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Congenital Myasthenic Syndromes

  • David Beeson
Chapter
Part of the Current Clinical Neurology book series (CCNEU)

Abstract

The congenital myasthenic syndromes (CMS) are rare hereditary disorders of neuromuscular transmission. They are characterized by fatiguable fluctuating muscle weakness that can vary in severity. Diagnosis is important since these are treatable conditions, and appropriate medication and interventions may avert life-threatening respiratory crises that occur in some syndromes. The advent of next-generation sequencing has facilitated the discovery of many genes that harbor CMS-associated mutations. Up to 30 different genes may be involved. These may encode proteins directly involved in signal transmission or in controlling the formation and maintenance of the neuromuscular synapse, and the severity of the disorders can vary from fatality in utero or the neonatal period to near-asymptomatic weakness. Recent findings show that many genes whose functions are not restricted to the neuromuscular junction can also cause myasthenic weakness, such as genes involved in protein glycosylation pathways or neurotransmitter release. In some of the more recently identified syndromes, the myasthenic weakness is only one component of a more complex phenotypic spectrum, which introduces additional challenges in patient treatment.

Keywords

Congenital myasthenic syndromes Neuromuscular junction Synaptic transmission Synaptic stability AChR Neurotransmitter release Glycosylation Ion channel kinetics 

Introduction

The congenital myasthenic syndromes (CMS) are rare inherited disorders of neuromuscular transmission characterized by fatiguable muscle weakness [1, 2]. Their overall prevalence is uncertain but is thought to be in the order of 1 in 100,000 of the population in the UK [3]. They are genetically determined (usually autosomal recessive, so a history of consanguinity is common), non-autoimmune disorders. Major clinical features include onset in infancy, fatiguable weakness, a decremental response to repetitive nerve stimulation, and absence of autoantibodies to the muscle acetylcholine receptor (AChR) or muscle-specific tyrosine kinase (MuSK) or low-density lipoprotein receptor-related protein 4 (LRP4), although a clear pathogenic role for LRP4 antibodies in myasthenia gravis patients has not yet been demonstrated. Remarkable differences in severity occur even within families harboring the same mutation. Although impairment of neuromuscular transmission may often give rise to similar clinical presentation, detailed analysis of intact biopsied muscle fibers from patients using electrophysiology, microscopy, and biochemical techniques demonstrates distinct molecular and cellular mechanisms. The syndromes may be classified on the basis of the site of the defect of neuromuscular transmission, but the ability to determine this may only be available at a few centers and is not always certain. Diagnosis depends upon electrophysiological tests, morphological studies of the endplate region in muscle biopsy specimens, and, increasingly, identification of the specific genetic defect (Table 16.1). Next-generation sequencing (NGS) is now helping to identify new CMS causative genes which are ubiquitously expressed, proving that protein defects not specifically confined to the neuromuscular junction (NMJ) can cause myasthenia [4, 5, 6, 7]. Moreover, in many of the more recently identified CMS-associated genes, aberrant neuromuscular transmission is only one component of a more complex phenotype in which muscle, the central nervous system, and other organs may also be affected [8, 9, 10, 11, 12].
Table 16.1

Classification of congenital myasthenic syndromes (CMS) and their genetic locia

Presynaptic CMS

Gene

CMS with episodic apnea

CHAT

Unconventional myosin 9

MYO9

Munc13-1

MUNC13

Synaptosome-associated protein 25

SNAP25B

High-affinity choline transporter 1 (CHT)

SLC5A7

Vesicular ACh transporter (VAChT)

SLC18A

Synaptobrevin 1 deficiency

VAMP1

Prolyl-endopeptidase-like gene

PREPL

Synaptic CMS

Congenital endplate acetylcholinesterase deficiency

COLQ

Agrin

AGRN

Collagen type XIII alpha 1 chain

COL13A1

Laminin α5 deficiency

LAMA5

Laminin ß2 deficiency

LAMB2

Postsynaptic CMS

AChR deficiency syndromes

CHRNA, CHRNB, CHRND, CHRNE

Multiple pterygium syndromes due to AChR γ-subunit mutations

CHRNG

AChR deficiency syndromes due to mutations in rapsyn

RAPSN

Slow-channel CMS

CHRNA, CHRNB, CHRND, CHRNE

Fast-channel CMS

CHRNA, CHRND, CHRNE

Low conductance syndrome

CHRNA, CHRNE

CMS due to voltage-gated sodium channel mutations

SCN4A

CMS due to mutations in MuSK

MUSK

CMS due to mutations in DOK7

DOK7

CMS due to mutations in LRP4

LRP4

Plectin deficiency

PLEC1

Ubiquitously expressed proteins (glycosylation)

Glutamine-fructose-6-phosphate transaminase-1

GFPT1

Dolichyl-phosphate-N-acetylglucosamine-phosphotransferase-1

DPAGT1

N-linked glycosylation protein 2

ALG2

N-linked glycosylation protein 13

ALG13

GDP-mannose pyrophosphorylase B

GMPPB

AChR muscle acetylcholine receptor, MuSK muscle-specific tyrosine kinase, DOK7 downstream of kinase 7, LRP4 low-density lipoprotein receptor-related protein 4

aThe genetic origin of further CMS cases is yet to be defined or not yet reported

Molecular Genetic Classification

For ease of classification , the CMS can be broadly grouped according to the location of the defective neuromuscular junction protein and thus are often classed as presynaptic, synaptic, and postsynaptic disorders [13]. Within these headings different genes may be defective. A diagrammatic representation of CMS-associated genes/proteins at the neuromuscular junction is shown in Fig. 16.1 and a list of proteins and their genetic loci in Table 16.1.
Fig. 16.1

Diagrammatic representation of the neuromuscular junction and the proteins in which mutations underlying congenital myasthenic syndromes have been found. Recently identified CMS genes are shown in red

CMS with episodic apnea was the first neuromuscular junction presynaptic disorder in which the genetic origin was defined and is due to mutations in the enzyme choline acetyltransferase (CHAT) [14, 15]. Other presynaptic disorders have been identified that have a paucity of synaptic vesicles, reduced quantal release, or resemble the autoimmune Lambert-Eaton myasthenic syndrome, and now, with the advent of NGS, the underlying genetics for these disorders are being uncovered [9, 16]. Acetylcholinesterase (AChE) deficiency is the most common CMS directly affecting proteins in the synaptic space and results from mutations in COLQ, which encodes an AChE-associated collagen tail that both anchors and concentrates AChE in the synaptic cleft [17, 18]. However, other proteins located in the basal lamina that are involved in interactions between the nerve terminal and the muscle membrane are being uncovered. Agrin initiates a signaling pathway that underlies the maintenance of pre- and postsynaptic structures, and other proteins in the synaptic cleft such as laminins or COL13A1 although not directly participating in the agrin pathway are thought also to be important for maintaining synaptic structure. Impaired function of these proteins has been found to result in a CMS. A series of other genes have been found to cause postsynaptic disorders, and these form the most common causes for CMS. Mutations in the genes encoding the AChR subunits were the first to be identified. These may give rise to kinetic abnormalities of AChR function or AChR deficiency or a combination of altered AChR kinetics and AChR deficiency [19, 20, 21, 22]. However, mutations of many other proteins located on the postsynaptic side of the NMJ that are involved in synaptic function, in AChR clustering, or in the maturation of the synaptic structure can commonly underlie CMS (Fig. 16.2). Mutations in MUSK [23, 24], AGRN [25, 26], LRP4 [27], and SCN4A [28] have proved to be rare, but mutations of RAPSN [29] or DOK7 [30] are common causes of CMS.
Fig. 16.2

