron microscopy, and electrophysiology, the skeletal muscle nicotinic acetylcholine receptor has become the best understood agonist-gated ion channel. The complete adult receptor contains two a subunits and one copy each of a {3, o, and e subunit, whereas the fetal acetylcholine receptor contains a distinct ysubunit in place of the e subunit. Each subunit is folded to yield four or more membrane-spanning a helices as well as extracellular and intracellular surfaces. Each a subunit possesses an acetylcholine binding site. The subunits are ar-
PHYSIOLOGY IN MEDICINE ranged in a pentamer forming a central pore (Figure 6). Predictions of the acetylcholine receptor structure must explain ion selectiVity, conductance properties, and gating. Acetylcholine receptors pass potassium and sodium ions and other cations, but are impermeable to anions. The acetylcholine receptor is a relatively nonselective channel compared with voltage-gated channels, such as sodium, calcium, and chloride channels. The selectiVity among channels is governed partly by the size of the pore. Based on the size of permeable cations, the size of the acetylcholine receptor pore at its narrowest point is approximately 0.65 nm, which is considerably larger than that of the voltagegated ion channels. Ions do not pass through the channel freely in a bulk-flow fashion, but instead bind to specific sites within the pore and traverse the channel by moving from one site to the next. The charge of the amino acid side chains that line the pore and the inner and outer vestibule contributes to the ion selectivity of the channel and may explain why anions cannot traverse the channel. The binding of acetylcholine to the channel probably leads to a change in pore structure that allows ions to traverse by opening the channel or changing properties of ion binding sites within the pore. Models of acetylcholine receptor quaternary structure predict that the extracellular entrance of the channel possesses a net negative surface charge because of the conformation of amino acid side chains. Site-directed mutagenesis studies have identified a relationship between conductance and net charge of the intracellular or extracellular entrance
Figure 5.1n patients with congenital paucity of synaptic clefts, nerve terminals are normal but postsynaptic junctional folds are immature and ill-formed, with markedly reduced acetylcholine receptor density.
to the acetylcholine receptor channel. When amino acids with negatively charged side chains are modified to have a nonpolar or a positively charged side chain, the conductance of the channel decreases. The internal surface of the pore is lined by uncharged, mostly nonpolar, amino acids. Accordingly, cations would be attracted to the entrance of the pore, but passage of ions would not be impeded by strong electrostatic interactions between the ion and the internal portion of the pore. Substitution of the y by the £ subunit produces a channel with a greater conductance and a shorter open time. The £subunit has fewer net positive charges in the region of the channel entrance, which may explain the difference in conductance. The specific differences between the £ andy subunits that account for
the different channel open time of adult- and fetal-type acetylcholine receptor have not yet been delineated. Clinical correlation. Several patients have been reported who appear to have had primary derangements of acetylcholine receptor function. High-conductance fast channel syndrome. A nine-year-old girl had delayed motor development and fatigable weakness since infancy. Her EMG revealed a decremental response, but her symptoms did not respond to anticholinesterases. Miniature end-plate potential amplitude was large, and the rate of decay was abnormally short. Singlechannel studies revealed increased conductance and decreased channel open time. The increase in conductance may be explained by a mutation Hospital Practice September 15. 1992
79
PHYSIOLOGY IN MEDICINE
Figure 6. The acetylcholine receptor is a pentamer consisting of two a subunits and one copy of each of a f3, 8, and £(or y) subunit. Acetylcholine binding to the a subunits is believed to open an ion channel through the pentamer's center. The narrowest region of the channel is wider than that of voltage-gated ion channels. Amino acids with negatively charged side chains that are clustered on the channel's extracellular and cytosolic surfaces contribute to cationic conductivity.
