congenital myasthenic syndromes

Congenital myasthenic syndromesDaniel Hanta a, Pascale Richardb, Jeanine Koeniga and Bruno Eymarda

Purpose of review

Congenital myasthenic syndromes are a heterogeneous group

of diseases caused by genetic defects affecting neuromuscular

transmission. In this article, a strategy that leads to the

diagnosis of congenital myasthenic syndromes is presented,

and recent advances in the clinical, genetic and molecular

aspects of congenital myasthenic syndrome are outlined.

Recent findings

Besides the identification of new mutations in genes already

known to be implicated in congenital myasthenic syndromes

(genes for the acetylcholine receptor subunits and the collagen

tail of acetylcholinesterase), mutations in other genes have more

recently been discovered and characterized (genes for choline

acetyltransferase, rapsyn, and the muscle sodium channel

SCN4A). Fluoxetine has recently been proposed as an

alternative treatment for slow channel congenital myasthenic

syndrome.

Summary

The characterization of congenital myasthenic syndromes

comprises two complementary steps: establishing the diagnosis

and identifying the pathophysiological type of congenital

myasthenic syndrome. Characterization of the type of congenital

myasthenic syndrome has allowed it to be classified as caused

by presynaptic, synaptic and postsynaptic defects. A clinically

and muscle histopathologically oriented genetic study has

identified several genes in which mutations cause the disease.

Despite comprehensive characterization, the phenotypic

expression of one given gene involved is variable, and the

aetiology of many congenital myasthenic syndromes remains to

be discovered.

Keywords

electromyography, genetic diagnosis, microelectrophysiology,

neuromuscular junction molecules, neuromuscular transmission,

treatment

Curr Opin Neurol 17:539551. # 2004 Lippincott Williams & Wilkins.

aInserm U582 and Unite Clinique de Pathologie Neuromusculaire, Institut deMyologie, and bUnite Fonctionnelle de Cardiogenetique et Myogenetique, Hopital dela Salpetrie`re, Paris, France

Correspondence to Daniel Hanta, Inserm U582, Institut de Myologie, Hopital de laSalpetrie`re, 47 Boulevard de lHopital, 75651 Paris Cedex 13, FranceTel: +33 1 42165706; fax: +33 1 42165700;e-mail: [email protected]

Current Opinion in Neurology 2004, 17:539551

Abbreviations

ChAT choline acetyltransferaseCMAP compound muscle action potentialCMS congenital myasthenic syndrome

# 2004 Lippincott Williams & Wilkins1350-7540

IntroductionCongenital myasthenic syndromes (CMSs) form aheterogeneous group of genetic diseases characterizedby a dysfunction of neuromuscular transmission. Thisdysfunction causes muscle weakness, which is increasedby exertion and usually starts during childhood. Theprevalence of CMS is estimated at one in 500 000 inEurope, and CMSs are much more uncommon thanautoimmune myasthenia [1].

Knowledge of the mechanisms underlying CMS hasincreased considerably in the past 25 years, because ofthe pioneering work undertaken by the group of Engel etal. [2]. Acetylcholinesterase deficiency was the first CMSidentified, based on the lack of the enzyme atneuromuscular junctions [2]. Progressively, the patho-physiological heterogeneity of CMS was demonstrated:besides synaptic CMS caused by acetylcholinesterasedeficiency, pre- and postsynaptic CMS were described,the latter including quantitative deficiency or kineticanomalies of the acetylcholine receptor. In the past 15years, many gene mutations responsible for CMS wereidentified, affecting the different acetylcholine receptorsubunits and the collagenic tail of acetylcholinesterase[3,4 ..]. Mutations in the genes for choline acetyltrans-ferase (ChAT) [5], rapsyn [6], and more recently thesodium channel SCN4A have been reported to causeCMS [7.].

Several reviews have been devoted to CMS, one of themore recent being that of Engel et al. [8 ..]. Theobjectives of the present review are to highlight theprincipal phenotypical and pathophysiological character-istics of CMS, to pinpoint the more recent advances inthe field, and to propose a strategy for the accuratecharacterization of these disorders.

Classification of congenital myasthenicsyndromes and recent findingsThe current classification of CMS is based on patho-physiology, i.e. on the precise identification of theneuromuscular transmission anomaly. The location ofthe dysfunction of neuromuscular transmission (Fig. 1)[9], which is specific to the different CMSs, is eitherpresynaptic (generally caused by an anomaly of ChAT),synaptic (corresponding to an anomaly of the acetylcho-linesterase collagen tail), or postsynaptic (secondary to ananomaly of acetylcholine receptor or rapsyn). In theexperience of Engels group, postsynaptic CMSs arethree times more frequent than acetylcholinesterasedeficiency and 10 times more frequent than presynaptic

539

CMS [4..]. The different classes and subclasses of CMSwill be described below with reminders of their firstdescriptions and main characteristics and with anemphasis on the latest findings.

Presynaptic congenital myasthenic syndromes

Among the presynaptic CMSs, only those caused bymutations in the ChAT gene have been fully character-ized, the others remain to be defined at the geneticlevel.