Illustrative diagram highlighting the classical pathway believed to be responsible for formation and maintenance of the neuromuscular junction

More recently, a series of genes encoding enzymes involved in the asparagine-linked (N-linked) glycosylation pathway have also been found to harbor CMS-causing mutations [4, 5, 6, 7] (Fig. 16.3). The phenotype for these subtypes often varies from the archetypal picture of myasthenia in that eye and facial muscles are often spared. As a result, many of these patients may go undiagnosed [2].
Fig. 16.3

Illustrative representation of the asparagine-linked (N-linked) glycosylation pathway in which glycan residues are stepwise added to dolichol phospholipid prior to transfer to a respective protein. CMS-associated genes are shown in red

Diagnostic Methods

CMS should be considered in any person presenting with fatiguable muscle weakness during infancy or early childhood. Clinical features may help to pinpoint which of the diverse range of genetic loci is involved and provide clues about the underlying molecular pathogenesis. Electromyography (EMG), the clinical phenotype, and, in some cases, muscle biopsies may all provide important diagnostic pointers.

Standard EMG can often detect impaired neuromuscular transmission, especially if the muscle under test is weak. Decrement in the compound muscle action potential (CMAP) elicited by repetitive nerve stimulation at low frequency (2–3 Hz) is suggestive of impaired neuromuscular transmission but is generally considered less sensitive than single-fiber electromyography (SFEMG). SFEMG revealing abnormal jitter and block can be operator-dependent and can occur in other disorders but is more sensitive than CMAP recording and will usually give an indication of defective neuromuscular transmission.

Phenotypic clues may be gleaned for many of the CMS and greatly facilitate targeted genetic screens, although now in nonspecialized services whole exome or whole genome sequencing may provide the quickest route to a genetic diagnosis. More detailed descriptions will be given later in this chapter, but illustrative examples are that a repetitive CMAP in response to a single nerve stimulus is frequently seen in syndromes involving over-excitation such as the slow-channel congenital myasthenic syndromes or that AChE deficiency syndromes and mild arthrogryposis multiplex congenita are a strong pointer to AChR deficiency due to RAPSN mutations [31, 32], although it can also occur rarely with mutations in other genes, such as some of the presynaptic disorders.

In specialist centers, muscle biopsy is essential for characterizing disorders where the underlying genetic cause remains unknown. Electrophysiology of endplates can determine quantal content and the amplitude of miniature endplate potentials and currents (MEPPs and MEPCs) and endplate potentials and currents (EPPs and EPCs). Their size and decay times may suggest abnormalities in the number and kinetic properties of the endplate AChR. Electron microscopy can define the ultrastructure of the pre- and postsynaptic apparatus of the neuromuscular junction, providing clues to whether the defect is pre- or postsynaptic. Binding of iodinated or fluorescence-labeled α-neurotoxins, such as α-bungarotoxin (α-BuTX), can be used to determine the localization and number of the AChR. Similarly, histochemistry may be used to establish the presence or absence of AChE at the endplate [1].

Response to treatments may provide additional clues about the disorder, although the tensilon test, where the short-term response to intravenous edrophonium is measured, may give misleading results. In all CMS, autoimmune myasthenia gravis should be excluded through testing for antibodies to AChR or to MuSK. Parental consanguinity and a positive family history are both suggestive of hereditary rather than autoimmune myasthenia, and the onset of myasthenia gravis at less than 1 year is very rare. Although most CMS first present in infancy or early childhood and show recessive inheritance, an exception to this generalization is the slow-channel myasthenic syndrome, which may present in infancy or adult life and is usually inherited as an autosomal dominant trait, and late-onset CMS associated with mutations in COLQ, RAPSN, and DOK7 have been reported [32, 33, 34]. Mutations affecting the N-linked glycosylation pathway tend to have a later onset in childhood or early adulthood.

Presynaptic Congenital Myasthenic Syndromes

These are the least well characterized of the myasthenic disorders. Electrophysiology and ultrastructure studies of the endplate regions in muscle biopsies identified apparent defects in the presynaptic apparatus in various CMS [1], and it is only with the advent of next-generation sequencing that identification of the genetic abnormalities has become apparent. These include disorders where the electrophysiology shows similarities to those seen for the Lambert-Eaton syndrome and others where there is a paucity of synaptic vesicles in the presynaptic bouton and reduced quantal release. Indeed a series of disorder have been identified that involve the process of neurotransmitter release into the synaptic cleft from the presynaptic vesicles. However, as might be expected, if there is a problem in the neurotransmitter release mechanism, then patients are likely to have problems at many sites other than at the neuromuscular junction; as a result, these patients will often suffer from a severe and often fatal multisystem disorder in which the myasthenic component only forms one small part. Thus, it is debatable as to whether they should truly be termed as a myasthenic syndrome.

Presynaptic CMS Associated with the Synthesis or Recycling of Acetylcholine

Clinical Features

The presynaptic CMS are autosomal recessive disorders that have previously been called familial infantile myasthenia and CMS with episodic apnea [1]. However, this terminology is not ideal since similar severe episodic apneic attacks are now known to occur both in presynaptic and other postsynaptic forms of CMS. The presynaptic disorders share common clinical symptoms that typically manifest at birth and consist of hypotonia, bulbar and respiratory muscle weakness, ptosis, and varying degrees of extraocular muscle weakness. Respiratory insufficiency with recurrent apnea is a hallmark of these disorders. The episodic crises, which may be life-threatening in early life, are frequently induced by infections and fever, stress, or overexertion but become less frequent with age. Patients usually respond well to anticholinesterase medication, which may be taken prophylactically in anticipation of a crisis. The majority of these presynaptic disorders are due to mutations in choline acetyltransferase (CHAT), though recently mutations in the high-affinity choline transporter (SLC5A7) [9] responsible for choline uptake into the nerve terminal and the vesicular acetylcholine transporter (VAChT encoded by gene SLC18A3) [10] have been identified. Mutations in SLC5A7 and SLC18A3 tend to result in more severe phenotypes that are often associated with arthrogryposis and neonatal death.

Diagnosis of patients with CHAT mutations may be helped by the characteristic EMG profile. In rested muscle, the CMAP elicited by repetitive nerve stimulation at 3 Hz and SFEMG may be normal. However, after exercise or repeated nerve stimulation at 10 Hz for up to 5 min, a decremental response and abnormal jitter can be seen. In vitro studies similarly show normal EPP and MEPP amplitudes in rested muscle that decrease after continuous 10-Hz stimulation [14]. Endplate AChR shows normal number and distribution. Patients with mutations in SLC5A7 or SLC18A3 may show a similar EMG profile but may also show significant decrement at a standard repetitive 3-Hz stimulation. They both can result in fatal disorders with death in utero or as neonates.

Molecular Basis

CHAT catalyzes the reversible synthesis of ACh from acetyl coenzyme A and choline at the synapse. It has also long been hypothesized that mutations in both the choline uptake transporter (CHT) and the vesicular acetylcholine transporter (VAChT) might also result in myasthenic syndromes. Impairment in the ability to resynthesize ACh sufficiently fast, and thus reduction of ACh within the presynaptic vesicles, is responsible for the activity-dependent weakness, evident as decrement and reduced MEPP amplitude following continuous stimulation at 10 Hz. Similarly either the reduced ability of the motor terminal to take up choline (CHT) or to transport synthesized choline into synaptic vesicles (VACHT) will affect neurotransmitter release and lead to myasthenic weakness. Frameshift mutations are likely to be lethal, and so most of mutations identified are mostly missense and cause either a reduction in enzyme/transporter expression or in activity or a combination of the two [14, 35, 36].

