Antibody-negative Myasthenia Gravis

Key Points at a Glance

Highlights of This Disease

• MG with both anti-AChR and anti-MuSK antibodies negative is called double-seronegative MG (dSNMG), accounting for about 10% of all MG.
• In ocular MG, the positivity rate of anti-AChR antibodies is 50% or less, with antibody-negative cases accounting for the majority.
• 82% of initial symptoms are ocular symptoms such as ptosis and diplopia, characterized by diurnal fluctuation, day-to-day variation, and easy fatigability.
• Even in antibody-negative cases, SFEMG (single-fiber electromyography) has a high sensitivity of 85–100%, making it the most important diagnostic tool.
• In Japan, anti-ChE drugs (pyridostigmine) are the first-line treatment, with steroids and tacrolimus added as needed.
• In anti-MuSK antibody-positive cases, pyridostigmine may be ineffective or harmful, leading to different treatment choices.

1. What is Antibody-Negative Myasthenia Gravis?

Myasthenia gravis (MG) is an autoimmune disease that affects the neuromuscular junction, causing fatigable muscle weakness. Most patients have anti-acetylcholine receptor (AChR) antibodies or anti-muscle-specific tyrosine kinase (MuSK) antibodies, but when both are negative, it is called double-seronegative MG (dSNMG). Furthermore, when anti-LRP4 antibodies are also negative, it is called triple-seronegative MG (tSNMG).

The distribution of antibody subtypes in MG is shown below.

Subtype	Approximate Frequency
Anti-AChR antibody positive	About 85% of generalized MG, 50% or less of ocular MG
Anti-MuSK antibody positive	Approximately 30-40% of anti-AChR-negative cases, about 5% of all MG
Double seronegative (dSNMG)	About 10% of all MG

In a systematic review of 45 dSNMG patients, the mean age at diagnosis was 52.4±20.5 years, 51% were female, 82% had ocular symptoms (ptosis, diplopia) as initial symptoms, 29% had pure ocular MG, and 71% had generalized MG ¹⁾. Malignancies were found in 15 of 45 patients (33%), of which 8 were associated with immune checkpoint inhibitors (ICIs) ¹⁾.

The annual incidence of MG is reported as 9-10 per million, and the prevalence as 150-250 per million ²⁾.

Q How common is antibody-negative myasthenia gravis?

A

dSNMG accounts for about 10% of all MG. However, retesting with live cell-based assay (Live CBA) has been reported to detect anti-AChR antibodies in up to 65% of patients who tested negative by standard assay (RIPA) ²⁾, suggesting that true “antibody-negative” cases may be even fewer.

2. Main symptoms and clinical findings

Subjective symptoms

• Ptosis: Accounts for about 70% of initial symptoms. Often starts in one eye and later becomes bilateral.
• Diplopia: About 50% of initial symptoms. May present as pseudo-trochlear nerve palsy (vertical strabismus) or pseudo-MLF syndrome (adduction deficit).
• Diurnal fluctuation: Best upon waking, worsens over the course of the day.
• Day-to-day variation: Symptoms vary from day to day.
• Fatigability: Characteristic pattern of worsening with repetitive activity and improvement with rest.
• Bulbar symptoms: dysphagia, dysarthria, dyspnea. Particularly common in anti-MuSK antibody-positive patients.

Clinical Findings (by antibody subtype)

Anti-AChR Positive Type

Features: Often presents as ocular MG, relatively mild.

Course: High remission rate, low incidence of myasthenic crisis.

Thymic abnormalities: May be associated with thymoma or thymic hyperplasia.

Anti-MuSK Positive Type

Features: More common in women. Predominantly bulbar symptoms and respiratory muscle involvement.

Course: Frequent myasthenic crises.

Thymic abnormalities: Rare. Pyridostigmine may be insufficient or worsen symptoms.

dSNMG

Features: Common in children and young adults, usually ocular MG.

Course: Generally mild. Low risk of thymoma.

Anti-LRP4 antibodies: Detected in 46% of cases, often mild at onset¹⁾.

Anti-LRP4 Positive Type

Characteristics: More common in women (male-to-female ratio 1:2.5). Onset is often mild.

Frequency: Detected in 18.7% of dSNMG cases²⁾. Detection rates vary from 2% to 50% depending on region and method²⁾.

Mechanism: LRP4 functions as a receptor for agrin and inhibits AChR clustering.

