There have been major advances in the understanding of the underlying causes of the various myasthenic syndromes, all of which involve abnormalities related to the neuromuscular junction ( Fig. 21.1 ). Their characteristic feature is muscle weakness with fatigue. Myasthenia gravis is an acquired autoimmune disorder caused in most cases by antibodies to the acetylcholine receptors (AChRs), or to a tyrosine kinase receptor, muscle-specific kinase (MuSK), or to low-density lipoprotein-related protein 4 (LRP4) ( ). Additional autoantibodies to other muscle proteins have also been identified, sometimes concomitantly with antibodies to AChRs ( ). These include agrin, cortactin, titin and the ryanodine receptor ( ). Most congenital forms of myasthenia are caused by mutations in genes encoding various key synaptic players in neuromuscular transmission (see Fig. 21.1 ; Table 21.1 ). In addition, neonates of mothers with autoantibodies may show transient myasthenia. Antibodies to voltage-gated calcium channels on the pre-synaptic membrane cause Lambert–Eaton syndrome, and neuromyotonia results from antibodies to a pre-synaptic voltage-gated potassium channel.
| Location/Function of Protein | Protein | Gene | Locus | Inheritance |
|---|---|---|---|---|
| Pre-synaptic | ||||
| Acetylcholine synthesis and recycling | Choline acetyltransferase (ChAT) | CHAT | 10q11.2 | AR |
| Loads acetylcholine into vesicles | Vesicular acetylcholine transporter (VAChT) | SLC18A3 | 10q11.23 | AR |
| Uptake of choline after breakdown of acetylcholine | High affinity choline transporter 1 | SCL5A7 | 2Q12.3 | AR |
| Priming of synaptic vesicles for release | MUNC13-1 | UNC13A | 9p13.3 | AR |
| Synaptic vesicle exocytosis, component of SNARE complex | Vesicular acetylcholine transporter (synaptobrevin1) | VAMP1 | 12p13.31 | AR |
| Vesicular exocytosis, component of SNARE complex | Synaptosomal-associated protein 25 | SNAP25B | 20p12.2 | AD |
| Calcium sensor for exocytosis of synaptic vesicles, component of SNARE complex | Synaptotagmin 2 | SYT2 | 1q32.1 | AD |
| Trafficking of vesicular ACh transporter | Prolyl endopeptidase-like protein | PREPL | 2p21 | AR |
| Trafficking of synaptic vesicles | Rabphilin 3A | RPH3A | 12q24.13 | AR |
| Role in axonal transport and/or neuronal branching and axon guidance | Unconventional myosin-IXA | MYO9A | 15q23 | AR |
| Synaptic | ||||
| Endplate AChE deficiency | Collagen tail of AChE | COLQ | 3p24.2 | AR |
| Reduced endplate potential quantal content | Laminin β2 chain | LAMB2 | 3p21 | AR |
| Reduced endplate potential quantal content/vesicle trafficking | Laminin α5 | LAMA5 | 20q13 | AR |
| Endplate AChE deficiency | Alpha chain of collagen XIII | COL13A1 | 10q22 | AR |
| Post-synaptic | ||||
| Fast channel syndromes | ACh receptor α subunit | CHRNA1 | 2q24-q32 | AR |
| ACh receptor δ subunit | CHRND | 2q33-q34 | AR | |
| ACh receptor ε subunit | CHRNE | 17p13 | AR | |
| Slow channel syndromes | ACh receptor α subunit | CHRNA1 | 2q24-q32 | AD |
| ACh receptor β subunit | CHRNB1 | 17p11-p12 | AD | |
| ACh receptor δ subunit | CHRND | 2q33-q34 | AD | |
| ACh receptor ε subunit | CHRNE | 17p13 | AD, AR | |
| ACh deficiency | ACh receptor β subunit | CHRNB1 | 17p11-p12 | AR |
| ACh receptor δ subunit | CHRND | 2q33-q34 | AR | |
| ACh receptor ε subunit | CHRNE | 17p13 | AR | |
| Abnormalities in clustering of ACh receptors | Rapsyn Muscle-specific kinase Agrin Low-density lipoprotein receptor-related protein 4 Downstream of tyrosine protein 7 | RAPSN MUSK AGRN LRP4 DOK7 | 11p11 9q31.3 1p36.33 11p11.2 4p1.3 | AR AR AR AR AR |
| Abnormalities of cytoskeleton | Plectin | PLEC | 8q24-qter | AR |
| Anomaly of muscle sodium channel | Sodium channel α-subunit | SCN4A | 17q23 | AR |
| DEFECTS IN GLYCOSYLATION | ||||
| Glutamine-fructose 6-phosphate transaminase 1 | GFPT1 | 2p12-p15 | AR | |
| Dolichyl-phosphate N -acetylglucosamine phosphotransferase | DPAGT1 | 11q 23.3 | AR | |
| Alpha-1,3-mannosyltransferase (homolog of yeast ALG2) | ALG2 | 9q22.33 | AR | |
