Introduction
Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction mediated
by autoantibodies. Clinically, pathological muscle fatigue occurs mainly in the eye
muscles, but can other muscle groups can likewise be affected. MG can be limited to
the eye muscles (ocular MG) or may extend to additional muscle groups (generalized
MG).
In addition to clinical diagnostics with provocation of muscle fatigue and pharmacological
testing, neurophysiological and laboratory tests are the most important investigations
used to confirm the suspected diagnosis of myasthenia gravis. The identification of
new antigens has not only changed antibody diagnostics; rather, MuSK antibody-positive
MG has been clinically distinguished as a separate subform of the disease.
Autoantibodies and myasthenia gravis
A neuromuscular junction consists of 3 components: (1) the terminal nerve ending where
acetylcholine is formed, deposited in vesicles and then released, (2) the synaptic
gap (3) the postsynaptic (muscle) membrane containing the acetylcholine receptor and
its helping proteins as well as cholinesterase. In myasthenia gravis, autoantibodies
can occur that affect various structures of the neuromuscular junction. Some of these
autoantibodies determine a separate clinical subtype, while others indicate comorbidities
such as thymomas.
Acetylcholine receptor antibodies (anti-AChR-ab)
The acetylcholine receptor antibody was the first pathogenic antibody identified in
MG patients, and can be detected in about 80–85% of MG patients; it does not generally
occur in healthy persons, and only rarely appears in patients with other autoimmune
diseases (overview in [1]). Its pathogenetic effects were identified early. AChR antibodies belong to the
complementary-binding IgG1 and IgG3 subclasses and thus mediate complement-dependent
damage to the postsynaptic muscle membrane [2]
[3]. In addition, the antibody links ACh receptors to each other, leading to internalization
of the receptors and depletion of AChR at the postsynaptic membrane (overview in [4]). These antibodies can also somewhat block AChR directly.
Antibodies against muscle-specific kinase (anti-MuSK-ab)
In 2000 it was first determined that approx. 50% of patients with seronegative MG
(no evidence of AChR-ab despite clinical myasthenia gravis) have autoantibodies against
a muscular surface protein that is not identical to the AChR [5]. This antigen was identified as MuSK, a transmembrane protein directly associated
with AChR [6]. The binding of antibodies to MuSK leads to a reduced clustering of AChR and thus
to a reduced number of AChR at the neuromuscular junction. Interestingly, anti-MuSK
antibodies belong to the IgG4 subclass and thus cannot activate a complement [7]. Clinically anti-MuSK-ab-positive MG patients frequently have an involvement of
facial, bulbar and axial muscles as well as muscular atrophy [8]. Patients with anti-MuSK antibodies suffer respiratory crises more frequently than
patients with anti-AChR antibodies. Thymus histology is as a rule normal; thymomas
are almost never observed among anti-MuSK patients [9]. The frequency of anti-MuSK among myasthenia patients appears to be 3–4% of all
cases and 25–30% of AChR-ab-negative cases of MG.
Antibodies against lipoprotein receptor-associated protein 4 (anti-LRP4)
In 2011 and 2012 two independent working groups first described antibodies against
the protein LRP4 in cases of seronegative MG [10]
[11]. According to these studies approx. 15–20% of seronegative MG patients, 7.5% of
AChR-ab-positive MG patients as well as 15% of MuSK antibody-positive MG patients
have anti-LRP4 antibodies. In Germany anti-LRP4-antibody-positive patients appear
to be a rarity, and it is estimated that they make up make up less than 1% of all
MG cases. In mice, passive transfer of the antibody leads to myasthenic symptoms.
Whether only LRP4-ab-positive patients are less severely affected by myasthenia is
a matter of controversy due to the low number of case histories. However, patients
with anti-LRP4 and an additional antibody were more severely affected [10]
[11]
[12].
Titin antibodies
In patients less than 50 years of age, titin antibodies are indicative of the presence
of a thymoma [13]. There is no clear selectivity here; thus a negative finding of titin antibodies
does not exclude the possibility of a thymoma in patents under 50. Therefore, thymoma
diagnosis by means of thoracic CT or MRI is a necessary part of a standard initial
investigation of myasthenia gravis. In patients older than 50 years of age, such antibodies
are more common even without presence of thymoma; the frequency of titin antibodies
in late-onset MG increases with age [14].
Antibodies against agrin and other proteins
Agrin antibodies have been demonstrated in some myasthenia gravis patients who generally
also had antibodies against AChR, MuSK or LRP4. The significance of these antibodies
is currently unclear. In addition, antibodies against the intracellular protein cortactin
have been detected; their relevance is likewise unexplained [1]
[15].
Detection methods for myasthenia-associated antibodies
The radioimmunoassay (RIA) is the standard method of detecting acetylcholine receptor
antibodies. With pertinent clinical symptoms, a positive test result confirms the
diagnosis; however, approx. 50% of all purely ocular myasthenia gravis and 15–20%
of generalized MG cases are negative for AChR antibodies. A so-called cell-based assay,
in which cells are transfected with the acetylcholine receptor, is clearly more sensitive
with the same specificity, but the test is currently not yet commercially available
(as of 09/2017). Introduction of this test could make antibodies to the acetylcholine
receptor detectable in up to 50% of previously seronegative MG patients. Standard
tests for anti-MuSK are radioimmunoassay or ELISA; in this case cell-based tests can
achieve higher sensitivities. Currently such tests have been established only in the
context of scientific inquiries. Titin antibodies can be detected using a commercially-available
ELISA.