Congenital myasthenic syndromes caused by ChAT mutations

These CMSs usually manifest at birth or in the neonatalperiod with bulbar disorders and respiratory insufficiencywith apnoea [10,11] or even sudden death [11,12]. Theseepisodes are triggered by fever, fatigue and overexertion.Apart from these bouts, the myasthenic signs are oftenmodest or not present. Cholinesterase inhibitors areeffective. Microelectrophysiology shows, after prolonged10 Hz repetitive stimulation, a reduction in amplitude of

the miniature endplate potentials. These anomalies arecharacteristic of a defect in the resynthesis of acetylcho-line or in the filling of synaptic vesicles [10]. Ultra-structural examination shows that, when muscle is atrest, the synaptic vesicles are of reduced size.

Ohno and collaborators [5] described the first mutationsin CHAT, the gene encoding ChAT and located in10q11.2. ChAT is a presynaptic protein localized in thenerve terminals, where it catalyses acetylcholine produc-tion. As shown in knockout mice, ChAT affectssynaptogenesis and coordinates synaptic maturation[13]. Mutations lead to a reduction or even abolition ofthe catalytic capacity of the enzyme [5]. Fourteenmutations have been reported to date, mostly of themissense type [5,14 .,15.]. Recent structural studiesindicate that whereas half of the missense mutationsare positioned in the molecule such that they affectenzyme activity directly, the remaining mutations aredistant from the active site and must exert indirect

Figure 1. Pathophysiological classification of congenital myasthenic syndromes

axon terminal

basal lamina

AChR

rapsyn

muscle fibre

Na+ channel

ACh

ChAT

AChE Q, T

Presynaptic defectsDefects in ACh resynthesis (AR) CHATPaucity of synaptic vesiclesLambert-Eaton like CMS

Synaptic defectsEndplate AChE deficiency (AR) COLQCOLQ

CHAT

Postsynaptic defectsAChR kinetic anomalies slow channel syndrome (AD) >, . fast channel syndrome (AR)AChR deficiency (AR) AChR >, . rapsyn (AR) RAPSNAnomaly of muscle Na+ channel -subunit SCN4A

RAPSN

SCN4A

> , ,

> ,,

Incompletely characterized CMSCMS with plectin deficiencyFamilial limb girdle myastheniaCMS with tubular aggregates

No identified defects

CHRNB1CHRND

CHRNA1CHRNE

The eight genes involved in congenital myasthenic syndromes (CMSs) are named CHRNA1, CHRNB1, CHRND, CHRNE, COLQ, CHAT, RAPSNand SCN4A. The coded protein and the gene location are, respectively, as follows: (1) a (2q24q32), b (17p11p12), d (2q33q34), e (17p13)subunits of acetylcholine receptor (AChR); (2) collagenic tail (ColQ) (3p242) of acetylcholinesterase (AChE); (3) ChAT (10q11.2); (4) rapsyn(11p11). The endplate species of acetylcholinesterase is composed of one, two, or three homotetramers of T globular catalytic subunits attached to acollagenic tail (ColQ), anchoring them in the synaptic basal lamina. Rapsyn stabilizes the acetylcholine receptor aggregates and links them to thepostsynaptic cytoskeleton. Heredity is either autosomal recessive (AR) or autosomal dominant (AD). Modified from the classification proposed by theEuropean Neuromuscular Center (ENMC) [9].

Neuromuscular disease: muscle540

effects [16 ..]. The possibility that the I336T ChATmutation found in three consanguineous Turkishfamilies was a founder was postulated [14 .].

Other presynaptic myasthenic syndromes still incompletely

characterized

The paucity of synaptic vesicles was described in onepatient with early-onset CMS. The density of acetylcho-line synaptic vesicles was reduced by 80% and thenumber of quanta released was drastically reduced [17].The exact cause of this CMS is still unknown.

The first case of LambertEaton-like CMS was firstreported in a child [18]. His myasthenic syndrome wascharacterized by a good response to guanidine and byelectrophysiological anomalies identical to those of aLambertEaton syndrome, i.e. diminished action poten-tials markedly potentiated by tetanic stimulation. Asecond case presented with severe hypotonia andrespiratory distress at birth [4..]. No mutation was foundin the gene coding for the presynaptic calcium channel.

Three patients were reported with a sporadic myasthenicsyndrome with associated signs of attack of the centralnervous system (cerebellar ataxia or nystagmus) [19].None presented, as in the LambertEaton syndrome,with a reduction of the action potentials or withpotentiation after high frequency stimulation. Microelec-trophysiology revealed a marked reduction in thespontaneous or nerve stimulation-induced release ofacetylcholine quanta.

Synaptic congenital myasthenic syndrome:

acetylcholinesterase deficiency

CMSs caused by acetylcholinesterase deficiency werefirst described in 1977 [2]. Since then, many cases ofpartial or complete deficiency of the enzyme located inthe synaptic basal lamina have been reported [20]. Thefirst symptoms usually arise in the neonatal period, andthe symptoms are severe with a significant lethal risk.However, the disease may start later, during infancy, andis not so severe. Several observations point to thediagnosis of acetylcholinesterase deficiency: autosomalrecessive heredity, repetitive compound muscle actionpotential (CMAP) after single stimulation (Fig. 2), theabsence of response to cholinesterase inhibitors, andslowed [20] but inconstant [21] pupil responses to light.