| UDP- N -acetyl-glucosaminyltransferase subunit (homolog of yeast ALG14) | ALG14 | 1p21.3 | AR | |
| GDP-mannose pyrophosphorylase, ß- subunit | GMPBB | 3q21.31 | AR | |
Myasthenia is usually suspected on clinical history. Muscle weakness and fatigability may be demonstrated by the inability to sustain a particular movement such as upward gaze, or speech, or holding out the arms, in addition to characteristic features such as the myasthenic snarl and ptosis. Confirmation is usually provided by pharmacological tests such as the response to acetylcholinesterase (AChE) inhibitor, either intravenous edrophonium (Tensilon), or a trial of oral pyridostigmine, by electrophysiological studies, such as the response decrement of the motor action potential to repetitive stimulation of a nerve, and/or by the presence of ‘jitter’ on single fibre electromyography, or by the finding of serum antibodies to AChRs, MuSK or LRP4. In addition to AChE inhibitors, treatment may involve the use of steroids, immunosuppressive drugs, drugs that deplete B cells, thymectomy or plasmapheresis ( ). Routine muscle biopsy usually has very little diagnostic role to play, although pathological features may be present, and specialized studies of the nerve terminals and the neuromuscular junction have contributed substantially to our understanding of the pathophysiology.
Myasthenia Gravis
This rare, acquired autoimmune disorder is caused by circulating antibodies to AChRs, MuSK or LRP4. It most frequently has onset in adulthood at 45–50 years of age. It has a higher prevalence in females than males (6:4), but later onset can be more common in men and onset in childhood can also occur ( ). The majority of cases are due to antibodies to AChR, and most of those previously described as ‘seronegative’ have either antibodies to MuSK or low-affinity AChR antibodies not detected on routine assay, or antibodies to LRPP4 ( ). In addition to these, antibodies to various muscle proteins have also been identified in some patients, including agrin, cortactin, myosin, actin, α-actinin, titin, filamin, vinculin, tropomyosin and the ryanodine receptor ( ). Also, patients with Hashimoto disease and antibodies to the thyroid gland can show myasthenic symptoms ( ). Graves’ disease, an autoimmune disorder caused by autoantibodies that stimulate the thyroid-stimulating hormone receptor, resulting in stimulation of thyroid hormone synthesis, is also known to be associated with myasthenia ( ). There are also reports of cases with an autoimmune disorder of caveolin-3 that also have antibodies to AChR and myasthenia gravis ( ). Autoantibodies are associated with inflammatory myopathies and some patients can show concomitant myasthenic symptoms and myositis (see Ch. 22 ; ).
A significant proportion of myasthenia gravis patients with AChR autoantibodies show hyperplasia of the thymus or have a thymoma. Thymectomy, immunosuppression and plasma exchange are therapeutic approaches resulting from the pioneering clinical and research work by John Newsom Davies and his team ( ). The muscle weakness and fatigability associated with antibodies to AChR are generalized, with weakness of the ocular muscles and ptosis being the most common presenting symptoms. Ocular involvement is common and in a proportion of patients the weakness remains isolated to the ocular muscles and eyelids. However, muscle weakness and fatigability can be generalized, variably involving, sometimes asymmetrically, all muscle groups including bulbar, respiratory and axial muscles. The weakness characteristically worsens with sustained exertion and during the course of the day. Spontaneous remissions of variable length can occur.
The clinical features associated with antibodies to MuSK are often severe and include involvement of facial, bulbar and respiratory muscle. Ocular symptoms are less prominent than in patients with antibodies to AChR. The thymus histology is usually normal and thymoma uncommon. Patients with LRP4 autoantibodies are more mildly affected. Although thymic changes have been reported, the involvement of the thymus is not clear.