Congenital Myasthenic Syndromes Associated with Impaired ACh Release

The mutations in the ACh recycling pathway clearly cause reduced ACh quantal release, but a second category of presynaptic disorders is formed by proteins involved in the actual docking of the synaptic vesicles and release of the neurotransmitter from the nerve terminals. This exocytosis is controlled by the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex . With the advent of next-generation sequencing, human mutations have been identified in synaptotagmin [37], SNAP25B [8], synaptobrevin [12], and Munc13-1 [11], which all feature in the docking and Ca2+-triggered fusion of synaptic vesicles with the presynaptic membrane at both central and neuromuscular synapses.

Clinical Features

To date these can be classified as very rare disorders, and so it is difficult to generalize about the clinical features other than to state that they are often severe or fatal and are likely to involve developmental and central abnormality. Comorbidities may involve cortical hyperexcitability, cerebellar ataxia, developmental delay, dysmorphic features, arthrogryposis, and intellectual disability. Unusual for CMS, the mutations identified in synaptotagmin 2 (SYT2) and SNAP25B are dominant. Mutations in SYT2 result in both a motor neuropathy and a Lambert-Eaton myasthenia-like syndrome, with small CMAPs and post exercise facilitation. The patients respond to 3,4-DAP. For SNAP25B, neurophysiological studies show reduced MEPP frequencies and quantal content. Recessive mutations for VAMP1 (synaptobrevin) led to hypotonia, feeding difficulties, and ophthalmoparesis presenting at birth and again show a reduction of CMAP amplitude with facilitation at high-frequency stimulation, and patients were reported to respond to pyridostigmine medication.

Molecular Basis

The SNARE complex forms the principal component that controls the docking, priming, and then fusion of synaptic vesicles with the presynaptic plasma membrane. Synaptotagmin acts as a sensor of the synaptic vesicles for the calcium influx that results from the activation of the presynaptic voltage-gated calcium channels, and Munc13 interacts with syntaxin-1 in the SNARE complex. This complex for exocytosis is common to release mechanisms at many different synapses and in endocrine tissues, and thus it is not surprising that mutations in these proteins can cause multiple neurological problems and are often fatal, particularly if frameshift mutations are present. Further reports describe a presynaptic syndrome due to mutations in MYO9A, which encodes an atypical myosin, but it is not yet clear how this protein is involved in presynaptic neurotransmitter release.

Synaptic Congenital MyasthenicSyndrome

Endplate Acetylcholinesterase Deficiency

Clinical Features

Endplate acetylcholinesterase deficiency (EAD) is an autosomal recessive disorder with onset at birth or in early childhood. Weakness is often severe affecting facial, cervical axial, and limb muscles that may result in lordosis and kyphoscoliosis. Difficulty feeding, respiratory distress, and delayed motor milestones are common. In some patients delayed papillary light reflexes are evident and may be used to distinguish EAD from other CMS. Weakness is refractory to anticholinesterase medication, and patients do not respond well to AChR open-channel blockers, such as quinidine sulfate or fluoxetine, that have been used for slow-channel syndrome. Ephedrine and salbutamol have been reported to be beneficial [1, 38, 39].

EMG shows features associated with defective neuromuscular transmission, with CMAP decrement on repetitive stimulation, and jitter and block evident with SFEMG. Single nerve stimulation elicits a repetitive CMAP, which is another diagnostic hallmark and is only seen in EAD or the slow-channel syndrome. On muscle biopsy microelectrode studies show prolonged decay of the MEPPs and EPPs indicating prolonged activation of the AChR ion channels owing to the absence of AChE, and histochemical staining of the neuromuscular junctions shows this absence or severe reduction of AChE at the endplates. Further analysis of the endplate region often demonstrates a compensatory reduction in nerve terminal size and extension of Schwann cells into the synaptic cleft.

Molecular Basis

The COLQ gene encodes the collagen-like tail (ColQ) that attaches the asymmetric form of AChE to the basal lamina at the neuromuscular junction. Mutations in ColQ rather than AChE itself have been found to cause endplate EAD. The ColQ protein contains an N-terminal proline-rich domain (PRAD) that binds AChE tetramers, a collagen domain with 63 Gxy repeats, and a C-terminal domain responsible for anchoring in the basal lamina and for initiating the collagen triple-helix structure. Many mutations have been identified in COLQ [1, 40]. They occur in all the putative functional domains, although there is some evidence that mutations in the C-terminal domain that effect the anchoring to the basal lamina are less severe [41]. The result of the mutations is the loss of the asymmetric form of AChE from the synaptic cleft and consequently the increase in the time that ACh is available to bind the AChR. Prolonged exposure to ACh will cause desensitization of AChR, and the persistent depolarization of the endplate will inactivate the voltage-gated sodium channels in the depths of the postsynaptic folds and thus block signal transmission. In addition, the prolonged stimulation of the endplates may lead to overload of calcium ions at the endplate and an endplate myopathy.

Rare Synaptic Forms of CMS

The formation and maintenance of the synapse involves cross talk between the pre- and postsynaptic sides. Basal lamina proteins help form scaffold structures that ensure the correct alignment of the functional components, but several may also play a role in anterograde or retrograde signals important for synaptic structure.

COL13A1 CMS Clinical Features

Patients identified to date with mutations in COL13A1 [42] have all had onset of weakness at birth including respiratory and feeding difficulties, slight dysmorphic facial features, ptosis but normal eye movements, and marked weakness of neck flexion. All have shown a decrement of compound muscle action potentials (CMAP) on repetitive nerve stimulation. A common feature for COL13A1 patients that might help distinguish them from other forms of CMS is the presence of pectus carinatum, which may suggest that the mutant COL13A1 is affecting a separate developmental respiratory pathway and would be in keeping with high levels of COL13A1 expressed in the lung, and with age the fatigability of the ptosis becomes limited and it may appear as a fixed ptosis. There also appears to be a natural improvement of the overall myasthenic weakness over time through childhood. It is also notable that there is a lack of a beneficial response to anticholinesterase medication, but beneficial effect for 3,4-DAP and salbutamol has been reported.

Molecular Basis

COL13A1 has three extracellular collagenous domains but is anchored by a transmembrane domain in the N-terminal region. It has a proprotease recognition site just outside of the transmembrane domain and exists as an anchored form and a cleaved ectodomain form [43]. Next-generation sequencing of individuals with suspected CMS revealed three patients in two independent kinships with mutations in COL13A1 [42]. A woman, without known consanguinity in the family, is homozygous for c.1171delG (p.Leu392Serfs*711), and two siblings from a consanguineous family were found to be homozygous for c.5231delG (p.Gly175Valfs*20). Much of our knowledge of the role of COL13A1 at the neuromuscular junction comes from animal models. Mice lacking col13a1 (col13a1−/−) show abnormal endplates which remained small, immature, and fragmented when compared to wild-type animals [44]. It is thought that it is the membrane-bound form that plays the major role in neuromuscular junction development. The COL13A1 ectodomain is thought to interact with fibronectin, heparin, and basal membrane proteins nidogen-2 and perlican, and col13a1−/− mice also show abnormalities in the presynaptic motor terminal with aberrant localization of vesicles and ACh release, as well as showing differences in the positioning and form of the terminal Schwann cells. There is also the suggestion that COL13A1 might interact with COLQ and that it therefore may have a role in the precise localization of the asymmetric form of acetylcholinesterase (AChE) in the synaptic cleft [45].

