doi:10.1093/brain/awl200 Brain (2006), 129, 2061–2076

Pre- and post-synaptic abnormalities associatedwith impaired neuromuscular transmission in agroup of patients with ‘limb-girdle myasthenia’

C. R. Slater,1 P. R. W. Fawcett,2 T. J. Walls,4 P. R. Lyons,5 S. J. Bailey,6 D. Beeson,7 C. Young1

and D. Gardner-Medwin3

1School of Neurology, Neurobiology and Psychiatry, University of Newcastle upon Tyne, Departments of2Clinical Neurophysiology and 3Paediatric Neurology, 4Department of Neurology, Regional Neurosciences Centre,Newcastle General Hospital, Newcastle upon Tyne, 5Department of Neurology, Royal United Hospital, 6Department ofPharmacy and Pharmacology, University of Bath, Bath and 7Neurosciences Group, Weatherall Institute ofMolecular Medicine, The John Radcliffe Hospital, Headington, Oxford, UK

Correspondence to: Prof. C. R. Slater, School of Neurology, Neurobiology and Psychiatry, University of Newcastleupon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UKE-mail: c.r.slater@ncl.ac.uk

The properties of neuromuscular junctions (NMJs) were studied in motor-point biopsy samples from eightpatients with congenital myasthenic syndromes affecting primarily proximal limb muscles [‘limb-girdlemyasthenia’ (LGM)]. All hadmoderate to severe weakness of the proximalmuscles, without short-term clinicalfatigability but with marked variation in strength over periods of weeks or months, with little or no facialweakness or ptosis and no ophthalmoplegia. Most had a characteristic gait and stance. All patients showeddecrement of the compound muscle action potential (CMAP) on repetitive stimulation at 3 Hz, and increasedjitter 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 hadsimilarly affected siblings. Intracellular recording from isolated nerve–muscle preparations revealed that thequantal 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 synapticpotentials and currents were similar to control values. The area of synaptic contact and extent of post-synapticfolding 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 proteinsnormally concentrated at the NMJ, including components of the basal lamina, post-synaptic membrane andpost-synaptic cytoskeleton. DNA analysis failed to detect mutations in the genes encoding any of the followingproteins: 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 withtreatments, 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. Weconclude 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;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 consecutivedifferences; 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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IntroductionLimb-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

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 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 methodsPatientsThe 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.

DiagnosisA 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.

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Control subjectsControl 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).

Clinical neurophysiologyNerve conduction studiesStandard motor and sensory nerve conduction studies were per-

formed as described previously (Fawcett et al., 1982).

ElectromyographyConventional 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 stimulationThese 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.

Single-fibre EMGThese 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 procedureMuscle 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 methodsThe 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 and their associated

nerve were isolated for intracellular recording and other samples

were removed for morphological studies.

Intracellular recordingFunctioning 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 EPCt. See Slater et al. (1992)

for further details.

Light microscopyAdditional 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-

bungarotoxin (a-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 microscopyAfter 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.

ImmunocytochemistryAntibody 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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DNA analysisGenomic 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-

moter regions ofCHRNA,CHRNB,CHRND andCHRNE (the AChR

subunit genes), RAPSN (the rapsyn gene), COLQ, CHAT andMUSK

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, 15 mM (NH4)2SO4, 1.5–2.0 mMMgCl2, pH 8.5 (Invi-

trogen, Paisley, Scotland, UK)], 1.25mMeach primer, 200mMdCTP,

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.

Data analysisDetails of analysis of electrophysiological, morphometric and a-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.

ResultsClinical featuresPatientsThis 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

Subject Sex Age atbiopsy (years)

Family history Age at onset Weakness: distribution Sinuousgait

Oculo motor Ptosis Facial Limb girdle

LGM1 F 12 No 15 months No No No Moderate NoLGM2 M 19 No 2.5 years No No Slight Moderate YesLGM3 M 14 No 11 months No No No Moderate YesLGM4 F 26 No 14 months No Slight Slight Moderate YesLGM5 M 22 Affected sister 9 years No No Slight Moderate SlightLGM6 F 16 No 6 months No Slight Slight Moderate YesLGM7 M 20 No 10 months No Slight Slight Moderate YesLGM8 F 38 Affected brother 5–6 years No No No Moderate Yes

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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.

