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Department of Clinical
Medicine, University of
Bergen, Postboks 7804,
5020 Bergen, Norway.
2
Department of Neurology,
Haukeland University
Hospital, Postbox 1400,
5021 Bergen, Norway.
3
Department of Neurobiology,
Hellenic Pasteur Institute,
127Vassilissis Sofias
Avenue115 21, Ampelokipi,
Athens, Greece.
Correspondence to N.E.G.
nils.gilhus@uib.no
1
doi:10.1038/nrneurol.2016.44
Published online 22 Apr 2016
REVIEWS
Key points
The characteristic muscle weakness in myasthenia gravis (MG) is caused by antibodies
directed against the neuromuscular junction
MG is divided into subgroups on the basis of specific antibodies, other biomarkers,
and clinical characteristics, such as age of onset, presence of thymoma, and
involvement of ocular muscles
The most common antibodies detected in MG are antibodies against acetylcholine
receptors (AChRs), muscle-specific kinase (MuSK) and low-density lipoprotein
receptor-related protein4 (LRP4)
Additional antibodies of interest in MG are directed against agrin, titin, KV1.4,
ryanodine receptors, collagenQ, and cortactin
Therapy should be tailored to the individual patient and guided by MG subgroup, and
can include symptomatic drug therapy, immunosuppressive drug therapy,
thymectomy and/or supportive therapy
The aim of treatment should be normal or near-normal function, which in most
patients requires long-term immunosuppressive treatment with a drug combination
that is individualized for the patient for optimal effectiveness
AChR antibodies
Prevalence. AChR antibodies can be detected with routine assays in 70% of all patients with MG14. In another
510% of patients, AChR antibodies can only be detected
with more-sensitive, cell-based assays15,16.
Antigenic modulation
Bivalent antibodies can
causecrosslinking of
receptorsand subsequent
receptorinternalization.
Epitope pattern
An epitope is a localized region
on an antigen capable of
eliciting an immune response,
and the epitope pattern refers
to all epitopes involved in an
immune response.
exacerbations19. Such correlations remain to be convincingly demonstrated, but repeated testing for antibodies
can influence therapeutic decisions: an increase in antibody concentration is thought to indicate exacerbation of
MG, whereas a stable or decreasing concentration could
indicate stable disease. However, loss of functional AChRs,
not the AChR antibody concentration, is what leads to the
symptoms of MG: reduced numbers of receptors correlate
with disease severity, and the loss of receptors depends not
only on total AChR-antibody concentration, but also on
autoantibody pattern and non-antibody factors14.
Testing. A radioimmunoprecipitation assay based on
a mixture of solubilized embryonic and adult AChRs
is the most rigorously validated test for AChR antibodies, and is the most reliable among the validated AChR
antibody tests. These testing kits are commercially available. Positive test results have a near 100% specificity for
MG in symptomatic individuals14. Such specificity is
crucial for a first-line screening test that is used also in
patients with vague muscular symptoms or fatigue as the
primarysymptom.
Although ELISA and immunofluorescence assays
have been developed as non-radioactive alternatives
with good sensitivity and specificity, they are inferior to
radioimmunoprecipitation20 and have not been widely
adopted in clinical practice. Cell-based assays, in which
AChR molecules are clustered on the membranes of cultured test cells, offer better sensitivity, and enable detection of low-affinity antibodies: they can detect antibodies
in 466% of patients with generalized MG in whom antibodies cannot be detected with radioimmunoassay15,21,22.
Antibodies that are detected with different tests all belong
to the same IgG subclasses, indicating that the aetiology
and pathogenesis is the same in patients with AChR antibodies that can only be detected in cell-based assays as
in those with antibodies that are detected in routine tests.
The radioimmunoassay is recommended as the firstline test because it has been used successfully in clinical practice; moreover, it not only detects the presence
of AChR antibodies but can also quantify their levels.
Supplementary cell-based testing should be performed
when MG is suspected but the patient is negative for
anti-AChR and anti-MuSK antibodies.
