New diagnostic maker for seronegative MG
__________________________________________________________________________________

Journals@Ovid Full Text Anti-LRP4 autoantibodies in AChR- and MuSK-antibody-negative myasthenia gravis.
Pevzner, Alexandra 1; Schoser, Benedikt 2; Peters, Katja 1; Cosma, Nicoleta-Carmen 1; Karakatsani, Andromachi 1; Schalke, Berthold 3; Melms, Arthur 4; Kroger, Stephan 1

Journal of Neurology. 259(3):427-435, March 2012.
[Article.][Original Communication: PDF Only]

AN: 00005068-201203000-00004.

AB : Myasthenia gravis (MG) is an autoimmune disorder characterized by a defect in synaptic transmission at the neuromuscular junction causing fluctuating muscle weakness with a decremental response to repetitive nerve stimulation or altered jitter in single-fiber electromyography (EMG). Approximately 80% of all myasthenia gravis patients have autoantibodies against the nicotinic acetylcholine receptor in their serum. Autoantibodies against the tyrosine kinase muscle-specific kinase (MuSK) are responsible for 5-10% of all myasthenia gravis cases. The autoimmune target in the remaining cases is unknown. Recently, low-density lipoprotein receptor-related protein 4 (LRP4) has been identified as the agrin receptor. LRP4 interacts with agrin, and the binding of agrin activates MuSK, which leads to the formation of most if not all postsynaptic specializations, including aggregates containing acetylcholine receptors (AChRs) in the junctional plasma membrane. In the present study we tested if autoantibodies against LRP4 are detectable in patients with myasthenia gravis. To this end we analyzed 13 sera from patients with generalized myasthenia gravis but without antibodies against AChR or MuSK. The results showed that 12 out of 13 antisera from double-seronegative MG patients bound to proteins concentrated at the neuromuscular junction of adult mouse skeletal muscle and that approximately 50% of the tested sera specifically bound to HEK293 cells transfected with human LRP4. Moreover, 4 out of these 13 sera inhibited agrin-induced aggregation of AChRs in cultured myotubes by more than 50%, suggesting a pathogenic role regarding the dysfunction of the neuromuscular endplate. These results indicate that LRP4 is a novel target for autoantibodies and is a diagnostic marker in seronegative MG patients. (C) 2012 Springer. Part of Springer Science Business Media

Here is an article discussing MG presenting primarily as dysphonia, EMG positive but largely seronegative with current testing.

http://scinet.dost.gov.ph/union/UploadFiles/download.php?b=sdarticle_011_174587.pdf&f=../Downloads/sdarticle_011_174587.pdf&t=application/pdf

elinora also found an excellent article in Brain:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2442426/

The first link no longer takes you to the full article, so it is copied here:

Current approach to seronegative myasthenia Zohar Argov
Received: 18 June 2010 / Accepted: 3 September 2010 / Published online: 18 September 2010 Ó Springer-Verlag 2010
Abstract The group of patients with weakness or fatigue who have electrophysiological evidence of neuromuscular transmission defects, but no antibodies against either acetyl choline receptor or muscle specific kinase, need special evaluation and therapeutic consideration. Such seronega- tive patients may have low affinity antibodies but may also be late onset of a congenital myasthenic syndrome. This review discuss the practical approach toward the condition of seronegative myasthenia.
Keywords Myasthenia Congenital myasthenic syndrome Repetitive nerve stimulation Acetyl choline receptor antibodies
Introduction
About 80% of patients with a clinical picture of generalized myasthenia who have combined electrophysiological and pharmacological evidence for neuromuscular junction (NMJ) transmission failure will have increased titers of anti-acetylcholine receptor (AChR) antibodies in their serum. These auto-antibodies decrease the number of postsynaptic receptors by complement-mediated degrada- tion. This condition was defined as seropositive myasthenia gravis (MG). For the patients without such antibodies, the term seronegative MG was applied. After the discovery of anti-muscle specific kinase (MuSK) antibodies in the serum of many ‘seronegative’ patients, this term became more blurred. In some early reports, this condition was rather
Z. Argov (&)
Department of Neurology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel
e-mail: zohara@ekmd.huji.ac.il
awkwardly defined as seronegative myasthenia with anti- MuSK antibodies (clearly a misnomer). It is now clear that myasthenia with anti-MuSK antibodies is a different auto- immune NMJ disease with several unique clinical and pathological features [1]. The mechanisms by which anti- bodies against MuSK cause myasthenia remain unresolved in part.
Of the 20% myasthenic patients without evidence for anti-AChR antibodies in their serum, up to one half have anti-MuSK antibodies, the frequency depending partly on ethnic origin [2]. The remaining 10–12% of the myasthenic patient population are those discussed in this review of seronegative myasthenia. It should immediately be noted that the notion that these patients have no serum antibodies against component(s) of the AChR has recently been challenged; this will be discussed in the immunology sec- tion below. However, although the title of this review suggests that the term seronegative myasthenia may not remain a valid diagnosis, in current practical life there are still numerous myasthenic patients without detectable antibodies in their serum.
Definition of seronegative myasthenia
For this review I chose the definition of seronegative myasthenia to be:
Patients with weakness or fatigue who have electro- physiological evidence of neuromuscular junction impaired transmission, but no abnormal auto-antibodies against neuromuscular junction components can be detected in their serum by routine tests.
The above definition is as wide as possible in order to help the clinician with the approach toward such patients. The above definition includes patients who have isolated
123


ocular myasthenia, since these patients are included in some of the series describing seronegative myasthenia. The definition also does not necessarily exclude patients who do not have a primary NMJ disease. Such patients may at times present a difficult diagnostic challenge; this is dis- cussed in the differential diagnosis section. The electro- physiological method by which NMJ impairment is demonstrated is not determined in the above definition, this could be either repetitive nerve stimulation (with or with- out additional techniques to enhance the NMJ failure) or single fiber EMG (SFEMG); this is discussed in the elec- trophysiology part. However, the definition excludes patients whose only evidence for myasthenic covndition is a positive edrophonium (Tensilon) test. This is because the test has many pitfalls and inaccuracies (both false negative and false positive percentages are high) and has been abandoned by many physicians. It should also be noted that absence of auto-antibodies relates both to the ‘classical’ anti-AChR antibodies as well as to the auto-antibodies against MuSK when tested by routine diagnostic tech- niques (see immunology).
