Seronegative Myasthenia Gravis

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Article initiated by:
Andrew Go Lee, MD, Bohan Kim, Kais M. Karowadia, Melody Ziari, Molly Aldred, Nagham Al-Zubidi, MD, Noor Laylani, MD, Pamela Davila-Siliezar, MD
All contributors:
Andrew Go Lee, MD, Bohan Kim, Kais M. Karowadia, Melody Ziari, Molly Aldred, Nagham Al-Zubidi, MD, Noor Laylani, MD, Pamela Davila-Siliezar, MD, Osama Al Deyabat, MBBS, Dalaa Khaled Abuelshih, Nagham Al-Zubidi, MD, Noor Laylani, MD, Osama Al Deyabat, MBBS, Sanjana Jaiswal
Assigned editor:
Osama Al Deyabat, MBBS
Review:
Assigned status Update Pending
 by Osama Al Deyabat, MBBS on November 23, 2025.


Disease Entity

Myasthenia Gravis (MG) is an autoimmune disorder that affects the neuromuscular junction, leading to fatigable muscle weakness. The three most common autoantibodies associated with MG are acetylcholine receptor (AchR) antibodies, muscle-specific tyrosine kinase (MuSK) antibodies, and lipoprotein receptor-related protein 4 (LRP4) antibodies. AchR antibodies are found in approximately 85% of patients with generalized MG. Patients who lack AchR antibodies but have MuSK antibodies are classified as having seronegative MG, which accounts for about 5% of cases. Double-seronegative MG refers to patients who test negative for both AchR and MuSK antibodies, comprising about 10% of cases. However, LRP4 antibodies have also been detected in varying degrees in some patients.

Etiology

MG is primarily associated with IgG1 antibodies against acetylcholine receptors (AChR) in the neuromuscular junction.[1] These antibodies activate the complement cascade, leading to the destruction of the postsynaptic membrane. They also cause internalization and degradation of AChRs, a process known as antigenic modulation, and may inhibit receptor activation following acetylcholine binding.[2]

In patients with seronegative MG, about 70% have anti-MuSK antibodies, which are typically IgG4.[1] Unlike AChR antibodies, anti-MuSK antibodies disrupt cellular signaling pathways without activating the complement system. [3] Meanwhile, LRP4 antibodies are thought to interfere with AChR clustering and agrin interactions, further contributing to impaired neuromuscular function. [4]

Clinical Manifestations

Seronegative MG (SnMG) shares many clinical characteristics with seropositive MG, including muscle weakness distribution, disease severity, and response to immunomodulatory therapies like plasmapheresis or intravenous immunoglobulin.[5]  This suggests a similar immune-mediated postsynaptic mechanism across both forms of MG. In general, seronegative myasthenia gravis presents with predominant ocular and bulbar weakness rather than widespread limb fatigue. The presence or absence of specific antibodies helps define distinct MG subtypes with varying clinical features.

Patients with anti-MuSK antibodies, for instance, tend to be predominantly female and often present with bulbar and respiratory muscle involvement. This subgroup frequently experiences more severe symptoms, such as myasthenic crises, but thymic abnormalities like hyperplasia or thymoma are rare.[6][7] In contrast, those with anti-AChR antibodies usually have milder symptoms, often presenting with ocular MG, and tend to have better outcomes, including higher remission rates and lower incidences of myasthenic crises.[5][8]

Double-seronegative MG (dSNMG) is more common in children or young adults, and these patients typically have ocular MG with a generally milder disease course. The risk of developing thymoma is lower in this group.[8] Those with anti-LRP4 antibodies—a subgroup of dSNMG—often have mild symptoms at disease onset, and while some may present with thymic hyperplasia, thymomas are rare. The prevalence of LRP4-MG varies, with higher frequencies observed in women and an overall female-to-male ratio of 2.5 to 1.[8]

Recent studies have also shed light on the clinical characteristics of triple-seronegative MG (tSNMG). In Chinese populations, adult tSNMG patients are more likely to show generalization from ocular MG, while juvenile patients experience higher relapse rate.[9] These findings underscore the variability in clinical presentations across different MG subtypes and patient demographics.

Clinical Features of MuSK-Associated Myasthenia Gravis

Patients with MuSK-associated MG commonly have notable respiratory and bulbar muscle Involvement during the disease course existing with more severe generalized weakness than double-seronegative MG. Initially some ocular symptoms may occur ,but persistent isolated ocular involvement is uncommon. Different approaches of treatments may end up with some improvement in overall disease severity. However , bulbar manifestations may still persist despite following treatment . These clinical features point out the importance of separating double-seronegative MG MuSK-positive disease.