Diagnosis using muscle biopsies is indicated by no orpoor visualization of acetylcholinesterase at the neuro-muscular junction. Acetylcholinesterase deficiency isrelated to mutations in the COLQ gene coding for thecollagenic tail of acetylcholinesterase [2224]. At theneuromuscular synapse, acetylcholinesterase is presentas asymmetric acetylcholinesterase, which is made up ofthree homotetramers each comprising four globular

catalytic subunits linked together by a collagenic tail(ColQ; Q for queue in French, which means tail) oftrimeric helicoidal structure. The collagenic tail concen-trates and anchors the enzyme within the synaptic basallamina.

Twenty-four recessive mutations have been described todate (Fig. 2). They are more often homozygous thanheterozygous, and nonsense than missense [4 ..]. Thefact that the same homozygous G240X mutation wasdetected in several Arab families and in one Iraqi Jewishpatient suggests that it is not uncommon in Near andMiddle East countries [25]. Depending on their localiza-tion, COLQ mutations have different consequences: inthe N-terminal proline-rich attachment domain, theyprevent attachment of the acetylcholinesterase catalyticsubunits to the collagenic tail; in the mid-part theyprevent the trimerization of the collagenic tail; in the C-terminal domain they most often impair anchoring of theenzyme within the synaptic basal lamina [24,26,27.,28.].This impaired attachment of the collagenic tail to thesynaptic basal lamina has recently been elegantly andcomprehensively tested using purified C-terminal do-main mutant ColQ applied to frog neuromuscularjunctions [29 ..].

To date there is no effective treatment for this type ofCMS.

Postsynaptic congenital myasthenic syndromes

Among these postsynaptic CMSs, two categories aredescribed: CMS in connection with a kinetic anomaly ofthe acetylcholine receptor; and CMS with a decreasednumber of acetylcholine receptors at the neuromuscularjunction. In the latter category, besides the CMSs withacetylcholine receptor deficiency as a result of numerousmutations in the different acetylcholine receptor subunitgenes, those caused by mutations in the rapsyn genewere identified recently [6] and are far from infrequent.

Congenital myasthenic syndrome caused by acetylcholine

receptor kinetic anomalies

Slow channel syndrome is the most frequent kineticanomaly of the acetylcholine receptor. This entity, ofautosomal dominant inheritance, is characterized by aprolonged opening time of the acetylcholine receptor[30]. Fifteen autosomal dominant missense point muta-tions causing a gain of function of the acetylcholinereceptor were identified [4..]. Although most of themutations were found in the acetylcholine receptor asubunit [31], other subunits are also concerned [32]. Themutations are located in two transmembrane domainstaking part in the formation of the acetylcholine receptorpore through which passes the sodium flux [33], M1 formutations of the a and b subunits and M2 for those,more frequent, affecting the a, b, d and e subunits [34];

Congenital myasthenic syndromes Hanta et al. 541

an area of the extracellular domain of the a subunit closeto the acetylcholine binding site (mutations aG153S andaV156M) [4 ..].

The functional consequences of the various mutationswere studied in intercostal muscle biopsy or byexpressing the mutation in cell systems [35]. Theprolonged opening time of the acetylcholine receptor isdependent either on the slowed closing of the channel oron the increased affinity of the acetylcholine receptor forits ligand [36]. In addition, a new mechanism thatinvolves not only delayed closure but also delayedopening of the channel in the case of a dS268F mutationwas recently published [37].

Clinical expression may vary from early onset andsevere to late onset and moderate [30,38]. Thearguments in favour of the diagnosis are autosomal

dominant heredity, no response to cholinesteraseinhibitors, and repetitive CMAP after a single stimula-tion. The last two characteristics are also found inacetylcholinesterase deficiency (Fig. 2). The selectivityof muscle involvement with a prevalent atrophic deficitof the finger extensors and of the cervical muscles issuggestive of slow channel syndrome. Remodelling ofthe ultrastructure of the endplate is observed withcalcium deposits, destruction of the postsynaptic folds,vacuolizations and tubular aggregates [30]. The diag-nosis leads to the therapeutic use of quinidine, a blockeragent able to normalize the acetylcholine receptoropening time [39]. Fluoxetine has recently been shownto be an alternative to quinidine when the latter is nottolerated by the patient [40 .].