LAMB2 and LAMA5 CMS

Single patients have been reported with myasthenic syndromes caused by mutations in laminin β2 (LAMB2) [46] and laminin α5 (LAMA5) [47]. Laminins are large heterotrimeric extracellular proteins that self-assemble into a cruciform structure but also interact with cell surface receptors. The patient with LAMB2 mutations suffered from congenital nephrosis, and the myasthenia became truly apparent following kidney transplants. The LAMA5 CMS patient had myopia and facial tics and showed neurophysiological features in keeping with a LEMS-like presynaptic disorder. Overall these studies emphasize the role of extracellular matrix proteins, distinct from those of the AGRN-LRP4-MUSK-DOK7 pathway, in the formation and maintenance of the neuromuscular synapse and further emphasize the importance of retrograde signaling across the synapse, although this process still remains poorly understood.

Postsynaptic Congenital Myasthenic Syndromes

The majority of CMS are caused by mutations in genes that encode postsynaptic proteins. Initial studies identified mutations in the genes encoding the AChR subunits that impair ion channel gating or reduce the number of endplate receptors or a combination of the two, giving rise to “slow-channel,” “fast-channel,” “reduced conductance,” or AChR deficiency syndromes [19, 20, 21, 22, 48, 49, 50]. However, CMS also arise from mutations in proteins involved in the formation and maintenance of the neuromuscular junction such as RAPSN [29, 31, 32], AGRN [25], LRP4 [27], MUSK [23], or DOK7 [30].

Genetic Disorders of the AChR

Muscle AChRs are glycosylated transmembrane molecules that mediate synaptic transmission. They are allosteric (existing in several different conformations), and the binding of two ACh to each receptor is thought to favor a conformational change that results in a brief activation of the channel and the influx of cations. Each AChR is made up of five subunits arranged in a pentameric structure around the central ion pore. In mammalian muscle there are two types of AChR: a form found in fetal muscle that consists of α2βγδ and in adult muscle that consists of α2βδε. The subunits are homologous, vary in size from 437 to 495 amino acids, and are encoded by separate genes of between 10 and 12 exons. In embryonic muscle, before innervation, fetal AChRs are distributed along the length of the muscle fibers. During innervation the AChRs are clustered on the postsynaptic membrane and are lost from extrasynaptic sites. At the same time, expression of the γ-subunit (fetal) mRNA is repressed, and it is replaced by ε-subunit (adult) mRNA transcribed from subsynaptic nuclei [50]. In humans the γ-subunit is readily detectable in neuromuscular junctions of fetal muscle up to around 31 weeks of gestation [51] and continues to be expressed at extremely low levels in adult muscle [52].

AChR Deficiency Due to Mutations in the AChR Subunits

Clinical Features

Hereditary AChR deficiency is the most common CMS [1, 2]. It is as an autosomal recessive disorder in which mutations in the AChR subunit genes cause a primary deficiency of AChR at the endplate. The phenotype may vary from mild to severe. Weakness is usually evident at birth or within the first year of life and is characterized by feeding difficulties, ptosis, impaired eye movements, and delayed motor milestones. Patients sometimes show improvement in adolescence, and in general the disease course is not progressive. In general, patients improve with anticholinesterase medication or 3,4-diaminopyridine. Electromyography typically shows decrement of CMAPs at 3-Hz stimulation, and single-fiber EMG shows increased jitter and block. Intracellular microelectrode recordings from the endplate region of biopsied muscle show that MEPPs and MEPCs may be decreased to around 8–26% and 20–42% of normal values. Consistent with MEPP and MEPC findings, staining with 125I-α-BuTx or Alexa Fluor-conjugated α-BuTx shows reduced numbers of AChR that are often distributed abnormally along the muscle fiber [53]. In addition, electron microscopy shows a severe reduction of the postsynaptic folds.

Molecular Basis

Mutations in each of the AChR subunits, CHRNA, CHRNB, CHRND, and CHRNE, may underlie AChR deficiency syndromes [1]. However, the overwhelming majority are in CHRNE. At least 100 different mutations, located along the length of the ε-subunit gene, have been identified and may cause premature termination of translation, affect the promoter [54, 55, 56] or the signal peptide, or affect the assembly of the AChR pentamer. Many are null mutations [57, 58]. It is thought that in these patients residual expression of the γ or fetal subunit is incorporated into the endplate receptors, which accumulate at low levels at the endplate, are able to mediate synaptic transmission, and partially compensate for the loss of adult AChR. Evidence to support this hypothesis includes recordings from endplates that demonstrate the receptors have functional properties of fetal AChR and that animal models in which the adult AChR is replaced by fetal AChR mimic the human condition [57]. The partial compensation through γ-subunit expression explains why the majority of AChR mutations are located in the ε-subunit gene. Most CHRNE mutations are limited to a few families although some evidence for founder effects is evident within European populations. In particular, the mutation ε1267delG [59, 60], which tends to manifest a relatively mild phenotype, is common in Southeastern Europe, particularly in the Romany gypsies [61]. Mutations CHRNA, CHRNB, and CHRND underlying AChR deficiency tend to cause a severe phenotype probably because they cannot be compensated by expression of an alternative subunit. In these cases the mutations usually affect assembly of the AChR pentamer [62].

Kinetic Abnormalities of the AChR

Mutations that underlie AChR deficiency may often also change the kinetics or functional properties of the AChR. In these cases the primary pathogenic defect is loss of endplate AChR, and the altered channel kinetics has a secondary effect. However, if the numbers of endplate AChR are not severely reduced, the phenotype is determined by the altered ion channel gating.

Slow-Channel Congenital Myasthenic Syndrome

Clinical Features

This syndrome was first described by Andrew Engel and colleagues [63]. It is an autosomal dominant disorder, with an age of onset of weakness that may occur neonatally or may not arise until adolescence and adulthood or during pregnancy. There is also a wide range of severity and in some patients variable penetrance [64]. Weakness and associated wasting of cervical and scapular muscles and of finger extensors are often early features. Frequently, there is only mild ptosis and extraocular muscle involvement. Unlike other CMS , this disorder is commonly slowly progressive, involving the respiratory, limb, and bulbar muscles.

On EMG decrement of CMAPs in response to 3-Hz stimulation may only be seen in affected muscles. However, as for endplate AChE deficiency, a characteristic repetitive CMAP response to a single nerve stimulus is often, but not always, present. In vitro microelectrode studies show prolongation of the EPPs and EPCs and of the MEPPs. Single-channel recordings directly from endplate regions show AChR with abnormally prolonged activations that account for the abnormally long decay of the EPPs and EPCs. Ultrastructural studies show an “endplate myopathy,” in which there is a widening of the postsynaptic cleft, areas of degenerating junctional folds, degenerating and swollen mitochondria, apoptotic subsynaptic nuclei, calcium deposits, and vacuole formation [65].

Molecular Basis

At least 32 different mutations have been identified that give rise to slow-channel congenital myasthenic syndromes [1]. They occur not only in each of the AChR subunits [66, 67] but also in different functional domains within each subunit [68], although mutations within the M2 channel pore region are most common [69]. They are single amino acid changes that result in a pathogenic gain of function for the AChR, explaining the dominant inheritance, although a single amino acid deletion may also cause a slow-channel syndrome [70].

The AChR is thought to adopt multiple conformations, and at least three interconvertible functional states have been recognized: a resting state in the absence of ACh in which the probability of opening is small, an active state in the presence of ACh in which the probability of opening is high, and a closed state that results from prolonged exposure to high ACh concentrations in which the AChR is “desensitized.” A simplistic but illustrative mechanism for the activation of the AChR (derived from recordings of single-channel currents) is given in Fig. 16.4.

