Pre- and post-synaptic abnormalities associated with impaired neuromuscular transmission in a group of patients with ‘limb-girdle myasthenia’
1 School of Neurology, Neurobiology and Psychiatry, University of Newcastle upon Tyne Bath 2 Department of Clinical Neurophysiology, Regional Neurosciences Centre, Newcastle General Hospital, Newcastle upon Tyne Bath 3 Department of Paediatric Neurology, Regional Neurosciences Centre, Newcastle General Hospital, Newcastle upon Tyne Bath 4 Department of Neurology, Regional Neurosciences Centre, Newcastle General Hospital, Newcastle upon Tyne Bath 5 Department of Neurology, Royal United Hospital Bath 6 Department of Pharmacy and Pharmacology, University of Bath Bath 7 Neurosciences Group, Weatherall Institute of Molecular Medicine, The John Radcliffe Hospital Headington, Oxford, UK
Correspondence to: Prof. C. R. Slater, School of Neurology, Neurobiology and Psychiatry, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK E-mail: c.r.slater{at}ncl.ac.uk
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Summary |
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The properties of neuromuscular junctions (NMJs) were studied in motor-point biopsy samples from eight patients with congenital myasthenic syndromes affecting primarily proximal limb muscles [‘limb-girdle myasthenia’ (LGM)]. All had moderate to severe weakness of the proximal muscles, without short-term clinical fatigability but with marked variation in strength over periods of weeks or months, with little or no facial weakness or ptosis and no ophthalmoplegia. Most had a characteristic gait and stance. All patients showed decrement of the compound muscle action potential (CMAP) on repetitive stimulation at 3 Hz, and increased jitter and blocking was detected by SFEMG, confirming the presence of impaired neuromuscular transmission. None of the patients had serum antibodies against acetylcholine receptors (AChRs). Two of the patients had similarly affected siblings. Intracellular recording from isolated nerve–muscle preparations revealed that the quantal content (the number of ACh quanta released per nerve impulse) was only
50% of that in controls. However, the quantal size (amplitude of miniature end-plate currents) and the kinetic properties of synaptic potentials and currents were similar to control values. The area of synaptic contact and extent of post-synaptic folding were 50% of control values. Thus, the quantal content per unit area of synaptic contact was normal. The number of AChRs per NMJ was also reduced to 50% of normal, so the local AChR density was normal. Immunolabelling studies revealed qualitatively normal distributions and abundance of each of 14 proteins normally concentrated at the NMJ, including components of the basal lamina, post-synaptic membrane and post-synaptic cytoskeleton. DNA analysis failed to detect mutations in the genes encoding any of the following proteins: AChR subunits, rapsyn, ColQ, ChAT or muscle-specific kinase. Response of these patients to treatment was varied: few showed long-term improvement with pyridostigmine and some even deteriorated with treatments, while others had intolerable side-effects. Several patients showed improvement with 3,4-diaminopyridine, but this was generally only transient. Ephedrine was helpful in half of the patients. We conclude that impaired neuromuscular transmission in these LGM patients results from structural abnormalities of the NMJ, including reduced size and post-synaptic folding, rather from any abnormality in the immediate events of neuromuscular transmission.
Key Words: congenital myasthenic syndrome; human; limb-girdle myasthenia; neuromuscular transmission; neuromuscular junction
Abbreviations: AChE, acetylcholinesterase; AChR, acetylcholine receptor; ADM, abductor digiti minimi; ANC, anconeus; CMAP, compound muscle action potential; DELT, deltoid; EDC, extensor digitorum communis; EPC, end-plate current; EPP, end-plate potential; LGM, limb-girdle myasthenia; MCD, mean of consecutive differences; mEPC, miniature EPC; mEPP, miniature EPP; NMJ, neuromuscular junction; VL, vastus lateralis
Received March 20, 2006. Revised April 25, 2006. Accepted May 3, 2006.
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Introduction |
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TopSummaryIntroductionPatients and methodsResultsIn vitro electrophysiologyLight microscopyStructure-function relationshipsProtein localization studiesDNA analysisResponse to treatmentDiscussionReferences |
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Limb-girdle myasthenias (LGMs) are rare conditions in which impaired neuromuscular transmission leads to weakness of proximal limb muscles with little or no involvement of the ocular, bulbar or facial muscles (Beeson et al., 2005
). This pattern of weakness differs from that in many other forms of myasthenia and gives rise to a characteristic ‘waddling’ gait. The term ‘limb-girdle myasthenia’ was introduced by McQuillen (1966) and since then a number of patients with these features have been reported (Morgan-Hughes et al., 1970; Johns et al., 1973; Dobkin and Verity, 1978; Oh and Kuruoglu, 1992; Azulay et al., 1994; Vasant et al., 1994; Sieb et al., 1996; Furui et al., 1997; Rodolico et al., 2002; Shankar et al., 2002). In most of these patients the onset of muscle weakness was first noticed in infancy or childhood, but in others a later onset was described. In at least one study, 20% of the LGM patients described had elevated serum levels of acetylcholine receptor (AChR) antibodies, suggesting an autoimmune aetiology (Rodolico et al., 2002). In most other cases, consanguineous parents or affected siblings suggested a genetic origin of the condition (McQuillen, 1966; Morgan-Hughes et al., 1970; Johns et al., 1973; Dobkin and Verity, 1978; Gardner-Medwin, 1993; Vasant et al., 1994; Sieb et al., 1996; Furui et al., 1997; Rodolico et al., 2002; Shankar et al., 2002). It is clear that the term LGM, as currently used, may encompass a heterogeneous group of conditions. As yet, however, there are no reports of studies that have tried to determine the underlying defects of neuromuscular transmission.