Clinical features of seronegative myasthenia (Table 1)
Clinical information about 150–200 patients with acquired myasthenia and ‘double’ seronegativity is available in the literature [1, 3–8] and there are certainly many more unreported patients. The above reports are not uniform and do not contain all patients’ details. A few of these patients may appear in more than one series report. Although as a whole the group is heterogeneous, some common features do emerge. The features summarized in Table 1 apply mainly to generalized myasthenia, although it is unclear in some series whether patients with isolated ocular myas- thenia were included, too.
The age of onset is very variable, but it is not clear if all the reported patients have indeed an acquired myasthenia.
Table 1 Features of acquired, ‘double’ seronegative generalized myasthenia
Age of onset (years): 1–83; mean = 40.5
Gender distribution: 1:1–2:1 (F:M)
Severity: usually mild disorder (MGFA score averaging around 2)
Ocular involvement: 30–70% at onset, most of patients during the course
Limb involvement: common but without atrophy Bulbar involvement: infrequent
Thymus: mostly normal
Good response to AChEI in most
Abnormal Repetitive nerve stimulation: 25–83% Response to therapy: usually good
For example, early childhood onset in some cases of Lavrnic et al. [5] may represent a still undiagnosed form of congenital myasthenic syndrome. Most of the patients had adult onset with a mean age of about 40 years. The gender distribution differs between equal female and male in some reports to some degree of female predominance in others. Many patients have ocular involvement at onset and most have it through the disease course, even when it does not remain restricted to the eye musculature. Limb involve- ment is common, but bulbar and respiratory muscle affliction is much less frequent than in both types of seropositive MG. In most series, the patients have a milder disease severity at its maximum compared to seropositive MG. Also, there seems to be a tendency toward good response to therapy in seronegative myasthenia, although because of the non-uniform approach to therapy in MG in general, it is very hard to compare data. Response to ace- tylcholine esterase inhibitors (AChEI) is usually favorable and hypersensitivity to these agents is extremely rare.
The thymus is usually normal or atrophied in seroneg- ative myasthenia (several patients had thymectomy, even though they had no clear increase in size of thymus on imaging), however, in the report of Leite et al. [9] some of the seronegative patients had mild thymic hyperplasia. Few patients had associated thyrogid or other immune mediated diseases, but the issue of associated autoimmune diseases and seronegative myasthenia requires further studies.
Thus, one can conclude that the features of the sero- negative myasthenia group of patients are more similar to the AChR antibody positive MG than to the anti-MuSK disorder, with possibly slightly milder course.
Electrophysiology
The definition of seronegative myasthenia in this review relies heavily on electrophysiological demonstration of NMJ transmission abnormality. NMJ transmission abnor- mality is defined as the presence of a decrement of more than 10% between the first and third and/or fifth compound muscle action potential upon low rate (usually 3 Hz) repetitive nerve stimulation. It should be noted that this test may be normal in up to 25% of all patients with seropos- itive generalized myasthenia. However, one should take care to have this test done on a weak muscle (not only a proximal muscle but a facial muscle if necessary) and look for decrement also after effort, which can increase the test sensitivity. SFEMG is considered the electrophysiological gold standard for demonstration of impaired neuromuscular transmission (more than 90% of patients with MG have abnormality) but is not widely available. An abnormal SFEMG is defined as an increase in jitter (expressed as mean consecutive difference or MCD) outside the upper