This scheme does not take into account channel openings of unliganded or monoliganded AChRs or various desensitized states and assumes the binding of the two ACh to a single receptor is equivalent. In neuromuscular transmission there is a transient saturating concentration of ACh, leading to rapid binding of two ACh (A2R, where the receptor is bound but closed), and the rapid open rate β ensures fast opening of the channel (A2R*). The hydrolysis of ACh by AChE rapidly clears ACh from the synaptic cleft, lowering the ACh concentration so that there is no rebinding once ACh dissociates from the receptor. Because the opening rate β and the dissociation rate k−2 are roughly similar, the receptor will oscillate between an open and a closed state before the ACh finally dissociates from the receptor. The activation of wild channels depends largely upon β (the opening rate), α (the closing rate), and k−2 (the dissociation rate). Mutations of the AChR that affect channel kinetics are likely to alter one or more of these rates [67].

Single-channel recordings of AChR harboring slow-channel mutations show prolonged ion channel activations, both from mutant AChR expressed in HEK 293 cells or Xenopus oocytes and recordings direct from muscle biopsies. For mutation αG153S [19, 68] that is located close to the predicted ACh binding site, kinetic analysis shows that prolonged activations arise primarily through a reduction in the rate of dissociation of ACh from the AChR (k−2), thereby increasing the number of channel openings during ACh occupancy. Thus, the primary effect of the αG153S mutation is to alter the affinity of AChR for ACh. However, the majority of slow-channel syndrome mutations are in the M2 transmembrane domains. Mutations in the M2 domain, such as εL264P [48], primarily slow the rate of channel closure (α), so that within activations the duration of individual openings is increased. Although severity of disease is variable, in general patients with mutations in the M2 region tend to be more severely affected. The prolonged ion channel activations explain the extended decay phase for the EPPs and MEPPs observed in slow-channel syndrome patients.

The evidence supports the theory that prolonged channel activations lead to excess entry of calcium or calcium overload which in turn activates a variety of enzymatic pathways leading to the degenerative changes on the postsynaptic side of the synapse. The endplate myopathy can lead to defective neuromuscular transmission through reducing the number of endplate AChR and reducing efficiency of transmission through widening of the synaptic cleft and decay of the postsynaptic folds. Neuromuscular transmission may also be compromised by an increased propensity of the mutant channels to desensitize and depolarization block of the voltage-gated sodium channels due to summation of the endplate potentials at physiological rates of stimulation [1].

Both quinidine and fluoxetine that, among other actions, block the AChR channel when it is open have been partially successful in treating patients and have improved symptoms [70, 71, 72, 73]. However, both of these drugs can have potentially serious adverse effects, and so patients should be carefully monitored.

Fast-Channel Congenital Myasthenic Syndrome

Clinical Features

The phenotypes of fast-channel congenital myasthenic syndromes, reduced conductance syndromes, and AChR deficiency syndromes share many features. Fast-channel syndromes show recessive inheritance, except in one reported case [74], with the fast-channel mutation usually found in combination with a low expressor or null allele. Weakness is usually evident at birth or in the first months and is characterized by feeding difficulties, ptosis, impaired eye movements, and delayed motor milestones [75]. Patients with fast-channel syndromes tend to be more severely affected than AChR deficiency syndromes due to ε-subunit mutations, and in one patient joint contractures at birth were reported [76]. EMG typically shows decrement at 3-Hz stimulation, and single-fiber EMG reveals an increase in jitter and block. Intracellular microelectrode recordings from the endplate of biopsied muscles show small MEPPs and MEPCs. However, biochemical and morphological analysis helps to differentiate the syndromes; endplates do not show a severe loss of receptors, postsynaptic folds, or other morphological changes associated with AChR deficiency. Fast-channel patients show a beneficial response to cholinesterase inhibitors to 3,4-diaminopyridine or to a combination of the two [75].

Molecular Basis

A series of mutations have been identified that alter the AChR channel properties causing abnormally brief channel activations, in direct contrast to the slow-channel mutations. The mutations have been identified in genes encoding each of the AChR subunits [20, 74, 76, 77]. They cause a loss of response to ACh and thus show recessive inheritance. When a fast-channel mutation segregates with a null mutation or a second fast-channel mutation, the fast-channel phenotypic footprint is uncovered. Mutations have been identified that reduce AChR affinity for ACh or alternatively effect the channel gating properties. For instance, εP121L has been identified in combination with εS143L, εG-8R, or Y15H. In each case the low-expressor second allele unmasks the phenotypic effects of εP121L that are generated by channel activations that are fewer and shorter than normal. εP121L slows the rate of channel opening (β) but has little effect on dissociation (k−2). Since channel opening depends upon β/k−2, εP121L will result in reduced channel reopening when ACh is bound and consequently shorter activations, resulting in a reduction in signal transmission. As perhaps might be expected, a third channel disease mechanism that causes a myasthenic syndrome is through reduced channel conductance. Like the fast-channel syndromes, these are recessive conditions in which the mutation causing reduced conductance is inherited alongside a null mutation, with the phenotype found to be similar to fast-channel syndrome [49].

Mutations Affecting AChR Clustering and Synaptic Structure

Efficient synaptic transmission depends upon the apposition of nerve terminal and postsynaptic apparatus and the correct localization of all the key functional components [78]. Just as studies of mutations underlying the fast- and slow-channel syndromes provide insights into AChR function, so studies of recently identified CMS are providing novel insights into synaptogenesis and the maintenance of the neuromuscular synapse. Rapsyn (RAPSN) is the crucial molecule that is thought to actually anchor AChR in the postsynaptic membrane, but the synaptic structure and AChR-rapsyn interaction are controlled by a pathway initiated from the nerve terminal by neural agrin and comprising of low-density lipoprotein receptor-related protein 4 (LRP4), MUSK, and DOK7 located at the postsynaptic membrane (see Fig. 16.2).

AChR Deficiency Due to RAPSN Mutations

Clinical Features

Mutations in the AChR-clustering protein rapsyn also cause endplate AChR deficiency [29]. Onset of manifestations is usually at birth, “early onset,” although occasional “late-onset” patients presenting from early adulthood through to middle age have been reported [31]. Early-onset cases are frequently associated with hypotonia and marked bulbar dysfunction often necessitating nasogastric feeding and may require assisted ventilation. Joint contractures (arthrogryposis multiplex congenita) of hands and ankles are common. In childhood the course of disease is associated with severe exacerbations often presenting with life-threatening respiratory failure. Patients tend to improve over time, severe apneic episodes are rarer over the age of 6, and in many cases in adulthood disability is minimal. “Late-onset” patients may be mistaken for “seronegative” immune-mediated myasthenia gravis. Bulbar, speech, and respiratory problems were not observed. Weakness of ankle dorsiflexion, which is uncommon in myasthenia gravis, may provide a clue that RAPSN mutations underlie the condition [32]. Both early- and late-onset cases show abnormal decrement on EMG and jitter on single-fiber EMG, although it is not always easy to detect. Patients with RAPSN mutations respond well to anticholinesterase medication, although some may gain further benefit from the addition of 3,4-diaminopyridine.