During a 20-year period starting in 1971, 27 cases of childhood myasthenia were seen by one of us (D.G-M.) in Newcastle upon Tyne (Gardner-Medwin, 1993). Of these, 12 were autoimmune in nature. Of the remaining 15, two had congenital myasthenia with episodic apnoea (‘familial infantile myasthenia’), one a largely ocular ‘congenital myasthenia’, one myasthenia with arthrogryposis and one a severe early form of myasthenia. The remaining 10 cases all had progressive but fluctuating muscle weakness with a predominantly limb-girdle distribution, and 7 of this group of 10 were described as having a characteristic clinical syndrome with a sinuous gait, described below (Gardner-Medwin, 1993, 1994). The disability in this group progressed from moderate to severe during the teens and twenties, some becoming unable to walk in their twenties or thirties. One patient, not included in the present study, but with full clinical and electromyographic confirmation of the diagnosis, developed respiratory failure and required nocturnal negative pressure ventilation from the age of 23 years, which has since been continued for 30 years. In many of these cases, the findings on initial investigation were surprisingly normal and suggested a progressive proximal ‘pseudomyopathic’ condition. Indeed, when these cases were first encountered, the presence of impaired neuromuscular transmission went unsuspected for months or years until detailed electromyographic studies were carried out. Later, once the clinical picture had become familiar, new cases were specifically investigated for myasthenia by electromyography at an early stage. While such studies provided clear evidence of impaired transmission, they could not address the question of the cellular, subcellular or molecular basis of that impairment. We have therefore undertaken detailed studies of the neuromuscular junctions (NMJs) in eight of the LGM patients seen in Newcastle. These include 6 of the original 10 cases and 2 others (LGM7 and LGM8) seen in the adult neurology department by one of us (T.J.W.) since 1993. This report describes the findings of those studies and provides, as far as we are aware, the first account of the properties of the NMJ in any patient with LGM.
Our initial aim was to identify any structural and functional abnormalities of the NMJ and, if possible, to determine whether the defects are predominantly pre- or post-synaptic. In most cases, we found structural abnormalities of both the pre- and post-synaptic components of the NMJ, whose functional consequences appear adequate to account for the decreased efficacy of neuromuscular transmission in these patients. At the same time, our findings appear to rule out abnormalities of a number of important molecular components of the NMJ, whose genes are now well-established targets of pathogenic mutations in many forms of congenital myasthenic syndrome (Engel et al., 2003b; Beeson et al., 2005). Recently, as we describe here, we have been able to undertake DNA analysis of most of the patients studied, and these have confirmed this impression, both in the two cases where there is a family history of the condition and in the others. We therefore raise the possibility that abnormalities of the regulatory events influencing the structure and maintenance of the NMJ account for impaired neuromuscular transmission in this group of patients. Brief accounts of this group of patients have already appeared (Fawcett et al., 1995; Slater et al., 1995).
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Patients and methods |
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TopSummaryIntroductionPatients and methodsResultsIn vitro electrophysiologyLight microscopyStructure-function relationshipsProtein localization studiesDNA analysisResponse to treatmentDiscussionReferences |
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Patients
The patients described in this report were part of an ongoing study of the structure and function of the NMJ in neuromuscular diseases that has been approved by the Joint Ethical Committee of the Newcastle Health Authority and the University of Newcastle upon Tyne. All patients included here underwent a motor-point muscle biopsy for diagnosis of the detailed mechanism of their defect in neuromuscular transmission after having given fully informed consent.
Diagnosis
A full history, clinical examination and functional assessment were performed at the time of biopsy on all LGM and control patients. On the basis of the clinical features, the results of neurophysiological studies (see below), appropriate biochemical investigations (including measurement of AChR antibodies and serum creatine kinase) and in many cases an earlier routine muscle histology, a diagnosis was established by the responsible physician.
Control subjects
Control data were obtained from patients with idiopathic muscle pain or adult-onset muscular dystrophy who had no obvious neurological involvement. Features of neuromuscular transmission in these patients have been described in detail previously (Slater et al., 1992).
Clinical neurophysiology
Nerve conduction studies
Standard motor and sensory nerve conduction studies were performed as described previously (Fawcett et al., 1982).
Electromyography
Conventional concentric needle electromyography was performed. Spontaneous activity and qualitative assessment of the motor unit potentials were assessed. The biceps brachii (BB), extensor digitorum communis (EDC), vastus lateralis (VL) and tibialis anterior (TA) muscles were examined in most of the patients.
Repetitive nerve stimulation
These studies were performed as described previously (Kennett and Fawcett, 1993). Surface bipolar stimulating electrodes were strapped over the appropriate motor nerve, and recordings were made with surface tin-plate 12 x 6 mm rectangular surface electrodes. The studies were carried out on the abductor digiti minimi (ADM), anconeus (ANC) and deltoid (DELT) muscles. Trains of 8–10 stimuli at a rate of 3 Hz were delivered at rest and immediately, and at intervals, after 20 s maximal voluntary contraction. Prolonged trains at rates of up to 30 Hz were also delivered in some of the cases. The ratio of the amplitudes of the fifth to the first responses was used as a measure of decrement.
Single-fibre EMG
These studies were performed on EDC and VL during weak voluntary contraction according to methods described previously (Stalberg and Trontelj, 1979). The jitter in at least 20 pairs was analysed and expressed as the mean of consecutive differences (MCD). Macro EMG was performed in the right VL according to methods described previously (Stalberg et al., 1982).