Neurophysiological studiesDetailed 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 conductionThe 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 stimulationSignificant 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

Subject Decrement SFEMG

Muscle Dec-3 Hz (%) Muscle Increased (%) Blocking (%) MCD (ms) MCD + block (ms)

LGM1 DELT 6 VL 14 36 48 87ADM 0 EDC 15 5 31 43

LGM2 DELT 49 VL 20 60 51 152ADM 56 EDC 33 33 68 93

LGM3 DELT 19 VL 16 6 29 35ANC 26 EDC 36 53 71 139ADM 5

LGM4 DELT 65 VL 35 39 54 84ANC 40 EDC 30 56 85 118ADM 7

LGM5 ADM 0 VL 18 70 68 122EDC 50 23 65 85BB 36 21 48 73

LGM6 DELT 27 VL 13 39 29 82ANC 32 EDC 36 54 94 130ADM 4

LGM7 ANC 86 VL 10 40ADM 22 EDC 0 100

LGM8 DELT 62 EDC 50 20 71 82ANC 40 Biceps 0 100 245

Fig. 1 Typical recordings demonstrating impairedneuromuscular transmission in LGM patients. (A) Decrementof CMAP (3 Hz) at rest is abolished 10 s after 20 s exercisebut returns later (‘2 min post’). (B) Administration ofedrophonium (2 mg followed rapidly by 8 mg) increasesCMAP amplitude at 30 s and reduces decrement 1 min later.(C) Three examples of abnormally increased ‘jitter’ (valuesare MCD) in SFEMG recordings from VL in patients withLGM. 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 studiesSingle-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 studiesThe 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 electrophysiologyFunctioning 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).

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.

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

Subject mEPPa(mV)

mEPPt(ms)

EPPa(mV)

EPPt(ms)

QC mEPCa(nA)

mEPCt(ms)

EPCa(nA)

EPCt(ms)

Rev(mV)

H(mV)

LGMLGM1 0.45 3.94 5.88 3.51 14.67 2.04 3.94 21.50 3.48 13.44 276.1LGM2 0.38 4.97 3.56 4.91 11.67 24.98 3.67 5.68 118.9LGM3 0.43 6.24 4.48 6.11 12.40 3.84 4.91 39.61 5.15 �4.06 134.1LGM4 0.51 6.64 5.07 5.04 11.48 4.41 4.26 37.12 4.11 �10.91 216.1LGM5 0.56 8.69 2.01 10.50 4.76 5.40 4.84 7.74 �0.99LGM6 0.58 5.47 7.65 5.69 16.54 4.25 3.56 50.96 3.47 �4.36 103.4LGM7 0.33 6.81 4.47 5.46 15.55 4.26 3.59 55.38 3.88 10.33 124.3LGM8 0.62 6.82 6.54 7.73 13.22 6.13 4.62 50.22 5.44 �11.28 119.1Ave 0.48 6.20 4.96 6.12 12.54 4.33 4.25 35.94 4.17 �0.27 156.0SD 0.10 1.43 1.76 2.13 3.63 1.28 0.57 16.67 0.80 9.27 64.5n 8 8 8 8 8 7 7 8 7 8 7

ControlPain1 0.52 5.16 10.11 5.26 22.18 5.28 3.94 77.65 4.83 3.12 105.2Pain2 0.66 6.17 7.38 5.98 14.99 4.69 3.74 48.75 3.86 34.69 160.5Pain3 0.53 7.33 10.51 6.88 22.47 3.69 4.06 61.99 3.43 �10.85 167.2Pain4 0.66 6.15 9.89 5.54 17.22 4.31 3.74 49.27 3.76 �8.85 134.2Pain5 0.90 4.46 13.70 4.81 20.33 4.55 3.27 50.33 3.68 �7.79 118.2Myo1 0.85 4.27 11.38 4.71 21.70 60.50 5.82Myo2 1.03 4.56 15.16 4.28 31.13Myo3 0.56 5.23 7.03 5.24 19.71 5.76 2.80 68.97 3.46 �0.29 150.0Ave 0.71 5.42 10.65 5.34 21.22 4.71 3.59 59.64 4.12 1.67 139.2SD 0.19 1.06 2.80 0.81 4.76 0.73 0.47 11.03 0.88 17.05 24.3n 8 8 8 8 8 6 6 7 7 6 6P (t-test) 0.009 0.22 0.0004 >0.9 0.001 >0.9 0.044 0.007 >0.9 >0.9 >0.9

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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.