On muscle biopsy endplates from patients with AChR deficiency due to ε-subunit mutations or AChR deficiency from rapsyn mutations appear similar; however, the two conditions differ in distinctive clinical features that may enable a targeted genetic screen [32]. The underlying cause of these differential features is unclear. However, whereas patients with ε-subunit null mutations most likely survive through maintained low-level expression of the fetal (γ) subunit, patients with rapsyn mutations express low levels of the ε-subunit. Thus, one clear difference between the rapsyn group and the ε-subunit group is the type of AChR that mediated synaptic transmission (Table 16.2).
Table 16.2

Distinguishing clinical characteristics of AChR deficiency due to mutations in CHRNE and RAPSN

Clinical feature

Early-onset rapsyn mutations

AChR deficiency

ε-subunit mutations

Arthrogryposis

Common

Absent

Episodic crises

Common

Rare

Ophthalmoplegia

Absent

Common

Spontaneous improvement

Common

Rare

Molecular Basis

In about a third of AChR deficiency patients, mutations are not detected in the AChR subunits. Many of these cases are due to the recessive inheritance of mutations with the AChR-clustering protein rapsyn [79, 80, 81]. Various functional domains have been proposed for rapsyn, including an N-terminal myristoylation signal involved in membrane association, a string of tetratricopeptide repeats involved in rapsyn self-association, a zinc finger/coiled-coil domain implicated in the interaction of rapsyn with the AChR, and a RING-H2 domain thought to be involved in binding to scaffold proteins. Mutations are observed along the length of the rapsyn protein. To date more than 30 mutations have been identified, but there are no clear phenotypic associations with their positions within rapsyn. However, the overwhelming majority of patients harbor the missense mutation N88K on at least one allele, suggesting an original founder mutation [82]. The observations suggest rapsyn-N88K retains at least partially function, whereas many of the others are null mutations. This is supported by cell culture experiments, in which several mutations were found to drastically inhibit rapsyn function and AChR-rapsyn association, whereas rapsyn-N88K was able to mediate agrin-induced AChR clusters but these clusters were found to be less stable than clusters formed with wild-type rapsyn [83]. Thus, it may be that in patients with the N88K mutation AChR deficiency is due to instability of the endplate rapsyn-N88K/AChR clusters.

Not all patients with AChR deficiency harbor N88K. Rarely, patients have other mutations in the coding region that result in partially functional rapsyn. In addition, a number of patients have been identified with mutations in the promoter region of the RAPSN gene. Some of these promoter mutations severely reduce rapsyn mRNA transcription, but one, −38A > G, has a less drastic effect on transcription and has been found homozygous in patients of Iranian-Jewish origin that commonly show facial malformation [84]. The facial malformation, high-arched palate, and joint contractures observed in patients with rapsyn mutations are all thought to result from akinesia in the womb presumably due to failed neuromuscular transmission at critical stages in fetal development. Thus, the rapsyn mutations must affect clustering of both the adult and the fetal AChR subtypes.

A mutation identified in the AChR δ-subunit gene, δΕ381Κ, does not affect AChR function but rather impairs rapsyn induced AChR clustering, presumably through impaired interaction with rapsyn [85]. The patient phenotype bears all the hallmarks of a “rapsyn deficiency” rather than an AChR ε-subunit deficiency. This mutation may shed light on the molecular basis for AChR-rapsyn interaction which has remained unresolved despite many years of study.

Congenital Myasthenic Syndromes Due to Mutations in the AGRN-LRP4-MUSK-DOK7 AChR-Clustering Pathway DOK7-CMS

Clinical Features

Muscle groups can be differentially affected by the CMS. A group of patients have been identified in which proximal muscles are more affected than distal muscle groups. These have been termed “limb-girdle” congenital myasthenia [86]. However, this term may lead to confusion with other non-myasthenic “limb-girdle” muscle disorders, and they may be better classified as CMS with proximal muscle weakness. Mutations in the AGRN-LRPR-MUSK-DOK7 AChR-clustering pathway give rise to hereditary “limb-girdle” myasthenic weakness, although mutations in DOK7 are by far the most common [30].

Clinical onset of disease is generally characterized by difficulty in walking after initial achievement of walking milestones. Patients occasionally have earlier signs of ptosis, floppy tone, and bulbar and respiratory problems, but even among these, walking difficulty is not appreciated. The walking and running impairment tends to worsen in childhood and is often accompanied by upper limb weakness and loss of ambulation in some patients. A waddling and lordotic gait is often seen associated with proximal lower limb and truncal weakness.

Ptosis is often present from an early age, though it may develop and progress in childhood. Eye movements are usually normal, while facial, jaw, and neck weakness is common and tongue wasting has been observed in around 50% of cases. Bulbar problems typically develop later in the clinical course than limb weakness. Features seen in patients with rapsyn mutations such as congenital joint deformity and weakness of ankle dorsiflexion, which are associated with reduced fetal movement in the womb, have not been observed. However, fluctuations in symptoms were common. In many cases studied diagnosis as a myasthenic disorder was delayed, and disorders such as muscular dystrophy and congenital myopathy were suggested [34, 87]. There is a remarkable improvement following treatment with the β2-adrenergic receptor agonists, ephedrine and salbutamol [88]. However, contrasting with the characteristic myasthenic response to pyridostigmine or 3,4-DAP which usually is felt within an hour, the response to β2-adrenergic receptor agonists occurs gradually over weeks and months, and it can take up to 2 years before the full benefit is achieved.

Molecular Basis

The classic view of the development of the neuromuscular synapse derives from a series of experiments in which components of the neuromuscular junction were “knocked out” [89, 90, 91, 92]. They highlighted a pathway in which agrin, released from the motor nerve terminal, binds to LRP4 which in turn interacts with and activates MuSK, a receptor tyrosine kinase, which in turn activates a kinase pathway in which the AChR β-subunit is phosphorylated, and rapsyn clusters and stabilizes the AChR on the postsynaptic membrane [93] (Fig. 16.3). The MuSK signaling is amplified by intracellular interaction with DOK7. DOK7 is found to bind specifically to MuSK and when expressed in cultured C2C12 cell-line myotubes is able to induce large AChR clusters in the absence of neural agrin. When the DOK7 gene was “knocked out” in mice, neuromuscular junctions failed to form, and offspring failed to survive past birth [94].

Splice site, missense, and frameshift mutations have been identified in DOK7, and recessive inheritance of these results in a myasthenic syndrome with the characteristic proximal or “limb-girdle”-type weakness described above. The majority of the mutations are located in the large 3′ exon of the gene that encodes the C-terminal region of DOK7. Mutation 1124_1127dupTGCC is common, occurring in at least one allele in 20/24 kinships reported in a recent study. It is thought that DOK7 activates MuSK through the DOK7 phosphotyrosine-binding domain interacting with the juxtamembrane phosphotyrosine-binding motif of MuSK. The binding can occur even when the C-terminal region is truncated [30]. Studies of the motor endplates from patients harboring DOK7 mutations found that components of the neuromuscular junction were present at normal density and showed normal function but that the size of the pre- and postsynaptic structures was reduced [95]. These observations, in combination with functional studies of the action of mutant DOK7 on the AChR-clustering pathway, show that mutations of DOK7 result in impaired maturation and maintenance of neuromuscular junction structure [30].