16
limit of normal range for a particular tested muscle or more
than 10% of fiber pairs showing abnormal MCD [10].
It is important to note that patients with neuromuscular diseases other than myasthenia may have abnormal NMJ physiology (either decrement or abnormal SFEMG or both). Such impairment is frequently found in motor neu- ron disease (ALS or other forms) [11, 12]. This is espe- cially of importance at the early stages of motor neuron disease when the diagnosis is not clear and the condition may mimic myasthenia. Abnormal decrement may also be found in myositis [13] and in mitochondrial disorders [14, 15] although this has been reported for only very few
patients with these conditions.
The definition used in this review for seronegative
myasthenia mandates an abnormal electrophysiological test, thus it is not suitable to discuss the frequency of abnormalities in this condition. However, it appears that in most series of seronegative myasthenia, repetitive nerve stimulation showed abnormal decrement in the majority (around 80%) of patients [1, 8]. SFEMG was the important diagnostic tool in most of the others. Interestingly, the SFEMG of the EDC muscle showed abnormality in only half of the patients [6] but in others series where SFEMG was applied to facial muscles, abnormal findings were found in the majority of patients.
Differential diagnosis (Table 2)
In conditions where the neuromuscular transmission is secondarily affected (motor neuron disease, mitochondrial disorders, myositis) diagnosis may not be immediately apparent (e.g. in the bulbar presentation of ALS) and care should be taken not to label these conditions as seronega- tive myasthenia too early. The correct diagnosis usually emerges with time as the clinical features become more typical and more testing, such as meticulous EMG, CPK and (when necessary) muscle biopsy, provide important diagnostic clues.
The situation is more problematic with the ‘focal’ forms of myasthenia. In many patients with ocular myasthenia at onset, the symptoms spread within the first year to other sites and antibodies are detected later in the course of their disease. However, in many others the condition remains limited to the eyes, even after prolonged follow up. In such patients the auto-antibody remains undetected through the disease course, and thus they are also grouped under the term of seronegative myasthenia. Since the course of ocular myasthenia is milder compared with all other forms of the disease, one should reserve the term seronegative myas- thenia only to cases where the symptoms become gen- eralized (this was not clearly defined in some of the patients’ series). A much more rare form of myasthenia
J Neurol (2011) 258:14–18
Table 2 Differential diagnosis of patients with abnormal NMJ electrophysiology and no detectable auto-antibodies
Secondary NMJ impairment
Motor neuron disease with early NMJ abnormality Myositis
Mitochondrial disorders
Hereditary NMJ disease (congenital myasthenic syndromes with late onset)
DOK 7*
Rapsin*
COLQ
Epsilon unit mutations
Limb girdle myasthenia with tubular aggregates* Slow channel disease*
Lambert Eaton Myasthenic syndrome (LEMS) ‘Localized’ myasthenia
Ocular myasthenia gravis
Dropped head syndrome
Seronegative myasthenia
Low affinity antibody myasthenia gravis Other antibodies?
* Described also with adult onset
presents with dropped head syndrome due to weakness localized to the extensor neck muscles. This rare clinical phenotype may also be the presentation form of ALS, late onset mitochondrial myopathy [16], and focal myositis [17]. Electrophysiological evaluation of proximal nerve- muscle units in dropped head syndrome may show decre- ment, suggesting myasthenia but not making it a definite diagnosis.
Lambert Eaton myasthenic syndrome (LEMS) has many feature of fatigue and weakness that are common for all NMJ diseases. It can be recognized clinically by the loss of reflexes and by the autonomic system involvement. Repetitive stimulations at low frequency may show a decrement but at high (tetanic) frequencies (or by other methods), the typical increment may emerge and point toward the right diagnosis and the need for different immune testing (antibodies against voltage gated calcium channels).
Most patients with congenital myasthenic syndromes (CMS) due to various hereditary defects in genes involved in the NMJ structure and function present during infancy or early childhood. Thus, the search for the cause of sero- negative myasthenia in childhood should start with molecular testing. However, some patients with CMS may present later in childhood, and even early adulthood, and pose a diagnostic challenge. Such patients have no abnor- mal auto-antibodies in their serum and could be errone- ously classified as acquired seronegative myasthenia. Specific features of certain CMS conditions may