Complication of thyroid eye disease: Approximately 15% of MG patients have thyroid eye disease. Enlargement of extraocular muscles on MRI suggests thyroid eye disease. MG does not show extraocular muscle hypertrophy (important distinguishing point). When ptosis is accompanied by mild strabismus, it is a diagnostic clue for MG.

Q What other symptoms besides ptosis and diplopia can occur?

A

Bulbar symptoms (dysphagia, dysarthria) may appear. In anti-MuSK antibody-positive type, respiratory muscle involvement is also common, with a high risk of myasthenic crisis. In dSNMG, ocular and bulbar symptoms tend to predominate over limb fatigue.

3. Causes and Risk Factors

MG is an autoimmune disease in which autoantibodies against the postsynaptic membrane of the neuromuscular junction impair neuromuscular transmission.

Mechanisms of action by antibody type:

• Anti-AChR antibodies (IgG1): Activate the complement cascade to destroy the postsynaptic membrane, and also promote internalization and degradation of AChR via antigen modulation.
• Anti-MuSK antibodies (IgG4): Do not activate the complement system, but inhibit MuSK function (signaling that stimulates AChR clustering).
• Anti-LRP4 antibodies: LRP4 functions as a receptor for agrin; anti-LRP4 antibodies inhibit AChR clustering and interaction with agrin.

Reasons for antibody negativity: Possible causes include limitations of testing methods (presence of low-affinity or conformation-dependent antibodies not detected by RIPA), low antibody production, effects of immunosuppressive therapy, immunodeficiency, antigen depletion, and immunosenescence²⁾.

Main risk factors:

• Thymoma: Occurs in 10–15% of MG patients.
• Autoimmune thyroid disease: Approximately 4–5% of MG patients have this complication.
• Immune checkpoint inhibitors (ICIs): Drugs such as pembrolizumab can induce dSNMG in some cases¹⁾.
• SARS-CoV-2 infection: There are case reports of new-onset dSNMG after infection³⁾⁴⁾.

Prevention and Daily Care

• If you experience diurnal fluctuations in ptosis or double vision, consider the possibility of myasthenia gravis and consult an ophthalmologist or neurologist early.
• Thymoma evaluation (chest CT) should always be performed after diagnosis is confirmed.
• If muscle weakness or ocular symptoms appear while using immune checkpoint inhibitors (cancer immunotherapy), promptly consult your primary physician.

4. Diagnosis and Examination Methods

In 2014, the Japanese Society of Neurology published diagnostic criteria in its clinical practice guidelines.

Clinical Evaluation

• Tensilon test (edrophonium test): Administer 10 mg of the anti-ChE drug edrophonium chloride intravenously in 2.5 mg increments and observe resolution of ptosis and double vision. Due to false positives and false negatives, only dramatic improvement is considered positive.
• Ice pack test: Apply a cold pack to the upper eyelid for 2 minutes; improvement of ptosis by 2 mm or more is positive. Sensitivity 80–92%, specificity 25–100%.
• Cogan’s lid twitch sign: Sensitivity 75%, specificity 99%.
• Forced eyelid closure test: Sensitivity 94%, specificity 91%.
• Upward gaze fatigue test: Have the patient look upward for 1 minute and observe worsening of ptosis or double vision.

Serological Tests

The characteristics of each test method are shown below.

Test Method	Sensitivity	Notes
RIPA (Radioimmunoprecipitation Assay)	Generalized type 80–85%, thymoma-associated nearly 100%, ocular type about 50%	Gold standard for AChR antibody detection2)
ELISA	Lower than RIPA (false negative +30%, false positive +5%)	Easy to perform2)
Live CBA (Cell-Based Assay)	Positive in up to 65% of RIPA-negative cases	Particularly useful in prepubertal and ocular MG2)

• Anti-MuSK antibody (RIPA): Detected in 30–40% of antibody-negative MG patients
• Anti-LRP4 antibody: Detection rate in dSNMG patients ranges from 2 to 50% depending on region and method²⁾

Neurophysiological Tests

• SFEMG (Single-Fiber Electromyography): Most sensitive for NMJ disorders. Sensitivity 80–100% for generalized MG, 85–100% for ocular MG (using frontalis or orbicularis oculi muscles)²⁾. If both RNS and SFEMG are abnormal in antibody-negative patients, it shows particularly high specificity for MG diagnosis.
• RNS (Repetitive Nerve Stimulation): Stimulation at 2–5 Hz; a decrement of 10% or more in CMAP amplitude between the first and fourth wave is considered positive. Sensitivity about 80% for generalized MG, less than 40% for ocular MG. Specificity is very high.