Disease Progression and Quality of Life

Patients with SNMG may experience fatigue , poor quality of life and face some difficulties in doing their daily activities and employment . This is why seronegative myasthenia gravis is considered one of the diseases that affect the patient’s daily activities and their quality of life . Ocular symptoms may be the initial presentation, as the disease progresses , it may evolve to be more generalized . Its impact on daily life becomes harder when bulbar involvement ,including dysphagia and dysarthria , start to appear during the disease course .

Diagnosis

Clinical Evaluations

Ice-Pack Test: Sensitivity 80 / Specificity 100

Cogan’s Eye Twitch: Sensitivity 75 / Specificity 99

Curtain Sign: Not very specific

Neurophysiological Testing

Neurophysiological testing plays a central role in diagnosing neuromuscular transmission abnormalities, particularly in cases of seronegative myasthenia gravis (SnMG). Two main electrophysiological techniques are used for evaluating neuromuscular junction (NMJ) function: repetitive nerve stimulation (RNS) and jitter analysis via single-fiber electromyography (SFEMG).[10]

SFEMG is regarded as the most sensitive diagnostic test for suspected NMJ disorders. Its sensitivity in diagnosing MG is between 80–100% for generalized MG and 93% for ocular MG.[10] In a study by Harrison et al., SFEMG identified MG mimics in 8 out of 61 patients, particularly in those negative for anti-AChR, anti-MuSK, and anti-LRP4 antibodies, highlighting SFEMG's importance in diagnosing SnMG.[11]

RNS is another critical neurophysiological test, depleting presynaptic acetylcholine, which an abnormal NMJ cannot compensate for. RNS has a sensitivity of about 80% for generalized MG but less than 40% for ocular MG. It is highly specific for diagnosing MG[12], and a combination of abnormal RNS and SFEMG findings is particularly specific for diagnosing MG in seronegative patients. RNS involves stimulating a nerve at 2–5 Hz and recording compound muscle action potentials (CMAP) from a corresponding muscle. A CMAP decrement of 10% between the first and fourth responses is commonly used to diagnose MG.

Serological Diagnosis

The diagnosis of seronegative MG heavily depends on which antibodies are tested and the methods employed. Approximately 85% of MG patients carry anti-AChR antibodies, making it the most prevalent antibody subtype.[13]

The radioimmunoprecipitation assay (RIPA) is considered the "gold standard" for detecting anti-AChR antibodies, with a sensitivity of 80–85% for generalized MG and nearly 100% for thymomatous MG.[14] However, its sensitivity drops to 50% in ocular MG and juvenile MG. RIPA's specificity approaches 100%, but its use is limited due to its reliance on radioactive reagents.

To address RIPA's limitations, enzyme-linked immunosorbent assays (ELISA) have been developed. While ELISA is easier to perform, it has lower sensitivity compared to RIPA.[15] [16]Recently, a novel Live Cell-Based Assay (CBA) has been introduced, which increases sensitivity by using human embryonic kidney cells to express AChR in its native conformation. This method detects clustered AChR antibodies, which are highly specific and pathogenic.[17] Live CBA has identified antibodies in up to 65% of RIPA-seronegative patients, particularly in prepubertal and ocular MG.[18] However, its use is limited to specialized centers.

In terms of anti-MuSK antibodies, RIPA remains the most reliable method, identifying these antibodies in 30–40% of seronegative MG patients. [19] A Live CBA test is also available, showing higher sensitivity for detecting anti-MuSK antibodies.

Anti-LRP4 antibodies are rare and primarily found in ocular or mild generalized MG. The detection rates of anti-LRP4 antibodies in double-seronegative patients range from 2% to 50%, depending on geographic and methodological factors.[20] Research on the clinical significance of anti-LRP4 antibodies remains limited.

Given the complexities of serological testing, clinicians should rely on a combination of clinical findings, neurophysiological tests, and antibody detection to avoid false-positive results. Testing for anti-AChR antibodies should be the first step, preferably with RIPA. If the patient is seronegative, anti-MuSK and anti-LRP4 testing may help further define the diagnosis.

