doi:10.1093/brain/awl200Brain (2006), 129, 2061–2076
Pre- and post-synaptic abnormalities associated
with impaired neuromuscular transmission in a
group of patients with ‘limb-girdle myasthenia’
C. R. Slater,1P. R. W. Fawcett,2T. J. Walls,4P. R. Lyons,5S. J. Bailey,6D. Beeson,7C. Young1
and D. Gardner-Medwin3
1School of Neurology, Neurobiology and Psychiatry, University of Newcastle upon Tyne, Departments of
2Clinical Neurophysiology and3Paediatric Neurology,4Department of Neurology, Regional Neurosciences Centre,
Newcastle General Hospital, Newcastle upon Tyne,5Department of Neurology, Royal United Hospital,6Department of
Pharmacy and Pharmacology, University of Bath, Bath and7Neurosciences 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
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 hadmoderateto severe weakness of the proximal muscles,withoutshort-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 treat-
ment 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 abnorm-
alities of the NMJ, including reduced size and post-synaptic folding, rather from any abnormality in the immedi-
ate events of neuromuscular transmission.
Keywords: congenital myasthenic syndrome; human; limb-girdle myasthenia; neuromuscular transmission;
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
#The Author (2006). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: firstname.lastname@example.org
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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 involve-
ment 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 intro-
duced 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 sug-
gested 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
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 clini-
cal 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 electromyo-
graphic 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 impair-
ment. 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 func-
tional 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 func-
tional 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 compo-
nents of the NMJ, whose genes are now well-established tar-
gets 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 mainte-
nance 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).
Patients and methods
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
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
2062Brain (2006), 129, 2061–2076 C. R. Slater et al.
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Control data were obtained from patients with idiopathic muscle
pain or adult-onset muscular dystrophy who had no obvious neuro-
logical involvement. Features of neuromuscular transmission in
these patients have been described in detail previously (Slater
et al., 1992).
Nerve conduction studies
Standard motor and sensory nerve conduction studies were per-
formed as described previously (Fawcett et al., 1982).
Conventional concentric needle electromyography was performed.
Spontaneous activity and qualitative assessment of the motor unit
potentials were assessed. The biceps brachii (BB), extensor digito-
rum 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 · 6 mm rectangular surface electro-
des. 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 imme-
diately, 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.
These studies were performed on EDC and VL during weak volun-
tary 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.
The laboratory methods used in this study have been described in
detail previously (Slater et al., 1992). The biopsy samples were main-
tained 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 andtheir associated
nerve were isolated for intracellular recording and other samples
were removed for morphological studies.
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 EPCt. See Slater et al. (1992)
for further details.
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 a-
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.
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.
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. Massoulie ´ (Marsh et al., 1984), 1 : 1000], s-
laminin (monoclonal C4 from J. R. Sanes, 1 : 100), a-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], b-dystroglycan [mono-
clonal 43DAG (Bewick et al., 1993), 1 : 100], b-spectrin [monoclonal
RBC2 (Bewick et al., 1992), neat], agrin (monoclonal AGR-520, 1 :
500), neuregulin (monoclonal, gift of A. Vincent).
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Genomic DNA from LGM and control samples was isolated
from peripheral blood using the Nucleon? II DNA extraction kit
(Tepnel Life Sciences, Manchester, UK). The coding exons and pro-
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
mMTris-HCl, 15mM(NH4)2SO4,1.5–2.0 mMMgCl2,pH8.5 (Invi-
dGTP and dTTP, 0.075 ml [a-35S]dATP (>1000 Ci/mmol) (GE
Healthcare, Little Chalfont, Bucks, UK), 50 mM dATP, with 50 ng
genomic DNA and 0.25 U AmpliTaq (Applied Biosystems, Warring-
ton, Cheshire, UK) in a 5 ml reaction. Samples were mixed with 5 ml
gel loading buffer (95% formamide, 20 mM EDTA, 0.05% bromo-
phenol 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 muta-
tions by bi-directional DNA sequencing (Oxford University DNA
sequencing facility) for all exons and promoter regions.