MuSK antibodies
Prevalence. Antibodies against MuSK can be detected in
110% of patients with MG14. These antibodies are present in patients from the Mediterranean area more often
than in those from Northern Europe, possibly owing to a
combination of genetic and environmental factors14.
Pathophysiology. In contrast with AChR antibodies, IgG
antibodies against MuSK belong mostly to the IgG4 subclass. Their mode of action also differs: AChR antibodies
bind to complement and crosslink their antigenic target,
whereas MuSK antibodies are directly pathogenic, even
if they do not bind to complement, and remain functionally monovalent23. Instead of modulating AChR
function, MuSK antibodies reduce the postsynaptic density of AChRs and impair their alignment between the
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motor nerve terminal and the postsynaptic membrane
(FIG.1b). Most MuSK antibodies bind to the extracellular
Nterminal Iglike domains of the AChR (BOX3).
Testing. In patients with MuSK-MG, concentrations of
anti-MuSK antibody tend to correlate with disease severity, and changes in antibody concentration over time
can reflect disease activity24. Immunoprecipitation with
radioimmunoassays is the standard technique for MuSK
antibody detection, and ELISA tests are also available,
although neither are as sensitive as cell-based assays23,25:
a large screening study showed that a cell-based assay
could detect MuSK antibodies in 13% of MG serum
samples that had been classified as seronegative using
radioimmunassay or ELISA11. Some of these patients
had ocular MG. The specificity of the cell-based assay is
estimated to be 9798%.
MuSKMG and AChRMG are distinct disease entities and rarely occur in the same patient. Nevertheless, a
few single-patient reports have demonstrated the existence of both AChR and MuSK antibodies in the same
patient, particularly when the most sensitive antibody
detection techniques are used16.
LRP4 antibodies
Prevalence. The likelihood of detecting LRP4 antibodies in MG depends on the assay and the examined
population. LRP4 antibodies are detected in 15%
ofpatients with MG of any type26,27, and in 733% of MG
patientswithout AChR and MuSK antibodies27,28.
Pathophysiology. In LRP4immunized mice, LRP4
antibodies are directly pathogenic and induce muscular weakness through disruption of the interaction
between LRP4 and agrin (BOX4), and inhibition of AChRmediated neuromuscular transmission29 (FIG1). The disease in the LRP4antibody mouse model was similar
to that seen in mice immunized with AChRs, and to
the muscle weakness seen in the human disease. LRP4
antibodies observed in these mice mainly belong to the
complement-binding IgG1 subclass30.
Other antibodies
Agrin antibodies. Agrin autoantibodies can be detected
in a minority of patients with MG, either with or without
antibodies against AChR, MuSK or LRP4 (REFS32,33).
Agrin antibodies have been detected only in patients
with MG, suggesting that these antibodies are specific to
thedisease.
Agrin is a heparan sulfate proteoglycan released from
the motor nerve terminal34. It regulates the formation,
maintenance and regeneration of the neuromuscular
junction34, and interference with agrin function leads to
insufficient neuromuscular transmission34. To date, no
directly pathogenic effect of agrin antibodies has been
established, although such antibodies inhibit MuSK
phosphorylation and AChR clustering invitro3234.
Titin antibodies. Antibodies against titin the largest of
all known proteins can be detected in 2030% of MG
patients with AChR antibodies, mostly in patients with
thymoma-associated disease or late-onset MG11,35. Titin
is located intracellularly and is essential for muscle contractility36. Given its intracellular location, titin antibodies
should not interfere with muscle function. Nevertheless,
their presence indicates a more severe form of MG with
mild myopathy and a need for immunosuppressive treatment11. Moreover, antibodies against titin are a sensitive
marker of thymoma in patients with MG whose symptom
onset occurs before the age of 50years11,35. Most titin antibodies target a 30kDa region of the protein, called myasthenia gravis titin30, that is located near the AI junction
in muscle, thereby potentially interfering with the role of
titin in mediating muscle elasticity37. A commercial kit is
available for titin antibody testing.