Neurophysiology
Repetitive stimulation represents the gold standard for the neurophysiological examination
to confirm myasthenia gravis. In principle, this method reproduces pathological muscle
fatigue through reiterated stimuli resulting in repeated muscle contractions (overview
in [16]). A further neurophysiological examination method is single-fiber electromyography
(SFEMG). This method utilizes differences in temporal blockages of different muscle
fibers of a motor unit and is usually only used when clinical symptoms, repetitive
stimulation and findings of autoantibodies do not provide a definite diagnosis. Neither
repetitive stimulation nor SFEMG are specific for autoimmune myasthenia gravis. A
pathological result only confirms a disturbance in neuromuscular transmission.
Repetitive stimulation
Repetitive stimulation relies on nerve stimulation and derivation of the potential
along the relevant muscle analogously to motor neurography. However, during this procedure,
after determination of the supramaximal threshold, stimulation is applied not once,
but repeated several times, as a rule 5 to 10 times at a frequency of 3 Hz. The percent
of decrement is measured between the 1st potential and the lowest of the first 5 potentials.
A decrement of greater than 8% is considered pathological. Suitable nerve-muscle pairs
for this examination are (1) facial nerve / nasalis muscle, (2) spinal accessory nerve
/ trapezius muscle (upper edge) and (3) axillary nerve / deltoid muscle [17]. When this examination is performed, care should be taken to sufficiently stabilize
the relevant extremity in order to avoid movement artifacts; non-supramaximal stimulation
intensity is an additional source of error.
In about 50–70% of myasthenia gravis patients repetitive stimulation is positive.
If repetitive stimulation does not exhibit a decrement, repetitive stimulation can
be used after applying stress, during which a one-minute load is applied over a period
of 4–5 min, and then 10 s afterward, repetitive stimulation is performed. It should
be noted that a decrement may also occur in other neuropathies or myopathies, and
in the case of ambiguous clinical symptoms, detailed neurography and myography have
to be carried out[Fig. 1.]
Fig. 1 Repetitive stimulation (3 Hz) of the right axillary nerve and derivation via the
right deltoid muscle of a patient with generalized myasthenia gravis. Decrement 31%
(amplitude reduction of 9.4 mV to 6.4 mV).
Increment test
The increment test is mainly performed for a Lambert-Eaton myasthenic syndrome (LEMS).
During normal 3 Hz repetitive stimulation, LEMS also exhibits a decrement; an increment
is detectable only at high stimulation frequencies of 30–50 Hz [16]. Since this test is very painful, nowadays a test with two individual supramaximal
stimuli is preferred before and after a 10–20 s muscle contraction. An increment greater
than 100% is demonstration of presynaptic neuromuscular transmission dysfunction;
incremental values between 60–100% are already highly suspicious, however. It should
be noted that in LEMS a significantly reduced amplitude of the starting MSAP can normally
be observed [16] [Fig. 2].
Fig. 2 Supramaximal stimulation of the ulnar nerve and derivation via the right abductor
digiti minimi muscle before and after 20 s of finger spreading in a patient with Lambert-Eaton
myasthenic syndrome (LEMS). Note the low final amplitude and the distinct increment
(360%).
Single-fiber electromyography
When a motor axon becomes depolarized, the stimulus is distended distally and excites
the individual muscle fibers almost simultaneously within the motor units. The variation
in the excitement interval from one muscle fiber to another is called jitter and is
an expression of the variability of neuromuscular transmission. If there is a functional
limitation of the neuromuscular junction, the jitter will be prolonged. A single-fiber
EMG needle has a smaller receiving radius than a concentric needle electrode. Using
this special needle it is possible to derive potential pairs of 2 fibers of the same
motor unit and thus determine the jitter. The muscles most frequently used are the
extensor digitorum muscle on the forearm or the frontalis muscle, since they can be
constantly innervated over a longer period and since these muscles are less subject
to age-related change (overview in [18]). In purely ocular forms, the SFEMG can also be performed by the orbicularis oculi
muscle, but which places higher demands on the examiner and the patient. A normal
SFEMG in a paretic muscle practically rules out myasthenia gravis [16]. Currently SFEMG is rarely employed due to the time factor and experience required
of the examiner.
Pharmacological tests
The Tensilon test, once regularly performed, is still occasionally used today. This
test uses the briefly active cholinesterase inhibitor edrophonium (Tensilon, Camsilon)
injected intravenously. The patient should be connected to an ECG monitor; initially
2 mg of Tensilon are administered as a test dose, if bradycardia does not occur, the
remaining 8 mg are subsequently injected. During the test, atropine should always
be available as an antidote. Muscle force generally improves after 30–45 s and continues
for about 4–5 min. The test can be combined with repetitive stimulation; after administration
of Tensilon the decrement should decrease. The clinical interpretation of the test
should take into account that Tensilon yields a false negative in approx. 25% of myasthenia
cases, and can produce false positive results in some muscular diseases or spinal
muscular atrophy.
Another option is the provisional administration of pyridostigmine bromide in a dosage
of 3–4×10 mg to 4×60 mg over several days.
The diagnosis of myasthenia gravis is based on clinical progression, diagnosis of
autoantibodies and, as needed, electrophysiological examinations.
In the case of a negative finding of acetylcholine receptor antibodies, anti-MuSK,
anti-LRP4 and anti-titin antibodies should be determined.
An electrophysiological examination is dispensable if the clinical symptoms are unambiguous
and there is a positive autoantibody test.