AGRN, LRP4, and MUSK CMS

Clinical Features

Mutations in AGRN, LRP4, and MUSK, which are the other key components of the AChR-clustering pathway, are rare, but they are large genes, and NGS is beginning to uncover more cases. There is considerable variation of disease severity, but early respiratory failure and feeding difficulties accompanied by mild ptosis, very mild ophthalmoparesis, and moderate to severe proximal weakness appear to be common. Agrin has several isoforms, and it is only the neural isoform that is critical for control of the synaptic structure of the neuromuscular junction. CMS due to AGRN mutations often have an accompanying mild distal myopathy [26], and it may be that this is due to effects of the mutations on AGRN on non-neural isoforms that are known to be expressed by muscle. Post-exercise increment on repetitive nerve stimulation has been noted in some but not all AGRN-CMS. Similarly mutations in LRP4 are commonly the cause of Cenani-Lenz syndactyly syndrome [96] and only very rarely cause a myasthenic syndrome [27]. In keeping with the findings from DOK7, patients with CMS-causing mutations in these genes do not improve and often worsen with anticholinesterase medication, but show a good response to β2-adrenergic receptor agonists [88], though patients with AGRN mutations tend to respond less well than LRP4-, MUSK-, or DOK7-CMS patients.

Molecular Basis

AGRN mutations occasionally occur in the N-terminal region of the protein [26] but are mostly found nearer to the C-terminus in the regions that give rise to the splice variants for the neuronal AGRN form. Typical mutants are p.Gly1675Ser, p.Gly1709Arg, and p.Val1727Phe [25, 26, 97]. This suggests that the CMS mutations are likely affecting the protein in the region where AGRN interacts with LRP4, and thus there is impaired activation of MUSK and severe reduction of the signal for AChR clustering.

LRP4 encodes low-density lipoprotein receptor-related protein 4. As stated above, most LRP4 mutations give rise to Cenani-Lenz syndactyly syndrome and have no effect on neuromuscular transmission. However, a few mutations have been identified in the third β-propeller domain that cause CMS, such as p.Glu1233Lys and p.Arg1277His [27]. The implication from these studies is that this domain is critical for the interaction between LRP4 and MUSK and that the mutations affect the interaction. MUSK is considered the major organizer of neuromuscular junction synaptic development, maintenance, and stability. MuSK mutations are extremely rare, and this may be because any severe loss-of-function mutations are not compatible with life. For the mutations that have been identified (p.Met605Ile, p.Ala727Val, p.Asp38Glu, and p.Pro344Arg), it has often been difficult to demonstrate a clear disease mechanism, although studies suggest that they affect processes that control the level of MUSK phosphorylation or may affect the interaction with DOK7 [98].

Prenatal Hereditary Myasthenia Due to Mutations in CHRNG

Neuromuscular transmission at nearly all normal adult muscle endplates is mediated by AChR consisting of α2βδε subunits. However, for crucial periods of fetal development, in utero transmission is mediated through the fetal form (α2βδγ) of the AChR [99]. Loss of fetal movement during these periods can lead to a series of developmental abnormalities.

Clinical Features

Multiple pterygia syndromes or Escobar’s syndrome is an autosomal recessive condition that manifests with orthopedic and cranial abnormalities. Characteristically, there is short stature, arthrogryposis multiplex congenita, pterygia of the neck, and anomalies of the head including low-set ears, ptosis, a pointed and receding chin, and high-arched palate [100]. Intrauterine death and stillbirths are common.

Molecular Basis

Mutations of the AChR γ-subunit gene CHRNG have been found to underlie many cases of Escobar’s syndrome [101, 102]. Mutations may be splice site, short duplication, missense, or nonsense mutations that result in either truncation or low expression levels of the γ-subunit. The loss of fetal AChR function associated with CHRNG mutations is thought to result in fetal akinesia, which in turn causes the associated multiple developmental abnormalities. Surprisingly, some patients that harbor γ-subunit null alleles can survive, suggesting early expression of the ε-subunit that partially compensates for loss of the γ-subunit. Similarly, the severity of the condition varies in patients with the same mutations. Following birth, neuromuscular transmission is mediated by the adult AChR, and patients show little or no progression of their condition. Since the disorder results from lack of neuromuscular transmission at crucial developmental phases, it might be expected that recessive inheritance of loss-of-function mutations in other essential components of the neuromuscular junction, such as RAPSN, MUSK, DOK7, or AChR subunits, would also result in fetal akinesia. This is indeed the case, but the resultant condition is not compatible with life.

CMS Due to Mutations in Glycosylation Pathways

The asparagine-linked (N-linked) glycosylation pathway is a ubiquitous process in eukaryotic cells involving to sequential addition of sugar moieties that are transferred to a protein at an asparagine residue at consensus sequence Asn-X-Ser or Asn-X-Thr [103]. Membrane-bound and exported proteins are modified in the endoplasmic reticulum in a process crucial for protein folding and multisubunit assembly. The glycans are often further processed during their intracellular transport through the Golgi to the plasma membrane. Mutations in components of this pathway produce a spectrum of severe multisystem disorders known as congenital disorders of glycosylation (CDGs) [104]. The neuromuscular junction is known to be highly glycosylated, and therefore it is perhaps not surprising that mutations affecting glycosylation can impair neuromuscular transmission [105]. What is surprising is that there are a considerable number of cases in which a defective neuromuscular transmission is the only presenting manifestation for mutations within the N-linked glycosylation pathway. Also surprising is that it is mutations in genes encoding the enzymes/subunits for the initial steps of the pathway or providing substrates that feed into the early stages of N-linked glycosylation that manifest as a myasthenia. Mutations in proteins involved in later steps of the pathway causing myasthenia have so far not been identified.

GFPT1, DPAGT1, ALG2, and ALG14 CMS Clinical Features

CMS due to “glycosylation mutations” share many similar phenotypic features [4, 5, 6, 7]. Onset of symptoms tends to be in childhood rather than at birth. Proximal muscles are affected more than distal with a typical limb-girdle pattern of weakness. Whereas fatiguable ptosis may or may not be present, eye and facial muscles are usually unaffected. Indeed, a characteristic of these patients is the lack of cranial and bulbar muscle involvement, and ptosis may be completely absent. Biopsies in many but not all of the patients show the presence of tubular aggregates, and this is a useful pointer to the underlying genetics although they can be seen rarely in some other forms of CMS. In some cases, modestly elevated serum creatine kinase (CK) levels and additional myopathic changes on needle EMG suggest a concomitant myopathy. Cognitive manifestations can vary from none to mild learning disabilities or major intellectual disability [106]. Mutations in these genes, in particular in DPAGT1, can also cause a severe congenital disorder of glycosylation type Ij characterized by severe hypotonia, intractable seizures, mental retardation, and microcephaly [107].

GFPT1, DPAGT1, ALG2, and ALG14 CMS Molecular Basis

GFPT1 encodes glutamine-fructose-6-phosphate transaminase-1, which catalyzes the first step in the biosynthesis of UDP-N-acetylglucosamine, an essential substrate for N- and O-glycosylation of protein [108]. Thus, it provides the rate-limiting step for feeding substrate into the first step of the N-glycosylation pathway. As with the majority of CMS forms, GFPT1-CMS is a recessive condition. Mutations are located through the length of the gene, are mostly missense, and are likely to affect either enzyme levels or catalytic activity. Knockdown of GFPT1 using siRNAs causes reduced surface expression of AChR suggesting that this may be at least one mechanism through which neurotransmission is affected [109]. DPAGT1 encodes the enzyme dolichyl-phosphate-N-acetylglucosamine-phosphotransferase-1, a transmembrane ER protein essential for N-linked glycosylation that catalyzes the first step in the dolichol oligosaccharide pathway for glycoprotein biosynthesis [110]. ALG14 encodes a membrane protein that together with DPAGT1 and ALG13 forms a functional multienzyme complex involved in the initial steps of N-glycosylation [111]. ALG2 encodes an alpha-1,3-mannosyltransferase that catalyzes the second and third mannosylation steps for the elongation of the carbohydrate chain linked to dolichol [112].