There is a high incidence of miscarriages in females with myasthenia gravis. Transient neonatal myasthenia affects about one in seven infants born to myasthenic mothers and may produce life-threatening weakness, requiring urgent treatment. The infant is usually affected at birth but the symptoms may sometimes be delayed for some days. The infant is usually floppy, with general hypotonia and weakness, and there may be associated swallowing and breathing problems. Arthrogryposis is a severe complication in some cases. The condition is self-limiting, with gradual recovery of the infant, usually within 2–4 weeks, but can be lethal. The non-lethal syndrome is often referred to as Escobar syndrome and can also be caused by mutations in genes encoding proteins at the neuromuscular junction. The autoimmune form results from maternal antibodies to the embryonic γ subunit of the acetylcholine receptor (see below; ).
Pathology
Abnormalities in muscle biopsies are mild or minimal, except at the neuromuscular junction. If present, the changes may be focal and are usually non-specific. They include atrophy of type 2 fibres, and sometimes atrophy of type 1 fibres, which may be accompanied by the presence of small dark angulated fibres with the oxidative enzymes, suggestive of denervation. Although lymphocytes have been reported in myasthenia gravis, they are not a consistent feature.
The pioneering studies of A G Engel have made a major contribution to our understanding of the pathogenesis of myasthenia gravis ( ). The folds of the junction are reduced, or absent, and debris from them accumulates between the nerve and the muscle membrane. Complement (C3, C9 and the C5b-9 membrane attack complex) and immune complexes can be demonstrated on the post-synaptic membrane, and binding of the autoantibodies in the serum from affected patients to neuromuscular junctions can be demonstrated.
Neuromuscular junctions are only occasionally present in a muscle biopsy, unless a motor point sample is specifically taken. In practice, the diagnosis of myasthenia is usually made without the need for a muscle biopsy, from clinical and electrophysiological examinations, estimation of AChR, MuSK, and LRP4 autoantibodies, and a response to AChE inhibitors.
Pathogenesis
The nerve impulse results in the release of acetylcholine, which binds to its receptor on the peak of the post-synaptic folds of the muscle fibre; this induces depolarization of the muscle membrane, the release of calcium, and results in an action potential. The AChRs in skeletal muscle are formed from five homologous subunits organized round a central ion channel, two α1, a β1, the ε or γ, and the δ subunits. In fetal muscle the γ subunit is present and is replaced at 31 weeks’ gestation by the ε subunit. Denervated muscle reverts to the fetal type, with the γ subunit. In myasthenia gravis the anti-AChR antibodies bind mainly to one site on an extracellular region of the α1 subunit. This is referred to as the main immunogenic region and is distinct from the cholinergic binding site. The bound antibodies cross-link AChRs, as well as inducing receptor loss due to complement-mediated lysis. The net result is a reduction in the number of functional AChRs and disruption of the structure of the post-synaptic membrane. Weakness and fatigue become obvious when a threshold number of affected receptors is exceeded.
Sera from patients with myasthenia gravis often also react with fetal muscle, suggesting that the γ subunit of AChR is also involved. It has been suggested, but not substantiated, that the involvement of the extraocular muscles might relate to the presence of this subunit in these muscles, as ocular muscles express several proteins found in fetal muscle. Rare patients who show no symptoms of myasthenia gravis have been identified in whom antibodies to the γ subunit cross the placenta and severely affect the fetus, causing death or arthrogryposis multiplex congenita ( ).
MuSK is a post-synaptic transmembrane tyrosine kinase that is activated by binding to LRP4 and acts as a signal for agrin-mediated clustering of AChRs. Agrin, together with MuSK and LRP4, forms a complex that is blocked by MuSK autoantibodies and interferes with AChR clustering ( ). It has been reported that MuSK is also important in the clustering of AChE by the formation of a complex with perlecan and the collagen Q tail of AChE ( ). MuSK autoantibodies are mainly of the IgG4 subtype, and, unlike those to AChR and LRP4, they do not activate complement.
Lambert–Eaton Syndrome
The characteristic features of Lambert–Eaton myasthenic syndrome are progressive weakness and fatigability, principally of the limb muscles ( ). Bulbar and ocular muscles are usually spared. Many patients also present with varying degrees of autonomic dysfunction, and there is strong association with lung neoplasms. It can be distinguished from myasthenia gravis by the augmentation in strength following voluntary contraction, due to pre-synaptic facilitation, and by depressed tendon reflexes. It results from immunoglobulin G (IgG) antibodies to voltage-gated calcium channels, which reduce their number and impair the calcium-dependent release of acetylcholine ( ).