Details of analysis of electrophysiological, morphometric and a-BgTx
binding data were as described previously (Slater et al., 1992). All
comparisons ofmeans were made byStudent’s t-test (two-tailed)with
appropriate correction when variances were unequal.
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.
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 mus-
cle 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 pre-
sent 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 stand-
ing, 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 electro-
myography 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
Table 1 Clinical features of limb-girdle patients
SubjectSex Age at
Family history Age at onsetWeakness: distributionSinuous
Oculo motorPtosis FacialLimb girdle
2064 Brain (2006), 129, 2061–2076 C. R. Slater et al.
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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.
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.
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 stimula-
tion 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
Table 2 Clinical neurophysiological findings in LGM patients
MuscleDec-3 Hz (%)Muscle Increased (%) Blocking (%)MCD (ms)MCD + block (ms)
Fig. 1 Typical recordings demonstrating impaired
neuromuscular transmission in LGM patients. (A) Decrement
of CMAP (3 Hz) at rest is abolished 10 s after 20 s exercise
but returns later (‘2 min post’). (B) Administration of
edrophonium (2 mg followed rapidly by 8 mg) increases
CMAP amplitude at 30 s and reduces decrement 1 min later.
(C) Three examples of abnormally increased ‘jitter’ (values
are MCD) in SFEMG recordings from VL in patients with
LGM. All illustrations from same patient (LGM4).
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(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 nor-
mal 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.
In vitro electrophysiology
Functioning nerve–muscle preparations were made from
biopsy samples from each of the eight LGM patients.
Intracellular recording techniques were used to assess a num-
ber 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).
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.
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.
Table 3 Properties of synaptic events at NMJs in LGM patients
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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.
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 asso-
ciated with increases in diameterof 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 6 9.4% versus 30.3 6 7.3%,
P < 0.02). No significant fibre-type grouping was observed.
Fig. 2 Evoked (EPP, EPC) but not spontaneous (mEPP,
mEPC) synaptic responses are reduced at NMJs from LGM
patients. Synaptic events recorded in vitro from NMJs in biopsy
samples from control and LGM patients. Each panel shows a
number of superimposed traces to indicate observed variability.
Fig. 3 The extent of ‘blocking’ seen in SFEMG in LGM
patients (open symbols) is related to EPP amplitude. Filled
symbol shows average values for eight control subjects.
Fig. 4 Quantal content is reduced at NMJs in LGM patients
relative to that in controls. Each symbol shows average value
for one patient.
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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.
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 term-
inals, 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 mm 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).
AChR distribution and abundance
rhodamine-a-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 mm in diameter. There was no significant dif-
ference 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 a-BgTx binding sites per NMJ, the binding of
125I-a-BgTx was studied in two controls and six LGM sam-
ples (Table 5). The mean value of125I-a-BgTx binding per
NMJ in the LGM samples was ?40% of that in controls.
of AChRswith fluorescent
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
Table 4 Properties of muscle and its innervation in LGM patients
Subject Sprouting (%) Collaterals (%)FTIRChEA (mm2) ChEL (mm) MFDiam (mm)Type 1 (%)
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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 6 SD: LGM, 1.77 6 0.37;
control, 3.46 6 0.77, P < 0.05).
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 reduc-
tion 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 substan-
tial part of the folded cell surface lies outside of the region of
close contact with the nerve (e.g. Fig. 6B).
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 fre-
quency of openings of folds into the synaptic cleft (1.95 6
0.39 SD openings per micrometre in controls versus 1.35 6
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
Fig. 5 Appearance of NMJs in control and LGM muscle. (A and B) Motor axon terminals (silver/ChE stain). (C and D) AChRs
(R-aBgTx). (E and F) AChE activity (histochemical reaction).