Advanced Antibody Testing

The diagnosis of seronegative MG may be challenging because routine antibody tests do not detect all pathogenic antibodies. Cell-based assays (CBAs) have enhanced the identification of antibodies against clustered acetylcholine receptors and MuSK receptors in patients who previously tested negative using routine methods. Therefore, these assays may help diagnose patients who would otherwise be classified as double-seronegative. Electrophysiological studies, such as repetitive nerve stimulation and single-fiber electromyography should be included to ensure broad diagnostic evaluation. Avoid misdiagnosis and consider other conditions that may mimic SNMG, such as congenital myasthenic syndromes, before establishing the diagnosis.

Differential diagnosis

Myasthenia gravis can have a multitude of signs and symptoms that can mimic other disease processes. The differential diagnosis for seronegative myasthenia gravis is the same as generalized and ocular myasthenia gravis and includes:

  • Thyroid Eye Disease
  • Lambert-Eaton Syndrome
  • Multiple Sclerosis
  • Brainstem Gliomas
  • Botulism
  • Polymyositis and dermatomyositis

Management

The treatment of Myasthenia Gravis (MG) begins with acetylcholine esterase inhibitors (AChE-is), most notably oral pyridostigmine, which remains the primary symptomatic treatment for MG. AChE-is are effective for the majority of patients with anti-AChR antibodies, and positive outcomes have also been observed in patients with anti-LRP4 and anti-Agrin antibodies, as well as those with triple or quadruple seronegative MG (SN-MG).[21] However, MuSK-MG patients typically do not benefit from pyridostigmine and may experience cholinergic side effects such as muscle cramps and fasciculations, with potential symptom worsening in some cases.[22]

For patients who do not achieve satisfactory symptom control with AChE-is, glucocorticoid therapy, often in combination with nonsteroidal immunosuppressive agents, is introduced. Oral prednisone or prednisolone is the most common first-line immunotherapy, but most patients require a combination of immunosuppressants to prevent relapse and minimize the side effects of prolonged steroid use. [23] Azathioprine, mycophenolate mofetil (MMF), cyclosporine, and tacrolimus are the main immunosuppressive agents used, with response rates exceeding 80%.[24]

Immunosuppressive therapy is effective in seropositive MG as well as in double and triple seronegative MG, although the response in anti-MuSK MG is less robust. Romi et al. examined long-term outcomes in a Norwegian cohort of MG patients, comparing treatment responses between seropositive and double-seronegative MG patients. Both groups received AChE-is, with about 24% of seronegative and 32% of seropositive patients requiring immunosuppressive drugs. Thymectomy was more common among seropositive patients, with some displaying thymic hyperplasia or atrophy, while seronegative patients rarely underwent thymectomy. Treatment with plasmapheresis was similarly effective for both seropositive and seronegative patients during acute exacerbations.[7]

Emerging treatments include monoclonal antibodies like rituximab, which targets CD20 and has shown promise in MG management, particularly in refractory cases. Additionally, new therapies such as belimumab, eculizumab, and granulocyte-macrophage colony-stimulating factor are being explored. Thymectomy, while commonly performed in younger MG patients, requires further investigation to confirm its long-term benefit in both seropositive and seronegative groups.[23]

Treatment Outcomes and Refractory Disease

Treatment response in seronegative MG may vary among patients, some patients continue to experience significant symptoms despite conventional immunotherapy and other treatments approaches . Other patients may require additional treatment methods , including intravenous immunoglobulin, biological therapies , or plasma exchange .Refractory disease remains an important challenge, particularly in patients with remarkable bulbar involvement. Misdiagnosed cases and limited support regarding targeted treatments in this patient population may worsen and affect the disease management. Individualized treatment approaches and further clinical research are needed to enhance clinical outcomes over time .

Conclusion

Although autoantibodies are an important and high specificity biomarker in both generalized and ocular MG, the sensitivity may be lower in ocular MG. Seronegative MG may require further testing for other antibodies associated with MG (e.g., MuSK and LRP4). Additional testing (e.g., electrophysiology, CT chest) may be necessary in seronegative MG and empiric treatment trial for MG may also be supportive evidence for the diagnosis. Clinicians should be aware that the antibody testing may be negative (seronegative) in both generalized and ocular MG.