KV1.4 antibodies. Antibodies against the subunit of
the voltage-gated K+ channel KV1.4 in skeletal muscle
are detected in 1020% of patients with MG14,38. KV1.4
channels are widely expressed in the CNS, where they are
concentrated in axonal membranes or near axons, and are
also present in the endocardium. In MG, KV1.4 antibodies seem to cross-react with voltage-gated K+ channels in
the heart muscle14,38. In a Japanese patient cohort39, KV1.4
antibodies were associated with severe MG and heart
complications, but the findings could not be confirmed
in European patients38. Invivo antibody binding to KV1.4
in MG has not been confirmed.
REVIEWS
a
Axonal terminal
Pathogenic
Present in MG
(unproven pathogenicity)
Action potential
Agrin
Ca2+
Ca2+
Choline
Acetic acid
Acetylcholine
Acetylcholine
Acetylcholinesterase
K+
Muscle
KV1.4
LRP4
MuSK
MuSK
AChR
clustering
Folding
Na2+
Cortactin
Actin
Myosin
Titin
ColQ
AChR
RyR
Muscle contraction
Muscle bre
Sarcoplasmic
reticulum
Auto-reactive T cell
Thymoma
Action potential
B cell activation
and auto-antibody
production
Ca2+
Ca2+
Complement
activation
Antigenic
Fold
modulation
destruction 2
C1 complement
1
MAC
AChR
internalization
Blocking
antibody
Clustering
Folding
Muscle contraction
Cell lysis
AChR degradation
c Thymectomy
Thymoma
B cell activation
and auto-antibody
production
Azathioprine
Mycophenolate
mofetil
Methotrexate
Cyclosporine
Plasma exchange
immunoadsorption
Ca2+
Ca2+
Complement
activation
Rituximab
Action potential
Acetylcholinesterase
inhibitor
Pyridostigmine
Ambenonium chloride
Neostigmine
Fold
destruction
Antigenic
modulation
AChR
internalization
Clustering
Folding
Cell lysis
Muscle contraction
AChR degradation
Nature Reviews | Neurology
4 | ADVANCE ONLINE PUBLICATION
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Ryanodine receptor antibodies. Antibodies against
the ryanodine receptor (RyR) are present in 70%of
AChR-MG patients with thymoma and in 14%
ofpatients with late-onset AChR-MG8,40. The RyR is
the Ca2+ channel in sarcoplasmic reticulum12. It opens
upon depolarization of the sarcolemma and participates in muscle contraction through release of calcium from the sarcolemma into the cytoplasm12. The
presence of anti-RyR antibodies indicates severe MG,
though the pathogenic role of these antibodies has not
been established12.
Collagen Q antibodies. CollagenQ is a protein that
concentrates and anchors acetylcholinesterase at the
neuromuscular junction, where it is localized in the
extracellular matrix and is, thus, accessible to antibodies; collagenQ is found only at the neuromuscular
junction. Antibodies against collagenQ were recently
detected in the serum of 12 of 415 (3%) patients with
MG41. In seven of these patients, no other antibodies
were found. As mutations in collagenQ can lead to
myasthenic syndromes41, anti-collagenQ antibodies
might be pathogenic. However, anti-collagenQ antibodies have also been detected in healthy controls11, and
any diagnostic or pathogenic significance of these antigens, or a capacity to interfere with anticholinesterase
therapies, remains to be proven.
with various autoimmune disorders, including myositis11. Any relevance to MG pathogenesis, diagnosis or
treatment remains to beproven.
Autoantibody-based subgroups
AChR-MG. MG with anti-AChR antibodies is divided
into early-onset MG (symptom onset before 50years)
and late-onset MG (onset after 50years). In both subgroups, symptoms are generalized. In non-AChR-MG
subtypes, the distinction between early and late onset
does not have any proven value for prognostication
ortreatment.