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any of the controls (Fig. 7). Studies of a-BgTx binding
revealed that even when folding was greatly reduced,
AChR labelling was restricted to the regions of post-synaptic
The extent of ‘occupancy’ of the folded region by the nerve
was similar in control and LGM samples.
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.Oneof thetwo LGM subjects, LGM2,hadnumerous
tubular aggregates (Fig. 6E) that were also seen in the light
microscope (see above). It may be significant that LGM2 was
folding. The other subject with tubular aggregates was
LGM1, in whom a single small aggregate was seen (Fig. 6G).
The results presented so far provide evidence of both func-
tional 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 functionalabnormalities observed in NMJs of
LGM patients might be more specifically correlated with the
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 signi-
ficant 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 6
SD: LGM, 0.44, 6 0.26 quanta/mm2, n = 8; control, 0.35 6
0.10 quanta/mm2, 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.
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
However, the ACh in a quantum acts very locally, within
<1 mm 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
125I-a-BgTx binding sites per NMJ observed.
AAChR¼ AChE· ððLPostþ ðLFold? LPostÞ/3Þ/LPostÞ:
The result of dividing the amount of125I-a-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
Table 5125I-a-BgTx binding at NMJs in LGM patients and controls
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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
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
Fig. 6 Post-synaptic folding is reduced at NMJs in muscle from LGM patients. (A and B) NMJs from control subjects showing abundant post-
synaptic folds. (C and D) NMJs from patient LGM2 showing greatly reduced folding. Note the abundant synaptic vesicles and mitochondria.
(E) Tubular aggregates in LGM2. (F and G) LGM1, showing variation of extent of folding in the same biopsy sample. (H and I) Distribution of
AChRs, labelled with a-BgTx, in LGM7 and a control sample. The clear labelling of post-synaptic membrane in both may be noted.
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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
Protein localization studies
Antibody labelling studies were made in an effort to identify
molecular defects that might underlie the structural and
Table 6 Ultrastructural features of NMJs in LGM patients
Fig. 7 Post-synaptic folding is significantly less at NMJs from LGM
patients than at those from controls (Pain, Myo). Fold index
defined in Patients and methods.
Fig. 8 Quantal content at NMJs in LGM (open symbols)
and control patients (filled symbols) is correlated with NMJ
area. P < 0.001.
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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 summar-
ized in Table 7. The following proteins were studied: AChE,
s-laminin, AChR subunits, rapsyn, NaV1, ankyrinG, utro-
phin, dystrophin, syntrophin, b-dystroglycan, b-spectrin,
agrin and neuregulin1. In brief, the intensity of immunola-
belling 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
Analysis of DNA samples from the LGM patients was under-
taken to look for the possibility of abnormalities in those
genes in which mutations most commonly give rise to con-
genital myasthenic syndromes (Engel et al., 2003a; Beeson
et al., 2005). Coding exons and untranslated regions 50to the
ATG initiation codon for genes encoding the muscle AChR a,
b, d 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 a, b, d 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
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 com-
pounds for a number of years. In three other patients (LGM2,
5, 8), pyridostigmine and 3,4-diaminopyridine caused mod-
erate 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,
Fig. 9 Immunocytochemical labelling of post-synaptic proteins
at LGM NMJs. Position of NMJ labelled with DBA lectin.
‘Control’ in bottom row, no primary antibody.
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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.
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 weak-
ness 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
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
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 homeo-
stasis (Salviati et al., 1985). One possibility is that they repre-
sent 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
Table 7 Immunocytochemical studies on LGM NMJs
AbLGM1 LGM2LGM7 LGM8
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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 mm 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
The post-synaptic folds are believed to function as ampli-
fiers 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
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
Taken together, our findings suggest that the clinical
weakness in these LGM patients is primarily a result of struc-
tural 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 achiev-
ing 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.
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
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