Pathology
There are only a few pathological studies of muscle biopsies, and reported features are of only mild non-specific changes, such as a reduction of type 1 fibres and a progressive type 2 fibre predominance ( ). Type 2 atrophy has been observed in a few cases ( ), and tubular aggregates have been reported in one patient ( ). Freeze fracture electron microscopy of the pre-synaptic membrane shows a reduction in the number of active zone particles, which are believed to represent the voltage-gated calcium channels, and abnormalities in their distribution ( ).
Acquired Neuromyotonia
The clinical features of neuromyotonia include muscle hyperactivity, muscle stiffness, cramps, myokymia, insomnia, pseudomyotonia and muscle weakness. Excessive perspiration is also common ( ). Some patients have additional autonomic and central nervous system symptoms. The underlying pathogenesis relates to autoantibodies influencing voltage-gated potassium channel (VGKC) function, although the full involvement of this family of channels is not known ( ). Muscle pathology shows non-specific features with increased variability in fibre size, mild fibrosis, occasional fibres devoid of periodic acid–Schiff (PAS) staining and sometimes indistinct fibre typing ( ).
Congenital Myasthenic Syndromes
The congenital myasthenic syndromes are an expanding, heterogeneous group of inherited disorders caused by mutations in genes that encode various proteins involved in neuromuscular transmission (see Fig. 21.1 ; ) . The wide application of next-generation sequencing is making a significant contribution to the identification of novel genes associated with myasthenic syndromes. Classification of these disorders is based on the location of the defective protein, pre-synaptic, the synaptic cleft, or post-synaptic (see Table 21.1 ). As with autoimmune myasthenia, diagnosis is based on family history, careful clinical examination, particularly with regard to observing the weakness and fatigability, electrophysiology and the response to AChE inhibitors (edrophonium; Tensilon test). A negative response to AChE inhibitors does not exclude a diagnosis of congenital myasthenia as not all subtypes respond to AChE inhibitors. Electrophysiology and a response to therapy, such as pyridostigmine, have revealed an associated myasthenic-like syndrome in some patients with mutations in genes more commonly associated with a congenital myopathy than with neuromuscular junction defects. These include TPM2 , TPM3, BIN1 , DNM2, MTM1 and RYR1 ( ).
Pathology
Many of the recorded morphological features relate to abnormalities of the neuromuscular junction, which are not routinely available for study in muscle biopsies (see ). The features observed include small synaptic vesicles, small endplates, uneven distribution of AChRs, wide synaptic spaces and degeneration of the junctional folds (see Fig. 21.8 ). Examining the binding of bungarotoxin can be useful if endplates are present in the biopsy ( ). In some congenital myasthenias there are no morphological changes at the endplate.
Routine histological and histochemical studies of muscle biopsies often show minimal or no pathology or non-specific changes such as variation in fibre size ( Figs. 21.2–21.4 ), endomysial fibrosis, small clusters of atrophic fibres, atrophy of type 2 fibres (see Figs. 21.2 and 21.3 ) and predominance of type 1 fibres (see Fig. 21.4 ; ). In our experience, if a patient is clinically severely affected and the muscle pathology is minimal, a myasthenic or metabolic condition should be considered. We have, however, noted hypertrophy of muscle fibres in some cases ( Fig. 21.5 ). In a study of intercostal muscle biopsies from 14 patients with DOK7 mutations, often described as ‘limb-girdle myasthenia’ (although other forms can also have a limb-girdle phenotype), the pathological features observed included type 1 fibre predominance, atrophy of type 2 fibres, occasional necrotic and regenerating fibres, areas of uneven oxidative enzyme staining and target-like areas ( ). In our own studies of 16 quadriceps biopsies, we observed type 1 fibre predominance and core-like areas devoid of oxidative enzyme activity in 7 out of the 16 cases ( ). Core-like areas and unevenness of oxidative enzyme stains are quite a common finding in biopsies from DOK7 patients, albeit non-specific ( Fig. 21.6 ). The neuromuscular junctions studied by , which are abundant in intercostal muscles, often showed degenerative changes, and antibodies to the DOK7 protein showed abnormalities with multiple positive areas; however, the changes were not specific to this form of myasthenia.