In AChR-MG, the early-onset and late-onset disease types differ with respect to thymic pathology, HLA
genotype and other genetic variables, autoimmune
comorbidity, and response to therapy. For example,
thymectomy has clear clinical benefits in early-onset
Cortactin antibodies. Cortactin is a cytosolic actin- MG, but its benefits in late-onset MG are more quesbinding protein in the skeletal muscle that promotes tionable and possibly non-existent8,43. Moreover, the
actin assembly42. Moreover, it has a role as a signalling response to immunosuppressive drugs, as well as the
protein involved in AChR clustering mediated by the risks and clinical relevance of adverse effects, can differ
agrinMuSK complex 42. Cortactin antibodies were between early-onset and late-onset forms2,4,8 (TABLE1).
recently detected in 20% of MG patients without AChR
MG onset can occur in childhood. The clinical
or MuSK antibodies,but also in 5% of MG patients with course of childhood and juvenile MG is similar to
AChR antibodies, healthy controls, and 13% of patients that of early-onset MG in adults4. Juvenile MG is most
common in East Asian populations44.
Figure 1 | Neuromuscular junction in myasthenia gravis (MG). a | Normal function
of neuromuscular junction, with major components implicated in MG shown. Action
potential at the presynaptic nerve terminal causes opening of voltage-dependent
Ca2+ channels, triggering release of acetylcholine and agrin into the synaptic cleft.
Acetylcholine binds to acetylcholine receptors (AChRs), which promote sodium channel
opening, which in turn triggers muscle contraction. Agrin binds to the complex formed
by low-density lipoprotein receptor-related protein4 (LRP4) and muscle-specific kinase
(MuSK), causing acetylcholine receptor (AChR) clustering, which is required for
maintenance of the postsynaptic structures of the neuromuscular junction. b | Major
pathogenic mechanisms of the AChR antibodies in MG include complement activation
at the neuromuscular junction, which causes formation of membrane attack complexes
(MACs) on the muscle membrane and destruction of the typical folds in the sarcolemma
(1); antigenic modulation that results in internalization and degradation of surface
AChRs (2); and binding of AChR antibodies at the AChR ligand binding site (3), which
could directly block acetylcholine binding and, consequently, channel opening.
Anti-MuSK and anti-LRP4 antibodies have been shown to block the intermolecular
interactions of MuSK or LRP4 respectively, and could thus inhibit the normal mechanisms
for maintenance of the organization of the neuromuscular junction (4). Antibodies with
known pathogenic involvement in MG are shown in red. c | MG treatment can restore
function of the neuromuscular junction by increasing the levels of available acetylcholine
(acetylcholinesterase inhibitors; green), which improves signal transduction, or by
reducing the concentration of autoantibodies (immunosuppressive drugs, plasma
exchange/immunoadsorption, B-cell-targeting therapies; red), which alleviates the
pathogenic mechanisms described in (b). KV1.4, voltage-gated potassium channel1.4;
RyR, ryanodine receptor.
REVIEWS
Box 2 | Structure and function of AChR
The acetylcholine receptor (AChR; top and side views shown) is composed of five
homologous subunits (pictured in different colours) that form a cation pore (middle).
Subunit composition changes with the age of the individual: 2 is the predominant
subunit composition in embryonic muscle tissue, and 2 in adult muscle tissue17,68.
Although AChRs have several targeted epitopes, the majority of anti-AChR antibodies
are directed against the main immunogenic region (MIR [17), a group of overlapping
epitopes located on the AChR subunit; the central core of these subunits is formed by
amino acids 6776.
MIR
MIR
AChR
Therapies
Symptomatic drug therapy
The acetylcholinesterase inhibitor pyridostigmine
represents the first-choice treatment in all types of
autoi mmune MG 4,8. Neostigmine, another acetyl
cholinesterase inhibitor with a shorter half-life, can also
be used14. Ambenonium chloride is another acetyl
cholinesterase inhibitor, though for most patients, it is
less effective than pyridostigmine or neostigmine. All
these drugs inhibit acetylcholine degradation, thereby
increasing the availability of acetylcholine in the
synapse. All MG subgroups besides MuSK-MG usually
respond well to this treatment14, but individual variation
isconsiderable.