Motor-point biopsy procedure
Muscle biopsy samples were removed from the belly of VL under local or general anaesthesia as described previously (Slater et al., 1992). All patients were taken off medication for at least 24 h before the biopsy procedure.
Laboratory methods
The laboratory methods used in this study have been described in detail previously (Slater et al., 1992). The biopsy samples were maintained and studied at room temperature (21–23°C) in a suitable physiological bathing solution (Liley, 1956) medium, gassed with 95% O2–5% CO2, while bundles of muscle fibres and their associated nerve were isolated for intracellular recording and other samples were removed for morphological studies.
Intracellular recording
Functioning nerve–muscle preparations were maintained in gassed bathing medium at room temperature. One or 2 intracellular microelectrodes were inserted into individual muscle fibres to record membrane potential and/or to pass current. The location of the NMJ was determined by searching for the site along the muscle fibre where evoked end-plate potentials (EPPs) and spontaneous miniature EPPs (mEPPs) with the fastest time courses could be recorded. Synaptic currents [end-plate currents (EPCs) and miniature EPC (mEPCs)] were recorded using a two-electrode voltage-clamp configuration. All records were stored as analogue signals on magnetic tape for subsequent computer analysis as described previously (Slater et al., 1992). The following features were measured or calculated: amplitude (corrected for a resting potential of –80 mV) and exponential decay time constant of EPPs and mEPPs, amplitude and exponential decay time constant (at –80 mV) of EPCs and mEPCs, quantal content, EPC reversal potential and voltage dependence of EPC
. See Slater et al. (1992) for further details.
Light microscopy
Additional bundles of muscle fibres containing NMJs were isolated and labelled and/or fixed for subsequent observation as described previously (Slater et al., 1992). Intramuscular nerves were labelled with a combined silver–cholinesterase method, acetylcholinesterase (AChE) activity was demonstrated histochemically and AChRs were labelled with fluorescent or radio-labelled conjugates of 
-bungarotoxin (-BgTx).
The area occupied by AChE activity was determined by tracing around each of the separate stained regions at individual NMJs, using a camera lucida and a light-emitting diode cursor and digitizing tablet, and summing these areas using a computer-based morphometry system.
Electron microscopy
After fixation, muscle samples were reacted to demonstrate cholinesterase activity. Regions containing NMJs were cut out and processed and embedded for electron microscopy (Slater et al., 1992). All morphometric analyses were based on length and area measurements made with a cursor and digitizing tablet.
Immunocytochemistry
Antibody labelling of unfixed frozen transverse sections was done as described previously (Bewick et al., 1992, 1996; Wood and Slater, 1998). NMJs were labelled with FITC-Dolichos biflorus lectin (Sigma-Aldrich Company Ltd., Gillingham, UK, 1 : 200). The following proteins were examined (antibody type, source/reference, dilution): AChE [polyclonal from J. Massoulié (Marsh et al., 1984), 1 : 1000], s-laminin (monoclonal C4 from J. R. Sanes, 1 : 100), -AChR subunit (monoclonal 210 from J. Lindstrom, 1 : 1000), 
-AChR subunit (monoclonal 154 from J. Lindstrom, 1 : 500), rapsyn [monoclonal 1234 from S. C. Froehner (Sealock et al., 1984), 1 : 100], voltage-gated sodium channel (NaV1) [polyclonal from S. R. Levinson (Dugandzija-Novakovic et al., 1995), 1 : 30], ankyrinG [polyclonal from S. Lambert, 1 : 100), dystrophin (monoclonal Dy8/6C5 (Bewick et al., 1992), 1 : 100], utrophin [monoclonal DRP3/20C5 (Bewick et al., 1992), 1 : 10], syntrophin [monoclonal SYN1351 from S. C. Froehner (Peters et al., 1997), 1 : 500], ß-dystroglycan [monoclonal 43DAG (Bewick et al., 1993), 1 : 100], ß-spectrin [monoclonal RBC2 (Bewick et al., 1992), neat], agrin (monoclonal AGR-520, 1 : 500), neuregulin (monoclonal, gift of A. Vincent).
DNA analysis
Genomic DNA from LGM and control samples was isolated from peripheral blood using the NucleonTM II DNA extraction kit (Tepnel Life Sciences, Manchester, UK). The coding exons and promoter regions of CHRNA, CHRNB, CHRND and CHRNE (the AChR subunit genes), RAPSN (the rapsyn gene), COLQ, CHAT and MUSK were screened for mutations by PCR amplification of genomic DNA and single-strand conformational polymorphism (SSCP) analysis. A typical PCR reaction for SSCP analysis included reaction buffer [60 mM Tris-HCl, 15 mM (NH4)2SO4, 1.5–2.0 mM MgCl2, pH 8.5 (Invitrogen, Paisley, Scotland, UK)], 1.25 µM each primer, 200 µM dCTP, dGTP and dTTP, 0.075 µl [-35S]dATP (>1000 Ci/mmol) (GE Healthcare, Little Chalfont, Bucks, UK), 50 µM dATP, with 50 ng genomic DNA and 0.25 U AmpliTaq (Applied Biosystems, Warrington, Cheshire, UK) in a 5 µl reaction. Samples were mixed with 5 µl gel loading buffer (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% Xylene Cyanol FF), denatured and loaded on a 8.3% (w/v) polyacrylamide gel (100 : 1 acrylamide : bis) containing 5% (v/v) glycerol. Gels were run at 6 W for 19 h at 4°C, dried on 3 MM Whatman paper and exposed to autoradiography for 17 h or longer. Genes from selected patients were fully screened for mutations by bi-directional DNA sequencing (Oxford University DNA sequencing facility) for all exons and promoter regions.