Imaging and Differential Diagnosis

• Chest CT: To check for thymoma. About 70% of MG patients have thymic hyperplasia, and 10–15% have thymoma.
• Thyroid function tests: To rule out autoimmune thyroid disease.

Differential diagnosis: Thyroid eye disease (extraocular muscle hypertrophy on MRI; no hypertrophy in MG), Lambert-Eaton myasthenic syndrome, congenital myasthenic syndromes (CMS; hereditary, antibody-negative, not indicated for immunosuppressive therapy²⁾), oculopharyngeal muscular dystrophy (OPMD), chronic progressive external ophthalmoplegia (CPEO).

Q Can MG be diagnosed even if antibody tests are negative?

A

Yes, it can. SFEMG has high sensitivity of 85–100% for ocular MG and is the most reliable test²⁾. The ice pack test (sensitivity 80–92%) and Cogan’s sign (sensitivity 75%, specificity 99%) are also useful ancillary tests. Even if antibodies are negative, if both RNS and SFEMG are abnormal, specificity for MG diagnosis is high.

5. Standard Treatment

Standard Treatment Protocol in Japan

1. Evaluation for thymoma: Confirm thymoma (or thymic enlargement) on CT; if present, prioritize extended thymectomy. Generalized MG is treated by neurologists, ocular MG by ophthalmologists.

2. First-line: Anticholinesterase drugs (pyridostigmine)

   • Mestinon®: Start at 2 tablets per day in two divided doses (morning and noon, taken at least 4 hours apart), can be increased up to 4 tablets per day.
   • Side effects: diarrhea, abdominal pain (muscarinic effects)
   • Symptomatic treatment, not a curative therapy
   • If remission can be achieved over six months or more, the course is mildest and favorable

3. Second-line: Steroid combination (when Mestinon® is difficult to take orally or insufficient alone)

   • Choose one of steroid pulse therapy, high-dose alternate-day steroid therapy, or low-dose oral steroid gradual increase method
   • During oral steroid therapy, bone density measurement and concomitant use of osteoporosis preventive medication (alendronate sodium hydrate, Bonaron® 35 mg once weekly after waking up) are necessary

4. Third-line: Immunosuppressant tacrolimus (Prograf®) (when steroids are insufficient, difficult to withdraw, or have strong side effects)

   • Start at 2 mg once daily after dinner
   • Regularly monitor tacrolimus blood concentration, glucose tolerance, and renal function
   • If blood concentration is ≤5 ng/mL and renal function is normal, increase to 3 mg/day
   • Reduce and discontinue steroids at a rate of prednisone equivalent 5 mg every 1 to 3 months

Prognosis: In ocular type, the non-steroid group is more likely to progress to generalized type than the steroid group. In the treated group in Japan, the rate of progression from ocular to generalized type is less than 10%.

Overseas Treatment Guidelines (Supplementary)

In a systematic review of dSNMG, the most frequently used treatments were pyridostigmine (84%), corticosteroids (76%), IVIG (27%), and azathioprine (18%)¹⁾. High improvement rates have been reported with rituximab (3/3 cases), plasma exchange (5/6 cases), and tacrolimus (5/6 cases)¹⁾. The response rate of immunosuppressants (azathioprine, mycophenolate mofetil, cyclosporine, tacrolimus) is over 80%²⁾.

The prognosis is poor in the group with malignant tumors, with a favorable outcome rate of 37.5%, significantly lower than 78.6% in the non-malignant tumor group (p=0.046)¹⁾.

Precautions in treatment

• When starting high-dose steroids, there is a risk of myasthenic crisis in up to 15% of patients.
• In anti-MuSK antibody-positive patients, pyridostigmine may not only be ineffective but can also worsen symptoms due to cholinergic side effects (muscle cramps, fasciculations).
• In ocular MG, only about 50% of patients have an adequate response to pyridostigmine alone, and ptosis responds better than diplopia.

Q Is the treatment different for anti-MuSK antibody-positive cases?

A

In anti-MuSK antibody-positive cases, pyridostigmine may be ineffective or cause worsening of symptoms. Plasma exchange and immunosuppressive therapy (especially rituximab) are considered effective, and combination with corticosteroids is often recommended. Referral to a specialized center is advisable.