In MuSK-MG, the response to acetylcholinesterase
inhibition is often insufficient, reflecting differences
between MG subgroups with respect to antibody-
induced pathology in the postsynaptic muscle membrane. A recent study reported a good response in
only 50% of patients with MuSKMG, and 10% did not
respond at all16. In such patients, 3,4diaminopyridine,
which increases presynaptic release of acetylcholine,
should be tried45. This drug blocks neuronal efflux
through K+ channels, which increases the duration of
action potentials and results in prolonged Ca2+ channel opening, thereby promoting acetylcholine release
at the muscle endplate45. The beneficial effect of pyridostigmine is specific to MG. The dose should be
adjusted to achieve the optimum according to therapeutic effect and adverse effects; most patients are able
to do this on their own. Unfortunately, 3,4diaminopyridine provides only a mild positive effect for most
patients14.
Reviews
| Neurology
anti-MuSK and anti-LRP4Nature
antibodies;
these
patients
16
tend to have more-severe MG and should, in our
opinion, be categorized according to the relevant AChR
antibody subgroup.
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patients with early-onset MG require pharmacological
immunosuppression; however, in many of them, this
need istemporary.
LRP4-MG is usually relatively mild, and immunosuppression is often not needed. For ocular MG, there
are two treatment aims: to improve symptoms (ptosis
and diplopia) and prevent generalized weakness.
Immunosuppression can do both46.
First-line treatments. First-line immunosuppressive
drug therapy for MG comprises either prednisone or
prednisolone, or the combination of prednisone or prednisolone with azathioprine47. These drugs have a broad
action on the immune system, and have been shown to
be beneficial in all MG subgroups, though the benefit
varies depending on the subgroup.
According to current recommendations, prednisolone alone should be given only as short-term treatment
(<1year)8,46. Long-term prednisolone monotherapy
could be considered for the treatment of ocular MG, but
for the majority of other patients with MG, combination immunosuppressive treatment is recommended to
obtain maximum effect with minimal adverse effects8,46.
Controlled studies of immunosuppression in MG
are scarce, and virtually none have described subgroup-
specific effects. However, uncontrolled studies and
clinical experience have proven the effect of this treatment for MuSK-MG, late-onset MG and thymoma MG,
as well as for early-onset MG and LRP4-MG, which
usually have milder symptoms. Whereas azathioprine
(23mg/kg daily) takes 615months to yield an optimal effect, prednisolone exerts its full effect during the
first few weeks and months of treatment. Alternate-day
dosing and gradual dose increases are widely used in
an attempt to avoid adverse effects. Once the optimal
improvement has been reached, the dose of prednisolone
should be slowly reduced to the lowest effective dose.
Two studies have indicated that prednisolone treatment
of ocular MG reduces the risk of MG generalization,
in addition to the beneficial effect on the ocular symptoms48,49. Any added value of azathioprine for ocular MG
has not beenshown.
If immunosuppressive therapy induces pharmacological remission or a marked improvement, it should
be maintained in the long-term, but the dose should be
reduced to avoid adverse effects. Full drug withdrawal
will often lead to new exacerbations, particularly in
MuSKMG, thymoma MG and late-onset MG. The
presence of additional antibodies, particularly against
RyR and titin, is an indication for long-term treatment, as these antibodies are more common in severe
MG8,11. Prednisolone and azathioprine can be safely
administered during pregnancy and lactation50.
Second-line treatments. Priorities for second-line
immunosuppressive therapy are debated. No controlled
studies have compared different drugs in MG. Formal
studies examining specific therapies in well-defined MG
cohorts, including different MG subgroups, are sparse.
For moderate and mild MG, mycophenolate mofetil is
an option after the failure of first-line therapy owing
Ig1+Ig2
Ig3
Frizzled
Frizzled
Transmembrane
segment
Juxtamembrane
segment
Kinase
Kinase
REVIEWS
MG crisis
Severe worsening of
myasthenic weakness that
requires intubation or
noninvasive ventilation
toavoidintubation.
or JCvirus-related progressive multifocal leukoencephalopathy. Treatmentsspecifically targeting Bcells represent an attractive strategy for all antibody-mediated
disorders, including all MG subgroups, and emerging monoclonal antibodies are promising. However,
concerns about the consequences of highly effective
immunosuppression persist.