Data analysis
Details of analysis of electrophysiological, morphometric and -BgTx binding data were as described previously (Slater et al., 1992). All comparisons of means were made by Student's t-test (two-tailed) with appropriate correction when variances were unequal.
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Results |
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Clinical features
Patients
This paper describes observations made on eight patients who were studied in detail. Relevant clinical details are given in Table 1. There were equal numbers of males and females. The age when muscle weakness was first noticed ranged from 6 months to 9 years. In all cases, limb-girdle weakness was moderate at the time of biopsy. Most patients developed severe difficulty in walking by the age of 20.
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All eight cases had progressive but fluctuating muscle weakness with a predominantly limb-girdle distribution but without significant muscle wasting or hypertrophy. A degree of muscle atrophy compatible with disuse occurred only in the more disabled cases. In contrast to the short-term variation and fatigability seen in myasthenia gravis, the muscle weakness in these patients varied over periods of weeks to months, nor was the fluctuation usually so great in degree as in the autoimmune disease. Exceptional exercise in some cases or pyrexial illnesses in others might result in increased weakness taking several days or longer to recover. Similar more gradual variation over a longer timescale occurred, apparently at random, without an obvious cause. Short-term fatigability could not be demonstrated by clinical exercise tests in any of these patients. No consistent pattern of selective involvement of limb or axial muscles was found on detailed muscle testing. Oculomotor weakness was absent but slight facial weakness and non-fatigable ptosis were present in some of the patients. On neurological examination, reflexes were normal and there were no signs of neurological abnormality apart from muscle weakness.
These patients typically had a distinctive abnormality of gait and stance that transformed this rather non-specific presentation into a recognizable clinical syndrome. All of the patients demonstrated the swaying or ‘waddling’ gait generally associated with proximal weakness of the lower limbs. But in seven of the eight cases, and in a lesser degree in the eighth (LGM1), the gait was exaggerated by an unusual degree of rotation of the trunk and inward rotation at the hips, without footdrop, giving rise to a sinuous movement of the trunk and limbs well beyond the degree familiarly seen in such proximal myopathies as the muscular dystrophies. Furthermore, the inward rotation of the lower limbs was, in the weaker patients, maintained and increased during standing, so that the patellae rested against each other and the feet were directed inwards beyond 45°. These features, together with the long-term fluctuation in weakness, and especially when seen in conjunction with reports of normal results in initial investigations (serum creatine kinase, routine electromyography and routine muscle histology) made it possible to make a correct provisional clinical diagnosis in several of these cases. Indeed the diagnosis in case LGM6 was initially proposed after examination of the photograph of her stance and the clinical details published by Young and Anderson (1987) and was subsequently confirmed on investigation in her and in the second case in that paper (LGM7) by kind permission of Dr Young.
The results of initial diagnostic tests were generally and characteristically normal. In particular, serum antibodies for AChR were not detected and creatine kinase levels were within the normal range in all eight cases.
All the patients were from the north of England or Scotland. There was no evidence of consanguinity of any of their parents and none of the families are known to be related. Two patients (LGM5 and LGM8) had similarly affected siblings compatible with, but not diagnostic of, autosomal recessive inheritance.
Neurophysiological studies
Detailed electromyographic studies were made of all the patients. The results of these studies are summarized in Table 2 and typical records are shown in Fig. 1.
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Motor and sensory nerve conduction
The motor and sensory nerve conduction findings were normal in all the patients, with no repetitive discharges of the compound muscle action potential (CMAP) following stimulation of the motor nerve in any of the nerves examined.
Repetitive nerve stimulation
Significant decrement of the CMAP in response to stimulation at 3 Hz was found at rest in each of the eight cases, and the changes were more pronounced in the proximal muscles (ANC and DELT, 41% decrement) than distal muscles (ADM, 19% decrement) (Table 2). There was evidence of post-activation potentiation in all LGM patients (Fig. 1A). The amplitude of the CMAP and the degree of decrement were reduced following i.v. edrophonium in each of the six LGM patients in whom this test was made (Fig. 1B), and in each of the four patients tested with 3,4-diaminopyridine.
Single-fibre EMG studies
Single-fibre EMG studies were performed on EDC, VL and biceps (Table 2). Jitter was abnormal in all the muscles studied, and blocking was present in 5–100% of the pairs studied in each muscle (Fig. 1C).
Macro EMG studies
The median macro motor unit potential amplitude was normal in six and reduced in two cases, while the SFEMG fibre density was normal in seven patients and slightly increased in one. This implies that there was little if any change in motor unit size as defined by neurophysiology, and that the defect of neuromuscular transmission is therefore unlikely to be a secondary consequence of increased motor axon branching as, for example, occurs in spinal muscular atrophy.
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In vitro electrophysiology |
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Functioning nerve–muscle preparations were made from biopsy samples from each of the eight LGM patients. Intracellular recording techniques were used to assess a number of different aspects of neuromuscular transmission using methods described in detail previously (Slater et al., 1992
) and the results were compared with those obtained from patients with idiopathic muscle pain and late-onset myopathy in that earlier study (Table 3, Fig. 2).