A peculiar case has been reported of a slow channelsyndrome with recessive transmission, occurring in a

Figure 2. Main features of slow channel syndrome and of acetylcholinesterase deficiency

Acetylcholinesterase deficiency- autosomal recessive

- slowed pupil response

-BGT fasciculin

control

patient

AChR AChE

AChR AChE

COLQ

In common

- no response to cholinesterase inhibitors

- repetitive CMAP after single stimulation

1 2 3 4 5 6 7 8 9 102 mV 2 ms

Repetitive CMAP

Slow channel syndrome- autosomal dominant

CHRNA1

AChR

M1 M2 M3 M4

s-s

NH2COOH

In both diseases, cholinesterase inhibitors are inefficient, and a repetitive motor response is evidenced by electrophysiological study. Recessivelytransmitted endplate acetylcholinesterase deficiency is demonstrated by the absence of fluorescent fasciculin staining at the endplate. Collagenic tailgene mutations will be determined. Slow channel syndrome is transmitted as an autosomal dominant trait. The most common mutations involveacetylcholine receptor a subunit in the pore region (transmembrane M1 and M2 domains) or in the vicinity of the acetylcholine binding site. Othermutations not shown here are within the b, d and e subunits. AChE, Acetylcholinesterase; AChR, acetylcholine receptor; a-BGT, a-bungarotoxin;CMAP, compound muscle action potential.


consanguineous family in connection with a homozygousmutation of the e subunit (eL78P) located in theextramembrane region. This mutation was pathogeniconly if present on two alleles [41]. In addition, a slowchannel syndrome associated with a chromosomaltranslocation 2q319p27 was described [42].

Fast channel syndromes are of autosomal recessivetransmission, although a case of autosomal dominanttransmission was reported recently [43.]. The diagnosisis made by microelectrophysiology showing a shorteningof the acetylcholine receptor opening time [44]. Clinicalseverity is variable. Arthrogryposis was reported in onecase [45]. The patients are responsive to the combina-tion of 3,4-diaminopyridine and cholinesterase inhibitors.Eight mutations were identified affecting a, d and esubunits and are located either in the extracellulardomain, in the M3 transmembrane domain (mutationaV285I), or in the cytoplasmic loop between the M3 andM4 domains (e mutations only) [8 ..]. Of the twomutations present in the patient, one is a nonsensemutation whereas the other is responsible for the kineticanomalies. This second mutation can modify the kineticsof the receptor by various mechanisms that can bedetermined on intercostal biopsy or after in-vitroexpression in cell models. A recent article thus detailsthe detrimental effects of a V132L mutation located inthe acetylcholine receptor a subunit within the signaturecystine loop on acetylcholine binding and channel gating[46 ..]. The different mechanisms underlying fast chan-nel syndromes are the topic of a recent review [47.].

Congenital myasthenic syndromes with predominant

acetylcholine receptor deficiency (with absent or only slight

kinetic anomalies)

These account for approximately half of CMS patients[4..]. The majority are related to mutations of theacetylcholine receptor. No peculiar clinical findings pointto this type of autosomal recessive CMS, whose severityis variable. Nevertheless a founder effect in the Gypsypopulation of the e1267delG mutation has been pro-posed [48]. An extensive study on five disease loci in thedifferent Gypsy groups has demonstrated a strongfounder effect and a carrier rate of 3.74% for thismutation [49].

Cholinesterase inhibitors are most often active and 3,4-diaminopyridine can provide additional benefit.

The described mutations are numerous (60 or more),either homozygous or heterozygous [4 ..]. They are of alltypes: missense mutations, chromosomal deletions,insertions, deletions. The mutations are located on thewhole gene encoding the acetylcholine receptor esubunit, most being located in the extracellular domainand in the cytoplasmic loop between the M3 and M4

transmembrane domains [4 ..]. Recently, a chromosomalmicrodeletion was identified for the first time in CHRNE[50], showing that this type of mutation may be missedby standard screening techniques. Lately a frameshiftmutation in exon 7 of CHRNE (e553del7) was shown toprovoke skipping of the preceding exon both in muscletissue and when expressed in COS cells [51 .].

Mutations in the promoter were also described [52,53].Interestingly, the injection of the corresponding recom-binant in the rat allowed the authors to demonstrate thata mutation in an N-box of the CHRNE promoter leads toless acetylcholine receptor synaptic expression [50].Another experimental approach, namely the cell expres-sion of green fluorescent tagged acetylcholine receptor,allowed others to show that mutations affecting cysteine470 of the e subunit prevent acetylcholine receptorsurface expression [54].

More rarely, other subunits of acetylcholine receptor a, band d subunits are implicated [4..]. The preponderanceof mutations of the e subunit may be caused by thepossibility of the re-expression of the g fetal acetylcho-line receptor isoform in the case of null mutations ofCHRNE [55,56].

Curiously, CMS not only affects humans but also SouthAfrican Red Brahman calves. These calves suspected ofmyasthenia have been shown to bear a homozygous 20basepair deletion mutation in bovine CHRNE. Thismutation leads to a non-functional allele and a severephenotype [57,58.].

Congenital myasthenic syndromes with mutations of the rapsyn

gene (RAPSN)

These were first identified in 2002 [6]. Rapsyn is a43 000 Mr postsynaptic cytoplasmic protein, whichparticipates in acetylcholine receptor assembly at theneuromuscular junction [59] and allows its anchoring tothe cytoskeleton by b-dystroglycan among other mole-cules [60]. Most mutations of this gene located in 11p11were identified in the tetratricopeptide repeat domain,and cell expression studies revealed that the co-expression of mutant rapsyn and acetylcholine receptorsubunits impair the recruitment of acetylcholine recep-tors to rapsyn clusters, an essential step for the anchoringof the acetylcholine receptor to the cytoskeleton [6].These mutations are responsible for a reduction ofrapsyn and consequently of the acetylcholine receptoritself at the neuromuscular junction. The reduction inrapsyn expression is not specific, because it is alsoobserved in primary acetylcholine receptor deficiencies.The inheritance of this CMS is autosomal recessive.