Light microscopyMuscleThe 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 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 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 LGMpatients. Synaptic events recorded in vitro from NMJs in biopsysamples from control and LGM patients. Each panel shows anumber of superimposed traces to indicate observed variability.

Fig. 3 The extent of ‘blocking’ seen in SFEMG in LGMpatients (open symbols) is related to EPP amplitude. Filledsymbol shows average values for eight control subjects.

Fig. 4 Quantal content is reduced at NMJs in LGM patientsrelative to that in controls. Each symbol shows average valuefor 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.

Intramuscular nervesThe 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 terminalsHuman 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 abundancePost-synaptic labelling of AChRs with fluorescent

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 of125I-a-BgTx was studied in two controls and six LGM sam-

ples (Table 5). The mean value of 125I-a-BgTx binding per

NMJ in the LGM samples was �40% of that in controls.

Distribution of ChE activityChE 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 (%) FTIR ChEA (mm2) ChEL (mm) MFDiam (mm) Type 1 (%)

LGMLGM1 4.3 13.7 1.08 109.0 28.3 64.3 62.5LGM2 19.0 22.0 1.12 139.1 39.6 57.8 39.0LGM3 12.0 28.0 1.19 82.1 34.3 62.4 44.0LGM4 11.0 16.0 1.19 78.6 32.0 48.1 18.5LGM5 10.1 40.1 1.23 107.9 40.1 57.6 24.5LGM6 8.7 21.7 1.15 126.1 41.1 62.8 33.5LGM7 2.0 25.5 1.21 82.4 43.9 57.5 19.5LGM8 12.5 33.3 1.20 47.1 32.1Ave 10.0 25.0 1.17 96.5 36.4 58.6 34.5SD 5.2 8.7 0.05 29.6 5.5 5.4 15.7n 8 8 8 8 8 7 7

ControlPain1 0.0 0.0 1.00 198.3 32.3 54.0 44.0Pain2 1.3 13.0 1.14 203.6 37.8 55.7 34.0Pain3 6.0 15.0 1.08 128.4 31.8 62.0 47.5Pain4 6.5 22.0 1.13 165.2 34.0 51.6 28.0Pain5 0.0 14.5 1.17 137.4 27.6 39.5 22.5Myo1 3.5 10.5 1.11 167.1 38.0 47.4 36.5Myo2 9.0 10.3 1.07 249.2 47.6 51.3 16.0Myo3 15.0 18.0 1.11 160.3 39.2 50.4 24.0Ave 5.2 12.9 1.10 176.2 36.0 51.5 31.6SD 5.1 6.5 0.05 39.3 6.1 6.5 10.9n 8 8 8 8 8 8 8P (t-test) 0.08 0.007 0.014 0.0006 >0.9 0.038 >0.9

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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).

Electron microscopyThe 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

membrane immediately adjacent to the nerve (Fig. 6G andH).

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. 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 themost strikingly reduced post-synaptic

folding. The other subject with tubular aggregates was

LGM1, in whom a single small aggregate was seen (Fig. 6G).

Structure–function relationshipsThe 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 functional abnormalities observed in NMJs of

LGM patients might be more specifically correlated with the

structural changes.

Transmitter releaseThe 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 amplitudeOne 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-a-BgTx binding sites per NMJ observed.