References

  1. ↑ 1.0 1.1 Binks S, Vincent A, Palace J. Myasthenia gravis: a clinical-immunological update. J Neurol. 2016 Apr;263(4):826-34. doi: 10.1007/s00415-015-7963-5. Epub 2015 Dec 24. PMID: 26705120; PMCID: PMC4826656.
  2. ↑ Lazaridis K, Tzartos SJ. Myasthenia Gravis: Autoantibody Specificities and Their Role in MG Management. Front Neurol. 2020 Nov 30;11:596981. doi: 10.3389/fneur.2020.596981. PMID: 33329350; PMCID: PMC7734299.
  3. ↑ Gilhus NE. Myasthenia Gravis. N Engl J Med. 2016 Dec 29;375(26):2570-2581. doi: 10.1056/NEJMra1602678. PMID: 28029925.
  4. ↑ Vincent, A., Bowen, J., Newsom-Davis, J., & McConville, J. (2003). Seronegative generalised myasthenia gravis: clinical features, antibodies, and their targets. Lancet Neurology, 2(2), 99–106. https://doi.org/10.1016/S1474-4422(03)00306-5
  5. ↑ 5.0 5.1 Evoli A., Spagni G., Monte G., Damato V. Heterogeneity in myasthenia gravis: Considerations for disease management. Expert. Rev. Clin. Immunol. 2021;17:761–771. doi: 10.1080/1744666X.2021.1936500.
  6. ↑ Park K.H., Waters P., Woodhall M., Lang B., Smith T., Sung J.-J., Kim K.-K., Lim Y.-M., Kim J.-E., Kim B.-J., et al. Myasthenia gravis seronegative for acetylcholine receptor antibodies in South Korea: Autoantibody profiles and clinical features. PLoS ONE. 2018;13:e0193723.
  7. ↑ 7.0 7.1 Romi F., Aarli J.A., Gilhus N.E. Seronegative myasthenia gravis: Disease severity and prognosis. Eur. J. Neurol. 2005;12:413–418. doi: 10.1111/j.1468-1331.2005.01137.x.
  8. ↑ 8.0 8.1 8.2 Zisimopoulou P., Evangelakou P., Tzartos J., Lazaridis K., Zouvelou V., Mantegazza R., Antozzi C., Andreetta F., Evoli A., Deymeer F., et al. A comprehensive analysis of the epidemiology and clinical characteristics of anti-LRP4 in myasthenia gravis. J. Autoimmun. 2014;52:139–145. doi: 10.1016/j.jaut.2013.12.004.
  9. ↑ Li W., Liu P., Cui W., Wang S., Ji Y., Zhang L., He X., Zhou S., Shen T., Zhao X., et al. Clinical characteristics of anti-AChR-MuSK-LRP4 antibody-negative myasthenia gravis in China. Muscle Nerve. 2023;67:481–488. doi: 10.1002/mus.27822.
  10. ↑ 10.0 10.1 Witoonpanich R., Dejthevaporn C., Sriphrapradang A., Pulkes T. Electrophysiological and immunological study in myasthenia gravis: Diagnostic sensitivity and correlation. Clin. Neurophysiol. 2011;122:1873–1877. doi: 10.1016/j.clinph.2011.02.026.
  11. ↑ Harrison P., Barton J., Winkel A. Chronic mimics of myasthenia gravis: A retrospective case series. Neuromuscul. Disord. 2023;33:250–256. doi: 10.1016/j.nmd.2023.01.006.
  12. ↑ Lo Y.L., Najjar R.P., Teo K.Y., Tow S.L., Loo J.L., Milea D. A reappraisal of diagnostic tests for myasthenia gravis in a large Asian cohort. J. Neurol. Sci. 2017;376:153–158. doi: 10.1016/j.jns.2017.03.016.
  13. ↑ Gastaldi M., Scaranzin S., Businaro P., Mobilia E., Benedetti L., Pesce G., Franciotta D. Improving laboratory diagnostics in myasthenia gravis. Expert. Rev. Mol. Diagn. 2021;21:579–590. doi: 10.1080/14737159.2021.1927715.
  14. ↑ Vernino S. Unraveling the Enigma of Seronegative Myasthenia Gravis. JAMA Neurol. 2015;72:630–631. doi: 10.1001/jamaneurol.2015.0205.
  15. ↑ Hewer R., Matthews I., Chen S., McGrath V., Evans M., Roberts E., Nute S., Sanders J., Furmaniak J., Smith B.R. A sensitive non-isotopic assay for acetylcholine receptor autoantibodies. Clin. Chim. Acta. 2006;364:159–166. doi: 10.1016/j.cccn.2005.05.035.
  16. ↑ Oger J., Frykman H. An update on laboratory diagnosis in myasthenia gravis. Clin. Chim. Acta. 2015;449:43–48. doi: 10.1016/j.cca.2015.07.030.
  17. ↑ Hong Y., Zisimopoulou P., Trakas N., Karagiorgou K., Stergiou C., Skeie G.O., Hao H.-J., Gao X., Owe J.F., Zhang X., et al. Multiple antibody detection in ‘seronegative’ myasthenia gravis patients. Eur. J. Neurol. 2017;24:844–850. doi: 10.1111/ene.13300.
  18. ↑ Cruz P.M.R., Al-Hajjar M., Huda S., Jacobson L., Woodhall M., Jayawant S., Buckley C., Hilton-Jones D., Beeson D., Vincent A., et al. Clinical Features and Diagnostic Usefulness of Antibodies to Clustered Acetylcholine Receptors in the Diagnosis of Seronegative Myasthenia Gravis. JAMA Neurol. 2015;72:642–649. doi: 10.1001/jamaneurol.2015.0203.
  19. ↑ Huda S., Waters P., Woodhall M., Leite M.I., Jacobson L., De Rosa A., Maestri M., Ricciardi R., Heckmann J.M., Maniaol A., et al. IgG-specific cell-based assay detects potentially pathogenic MuSK-Abs in seronegative MG. Neurol. Neuroimmunol. Neuroinflamm. 2017;4:e357. doi: 10.1212/NXI.0000000000000357.
  20. ↑ Tzartos J.S., Zisimopoulou P., Rentzos M., Karandreas N., Zouvelou V., Evangelakou P., Tsonis A., Thomaidis T., Lauria G., Andreetta F., et al. LRP4 antibodies in serum and CSF from amyotrophic lateral sclerosis patients. Ann. Clin. Transl. Neurol. 2014;1:80–87. doi: 10.1002/acn3.26.
  21. ↑ Zouvelou V., Zisimopoulou P., Rentzos M., Karandreas N., Evangelakou P., Stamboulis E., Tzartos S.J. Double seronegative myasthenia gravis with anti-LRP 4 antibodies. Neuromuscul. Disord. 2013;23:568–570. doi: 10.1016/j.nmd.2013.03.013.
  22. ↑ Deymeer F. Myasthenia gravis: muSK-MG, late-onset MG and ocular MG. Acta Myol. 2020;39:345–352.
  23. ↑ 23.0 23.1 Sanders D.B., Wolfe G.I., Benatar M., Evoli A., Gilhus N.E., Illa I., Kuntz N., Massey J.M., Melms A., Murai H., et al. International consensus guidance for management of myasthenia gravis: Executive summary. Neurology. 2016;87:419–425.
  24. ↑ Gilhus N.E., Skeie G.O., Romi F., Lazaridis K., Zisimopoulou P., Tzartos S. Myasthenia gravis—autoantibody characteristics and their implications for therapy. Nat. Rev. Neurol. 2016;12:259–268. doi: 10.1038/nrneurol.2016.44.