Since the first four cases were published, nearly 50 othercases have been reported [6163,64.,65,66 .,67.,68]. Half


of them bear the homozygous N88K. The other halfbears N88K on one allele and a second mutation on theother allele. This second mutation is localized all alongthe rapsyn molecule, and nearly 20 different mutationshave been identified to date (Fig. 3a) [6]. Missensemutations predominate (approximately two-thirds ofcases). When the second mutation is not identified bydirect sequencing, the search for a chromosomal micro-deletion of RAPSN is recommended [69].

Two E-box mutations were identified in the rapsynpromoter [64.] (Fig. 3b). Seven of the eight patientsreported originated from the Jewish population of Iraqand Iran and had already been described for theirpeculiar clinical phenotype: benign CMS with facialmalformations (mandibular prognathism, elongated face)[70].

A founder effect of the frequently identified N88Kmutation is likely at least in the European or Indo-European population [61,62,71..], although the exis-

tence of other founders has been proposed [72]. Thehigh frequency of the N88K mutation may lead to casesof pseudo-dominant inheritance.

Genotypephenotype correlation is not easy. Analysisof the corpus of clinical observations confirms theexistence of two phenotypes: (1) a neonatal form,even antenatal (with arthrogryposis multiplex congeni-ta), with major respiratory disorders and severeprogression of the disease; and (2) mild formsbeginning during childhood or in adulthood. On thebasis of 16 cases, a distinction between early and lateonset was proposed [66.]. The importance of theidentification of the late-onset cases is to avoidimproper immunotherapy. Patients with the rapsynmutation responded well to cholinesterase inhibitors[66 .] or to the combination of cholinesterase inhibitorsand 3,4-diaminopyridine [67.].

In summary, mutations in RAPSN and the resultingrapsyn deficiency appear to be an important cause of

Figure 3. Diagram depicting the main domains of rapsyn and the localization of the identified mutations

Tetratricopeptide repeats (TPR) necessaryto rapsyn self-association

AChR

TPR1 TPR2 TPR3 TPR4 TPR5 TPR6 TPR7 coiledcoil RING

Q3K

L14P

46in

sCA

25V

F81

LY

86X

N88

KR

91L

C97

X

Q12

4X

A14

2DR

151P

V16

5M

553i

ns5

IVS

4-2A

G

A24

6V

Y26

9X

G29

1D

E33

3X

1083

_108

4dup

CT

1177

delA

A

(a)

(b)

38AG 27CG

E box E box

-d

ystr

ogly

can

(a) Seven tetratricopeptide repeat domains (TPR17) are necessary for rapsyn to self-associate. The coiled-coil domain binds to the large cytoplasmicloop of the acetylcholine receptor (RACh) subunits. The RING domain binds rapsyn to b-dystroglycan. A serine phosphorylation site is located atcodon 406. Modified from Ohno et al. [6]. (b) Two E-box (CANNTG-type sequence, on grey background), to which myogenic factors can bind arelocated upstream of the transcription initiation site in the rapsyn gene promoter region. Localization of the two different rapsyn promoter mutations.Adapted from Ohno et al. [64.].


CMS associated with endplate acetylcholine receptordeficiency.

Congenital myasthenic syndrome with plectin deficiency

Plectin is a highly preserved structural protein of thecytoskeleton expressed in several cell types, includingskeletal muscle and the postsynaptic membrane. Plectindeficiency was described in a patient presenting withprogressive myopathy, associated with myasthenic syn-drome (involving facial, limb and oculomotor muscles),and epidermolysis bullosa [73]. The pathophysiology ofthis CMS is poorly understood.

Congenital myasthenic syndrome caused by a mutation in the

sodium channel SCN4A

The case was recently reported of a 20-year-old patientpresenting since birth with very short bouts (330 min)of respiratory distress and bulbar paralysis [7 .]. Thediagnosis was made by electrophysiology of the inter-costal muscle, which revealed the impossibility ofevoking an action potential after nerve stimulation.Two mutations of SCN4A were identified, includingonly one (V1442E) located in the S3/S4 extracellulardomain, which was found to be pathogenic whenexpressed in HEK cells. The clinical aspect is quitedifferent from that usually associated with a SCN4Amutation (dyskalemic paralysis, congenital paramyot-ony).

Incompletely characterized congenital myasthenic

syndromes

These CMSs are described on clinical or histologicalgrounds, but their molecular origin and more generallytheir pathophysiology remain unknown in the absence ofan exhaustive exploration.

Familial limb girdle myasthenia

Several families have been reported [74,75]. Thispreviously named myasthenic myopathy is of recessiveinheritance. Clinically, the absence of oculobulbar signswas remarkable. The weakness and fatigability involvedthe girdles. The peculiarity of this not yet understoodentity was recently stressed with the publication of fivecases, who all presented with tubular aggregates in theirmuscle biopsy and who all responded favourably tocholinesterase inhibitors [76].