Alternative second-line treatments and third-line treatments. Alternative second-line and third-line treatment
options for MG include methotrexate, cyclosporine, tacrolimus and cyclophosphamide14,8. Cyclosporine has a
proven effect in well-controlled studies, but its use has
been limited by a high risk of adverse effects. Response
rates for the different MG subgroups have not been
defined. The presence of antibodies that have been linked
to more-severe disease (that is, antibodies against RyR,
titin or KV1.4) increases the need for active treatment. The
effects of these treatments in the presence of antibodies
against agrin, collagenQ and cortactin are notknown.
Intravenous immunoglobulin (IvIg) is predominantly
used as short-term immunoactive therapy in acute situations. IvIg and plasma exchange have similar effects on
MG exacerbations5760. Such treatment should always be
given for an ongoing or imminent MG crisis, and is also
recommended shortly before situations in which muscle
weakness is expected to deteriorate or lead to complications, such as surgery. The effect of IvIg and plasma
exchange occurs 25days after treatment and lasts for
Thymectomy
Thymic abnormalities in MG, such as hyperplasia and
thymoma, strongly suggest a role for a thymus-mediated
immune response in MG43,63. Active germinal centres and
an ongoing export of disease-inducing Tlymphocytes
will be effectively stopped by thymectomy43,63.
Early-onset MG. Many controlled studies have shown
that patients with early-onset MG who are thymectomized have a more favourable outcome than those who
are not63,64. However, none of the studies have been prospective or included matched control groups. Present evidence clearly favours early and complete thymectomy in
patients with early-onset MG who are not symptom-free
on symptomatic drugs alone. The fact that thymic hyperplasia is common in this subgroup supports a therapeutic
effect of thymectomy performed early after MG onset.
Early thymectomy will prevent export of AChR-specific
Tcells from the thymus to lymph nodes and peripheral
lymphoid tissue43,63. For a positive outcome, removal of
all thymus tissue is essential; the results of various video-
assisted and robotic surgical procedures and traditional
methods are similar65.
Late-onset MG. Late-onset MG is traditionally regarded
as less responsive or nonresponsive to thymectomy63,64;
however, the evidence regarding thymectomy in patients
with late-onset MG is sparse. The fact that the thymus of
patients in this subgroup is usually atrophic (an age-normal finding) does not provide any support to the use of
thymectomy. However, the onset age of 50years implies
that thymus function has no strong influence on pathogenesis. In patients with MG onset at 5065years who have
thymic hyperplasia, the response to thymectomy might
be expected to be similar to that in early-onset MG, and
thymectomy should be considered in selectedpatients.
Antibodies against titin and RyR are most common in
late-onset MG. Although not tested in controlled studies,
such antibodies probably indicate that no response will be
seen to thymectomy. Biomarkers are needed to establish
indications for thymectomy in late-onset MG.
Thymoma-associated MG. In thymoma MG, the thymoma of the thymus gland should always be removed to
treat the cancer. The response of MG to thymectomy is
variable, and improvement of MG symptoms is usually
more limited than in early-onset MG.
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Table 1 | Characteristics of myasthenia gravis subgroups
Subgroup
Antibody
Additional Age at
antibodies onset
Proportion of Thymus
patients (%)
Clinical benefit
of thymectomy
Early-onset
Anti-AChR
Rare
<50years
1525
Hyperplasia
common
Proven
Late-onset
Anti-AChR
Common
>50years
3545
Atrophy common
Not proven
(butpossible)
Thymoma
Anti-AChR
Very
common
Any
10
Lymphoepithelioma Proven
MuSK
Anti-MuSK
Rare
Any
110
Normal
None
LRP4
Anti-LRP4
Rare
Any
15
Normal
None
Any
1015
Variable
None
Ocular
Any
15
Variable
None
Variable
Rare
AChR, acetyline choline receptor; LRP4, low-density lipoprotein receptor-related protein4; MuSK, muscle-specific kinase.
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Acknowledgements
Author contributions
www.nature.com/nrneurol
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