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Intracellular recordings demonstrated significantly impaired neuromuscular transmission, thus confirming the main finding of the clinical neurophysiological studies. The mean EPP amplitude in LGM patients was only 46% of that in the controls (Table 3). The EPP amplitude in individual LGM subjects was significantly correlated with the fraction of abnormal muscle fibre pairs recorded by SFEMG (Fig. 3; P < 0.002). This suggests that the reduction in EPP amplitude seen in the isolated biopsy samples accounted for observed weakness of muscle in vivo.
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Reduced EPP amplitude could result either from fewer transmitter quanta being released by a single nerve impulse or from a smaller effect of individual quanta. In the event, both effects were observed. The mean quantal content (the steady state number of quanta released by a single nerve impulse when stimulating at 1 Hz) was 59% of the control value (Table 3, Fig. 4). The patient with the greatest reduction in quantal content was one of the two familial cases (LGM5). The mean amplitude of the mEPPs was 68% of that in the controls. Together, these two effects could account for the overall reduction in EPP amplitude observed.
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The amplitude and kinetics of synaptic currents reflect more closely the action of ACh on the muscle fibre membrane than do those of synaptic potentials. While the amplitude of the EPCs was significantly reduced in LGM patients relative to control values, the amplitude of the mEPCs was not (Table 3, Fig. 2). It is likely that the observed reduction in mEPP amplitudes in LGM samples resulted in part from a decrease in muscle fibre input resistance associated with the slightly greater diameter of the muscle fibres in the LGM patients (see below).
The decay time of the EPCs was not different from that in controls, while that of the mEPCs was slightly, but only just significantly, longer. The reversal potential of the EPC and the dependence of the EPC decay time constant on membrane potential [‘H’, see Slater et al. (1992)] were also not different from those in controls. Taken together, these findings suggest that the local abundance and kinetic properties of AChRs in the post-synaptic membrane of LGM patients was close to normal. Further, the absence of any marked prolongation of the synaptic currents suggests that the function of AChE in the synaptic cleft was also normal.
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Light microscopy |
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Muscle
The general appearance of the muscles was uniformly normal. There were no signs of muscle fibre necrosis, inflammatory infiltration or any other signs of muscle degeneration. The average diameter of muscle fibres, measured from transverse frozen sections after ATPase staining, was 14% larger than in the control samples (Table 4, P < 0.05). This was mostly associated with increases in diameter of type 1 (1.29-fold increase) and 2b (1.15-fold increase) fibres. The fibre-type proportions were nearly normal, with the only significant difference from the control group being a modest decrease in the fraction of type IIb fibres (18.5 ± 9.4% versus 30.3 ± 7.3%, P < 0.02). No significant fibre-type grouping was observed.
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Tubular aggregates have been observed in about half of the cases of LGM reported in the literature (see above). These were detected in the light microscope in only one case (LGM2). Their ultrastructure is described below.
Intramuscular nerves
The intramuscular nerves were studied after impregnation with silver. While there was no significant increase in axonal sprouting from nodes of Ranvier or from the synaptic terminals, there was a significant increase in the fraction of NMJs innervated by collateral axonal branches (Table 4). Associated with this, the functional terminal innervation ratio, a measure of intramuscular branching (Coers and Woolf, 1959), was also significantly increased relative to control values. These findings suggest that a moderate degree of axonal sprouting and adaptive re-innervation may have occurred in the early stages of the condition.
Motor nerve terminals
Human motor nerve terminals typically consist of a number of varicosities, some 2–3 µm in diameter, connected by much thinner unmyelinated axons (Fig. 5A). Those at NMJs of LGM patients have a similar appearance (Fig. 5B), but often appeared smaller and less compact. Very few examples were ever seen of the highly elongated terminals typical of some forms of inherited AChR deficiency (Vincent et al., 1981; Slater et al., 1997).
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AChR distribution and abundance
Post-synaptic labelling of AChRs with fluorescent rhodamine-
-BgTx was clearly observed at the NMJs in all LGM and control samples studied (7 out of 8 and 6 out of 8, respectively; Fig. 5C and D). At both LGM and control NMJs, the labelling was frequently concentrated in spot-like regions, typically 4–8 µm in diameter. There was no significant difference between NMJs in LGM patients and controls in the areas of the individual spot-like regions of high AChR density (P > 0.9), nor, at a qualitative level, was there any obvious difference in the intensity of labelling. To determine the number of -BgTx binding sites per NMJ, the binding of 125I--BgTx was studied in two controls and six LGM samples (Table 5). The mean value of 125I--BgTx binding per NMJ in the LGM samples was 40% of that in controls.
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Distribution of ChE activity
ChE activity was readily demonstrated at the NMJs of all LGM patients (Fig. 5E and F). There was no obvious difference in the intensity of the reaction product between patients and controls. The area of increased ChE activity was measured to provide a basis for calculating synaptic area (see below), since the labelling was often more clearly defined than the fluorescent labelling of AChRs. In LGM samples this ChE area was only 55% of that in the control samples (Fig. 5E and F, Table 4). In contrast, there was no significant increase in the length along the muscle fibre of the region containing ChE activity, such as occurs in some AChR deficiencies (see above). A number of animal studies have shown that the size of the NMJ as revealed by ChE labelling is roughly proportional to the muscle fibre diameter (Kuno et al., 1971
; Harris and Ribchester, 1979). The ratio of ChE area to muscle fibre diameter (determined from the same teased fibres used for ChE area measurements) in the LGM sample was only 44% of that in the controls (mean ± SD: LGM, 1.77 ± 0.37; control, 3.46 ± 0.77, P < 0.05).