Congenital myasthenic syndrome with tubular aggregates

This CMS is associated with tubular aggregates at thehistological muscle examination. The case of threesisters presenting with a slowly progressive myopathybeginning in early childhood associated with cardio-myopathy was reported. A favourable response tocholinesterase inhibitors was noted [77]. Similar char-acteristics were described in another family [75]. Asporadic case was reported recently [78]. In the absence

of thorough investigations of neuromuscular transmis-sion, the classification of these cases remains delicate,more especially as the presence of tubular aggregates isnot specific and can be associated with isolatedmyopathy, painful cramps [79] and with slow channelsyndromes [31].

Approach to the diagnosis of congenitalmyasthenic syndromesOn the basis of these historical advances in the knowl-edge of CMS, the diagnostic strategy includes roughlytwo successive steps: (1) the association of a clinical-electrophysiological picture of a myasthenic syndrome,and data in favour of a congenital origin; and (2) therecognition of the pathophysiological type, which isbased on clinical data, the type of hereditary transmis-sion, the response to cholinesterase inhibitors, the resultsof electromyography, and finally the muscle biopsy andmolecular genetics. The sequential order of these twolast investigations depends upon the initial clinical-electromyographical data.

Clinical presentation

The various CMSs share a common clinical presenta-tion. The onset is in general early. Late appearance ofthe symptoms during adolescence, or even in the adult,is more rarely reported. Some clinical signs suggest ananomaly of neuromuscular transmission: ophthalmople-gia and ptosis, dysphonia and swallowing disturbance,facial paresis, and muscle fatigability. In the young child,the ptosis is not easy to recognize because hypotonia,poor mimicry, suction disorders, and weakness of the cryare in the foreground. The occurrence of bouts andworsening by exertion are characteristics of the disease.The favourable effect of cholinesterase inhibitors is asignificant argument in favour of a myasthenic syn-drome. However, two types of CMS are worsened bycholinesterase inhibitors: slow channel syndrome andacetylcholinesterase deficiency. With the propermyasthenic signs, myopathic signs are often associated:amyotrophy, tendinous retractions, facial malformationand scoliosis. The severity of the CMS is variable,depending upon the severity of the walking deficit, thebulbar disorders and the respiratory difficulties. Acuterespiratory failure may occur, triggered by infectiousepisodes, and is frequent in the first months of life. Inthe absence of respiratory assistance, the risk of death ishigh [12].

A family history of the disease is an essential argument infavour of the genetic origin of myasthenic syndrome.Most CMSs are of autosomal recessive inheritance. Slowchannel syndrome is the only autosomal dominant CMScharacterized hitherto. The progression patterns ofCMSs are highly variable, including in a given patient,at various periods of life. Myasthenic bouts are fre-


quently triggered by infectious episodes, pregnancy andeven periods. Progressive aggravation of the disease maysometimes occur late in adulthood, with the appearanceof respiratory insufficiency [27 .]. A favourable progres-sion is possible after a severe neonatal onset [4..].

Titration of anti-acetylcholine receptor antibodies in the

serum

The absence of antibodies against acetylcholine receptorand muscle-specific receptor tyrosine kinase [80,81.] is aprerequisite for the diagnosis of CMS, although anexception was reported [82 .].

Electromyography

The electrophysiology of neuromuscular transmission isthe determinant for the diagnosis of CMS. This includessearching for neuromuscular block, repetitive motorresponses and increments [83,84]. The observation of aneuromuscular transmission block affirms the myasthe-nic syndrome. The decrement can be absent in CMS,particularly in patients who are not highly symptomatic,and in cases of CMS caused by mutations in ChAT [5] orrapsyn [6]. In the case of a ChAT deficiency, thedecrement may appear only after an initial 5-min 10 Hzstimulation [14 .]. Repetitive CMAPs are pathognomonicof two varieties of CMS: slow channel syndrome andacetylcholinesterase deficiency (Fig. 2). The search foran increment is imperative. An increment greater than100% in amplitude and in area is suggestive of apresynaptic origin.

Muscle biopsy

A first role of the muscle biopsy is to eliminate thediagnosis of myopathy (congenital myopathy or mito-chondrial cytopathy). Although non-specific, the pre-dominance of type I fibres and the marked atrophy oftype II fibres is suggestive of CMS. The presence oftubular aggregates is frequent, but poses the problem ofthe group of CMSs with tubular aggregates. Theneuromuscular junctions are visualized for (acetyl)choli-nesterase by the histochemical technique of Koelle,fasciculin or specific antibodies, and for acetylcholinereceptor by fluorescent a-bungarotoxin, which binds toit. The neuromuscular junctions frequently exhibitvariable anomalies: reduced size, the disappearance ofsynaptic folds, all modifications are not specific, however,to a given CMS.