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[2]

[3]

  1. ↑ Vincent A, Badi R, Barisic N, Berrih-Aknin S, Bonifati DM, Brenner T, Chalk C, Chang T, Chiu HC, Corbin D, Cortés-Vicente E, Deymeer F, Erdem-Ozdamar S, Evoli A, Eymard B, Gilhus NE, Gotkine M, Heckmann J, Illes Z, Jacobson L, Kleopa KA, Kostera-Pruszczyk A, Kuwabara S, Lisak R, Mantegazza R, Melms A, Motomura M, Nafissi S, Nicolle M, Oger J, Reddel S, Roda R, Rosales RL, Salutto V, Sanders D, Singh S, Spinazzi M, Verschuuren JJGM, Vissing J, Witoonpanich R. Relative frequencies of muscle specific kinase antibody myasthenia in 46 centres worldwide. Brain. 2026 Jun 23:awag215. doi: 10.1093/brain/awag215. Epub ahead of print. PMID: 42334385.
  2. ↑ Lehnerer S, Stegherr R, Grittner U, Stein M, Gerischer L, Stascheit F, Herdick M, Doksani P, Meisel A, Hoffmann S. The burden of disease in seronegative myasthenia gravis: a patient-centered perspective. Front Immunol. 2025 Apr 8;16:1555075. doi: 10.3389/fimmu.2025.1555075. PMID: 40264778; PMCID: PMC12011775.
  3. ↑ Verza MU, Schiavolin M, Beretta F, Cornacchini S, Lotti A, Spagni G, Damato V. Clinical Characteristics and Treatment Management of Seronegative Myasthenia Gravis: A Systematic Review of the Literature. Muscle Nerve. 2026 Sep;74 Suppl 1(Suppl 1):S76-S83. doi: 10.1002/mus.70130. Epub 2026 Jul 1. PMID: 42387811; PMCID: PMC13599449.
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