Electron microscopy
The ultrastructure of NMJs in the human VL, and our approach to quantifying it, have been described in detail elsewhere (Slater et al., 1992). While the general appearance of NMJs in LGM samples (Fig. 6C, D and F–H) was similar to that in controls (Fig. 6A, B and I), there were some clear differences (Table 6). The most striking of these was a reduction in the extent of post-synaptic folding at the LGM NMJs. At human NMJs, the pattern of folding is often very complex (Slater et al., 1992). The folds, and the high density of NaV1 channels contained in the membrane in their depths, are believed to amplify the EPC, thus enhancing neuromuscular transmission (Wood and Slater, 2001). Frequently, a substantial part of the folded cell surface lies outside of the region of close contact with the nerve (e.g. Fig. 6B).
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In the LGM patients the extent of folding, as reflected both by the total length of folded membrane at each NMJ in the section (FoldL) and by the increase in post-synaptic length caused by the folding [‘folding index’ (Slater et al., 1992
)] was 50–60% of the control value (Fig. 6, Table 6). This reduction was associated with a significant decrease in the mean frequency of openings of folds into the synaptic cleft (1.95 ± 0.39 SD openings per micrometre in controls versus 1.35 ± 0.53 SD per micrometre in LGM, P = 0.021) but not in the length of membrane associated with each opening. Although the extent of the reduction in folding index varied from patient to patient (e.g. Fig. 6D versus F), and even between the NMJs of a single patient (Fig. 6F versus G), the mean folding index was lower for each of the LGM patients than for any of the controls (Fig. 7). Studies of -BgTx binding revealed that even when folding was greatly reduced, AChR labelling was restricted to the regions of post-synaptic membrane immediately adjacent to the nerve (Fig. 6G and H). The extent of ‘occupancy’ of the folded region by the nerve was similar in control and LGM samples.
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Tubular aggregates, which have been observed in some patients with limb-girdle weakness (see above), were only seen in biopsy samples from two of the LGM subjects (LGM1 and 2), and none were seen in samples from the controls. One of the two LGM subjects, LGM2, had numerous tubular aggregates (Fig. 6E) that were also seen in the light microscope (see above). It may be significant that LGM2 was also the patient with the most strikingly reduced post-synaptic folding. The other subject with tubular aggregates was LGM1, in whom a single small aggregate was seen (Fig. 6G).
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Structure–function relationships |
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TopSummaryIntroductionPatients and methodsResultsIn vitro electrophysiologyLight microscopyStructure-function relationshipsProtein localization studiesDNA analysisResponse to treatmentDiscussionReferences |
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The results presented so far provide evidence of both functional and structural abnormalities associated with the pre- and post-synaptic components of the NMJs in patients with LGM. Since there is a close relationship between structure and function at the NMJ (Slater, 2003
), it was reasonable to ask whether the functional abnormalities observed in NMJs of LGM patients might be more specifically correlated with the structural changes.
Transmitter release
The quantal content of nerve-evoked transmitter release is generally proportional to the area of synaptic contact (e.g. Harris and Ribchester, 1979; Slater et al., 1992). To see if this is true in LGM patients, the mean area of ‘synaptic contact’ for each patient was estimated by multiplying the mean area occupied by high ChE activity by the mean ‘occupancy’ of the differentiated post-synaptic membrane by the nerve, determined from electron micrographs (Table 6). When the entire group of LGM and control subjects studied was considered, there was a highly significant correlation between the quantal content and synaptic area (Fig. 8). However, when the average ratios of quantal content/synaptic area for LGM and control samples were compared, there was no significant difference (mean ± SD: LGM, 0.44, ± 0.26 quanta/µm2, n = 8; control, 0.35 ± 0.10 quanta/µm2, n = 8). Thus, although the quantal content in LGM patients is lower than normal, the quantal release per unit area of synaptic contact is not.
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Factors that may influence mEPP amplitude
One factor that could contribute to the reduction in mEPP amplitude at LGM NMJs relative to controls would be a reduction in the density of post-synaptic AChRs. Such a reduction appears at first to be supported by the reduced number of 125I-
-BgTx binding sites per NMJ observed. However, the ACh in a quantum acts very locally, within <1 µm of the site of release (Salpeter, 1987). Thus, it is the local density of AChRs, not their total number at the NMJ, that determines quantal size. To determine the local density of AChRs, it is necessary to estimate the area in which the AChRs are concentrated. Previous studies indicate that the AChR-rich zone extends about one-third of the way down the synaptic folds (Fertuck and Salpeter, 1974). On this basis, the likely AChR-rich area can be estimated as
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The result of dividing the amount of 125I--BgTx bound/NMJ by this estimate of AChR area (Table 5) shows that a reduction in AChR density of 16% was observed in the LGM patients relative to controls but that this difference was not statistically significant with the small sample available. This is consistent with the small (8%) and statistically insignificant difference in mEPC amplitudes between LGM and control subjects.
A second factor that could influence mEPP amplitude is the input resistance of the muscle fibre (Katz and Thesleff, 1957). Other things being equal, the input resistance is inversely related to the (muscle fibre diameter)3/2. On this basis, the 14% increase in muscle fibre diameter observed in LGM patients, relative to controls, would result in a reduction of mEPP amplitude of 23%. In fact the observed reduction was 32%. It thus seems likely that most of the reduction in mEPP amplitude is a result of the slightly greater fibre diameter, with a slight reduction in AChR density as a further contributing factor.