Two types of information are decisive: (1) the absence ofacetylcholinesterase at the neuromuscular junctionestablishes the diagnosis of acetylcholinesterase defi-ciency; a study by ultracentrifugation on sucrose gradientwill generally reveal the absence of asymmetrical(synaptic) forms of the enzyme; and (2) a significantreduction in the number of acetylcholine receptors,further quantified by binding with iodinated a-bungar-

otoxin, points to a primary anomaly of acetylcholinereceptor or rapsyn.

The expression of the fetal g subunit of the acetylcho-line receptor argues in favour of a primary anomaly ofthe acetylcholine receptor e subunit [55,56]. Immuno-cytochemical study of the expression of other markers ofthe neuromuscular synapse can also be performed: agrin,muscle-specific receptor tyrosine kinase, rapsyn, neur-egulin, a-dystrobrevin or utrophin [85]. It is aetiologi-cally suggestive if there is a major and selectivereduction of the expression of a protein, but the primarynature of the deficit is, however, not established (adeficit in rapsyn is found in CMS with mutations in boththe rapsyn gene and the acetylcholine receptor subunitgenes).

Molecular genetics

The diagnosis of CMS can be confirmed by molecularanalyses in the eight genes whose mutations are so farknown to cause CMS: four genes encoding the variousacetylcholine receptor subunits (CHRNE, CHRNA1,CHRNB1, CHRND), the genes encoding rapsyn(RAPSN), the collagen tail of acetylcholinesterase(COLQ), choline acetyltransferase (CHAT), and thesodium channel (SCN4A). With the exception of theGypsy e1267del mutation [48] and the RAPSN N88Kmutation [6,71 ..], a search has been made to identifyprivate mutations. Analysis of the coding sequencesand flanking intronic regions by direct sequencing afterpolymerase chain reaction amplification of each fragmenton genomic DNA is required. Many mutations havebeen described to date, and the two predominant genesin postsynaptic CMS appear to be CHRNE and RAPSN.In approximately half of the cases, the analysis of thesegenes does not identify a mutation causing the disease,suggesting that other genes could be involved. When anew mutation is identified, its pathogenic character canbe demonstrated by expression studies of this mutationin HEK cells, COS cells or oocytes, but other experi-mental models can also be used.

Microelectrophysiology of neuromuscular transmission

Microelectrophysiology of the intercostal muscle can beused to specify the pre- or postsynaptic location of thedysfunction of neuromuscular transmission, and inpostsynaptic CMS, to find kinetic anomalies of theacetylcholine receptor [4..]. The complexity of thesetechniques (patch clamp) and the risks of generalanaesthesia in a myasthenic patient limit the indicationsfor this exploration, more especially because the expres-sion of the mutations in experimental cell systems byitself allows the pathophysiological characterization ofCMS. In addition, the study of other muscles under localanaesthesia was proposed: quadriceps [86] and ancone[15].


Difficulties of diagnosisThe diagnosis of CMS is not always easy. Faced withsporadic CMS beginning after the neonatal period, thediagnosis of seronegative autoimmune myasthenia maybe difficult to eliminate, more especially as long periodsof remission are possible in both afflictions and bouts canoccur in the adult CMS patient during pregnancy [38].Muscle-specific receptor tyrosine kinase (MuSK) anti-bodies have been detected in more thanhalf of the patients presenting with seronegative(no acetylcholine receptor antibodies) auto-immunemyasthenia [80,81 .]. In case of uncertainty, the absenceof MuSK antibodies must be verified before establishinga diagnosis of CMS.

Three recent observations have stressed that it issometimes difficult to draw clear boundaries betweenautoimmune myasthenia and CMS [82,87,88.]. The firstreported two sisters carrying heteroallelic mutations ofthe acetylcholine receptor a subunit, both presentingwith neonatal myasthenic syndrome, but one developedautoimmune myasthenia as an adult, attested by thetransitory presence of anti-acetylcholine receptor anti-bodies and a favourable response to plasmaphereses andcorticotherapy [82]. The authors suggested that thegenetic anomaly of the acetylcholine receptor couldconstitute the factor triggering autoimmune myasthenia.The second observation concerned a patient presentingwith acquired slow channel syndrome beginning at 30years of age. The passive transfer of the serum of thispatient to a mouse reproduced kinetic anomalies of theacetylcholine receptor, which demonstrated its autoim-mune origin and excluded a congenital affliction [87].The third observation reported an acetylcholine recep-tor-seronegative, MuSK-seropositive myasthenia gravispatient, in whom no acetylcholine receptor or MuSKdeficiency was found in muscle biopsies despite theelectrophysiological impairment. Mutation analysis ofMUSK did not reveal mutations but polymorphisms. Theauthors concluded that circulating anti-MuSK antibodiesmay not have caused the myasthenic syndrome in thispatient [88.].

Phenotypegenotype correlations andprognosisThe genotype and clinical phenotype are not correlatedin CMS. Mutations in different synaptic proteins givesimilar clinical pictures: the occurrence of apnoeicepisodes in early childhood was reported in CMSs witha deficit in ChAT, in those caused by primary anomaliesof the acetylcholine receptor, of the acetylcholinesteraseor of rapsyn. Arthrogryposis has been described in CMScaused by mutations in the gene encoding rapsyn [66.]and the acetylcholine receptor d subunit [45]. The samemutation could lead to very different clinical phenotypes:for example, the homozygous N88K rapsyn mutation

leads either to a very severe neonatal form or to a late-onset and benign form [6,66 .]. Finally, variability within afamily was noted in certain cases of CMS.