Studies with tunicamycin (an inhibitor of DPAGT1) show the clear role of DPAGT1 in glycosylation of the AChR, in particular of the AChR δ-subunit [5]. Moreover, as with GFPT1, siRNA knockdown of DPAGT1, ALG14, or ALG2 all reduces cell surface expression of the AChR [5, 6]. Thus, it appears that impaired function of the early steps of the N-linked pathway affect the surface expression of the AChR. Muscle biopsies from a patient with DPAGT1 mutations show loss of postsynaptic junctional folds characteristic of AChR deficiency syndromes, and the patients respond to anticholinesterase medication, both of which argue for loss of endplate AChR as a key mechanism of disease [5, 6]. However, it might also be expected that other glycosylated proteins at the neuromuscular junction are affected, and there is evidence that synaptic structure and the presynaptic terminal are also affected [106].

GMPPB CMS Clinical Features

GMPPB CMS provides a useful illustration of the forms of CMS that are now being uncovered by NGS. Mutations in this ubiquitously expressed gene disturb both downstream O- and N-glycosylation that may manifest as a dystroglycanopathy [113] or a combination of myasthenia and a dystroglycanopathy [7]. The myasthenic component responds to anticholinesterase medication with some additional improvement with the addition of β2-adrenergic receptor agonists, but this does not help the disease component due to the dystroglycanopathy, and therefore there is likely to be long-term progression of the disorder. Nevertheless, recognition is important since appropriate medication can significantly improve quality of life.

Patients with reduced α-dystroglycan O-mannosylation showed a wide range of disease severity varying from congenital muscular dystrophy with structural brain involvement, to a milder, later-onset limb-girdle muscular dystrophy [113]. Thus, mutations in GMPPB produce a wide phenotypic spectrum that includes CMS. CMS patients with GMPPB mutations usually present in adolescence or early adulthood; although some symptoms may be present at an earlier age, they have a predominantly limb-girdle pattern of weakness, more severe in lower than upper limbs, with associated waddling gait. As with the other “glycosylation CMS” subtypes, eye, facial, and bulbar muscles are frequently spared. Patients are defined as having CMS through the presence of fatiguable muscle weakness on examination and decrement of compound muscle action potentials. Surprisingly, many of the very severe congenital muscular dystrophy cases do not have decrement of CMAP, which appears largely restricted to the milder CMS presentations [114]. In addition, frequently no neurophysiological characteristics of CMS were identified when testing eye or facial muscles but were apparent when testing affected limb-girdle muscles. Thus, these cases reinforce the notion that for confirmation of a suspected myasthenia diagnosis, it is important to perform neurophysiological tests on muscles that show fatiguable weakness. In accordance with the dystroglycanopathy component, serum creatine kinase (CK) levels are markedly raised in GMPPB CMS (which is unusual in other forms of CMS) and therefore provide a pointer for genetic screening [114].

GMPPB CMS Molecular Basis

GMPPB encodes GDP-mannose pyrophosphorylase B that catalyzes the conversion of mannose-1-phosphate and GTP- to GDP-mannose. GMPPB contributes to both the N-glycosylation and the O-mannosylation pathways since mannose is a key glycan added in both. Mutations within the O-mannosylation pathway were originally identified in patients with a form of muscular dystrophy within the spectrum of dystroglycanopathies [113, 115]. These disorders are characterized by reduction in α-dystroglycan glycosylation, which is the most well characterized and functionally relevant O-mannosylated protein. Mutations are found throughout the gene, and there are no obvious genotype-phenotype correlations as yet. Indeed in some patients, the same mutations are present in severe dystroglycanopathies as are seen in mild CMS cases. Pathogenicity of genetic variants correlates well with a marked increase in the presence of protein aggregates following expression of mutant GMPPB in the TE671 muscle cell line [114].

Rare Congenital Myasthenic Syndromes

Patients have been reported of heteroallelic mutations in the postsynaptic voltage-gated sodium channel, SCN4A [28]. In a severely affected patient with mutations in SCN4A, endplate potentials of normal amplitude failed to activate the postsynaptic voltage-gated sodium channels. This is due to the rapid inactivation of the of Nav1.4 channels resulting from the presence of mutation Val1442Glu. This mutation may show dominant inheritance although it was identified in a patient who also harbored a clinically silent mutation S246L that causes small but detectable biophysical changes [28]. More recently a further case homozygous for Arg1457His has been reported [116].

The prolyl-endopeptidase-like gene (PREPL) encodes a protein that belongs to the prolyl-oligopeptidase subfamily of serine peptidases: proteolytic enzymes that cleave peptides, in which serine serves as the nucleophilic acid at the active site. PREPL is ubiquitously expressed, with highest levels in the brain, kidney, and muscle [117]. One report to date has identified a patient with CMS due to an isolated PREPL deficiency and no cystinuria [118]. A known function of PREPL at the NMJ is to act as an effector of the clathrin-associated adaptor protein 1 in the trafficking of the vesicular ACh transporter [119].

MYO9 encodes the protein myosin-IXA belonging to the superfamily of unconventional myosins. These are actin-based molecular motors implicated in diverse cellular processes [120]. The unconventional myosins are defined by myosin-like head (motor) domains attached to class-specific tail domains that differ greatly from myosin-II. In addition, they do not form bipolar thick filaments when binding to actin filaments [120]. There is increasing evidence that atypical myosins are expressed in peripheral neurons and might play an important role in axonal transport [121].

Three patients from two unrelated families with missense biallelic mutations in MYO9 have been reported [122]. All patients had severe neonatal onset with ptosis, hypotonia, respiratory, and bulbar involvement. Additional features included developmental delay, nystagmus, and oculomotor apraxia. There was a positive response to pyridostigmine and 3,4-diaminopyridine.

Treatment

Response to pharmacological treatment depends on the CMS subtype. Classic treatments include acetylcholinesterase inhibitors to inhibit the acetylcholinesterase from breaking down acetylcholine [123]; 3,4-diaminopyridine that works by blocking presynaptic potassium channels and thus increases the action potential duration and acetylcholine release [124]; and fluoxetine and quinidine that work as open-channel blockers to restore synaptic currents in slow-channel syndrome [71, 72]. More recently, a number of studies have reported great benefit of therapy with β2-adrenergic agonists such as salbutamol and ephedrine in DOK7-CMS [88, 125, 126]. The use of these drugs is increasing in other CMS subtypes such as acetylcholinesterase deficiency [127, 128] and CMS due to abnormal glycosylation [106, 129]. The molecular mechanism for salbutamol and ephedrine at the NMJ is unknown, although for patients with mutations in the AGRN-MUSK-DOK7 pathway, there is a slow but progressive response starting within weeks and increasing in effect before stabilizing at between 6 and 24 months. Additionally, patients with severe AChR deficiency syndrome on long-term anticholinesterase medication may also show remarkable improvement when β2-agonists are added [130]. Indeed β2-agonists are likely to be helpful to anyone on long-term anticholinesterase medication. Chronic anticholinesterase medication is known to be detrimental to synaptic structure, as is impaired signaling through the AGRN-LRP4-MUSK-DOK7 pathway, and therefore an attractive hypothesis is that β2-agonists act to rebuild and stabilize the neuromuscular junction synaptic structure [2].

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Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Nuffield Department of Clinical NeurosciencesUniversity of Oxford, Neurosciences Group, Weatherall Institute of Molecular Medicine, The John Radcliffe HospitalOxfordUK

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