that results in an increase in the net negative charge of the external surface of the acetylcholine receptor channel. The shorter channel open time could result if the mutation destabilized the open-state conformation of the acetylcholine receptor. Anatomic studies of the postsynaptic region demonstrated some degeneration and immaturity of the junctional folds, which may explain the weakness expressed clinically. The degenerative changes could have resulted in part from the increased influx of calcium through the larger conductance channel. Short channel open time and acetylcholine receptor deficiency. A single patient has been described. At birth, she required mechanical ventilation and was hypotonic and ophthalmoparetic. Her fatigable weakness improved with anticholinesterases. EMG showed a decremental re80
Hospital Practice September 15. 1992
sponse with repetitive stimulation. Miniature end-plate potential amplitude was low, but increased with neostigmine treatment. The acetylcholine receptor open time was decreased, but the conductance was normal. Bungarotoxin binding revealed a decrease in acetylcholine receptor density, although there were no abnormalities of the postsynaptic membrane. The reduced miniature endplate potential amplitude was probably secondary to fewer acetylcholine receptors. The shorter channel open time and miniature end-plate potential duration suggest a mutation that destabilized the open state. Low conductance, increased open time, and acetylcholine receptor deficiency. In another patient with acetylcholine receptor deficiency, a very different clinical presentation was described. A 48-year-old woman
had had generalized and extraocular muscle weakness since childhood, and a deeremental response was identified by EMG. Miniature end-plate potential amplitudes were decreased, and miniature endplate potential time course was prolonged. Single-channel conductance was decreased by 20%, and channel open time was nearly twice that of controls. Acetylcholine receptor density was decreased, but the pattern of the junctional folds was normal. The findings are similar to what one would expect if the ysubunit replaced the e subunit. However, the channel open time was much longer than would be expected by a simple subunit substitution. Slow channel syndrome. Patients with this syndrome have presented from infancy to adulthood with intermittent weakness and a history of a slowly progressive myopathy. Involvement appears to be greater in the arms and trunk than it is in the legs. Weakness does not improve with anticholinesterase treatment, and in some cases anticholinesterase medications actually exacerbate the condition. EMG shows decremental responses to repetitive stimulation, and single electrical stimulation produces repetitive compound muscle action potentials, as seen in acetylcholinesterasedeficient patients. Microelectrode studies have demonstrated prolonged miniature end-plate potential duration and a decreased amplitude. The electrophysiologic studies resemble those of acetylcholinesterase deficiency, but stains reveal the presence of functional acetylcholinesterase. Presumably, prolongation of the miniature end-plate potential results from prolongation of the acetylcholine receptor's open time.
PHYSIOLOGY IN MEDICINE The increased open time would therefore prolong depolarization of the membrane beyond the refractory period for action potential generation and produce the observed repetitive compound muscle action potentials. The only anatomic abnormality identified is degeneration of junctional folds; acetylcholine receptor density is normal. The cause of the membrane damage has been hypothesized to result from prolonged depolarization of the end plate. Prolonged depolarization could lead to increased influx of calcium and activation of calcium-sensitive proteases. The hypothesis remains speculative. Abnormality of acetylcholine and acetylcholine receptor interaction. From birth, a 21-year-
old woman had experienced severe fatigable generalized weakness, which responded partially to anticholinesterases. EMG revealed a decremental response to 2-Hz stimulation. Miniature endplate potential amplitudes were decreased. No reduction in acetylcholine receptor density or synaptic vesicle abnormality was identified to explain the reduction in miniature end-plate potential. Acetylcholine-induced end-plate current fluctuations were used to study acetylcholine receptor kinetic properties. These studies found normal single-channel conductance, but more detailed analysis indicated complex channel closure. Three possibilities may be considered to explain the current fluctuation data: 1) 1\vo populations of acetylcholine receptors with different open times may be present; 2) the open ion channel may be transiently blocked by acetylcholine or in some other way; or 3) a decrease in the rate of acetylcholine dissociation from the receptor, or a receptor
abnormality, may exist so that closure of the channel is no longer the rate-limiting step. Since the channel recordings revealed normal single-channel conductance, the first possibility would not explain the low miniature end-plate potential amplitude. Blockage of the channel by an agonist would not explain the clinical improvement with anticholinesterase treatment, and an increase in miniature endplate potential amplitude with neostigmine would not be expected. Reduced affinity of the receptor for acetylcholine could explain the decreased miniature end-plate potential in the setting of normal acetylcholine receptor conductance and a normal number of acetylcholine receptors, as well as the improvement with anticholinesterases. Acetylcholine receptor with altered d-tubocurartne binding.
J. A. Morgan-Hughes and coworkers described a 32-year-old man with a 14-month history of fluctuating generalized and bulbar weakness. The patient's parents were first cousins, and two siblings had died during infancy. An EMG showed a slight decrement in compound muscle action potential after 5-Hz stimulation. Light microscopic analysis of a triceps biopsy revealed tubular aggregates. Electron microscopy of some end plates revealed reduced synaptic vesicles and short, irregular junctional folds. Microelectrode recordings were not performed. The acetylcholine receptor density was reduced, as measured by a-bungarotoxin binding. The acetylcholine receptor affinity for binding of d-tubocurarine was increased 10-fold, compared with normal and myasthenic controls. Presumably, a change in acetylcholine receptor structure led to altered sensitivity to
d-tubocurarine, a reduction of acetylcholine receptor density, and alterations of the end plate.