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Protein localization studies |
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TopSummaryIntroductionPatients and methodsResultsIn vitro electrophysiologyLight microscopyStructure-function relationshipsProtein localization studiesDNA analysisResponse to treatmentDiscussionReferences |
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Antibody labelling studies were made in an effort to identify molecular defects that might underlie the structural and functional abnormalities at NMJs in LGM patients. Suitable samples of muscle containing NMJs were available for cases LGM1, 2, 7 and 8. These include the case with the most profound reduction in post-synaptic folding (LGM2) and one of the two cases with an affected sibling (LGM8). Typical results of these studies are illustrated in Fig. 9 and summarized in Table 7. The following proteins were studied: AChE, s-laminin, AChR subunits, rapsyn, NaV1, ankyrinG, utrophin, dystrophin, syntrophin, ß-dystroglycan, ß-spectrin, agrin and neuregulin1. In brief, the intensity of immunolabelling for all the proteins investigated was substantially increased at the NMJ, relative to the levels present in non-junctional regions of the muscle, in all the LGM and control samples studied.
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DNA analysis |
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TopSummaryIntroductionPatients and methodsResultsIn vitro electrophysiologyLight microscopyStructure-function relationshipsProtein localization studiesDNA analysisResponse to treatmentDiscussionReferences |
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Analysis of DNA samples from the LGM patients was undertaken to look for the possibility of abnormalities in those genes in which mutations most commonly give rise to congenital myasthenic syndromes (Engel et al., 2003a
; Beeson et al., 2005). Coding exons and untranslated regions 5' to the ATG initiation codon for genes encoding the muscle AChR , ß, 
and subunits, rapsyn, ColQ (the collagenous tail of AChE), cholineacetyltransferease and MuSK were screened for mutations. Samples from all but one of the cases (LGM6) were available for analysis. DNA from patients LGM1-5 and 7–8 was screened by SSCP analysis. In addition, PCR amplicons for genes encoding the AChR , ß, and subunits, rapsyn, cholineacetyltransferease, ColQ and MuSK were fully screened by bi-directional DNA sequencing of all coding exons for the two familial cases (LGM 5 and 8). No mutations were identified. |
Response to treatment |
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TopSummaryIntroductionPatients and methodsResultsIn vitro electrophysiologyLight microscopyStructure-function relationshipsProtein localization studiesDNA analysisResponse to treatmentDiscussionReferences |
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Various treatments have been tried in an effort to reduce the muscle weakness in this group of patients. These include pyridostigmine, 3,4-diaminopyridine and ephedrine. No consistent pattern of response to medication has been observed. In one patient (LGM2) pyridostigmine and 3,4-diaminopyridine both caused moderate improvement on their own, but when given together there was a dramatic improvement in muscle strength, allowing the previously wheel-bound patient to walk on his own. This patient has been successfully treated with a combination of these compounds for a number of years. In three other patients (LGM2, 5, 8), pyridostigmine and 3,4-diaminopyridine caused moderate improvement, but intolerable cholinergic side-effects prevented the long-term use of pyridostigmine in two of them (LGM5, 8). In three other patients, pyridostigmine caused increased muscle weakness (LGM1, 3, 4).
The same distinctive fluctuations in muscle strength observed before treatment (see above) are also seen in relation to trials of medication. Several patients have shown benefit in terms of improved muscle strength and a reduction in the decrement in the CMAP amplitude during repetitive stimulation when initially given various medications only to deteriorate subsequently, sometimes to a situation where they are weaker than before treatment. The time course of this secondary deterioration is typically days to weeks and, if treatment is stopped, it then usually takes a similar period of time for the muscle strength to improve.
Several of the patients have shown moderate, but sustained, improvement with ephedrine (LGM3, 4, 5, 8). These included some who failed to improve, or even deteriorated, on pyridostigmine.
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Discussion |
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TopSummaryIntroductionPatients and methodsResultsIn vitro electrophysiologyLight microscopyStructure-function relationshipsProtein localization studiesDNA analysisResponse to treatmentDiscussionReferences |
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Main findings
The patients we describe in this report share a combination of clinical features that sets them apart from many other patients with impaired neuromuscular transmission. These features include onset in infancy or childhood, weakness primarily of the proximal muscles and fluctuations in the severity of weakness on a timescale of weeks. Most cases also displayed a characteristic ‘sinuous’ or ‘waddling’ gait (Gardner-Medwin, 1993
). Because the onset and pattern of weakness of these patients are similar to those in patients described in the past as having ‘limb girdle myasthenia’, we chose to keep this name. However, we recognize that this is a descriptive name that may represent conditions of heterogeneous aetiology. While our patients varied in their severity of weakness, our laboratory studies revealed that they had two important abnormalities in common: reduced size of the NMJs, likely to contribute to the reduced ACh output, and reduced post-synaptic folding, likely to increase the threshold for muscle fibre action potential generation. Together, these effects appear likely to account for the impairment of transmission and the resulting clinical weakness observed in LGM patients.
Many other features of the NMJ we studied were normal in LGM patients. These included quantal release per unit area of synaptic contact, the size and kinetics of mEPCs and the presence of a number of key proteins of the NMJ and the genes that encode them. In particular, the best-known targets of gene mutations in congenital myasthenic syndromes (Engel et al., 2003a) and of autoantibodies in acquired myasthenias (Vincent et al., 2003) were present in apparently normal abundance and distribution. Our findings thus suggest that the primary targets of the abnormalities in these patients are not the proteins responsible for the immediate events of neuromuscular transmission but rather elements of the mechanisms that determine NMJ size and conformation.