6. Pathophysiology and detailed mechanism of onset

In normal neuromuscular transmission, a nerve impulse triggers Ca²⁺ influx into the presynaptic terminal, leading to exocytosis of acetylcholine (ACh) from synaptic vesicles. ACh binds to AChRs on the postsynaptic membrane, causing muscle contraction.

Mechanism of neuromuscular transmission impairment by anti-AChR antibodies (IgG1):

• Complement cascade activation → destruction of the postsynaptic membrane
• Antigenic modulation → increased internalization and degradation of AChRs
• Inhibition of receptor activation after ACh binding

Mechanism of anti-MuSK antibodies (IgG4): Because they are of the IgG4 subclass, they do not activate complement. They impair neuromuscular transmission by inhibiting MuSK function (signaling that stimulates AChR clustering).

Mechanism of anti-LRP4 antibodies: LRP4 functions as a receptor for agrin. Anti-LRP4 antibodies inhibit AChR clustering and interaction with agrin, impairing neuromuscular transmission.

Interpretation of antibody negativity: With live CBA, anti-AChR antibodies can be detected in 15–65% of RIPA-negative patients, and anti-MuSK antibodies in an additional 8% ²⁾. This suggests the possible presence of low-affinity, conformation-dependent antibodies that cannot be detected by standard tests.

Mechanism of ICI-induced MG: PD-1 inhibitors (e.g., pembrolizumab) cause dysregulation of T-cell self-tolerance, triggering an autoimmune response against the neuromuscular junction¹⁾.

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7. Latest Research and Future Perspectives (Investigational Reports)

For Patients: Please Read Carefully

The following information is currently under investigation or in clinical trials and is not standard treatment available at general hospitals. It is reference information for professionals regarding future medical developments.

Efgartigimod (FcRn Inhibitor)

Efgartigimod is a human IgG1 antibody Fc fragment that inhibits FcRn, which is responsible for IgG recycling, thereby reducing pathogenic autoantibodies in serum. It was approved for anti-AChR antibody-positive generalized MG in the ADAPT trial. In Japan, it is approved regardless of antibody status⁵⁾.

Sorrenti et al. (2024) administered efgartigimod 10 mg/kg for 5 cycles to a 56-year-old woman with refractory triple antibody-negative generalized MG who had a 28-year treatment history. Marked improvement was observed: MGFA classification IIIb→IIb, MG-ADL score 11→0, MG-QoL15 score 30→0, QMG score 28→6⁵⁾.

Complement Inhibitors / Rituximab

Eculizumab and ravulizumab are approved for anti-AChR antibody-positive generalized MG, but clinical trials in antibody-negative MG have not been conducted. Rituximab (anti-CD20 monoclonal antibody) is considered promising for refractory cases; a systematic review of dSNMG reported favorable outcomes in 2 of 3 cases¹⁾.

Expectations for “Reclassification” Using Live CBA

Live CBA can detect clustered AChR antibodies that are not detected by standard tests (RIPA). It has the potential to reclassify 15–65% of RIPA-negative patients as antibody-positive²⁾⁵⁾, which is expected to lead to more appropriate treatment selection. Currently, it is only available at specialized facilities.

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8. References

1. Abuhammad A, Ayyad M, Albandak M, et al. Double-seronegative myasthenia gravis: clinical characteristics, treatment, and outcomes — a systematic review of case reports and case series. BMC Neurology. 2026;26:107.
2. Vinciguerra C, Bevilacqua L, Lupica A, et al. Diagnosis and Management of Seronegative Myasthenia Gravis: Lights and Shadows. Brain Sci. 2023;13:1286.
3. Sadiq W, Waleed MS, Rizvi TA, et al. Myasthenia Gravis Associated With COVID-19 Infection. Cureus. 2023;15(5):e39506.
4. Castro Silva B, Saianda Duarte M, Rodrigues Alves N, et al. Seronegative Myasthenia Gravis: A Rare Disease Triggered by SARS-CoV-2 or a Coincidence? Cureus. 2024;16(8):e67511.
5. Sorrenti B, Laurini C, Bosco L, et al. Overcoming therapeutic challenges: Successful management of a supposedly triple seronegative, refractory generalized myasthenia gravis patient with efgartigimod. Eur J Neurol. 2024;31:e16306.