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

AAChR ¼ AChE · ððLPost þ ðLFold � LPostÞ/3Þ/LPostÞ:

The result of dividing the amount of 125I-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 5 125I-a-BgTx binding at NMJs in LGM patients and controls

Subject BgTx/NMJ ChEA BgTx/ChEarea AChRarea BgTx/AChRarea·107 sites mm2 ·105 sites/mm2 mm2 ·105 sites/mm2

LGMLGM2 0.24 139.1 0.173 174.35 1.38LGM4 0.90 78.6 1.146 189.40 4.75LGM5 1.26 107.9 1.168 297.93 4.23LGM6 2.04 126.1 1.618 309.75 6.59LGM7 0.46 82.4 0.559 183.93 2.50LGM8 0.65 47.1 1.381 103.36 6.29Ave 0.93 96.8 1.007 209.79 4.29SD 0.65 34.0 0.540 79.28 2.06n 6 6 6 6 6

ControlPain3 2.62 128.4 2.040 446.29 5.87Pain4 2.06 165.2 1.247 477.01 4.32Ave 2.34 146.8 1.644 461.65 5.09SD 0.40 26.0 0.561 21.72 1.10n 2 2 2 2 2P (t-test) 0.03 0.11 0.200 0.006 >0.9

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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

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

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 andG) LGM1, showing variation of extent of folding in the same biopsy sample. (H and I) Distribution ofAChRs, 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

contributing factor.

Protein localization studiesAntibody 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

Subject #NT NTA(mm2)

MITA/NTA PreL(mm)

PostL(mm)

PreL/PostL(‘occ’)

FoldL(mm)

FoldL/PostL(’FI’)

LGMLGM1 1.33 2.63 0.22 5.09 8.67 58.71 47.50 6.08LGM2 1.54 1.94 0.21 3.41 11.73 29.10 20.64 1.75LGM3 1.23 2.20 0.18 4.75 16.40 28.97 54.48 3.97LGM4 1.25 2.54 0.10 4.26 8.53 49.96 44.66 5.06LGM5 1.00 1.66 0.11 2.50 10.90 22.94 68.50 6.46LGM6 0.94 1.28 0.23 3.03 9.97 30.37 53.57 5.71LGM7 1.14 2.68 0.21 3.65 11.66 31.29 54.81 4.70LGM8 1.24 2.95 0.11 4.12 14.17 29.07 65.01 4.82

1.21 2.23 0.17 3.85 11.51 35.05 51.15 4.82SD 0.19 0.57 0.05 0.87 2.69 12.38 14.69 1.48n 8 8 8 8 8 8 8 8

ControlPain1 1.50 1.67 0.09 5.39 13.92 38.74 114.97 8.97Pain2 2.00 1.40 0.13 6.65 15.07 44.15 159.10 11.05Pain3 1.50 0.74 0.06 4.16 11.82 35.17 99.59 8.18Pain4 1.54 1.57 0.13 3.55 14.14 25.13 94.20 6.70Pain5 1.76 1.96 0.14 6.58 16.90 38.93 115.00 7.44Myo1 1.33 1.55 0.11 3.17 10.40 30.51 71.80 7.53Myo2 2.20 1.40 0.19 6.57 14.14 46.49 73.85 6.61Myo3 1.45 2.65 0.06 4.27 12.78 33.40 99.80 7.97

1.66 1.62 0.12 5.04 13.65 36.57 103.54 8.05SD 0.30 0.51 0.04 1.35 1.86 6.57 25.87 1.34n 8 8 8 8 8 8 8 8P (t-test) 0.002 0.039 0.042 0.052 0.083 >0.9 0.0004 0.0006

Fig. 7 Post-synaptic folding is significantly less at NMJs from LGMpatients than at those from controls (Pain, Myo). Fold indexdefined in Patients and methods.

Fig. 8 Quantal content at NMJs in LGM (open symbols)and control patients (filled symbols) is correlated with NMJarea. 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

samples studied.

DNA analysisAnalysis 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 50 to 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 treatmentVarious 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 proteinsat 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.

DiscussionMain findingsThe 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

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 othersOur 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 transmissionin 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

Subject

Ab LGM1 LGM2 LGM7 LGM8

AChE X X X Xs-laminin X X Xa-AChR X X X«-AChR X X XRapsyn X X XNaV1 X X XAnkyrin X X XDystrophin X X XUtrophin X X XSyntrophin Xb-Dystroglycan X Xb-Spectrin X X X XAgrin X X XNeuregulin1 X X X

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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

reduced size.

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

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 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.

AcknowledgementsWe 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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