Prognosis is difficult to assess. A favourable outcome ispossible in cases of CMS initially thought to be severebecause of respiratory or bulbar bouts. In contrast, motorand respiratory degradation occurring late in adulthoodhas been reported in patients initially only slightlyaffected [27.]. As indicated above, knowledge of theprimary molecular anomaly of CMS does not enable theprediction of disease progression. The response totreatments known to ameliorate neuromuscular transmis-sion is a significant prognostic factor: thus in acetylcho-linesterase deficiency, the absence of amelioration bycholinesterase inhibitors or any other drug may bealarming.

TreatmentNon-specific measures are essential: immediate treat-ment of respiratory distress, the prevention of infectionsand of malnutrition as a result of swallowing disorders,and orthopaedic surveillance of spinal complications andretractions. Drug contraindications must be respected asfor any other myasthenic syndrome. In the case of CMS,there is no reason to apply the immunosuppressivetherapy used for myasthenia gravis. Cholinesteraseinhibitors are efficient in all CMSs, with the exceptionof slow channel syndrome and acetylcholinesterasedeficiency, which they can even worsen. They exert apreventative effect on the respiratory decompensationsof CMS caused by ChAT mutations [4 ..]. 3,4-Diamino-pyridine, whose mode of action is presynaptic, issometimes effective in pre- or postsynaptic CMSs [39].Patients suffering from slow channel syndrome benefitfrom the regulatory action of acetylcholine receptorblockers: quinidine is effective by correcting theprolonged opening of the acetylcholine receptor [89],but is formally contraindicated in all the other forms ofCMS. A favourable effect of fluoxetine was recentlydemonstrated in some patients, and is of interest despitethe large amount needed [40 .]. At present, there is nospecific treatment for acetylcholinesterase deficiency.

ConclusionAlthough the epidemiology of the CMSs is poorlyunderstood, these disorders constitute the major causeof the myasthenic syndrome in the young child and are aminor cause of adult myasthenic syndrome. Thediagnosis is often difficult to ascertain because of thefrequent absence of a family history of the disease, andbecause of the pre-eminence of the myopathic signscompared with myasthenic signs. The early onset of thefirst symptoms, the presence of fluctuations, thedemonstration of a neuromuscular block, repetitiveCMAP after single stimulation, and the cholinesterase


inhibitor test all enable the rectification of the diagnosisand the proposal of an effective treatment and geneticcounseling. The numerous studies devoted to CMS overmore than 20 years have demonstrated the patho-physiological heterogeneity of CMS. Characterizationof the CMS is based on the mode of transmission, thesearch for a CMAP, the positive or negative response tocholinesterase inhibitors, the study of motor endplates,which is easily done on a deltoid muscle biopsy, andmolecular genetics. It will thus be possible to identifythe majority of CMSs: a primary anomaly of one of thevarious acetylcholine receptor subunits, of rapsyn,acetylcholinesterase, ChAT or even SCN4A. However,the origin of a significant fraction of CMSs remainsunknown. Numerous molecules of the neuromuscularjunction are potential candidates for CMS and may be

tested (Fig. 4) [90]. Therefore, in non-identified cases,various investigations will be used: genetic linkageanalysis in the case of large families, the demonstrationin the muscle biopsy of a selective deficit in one givensynaptic molecule, or microelectrophysiology of theintercostal muscle. Collaboration between clinicians,morphologists, geneticists, and neurobiologists is essen-tial for a complete characterization of the CMSs and forthe understanding of the fundamental mechanisms ofneuromuscular transmission based on human pathology.

AcknowledgementsWe thank Claire Legay and Hanns Lochmuller for critical reading of themanuscript. This study was supported by the Direction de la RechercheClinique de lAssistance Publique, Hopitaux de Paris (PHRC #AOM01036), Association Francaise contre les Myopathies, GIS-MaladiesRares, and Reseau Inserm de Recherche Clinique.

Figure 4. Neuromuscular junction molecules that are or might be involved in congenital myasthenic syndrome

-CGRP

acetylcholineChAT

2, 4, 2, 1 laminin 3, 4, 5 type IV collagenAChE Q, Tagrinneuregulin

ankyrindystrophin

Na+ channel

AChR- , , , 7A, B, 1 integrin , dystroglycanMuSKErbB2, ErbB4

rapsynutrophin

plectin

K+ channelCa++ channel

ErbB3

synaptobrevinsynaptotagminsynaptophysin

Molecules with proved implication in congenital myasthenic syndromes are indicated in bold letters. The molecules, the genes of which are potentialcandidates, are indicated in normal letters. AChE, Acetylcholinesterase; AChR, acetylcholine receptor; MuSK, muscle-specific receptor tyrosinekinase. Adapted from Sanes and Lichtman [90].


References and recommended readingPapers of particular interest, published within the annual period of review, havebeen highlighted as:. of special interest.. of outstanding interest

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