immediately point toward the neuromuscular protein that is mutated in these patients (see for review [18]). Those CMS conditions that may present later in childhood include: the limb girdle myasthenia due to DOK7 mutations [19, 20]; CMS with COLQ defects [21]; limb girdle myasthenia with tubular aggregates and no DOK7 mutations [19 and Lochmuller, personal communication]. CMS with rapsin mutations [22], and with mutations in Epsilon subunit [23] usually become apparent in the first decade of life. How- ever, at least three conditions may manifest only in ado- lescence and mimic the sporadic seronegative myasthenia: CMS with rapsin mutations [22], dominantly inherited slow channel disease [24] and limb girdle myasthenia with DOK7 mutations [21]. Symptoms that could be traced to early life (e.g. respiratory difficulties, even transient, at infancy), skeletal and facial deformities and a non-pro- gressive nature of the disease may be clues to the need for CMS testing in an apparent seronegative generalized myasthenia.
Immunology
It should first be noted that in some seropositive myas- thenic patients, even those with a generalized disease, the abnormal anti-AChR antibodies are not detected in the serum by a first test that is performed close to disease onset. Repeated tests should be done within the first months in such patients, especially when the clinical features are strongly indicative of classical MG. The reason for this ‘late’ appearance of antibodies is not clear. With the latest findings of low affinity antibodies (see below) it is possible that some of these patients had initially low affinity anti- bodies before their antibody levels rose to the range that could be detected by routine testing.
It was suggested that other forms of antibodies, e.g. blocking antibodies, which impair NMJ function but do not cause their destruction (or do so at very low level) may lead to myasthenic disease and, if undetected, may be the cause of seronegative myasthenia. Seronegative myasthenia could theoretically be the result of auto-antibodies against another, yet unidentified, NMJ protein, and this may still be true for a small fraction of these patients.
As described above, the clinical features of the sero- negative myasthenia patients more closely resembled those of the classical AChR antibody mediated disease than those with the anti-MuSK mediated myasthenia. This led to a hypothetical explanation: the antibodies against AChR in such patients have low levels or have low affinity for the receptor, and thus the routine tests cannot detect them. This hypothesis was confirmed in a recent study from Angela Vincent’s group in Oxford [9]. In this seminal work, the authors have aggregated AChR on cell surfaces in vitro and
tested the serum samples of their seronegative myasthenic patients for the presence of antibodies bound to the artifi- cially clustered system, using florescent labeled anti-human IgG antibodies. This newly devised test showed that about 2/3 of the seronegative patients had low affinity anti-AChR auto-antibodies in their serum. These antibodies were fur- ther determined to be complement-fixing IgG1 antibodies, which are presumed to be pathogenic by binding to extra cellular domains and probably causing lysis of the receptor. Also, these authors have shown in an earlier paper [25] that in such seronegative patients, thymus tissue (when avail- able) had histological changes similar to those of typical seropositive MG, unlike the normal thymus in the vast majority of patients with anti-MuSK myasthenia. This observation further advances the notion that an autoim- mune disease is active in double seronegative myasthenia.
Conclusions
The new immunological findings in patients with sero- negative myasthenia support the clinical impression that many of them have an autoimmune disorder which is very similar to the classical anti-AChR antibody mediated MG. Is this the end of seronegative myasthenia as one editorial comment suggested (‘Seronegative’ myasthenia is no longer seronegative [26])? Would a better designed test using the principles shown by the Oxford group eliminate the entity ‘‘seronegative myasthenia’’ from our diagnostic list? This remains to be seen, as one should remember that even in the Oxford series not all seronegative patients had low affinity antibodies. About 1/3 of those still await a better test or another explanation. It is still possible that in some of these patients the low affinity antibodies may not be the primary pathogenic cause of the disease. The importance of the findings of low affinity antibodies is in the recognition that AChR antibody-mediated MG may have different mechanisms. It also lends support for the use of some of the more costly treatments of myasthenia in these patients, as now plasmapheresis (and possibly IVIG) has a scientific basis for its application to seronegative myasthenic patients. A more complicated question is whether to do thymectomy in seronegative MG, since the issue of thymectomy has not been fully settled even in seropositive MG. It is the opinion of the reviewer that with current knowledge about this ‘new’ disorder, it should be considered only in seronegative patients with clearly enlarged thymus. A routine thymectomy policy (which may be different from center to center) may not be appli- cable for this low affinity antibody mediated MG.
Since a test for low affinity antibodies may take time to develop and may not be available to many treating physi- cians, many patients with seronegative myasthenia are still