Identification ofPatients Distinction between patients with autoimmune myasthenia gravis and forms of congenital disorders of neuromuscular transmission is of utmost importance for appropriate management. The treatment of myasthenia gravis often involves long-term administration of immunosuppressive agents, plasmapheresis, or thymectomy, which would not benefit patients with the congenital disorders and could be harmful. Several clinical and diagnostic features should alert the physician to the presence of a congenital disorder of neuromuscular transmission. Since myasthenia gravis and Lambert-Eaton myasthenic syndrome are rare in childhood, the physician should consider a congenital disorder of neuromuscular transmission in an infant or child with weakness and an EMG that shows a deeremental or incremental response to repetitive stimulation. A long history of muscle weakness, often from early childhood, is also suggestive. Distinct fluctuations of strength are common. Patients with myasthenia gravis usually have been symptomatic for a year or more before diagnosis and may have frequently been misdiagnosed, but their complaints tend to be of relatively recent onset. A diagnosis of myasthenia gravis in a family member may also serve as a diagnostic clue. A poor or oversensitive response to anticholinesterase treatment is seen in some of the congenital disorders, but improvement is consistent with the diagnosis. A posi(continues)
Hospital Practice September 15, 1992
81
PHYSIOLOGY IN MEDICINE (continued)
tive edrophonium chloride test is consistent with acquired or congenital diseases of neuromuscular transmission. An EMG is imperative; it will confirm a disorder of neuromuscular transmission and repetitive stimulations. An incremental response usually indicates the presence of Lambert-Eaton, so that a search for a neoplasm or autoimmune disorder should be initiated. Only one patient with a congenital form has been reported with an EMG response similar to that associated with Lambert-Eaton. The single most helpful finding may be the repetitive compound muscle action potential observed after a single nerve stimulation. This finding suggests maintenance of the endplate depolarization beyond the refractory period for action potential generation. The only acquired condition that could produce a prolonged end-plate potential is poisoning or overdose with anticholinesterase agents. All congenital disorders of neuromuscular transmission involve a lack of serum antibodies to the acetylcholine receptor; however, roughly 10% of patients with generalized myasthenia and 50% of those with ocular myasthenia also lack acetylcholine receptor antibodies. The majority of seronegative myasthenic patients probably have an autoimmune disorder and respond to standard therapy for myasthenia gravis. If the features we have described exist in a seronegative patient, however, a congenital neuromuscular transmission disorder should be strongly considered. When the history, physical examination, EMG, and serologic
tests (including acetylcholine receptor antibody titers) suggest a diagnosis of congenital disorder of neuromuscular transmission, the patient should be referred to a center that can perform specialized testing. Muscle biopsy is required in such cases, as is electron microscopic analysis of the neuromuscular junction, which allows for characterization of postsynaptic anatomy, quantitative estimates of acetylcholine receptor density, and synaptic vesicle morphology and concentration. Microelectrode studies of end-plate currents are helpful to further delineate the defect. Also, sufficient quantities of muscle should be removed and frozen at -70 oc to allow for future studies, including genetic analysis.
Future Prospects Congenital forms of myasthenia gravis were characterized clinically in the 1950s, but delineation of the underlying pathology has followed advances in microscopy and electrophysiology in the past 15 years. Application of molecular biology techniques to these diseases will further improve understanding of the neuromuscular junction and may be particularly important in the analysis of acetylcholine receptor function, as these disorders appear to offer natural site-directed mutagenesis studies. With the advent of gene therapy for inherited disorders, specific therapy for congenital disorders of neuromuscular transmission may be close at hand. D
Selected Reading Bady B, Chauplannaz G. Carrier H: Congenital Lambert-Eaton myasthenic syndrome. J Neurol NeurosurgPsychiatry 50:476, 1987 Dani JA: Site-directed mutagenesis and single-channel currents define the ionic channel of the nicotinic acetylcholine receptor. Trends Neurosci 12: 125, 1989 Engel AG eta!: Newly recognized congenital myasthenic syndromes: I. Congenital paucity of synaptic vesicles and reduced quanta! release; II. High conductance fast-channel syndrome; III. Abnormal acetylcholine receptor (AChR) interaction with acetylcholine; IV. AChR deficiency and short channel open time. ProgBrain Res 84: 125. 1990 Engel AG et al: Recently recognized congenital myasthenic syndromes: (A) End-plate acetylcholinesterase deficiency, (B) Putative abnormality of the ACh induced ion channel. (C) Putative defect of ACh resynthesis or mobilization: Clinical features, ultrastructure and cytochemistry. NY AcadSci377:614, 1981 Guy HR. Hucho F: The ion channel of the nicotinic acetylcholine receptor. TrendsNeurosci 10:318, 1987 Kaminski HJ et al: Why are eye muscles frequently involved in myasthenia graVis? Neurology 40: 1663, 1990 Morgan-Hughes JA et al: Alterations in the number and affinity of junctional acetylcholine receptors in myopathy with tubular aggregates: A newly recognized receptor defect. Brain 104 : 2 79. 1981 Ruff RL: Ionic channels: I. The biophysical basis for ion passage and channel gating; II. Voltage and agonist-modified channel properties and structure. Muscle Nerve 9: 675, 767. 1986 Salpeter MM (Ed): The Vertebrate Neuromuscular Junction. Alan R Liss. NewYork, 1987 Toyoshima C. Unwin N: Ion channel of acetylcholine receptor reconstructed from images of postsynaptic membranes. Nature 336: 24 7, 1988
Hospital Practice September 15, 1992
85