It is of interest that over two decades these patients represented nearly half of the patients seen with childhood myasthenia in Newcastle. While this may reflect some bias in our selection of patients for detailed study as our interest in these patients grew, it nonetheless suggests that this is an important, if sometimes difficult to identify, group of children.
Comparison of our patients with others
Our patients resemble some, but not all, of those that have been reported as having LGM. Some LGM patients have detectable serum antibodies to AChR (Rodolico et al., 2002), suggesting an autoimmune aetiology. None of our patients had such antibodies or any other indications of an autoimmune condition. In addition, tubular aggregates were present in the muscles of about half of the previously reported LGM patients (Johns et al., 1973; Dobkin and Verity, 1978; Azulay et al., 1994; Sieb et al., 1996; Furui et al., 1997; Rodolico et al., 2002). These structures appear to be rather non-specific, in that they occur in a number of conditions (reviewed in Pierobon-Bormioli et al., 1985) and were only prominent in one of our eight patients. The physiological significance of tubular aggregates is unclear. There is some evidence that these structures are involved in calcium homeostasis (Salviati et al., 1985). One possibility is that they represent part of the muscle's response to a changed pattern of activity, regardless of its origin.
Although our LGM patients vary in the severity of their abnormalities, as a group they are distinguishable clinically from many previously described patients with acquired and congenital myasthenias. In particular, while the ocular and facial muscles are frequently the most affected in many other myasthenias, the effect on these muscles is only minor in LGM patients. When our laboratory findings are added to these clinical considerations, we can confidently exclude many of the well-recognized conditions as the basis of impaired neuromuscular transmission in our patients. These include the acquired conditions myasthenia gravis and Lambert–Eaton myasthenic syndrome (LEMS) and the inherited conditions (CMS) resulting from mutations in ChAT, AChRs, AChE, rapsyn and MuSK, the signalling kinase that plays a central role in mediating the ability of agrin to induce post-synaptic differentiation.
What is the basis of impaired transmission in these LGM patients?
One of our main initial aims was to determine whether the defects that account for impaired neuromuscular transmission in LGM patients are pre- or post-synaptic. In the event, we found that both sides of the synapse are involved. Human NMJs, like those of a number of other species, normally consist of a number of roughly circular spot-like nerve–muscle contacts 5 µm in diameter (Walrond and Reese, 1985; Slater et al., 1992; Wilkinson and Teng, 2003). The quantal content per unit area is similar in humans to that in many species (Slater, 2003). However, the overall area of synaptic contact is relatively small in humans, compared with the size of the muscle fibres (Slater, 2003). As a result, the quantal content is relatively low in humans. In LGM patients, both the area of synaptic contact and the quantal content are reduced to 50% of their normal values. While there is no significant correlation between the area of contact and quantal content within the group of LGM patients, the sample is small and the electrophysiological and morphometric studies were made on different parts of the biopsy specimen. Since, on average, the quantal release per unit area is normal in the LGM patients but the NMJs are small, an obvious hypothesis is that the reduced ACh release of the NMJs is a result of their reduced size.
The post-synaptic folds are believed to function as amplifiers of ACh action (Martin, 1994; Wood and Slater, 1997; Wood and Slater, 2001). This effect results from both the high density of voltage-gated sodium channels in the membrane in the depths of the folds (Flucher and Daniels, 1989) and the high electrical resistance of the narrow sheets of cytoplasm bounded by the folded membrane (Vautrin and Mambrini, 1989; Martin, 1994). Thus, any reduction in the extent of folding, as seen in LGM, would be expected to increase the threshold for the initiation of action potentials in the muscle fibre and thus lower the safety factor of neuromuscular transmission. In mice, reduced folding is associated with a reduced abundance of a number of post-synaptic cytoskeletal proteins, such as dystrophin and utrophin (Torres and Duchen, 1987; Lyons and Slater, 1991; Grady et al., 1997; Deconinck et al., 1997). This suggests that the folding process depends on complex interactions between numerous proteins.
An interesting finding was the slightly increased muscle fibre diameter in LGM patients compared with controls. Muscle fibre diameter is well known to be sensitive to activity and load. This finding therefore suggests that in spite of the obvious impairment of neuromuscular transmission the level of excitation of muscles is great enough to maintain muscle fibre size. Nonetheless, the slight increase in diameter, by virtue of the associated reduction of electrical ‘input resistance’, would of itself have the effect of reducing EPP amplitude.
Taken together, our findings suggest that the clinical weakness in these LGM patients is primarily a result of structural abnormalities of the NMJ, rather than defects in the process of neuromuscular transmission itself. At present, very little is known about the molecular factors that determine the size and conformation of the NMJ. Structural and adhesion proteins of the extracellular matrix, as well as components of the signalling pathways that mediate interactions between nerve and muscle, for example MuSK (Chevessier et al., 2004), are all likely to be involved. It may well be that achieving a clearer understanding of the molecular defects in LGM patients will further advance understanding of these factors. An important further conclusion of our study is that clinically significant impairment of neuromuscular transmission can occur in the absence of abnormalities of some of the key molecules that mediate that transmission.
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Acknowledgements |
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We thank Dr M. A. Johnson for making the muscle fibre-type studies. Dr Guy Bewick did some of the early immunolabelling studies. The support of this work by the Muscular Dystrophy Group and Action Research is gratefully acknowledged. We are particularly grateful to Prof. John Harris who provided essential support for this project during his tenure as Director of the Muscular Dystrophy Group Laboratories in Newcastle.
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