presented to the clinic and no single agreed-upon policy for their management is yet available.
References
1. Evoli A, Tonali P, Padua L et al (2003) Clinical correlates with anti-MuSK antibodies in generalized seronegative myasthenia gravis. Brain 126:2304–2311
2. Vincent A, Leite MI (2005) Neuromuscular junction autoimmune disease: muscle specific kinase antibodies and treatments for myasthenia gravis. Curr Opin Neurol 18:519–525
3. Vincent A, McConville J, Farrugia ME, Newsome-Davis J (2004) Seronegative myasthenia gravis. Sem Neurol 24:125–133
4. Zhou L, McConville J, Chaudhry V et al (2004) Clinical com- parison of muscle-specific tyrosine kinase (MuSK) antibody- positive and–negative myasthenic patients. Muscle Nerve 30:55–60
5. Lavrnic D, Losen M, Vujic A et al (2005) The features of myasthenia gravis with antibodies to MuSK. J Neurol Neurosurg Psychiat 76:1099–1102
6. Nemoto Y, Kuwabara S, Misawa S et al (2005) Pattern and severity of neuromuscular transmission failure in seronegative myasthenia gravis. J Neurol Neurosurg Psychiat 76:714–718
7. Romi F, Aarli JA, Gilhus NE (2005) Seronegative myasthenia gravis: disease severity and prognosis. Eur J Neurol 12:413–418
8. Padua L, Tonali P, Aprile I et al (2006) Seronegative myasthenia gravis: comparison of neurophysiological picture in MuSK? and
MUSK- patients. Eur J Neurol 13:273–276
9. Leite MI, Jacob S, Viegas S et al (2008) IgGI antibodies to
acetylcholine receptors in ‘seronegative’ myasthenia gravis.
Brain 131:1940–1952
10. Sanders DB, Stalberg EV (1996) AAEM minimonograph #25:
single fiber electromyography. Muscle Nerve 19:1069–1083
11. Denys EH, Norris FH (1979) Amyotrophic lateral sclerosis. Impairment of neuromuscular transmission. Arch Neurol
36:202–205
12. Stalberg E, Schwartz MS, Trontelj JV (1975) Single fiber elec-
tromyography in various processes affecting the anterior horn cell. J Neurol Sci 24:403–415
13. Vasilescu C, Bucur G, Petrovici A, Florescu A (1978) Myas- thenia in patients with dermatomyositis: clinical, electrophysio- logical and ultrastructural studies. J Neurol Sci 38:129–144
14. Krendel DA, Sanders DB, Massey JM (1987) Single fiber elec- tromyography in chronic progressive external ophthalmoplegia. Muscle Nerve 10:299–302
15. Cruz-Martinez A, Arpa J, Santiago S et al (2004) Single fiber electromyography (SFEMG) in mitochondrial diseases (MD). Electromyogr Clin Neurophysiol 44:35–38
16. Johnson W, Karpati G, Carpenter S et al (1995) Late-onset mitochondrial myopathy. Ann Neurol 37:16–23
17. Biran I, Cohen O, Diment J et al (1999) Focal, steroid responsive myositis causing dropped head syndrome. Muscle Nerve 22:769–771
18. Schara U, Lochmuller H (2008) Therapeutic strategies in con- genital myasthenic syndromes. Neurotherapeutics 5:542–547
19. Palace J, Lashley D, Newsom-Davis J et al (2007) Clinical fea-
tures of the DOK7 neuromuscular junction synaptopathy. Brain
130:1507–1515
20. Muller JS, Herczegfalvi A, Vilchez JJ et al (2007) Phenotypical
spectrum of DOK7 mutations in congenital myasthenic syn-
dromes. Brain 130:1497–1506
21. Muller JS, Petrova S, Kiefer R et al (2004) Synaptic congenital
myasthenic syndrome in three patients due to a novel missense mutation (Taa1A) of the COLQ gene. Neuropedatrics 35:183–189
22. Burke G, Cossins J, Maxwell S et al (2003) Rapsyn mutations in hereditary myasthenia:distinct early- and late-onset phenotypes. Neurology 61:826–828
23. Abicht A, Stucka R, Karcagi V et al (1999) A common mutation (epsilon 1267delG) in congenital myasthenic patients of Gypsy ethnic origin. Neurology 53:1564–1569
24. Chauplannaz G, Bady B (1994) Hereditary myasthenic syn- dromes with late onset. Value of electrophysiological tests. Rev Neurol 150:142–148
25. Leite MI, Strobel P, Jones M et al (2005) Fewer thymic changes in MuSK antibody-positive than in MuSK antibody-negative MG. Ann Neurol 57:448–448
26. Lindstrom J (2008) ‘Seronegative’ myasthenia gravis is no longer seronegative. Brain 131:1684–1685

More links on LRP4 as confirmed 3rd cause of MG:

Press release: http://news.gru.edu/archives/10267
PubMed abstract of research ("Antibodies against low-density lipoprotein receptor-related protein 4 induce myasthenia gravis.", Journal of Clinical Investigations, 2013 Nov 8): http://www.ncbi.nlm.nih.gov/pubmed/24200689

another link from strizzlow re LPR4:

http://www.sciencecodex.com/new_cause_found_for_muscleweakening_disease_myasthenia_gravis-122755